TW202417504A - Anti-steap1 antigen-binding molecules and uses thereof - Google Patents

Abstract

The present invention provides monospecific and multispecific antigen-binding molecules and compositions thereof. The invention also features methods of treating a disease or disorder, such as cancer, methods of inhibiting tumor growth, kits for use with the one or more methods.

Description

Translated fromChinese

抗　STEAP1　抗原結合分子及其用途Anti-STEAP1 antigen binding molecules and their uses

本發明涉及與 STEAP1 結合的抗原結合分子，包括單特異性及多特異性抗體、其組成物，以及治療疾病諸如癌症之方法。The present invention relates to antigen binding molecules that bind to STEAP1, including monospecific and multispecific antibodies, compositions thereof, and methods for treating diseases such as cancer.

癌症為在遺傳、表型及病理位準上演變的異質性疾病。目前的療法包括化學療法、放射療法、手術、激素療法、靶向療法、免療療法及乾細胞移植。靶向療法及免疫療法在媒介有效毒殺腫瘤細胞方面顯示出前景。然而，腫瘤內異質性、癌症亞型及導致獲得性耐藥的突變的存在給標靶選擇帶來挑戰。仍然需要開發識別在不同癌症亞型及那些藉由突變獲得耐藥性的癌症亞型中共享的一種或多種標靶的治療劑。Cancer is a heterogeneous disease that evolves genetically, phenotypically, and pathologically. Current treatments include chemotherapy, radiation therapy, surgery, hormonal therapy, targeted therapy, immunotherapy, and stem cell transplantation. Targeted therapy and immunotherapy show promise in mediating effective killing of tumor cells. However, the presence of intratumor heterogeneity, cancer subtypes, and mutations that lead to acquired resistance present challenges for target selection. There remains a need to develop therapeutics that recognize one or more targets shared among different cancer subtypes and those cancer subtypes that acquire resistance through mutations.

本文所述之本發明的至少一個態樣涉及一種多特異性抗原結合分子，該多特異性抗原結合分子包含：(A) 第一抗原結合域，其與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含選自 SEQ ID NO: 7、17 至 25、30 至 34、38 及 68 的 VH 序列之 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含選自 SEQ ID NO: 8、26 至 29、39 及 69 的 VL 序列之 CDR-L1、CDR-L2 及 CDR-L3；以及 (B) 第二抗原結合域，其與 T 細胞受體結合。At least one aspect of the invention described herein relates to a multispecific antigen-binding molecule comprising: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3 of a VH sequence selected from SEQ ID NOs: 7, 17 to 25, 30 to 34, 38 and 68; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3 of a VL sequence selected from SEQ ID NOs: 8, 26 to 29, 39 and 69; and (B) a second antigen-binding domain that binds to a T cell receptor.

本文所述之本發明的至少另一態樣涉及一種多特異性抗原結合分子，該多特異性抗原結合分子包含：(A) 第一抗原結合域，其與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3；以及 (B) 第二抗原結合域，其與 T 細胞受體結合；其中： CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)；其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)；其中 Xaa³為 Asp (D) 或 Ser (S)；Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)；Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)；其中 Xaa⁷為 Phe (F) 或 Tyr (Y)；Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。At least another aspect of the invention described herein relates to a multispecific antigen-binding molecule comprising: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; and (B) a second antigen-binding domain that binds to a T cell receptor; wherein: CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Ser (S); Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (N); and Xaa⁶ is Ser (S) or Tyr (Y); CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，CDR-H1 包含 SEQ ID NO: 10、1 或 9 之胺基酸序列。In some embodiments, CDR-H1 comprises the amino acid sequence of SEQ ID NO: 10, 1 or 9.

在一些實施例中，CDR-H2 包含 SEQ ID NO: 2、11、12 或 13 之胺基酸序列。In some embodiments, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 2, 11, 12 or 13.

在一些實施例中，CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列。In some embodiments, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 16, 3, 14 or 15.

在一些實施例中，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列；CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列；CDR-H3，其包含 SEQ ID NO: 16 之胺基酸序列；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；以及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。In some embodiments, the first antigen-binding domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 16; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 1 之胺基酸序列；CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；以及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。In some embodiments, the first antigen-binding domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列；CDR-H2，其包含 SEQ ID NO: 11 之胺基酸序列；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；以及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。In some embodiments, the first antigen-binding domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列；CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；以及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。In some embodiments, the first antigen-binding domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列；CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列；CDR-H3，其包含 SEQ ID NO: 14 之胺基酸序列；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；以及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。In some embodiments, the first antigen-binding domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列；CDR-H2，其包含 SEQ ID NO: 13 之胺基酸序列；CDR-H3，其包含 SEQ ID NO: 15 之胺基酸序列；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；以及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。In some embodiments, the first antigen-binding domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，多特異性抗原結合分子為人源化抗體。In some embodiments, the multispecific antigen-binding molecule is a humanized antibody.

在一些實施例中，第一抗原結合域及該第二抗原結合域各自獨立地包含 IgG 骨架區，視情況為 IgG1 或 IgG4 骨架區。In some embodiments, the first antigen-binding domain and the second antigen-binding domain each independently comprise an IgG framework region, optionally an IgG1 or IgG4 framework region.

在一些實施例中，第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列。In some embodiments, the first antigen binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 7, 17 to 25, 30 to 34, 38 or 68.

在一些實施例中，第一抗原結合域包含：抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 8、26 至 29、39 或 69 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the first antigen binding domain comprises: an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 8, 26 to 29, 39 or 69.

在一些實施例中，第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 68 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 69 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 68; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 69.

在一些實施例中，第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 34 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 34; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.

在一些實施例中，第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 30 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 30; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.

在一些實施例中，第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 31 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 31; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.

在一些實施例中，第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 32 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 32; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.

在一些實施例中，第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 33 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 33; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.

在一些實施例中，T 細胞受體為分化簇 3 (CD3)。In some embodiments, the T cell receptor is cluster of differentiation 3 (CD3).

在一些實施例中，與 CD3 結合的第二抗原結合域包含含有 CDR-H1、CDR-H2 及 CDR-H3 的抗 CD3 重鏈可變區 (VH) 及含有 CDR-L1、CDR-L2 及 CDR-L3 的抗 CD3 輕鏈可變區 (VL)，其中該 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3 分別包含 SEQ ID NO: 48 至 53 之序列。In some embodiments, the second antigen-binding domain that binds to CD3 comprises an anti-CD3 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3 and an anti-CD3 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 48 to 53, respectively.

在一些實施例中，與 CD3 結合的第二抗原結合域包含：抗 CD3 重鏈可變區，其包含與 SEQ ID NO: 54 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及與 SEQ ID NO: 55 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the second antigen binding domain that binds to CD3 comprises: an anti-CD3 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 54; and a VL sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 55.

在一些實施例中，與 CD3 結合的第二抗原結合域包含含有 CDR-H1、CDR-H2 及 CDR-H3 的抗 CD3 重鏈可變區 (VH) 及含有 CDR-L1、CDR-L2 及 CDR-L3 的抗 CD3 輕鏈可變區 (VL)，其中該 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3 分別包含 SEQ ID NO: 40 至 45 之序列。In some embodiments, the second antigen-binding domain that binds to CD3 comprises an anti-CD3 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3 and an anti-CD3 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 40 to 45, respectively.

在一些實施例中，與 CD3 結合的第二抗原結合域包含：抗 CD3 重鏈可變區，其包含與 SEQ ID NO: 46 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 CD3 輕鏈可變區，其包含與 SEQ ID NO: 47 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the second antigen binding domain that binds to CD3 comprises: an anti-CD3 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 46; and an anti-CD3 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 47.

在一些實施例中，多特異性抗原結合分子包含一個或多個重鏈恆定域，其中該一個或多個重鏈恆定域係選自第一 CH1 (CH1₁) 域、第一 CH2 (CH2₁) 域、第一 CH3 (CH3₁) 域、第二 CH1 (CH1₂) 域、第二 CH2 (CH2₂) 域及第二 CH3 (CH3₂) 域。In some embodiments, the multispecific antigen-binding molecule comprises one or more heavy chain constitutive domains, wherein the one or more heavy chain constitutive domains are selected from a first CH1 (_CH11 ) domain, a first CH2 (_CH21 ) domain, a first CH3 (_CH31 ) domain, a second CH1 (_CH12 ) domain, a second CH2 (_CH22 ) domain, and a second CH3 (_CH32 ) domain.

在一些實施例中，該一個或多個重鏈恆定域中之至少一個與另一個重鏈恆定域配對。In some embodiments, at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain.

在一些實施例中，CH3₁及 CH3₂域各自包含一個隆凸或腔窩，且其中 CH3₁域中的隆凸或腔窩分別位於 CH3₂域的腔窩或隆凸中。In some embodiments, the_CH3.1 and_CH3.2 domains each comprise a protuberance or a cavity, and wherein the protuberance or the cavity in the_CH3.1 domain is located in the cavity or the protuberance of the_CH3.2 domain, respectively.

在一些實施例中，CH3₁及 CH3₂域在該隆凸與腔窩之間的界面處相接。In some embodiments, the_CH31 and_CH32 domains meet at the interface between the protuberance and the cavity.

在一些實施例中，CH2₁及 CH2₂域各包含隆凸或腔窩，且其中該 CH2₁域中的該隆凸或腔窩分別可定位於該 CH2₂域中的該腔窩或隆凸中。In some embodiments, the_CH2.1 and_CH2.2 domains each comprise a protuberance or a cavity, and wherein the protuberance or the cavity in the_CH2.1 domain can be positioned in the cavity or the protuberance in the_CH2.2 domain, respectively.

在一些實施例中，CH2₁及 CH2₂域在該隆凸與腔窩之間的界面處相接。In some embodiments, the_CH21 and_CH22 domains meet at the interface between the protuberance and the cavity.

在一些實施例中，第一抗原結合域包含與 SEQ ID NO: 83 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 重鏈恆定區序列及與 SEQ ID NO: 82 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 輕鏈恆定區序列；且第二抗原結合域包含與 SEQ ID NO: 85 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 CD 重鏈恆定區序列及與 SEQ ID NO: 84 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 CD 輕鏈恆定區序列。In some embodiments, the first antigen binding domain comprises an anti-STEAP1 heavy chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 83 and an anti-STEAP1 light chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 82; and the second antigen binding domain comprises an anti-CD heavy chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 85 and an anti-CD light chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 84.

在一些實施例中，第一抗原結合域包含與 SEQ ID NO: 85 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 重鏈恆定區序列及與 SEQ ID NO: 84 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 輕鏈恆定區序列；且第二抗原結合域包含與 SEQ ID NO: 83 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 CD3 重鏈恆定區序列及與 SEQ ID NO: 82 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 CD3 輕鏈恆定區序列。In some embodiments, the first antigen binding domain comprises an anti-STEAP1 heavy chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 85 and an anti-STEAP1 light chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 84; and the second antigen binding domain comprises an anti-CD3 heavy chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 83 and an anti-CD3 light chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 82.

在一些實施例中，多特異性抗原結合分子包含含有 SEQ ID NO: 73 的抗 STEAP1 重鏈、含有 SEQ ID NO: 72 的抗 STEAP1 輕鏈、含有 SEQ ID NO: 79 的抗 CD3 重鏈及含有 SEQ ID NO: 78 的抗 CD3 輕鏈。In some embodiments, the multispecific antigen-binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 73, an anti-STEAP1 light chain comprising SEQ ID NO: 72, an anti-CD3 heavy chain comprising SEQ ID NO: 79, and an anti-CD3 light chain comprising SEQ ID NO: 78.

在一些實施例中，多特異性抗原結合分子包含含有 SEQ ID NO: 73 的抗 STEAP1 重鏈、含有 SEQ ID NO: 72 的抗 STEAP1 輕鏈、含有 SEQ ID NO: 81 的抗 CD3 重鏈及含有 SEQ ID NO: 80 的抗 CD3 輕鏈。In some embodiments, the multispecific antigen-binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 73, an anti-STEAP1 light chain comprising SEQ ID NO: 72, an anti-CD3 heavy chain comprising SEQ ID NO: 81, and an anti-CD3 light chain comprising SEQ ID NO: 80.

在一些實施例中，多特異性抗原結合分子包含含有 SEQ ID NO: 71 的抗 STEAP1 重鏈、含有 SEQ ID NO: 70 的抗 STEAP1 輕鏈、含有 SEQ ID NO: 79 的抗 CD3 重鏈及含有 SEQ ID NO: 78 的抗 CD3 輕鏈。In some embodiments, the multispecific antigen-binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 71, an anti-STEAP1 light chain comprising SEQ ID NO: 70, an anti-CD3 heavy chain comprising SEQ ID NO: 79, and an anti-CD3 light chain comprising SEQ ID NO: 78.

在一些實施例中，多特異性抗原結合分子包含含有 SEQ ID NO: 71 的抗 STEAP1 重鏈、含有 SEQ ID NO: 70 的抗 STEAP1 輕鏈、含有 SEQ ID NO: 81 的抗 CD3 重鏈及含有 SEQ ID NO: 80 的抗 CD3 輕鏈。In some embodiments, the multispecific antigen-binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 71, an anti-STEAP1 light chain comprising SEQ ID NO: 70, an anti-CD3 heavy chain comprising SEQ ID NO: 81, and an anti-CD3 light chain comprising SEQ ID NO: 80.

在一些實施例中，抗原結合分子為單鏈 Fv (scFv)、三特異性 (Fab3)、雙特異性 (Fab2)、雙功能抗體 (diabody) ((VL-VH)2 或 (VH-VL)2)、三功能抗體 (triabody) (三價)、四抗體 (tetrabody) (四價)、微抗體 (minibody) ((scFV-CH)2)、雙特異性單鏈 Fv (雙-scFv)、IgGδCH2、scFv-Fc 或 (scFv)2-Fc。In some embodiments, the antigen binding molecule is a single chain Fv (scFv), a trispecific (Fab3), a bispecific (Fab2), a diabody ((VL-VH)2 or (VH-VL)2), a triabody (trivalent), a tetrabody (tetravalent), a minibody ((scFV-CH)2), a bispecific single chain Fv (bi-scFv), IgGδCH2, scFv-Fc or (scFv)2-Fc.

在一些實施例中，多特異性抗原結合分子為多特異性抗體，較佳為雙特異性或三特異性抗體。In some embodiments, the multispecific antigen-binding molecule is a multispecific antibody, preferably a bispecific or trispecific antibody.

在一些實施例中，多特異性抗原結合分子進一步包含與腫瘤相關抗原結合的第三抗原結合域。在一些實施例中，腫瘤相關抗原為在前列腺癌細胞或尤文氏肉瘤 (Ewing sarcoma) 上表現的受體。在一些實施例中，腫瘤相關抗原為前列腺特異性膜抗原 (PSMA)、STEAP2、前列腺幹細胞抗原 (PSCA)、上皮細胞黏著分子 (EpCAM)、前列腺特異性抗原 (PSA)、前列腺酸性磷酸酶 (PAP) 或 HBA-71。In some embodiments, the multispecific antigen binding molecule further comprises a third antigen binding domain that binds to a tumor-associated antigen. In some embodiments, the tumor-associated antigen is a receptor expressed on prostate cancer cells or Ewing sarcoma. In some embodiments, the tumor-associated antigen is prostate-specific membrane antigen (PSMA), STEAP2, prostate stem cell antigen (PSCA), epithelial cell adhesion molecule (EpCAM), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP) or HBA-71.

在一些實施例中，多特異性抗原結合分子與人 STEAP1、石蟹獼猴 STEAP1 或其組合結合，較佳地其中該多特異性抗原結合分子與 SEQ ID NO: 65 之人 STEAP1 結合。In some embodiments, the multispecific antigen-binding molecule binds to human STEAP1, red macaque STEAP1, or a combination thereof, preferably wherein the multispecific antigen-binding molecule binds to human STEAP1 of SEQ ID NO: 65.

在一些實施例中，第一抗原結合域與選自 STEAP1 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的至少一個、至少兩個、至少三個、至少四個或至少五個殘基結合，其中殘基位置 101、102、103、195、198、202 及 281 對應於 SEQ ID NO: 65 中所示之位置 101、102、103、195、198、202 及 281。在一些實施例中，第一抗原結合域與 STEAP1 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 結合。在一些實施例中，第一抗原結合域之重鏈可變區與選自 STEAP1 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的至少一個、至少兩個、至少三個、至少四個或至少五個殘基形成氫鍵。在一些實施例中，第一抗原結合域之重鏈可變區的選自 Leu56、Ser73、Asn74、Gly101、Tyr103 及 Tyr107 的至少一個、至少兩個、至少三個、至少四個或至少五個選殘基與選自 STEAP1 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的至少一個、至少兩個、至少三個、至少四個或至少五個殘基形成氫鍵，其中第一抗原結合域之重鏈可變區的殘基位置對應於 SEQ ID NO: 18 中所示之位置 56、73、74、101、103 及 107。In some embodiments, the first antigen-binding domain binds to at least one, at least two, at least three, at least four, or at least five residues selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202, and Lys281 of STEAP1, wherein residue positions 101, 102, 103, 195, 198, 202, and 281 correspond to positions 101, 102, 103, 195, 198, 202, and 281 as shown in SEQ ID NO: 65. In some embodiments, the first antigen-binding domain binds to Ser101, His102, Gln103, Trp195, Gln198, Gln202, and Lys281 of STEAP1. In some embodiments, the heavy chain variable region of the first antigen binding domain forms a hydrogen bond with at least one, at least two, at least three, at least four, or at least five residues selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202, and Lys281 of STEAP1. In some embodiments, at least one, at least two, at least three, at least four or at least five residues selected from Leu56, Ser73, Asn74, Gly101, Tyr103 and Tyr107 of the heavy chain variable region of the first antigen binding domain form hydrogen bonds with at least one, at least two, at least three, at least four or at least five residues selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202 and Lys281 of STEAP1, wherein the residue positions of the heavy chain variable region of the first antigen binding domain correspond to positions 56, 73, 74, 101, 103 and 107 shown in SEQ ID NO: 18.

在一些實施例中，第一抗原結合域與選自 STEAP1 之 Gln201、Gln202、Asn203 及 Lys204 的至少一個、至少兩個或至少三個殘基結合，其中殘基位置 201、202、203 及 204 對應於 SEQ ID NO: 65 中所示之位置 201、202、203 及 204。在一些實施例中，第一抗原結合域與 STEAP1 之 Gln201、Gln202、Asn203 及 Lys204 結合。在一些實施例中，第一抗原結合域之輕鏈可變區與選自 STEAP1 之 Gln201 及 Gln202 的至少一個殘基形成氫鍵。在一些實施例中，第一抗原結合域之輕鏈可變區的選自 Tyr53 及 Tyr54 的至少一個殘基與選自 Gln201 及 Gln202 的至少一個殘基形成氫鍵，其中第一抗原結合域之輕鏈可變區的殘基位置對應於 SEQ ID NO: 18 中所示之位置 53 及 54。在一些實施例中，第一抗原結合域之輕鏈可變區的殘基與選自 STEAP1 之 Asn203 及 Lys204 的至少一個殘基形成凡得瓦交互作用。In some embodiments, the first antigen-binding domain binds to at least one, at least two, or at least three residues selected from Gln201, Gln202, Asn203, and Lys204 of STEAP1, wherein residue positions 201, 202, 203, and 204 correspond to positions 201, 202, 203, and 204 as shown in SEQ ID NO: 65. In some embodiments, the first antigen-binding domain binds to Gln201, Gln202, Asn203, and Lys204 of STEAP1. In some embodiments, the light chain variable region of the first antigen-binding domain forms a hydrogen bond with at least one residue selected from Gln201 and Gln202 of STEAP1. In some embodiments, at least one residue selected from Tyr53 and Tyr54 of the light chain variable region of the first antigen binding domain forms a hydrogen bond with at least one residue selected from Gln201 and Gln202, wherein the positions of the residues in the light chain variable region of the first antigen binding domain correspond to positions 53 and 54 as shown in SEQ ID NO: 18. In some embodiments, the residues in the light chain variable region of the first antigen binding domain form a van der Waals interaction with at least one residue selected from Asn203 and Lys204 of STEAP1.

在一些實施例中，多特異性抗原結合分子具有約 11、11.5、12、12.6、13、13.5、15、18、20、23.4、25、27.9、29.1 或 30 µg/mL 之 C_max。In some embodiments, the multispecific antigen-binding molecule has a_Cmax of about 11, 11.5, 12, 12.6, 13, 13.5, 15, 18, 20, 23.4, 25, 27.9, 29.1 or 30 μg/mL.

在一些實施例中，多特異性抗原結合分子具有約 11 µg/mL 至約 30 µg/mL 之 C_max。In some embodiments, the multispecific antigen-binding molecule has a C_max of about 11 μg/mL to about 30 μg/mL.

在一些實施例中，多特異性抗原結合分子具有約 20 µg/mL 至約 28 µg/mL 之 C_max。In some embodiments, the multispecific antigen-binding molecule has a_Cmax of about 20 µg/mL to about 28 µg/mL.

在一些實施例中，多特異性抗原結合分子具有約 24 µg/mL 之 C_max。In some embodiments, the multispecific antigen-binding molecule has a C_max of about 24 μg/mL.

在一些實施例中，多特異性抗原結合分子具有約 0.6、0.56、0.5、0.45、0.4、0.35、0.3、0.25、0.2、0.15、0.1、0.09、0.05 或更低之 EC₅₀。In some embodiments, the multispecific antigen-binding molecule has an_EC50 of about 0.6, 0.56, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.09, 0.05 or less.

在一些實施例中，多特異性抗原結合分子具有約 0.05 至約 0.8 之 EC₅₀。In some embodiments, the multispecific antigen-binding molecule has an_EC50 of about 0.05 to about 0.8.

在一些實施例中，EC₅₀係用人 CD8+ T 細胞及表現 STEAP1 之 LNCaP-X1.2 細胞在細胞毒殺測定中在 72 小時所判定，且該 EC₅₀為約 0.05 至約 0.4。In some embodiments,_theEC50 is determined in a cytotoxicity assay using human CD8+ T cells and LNCaP-X1.2 cells expressing STEAP1 at 72 hours and is about 0.05 to about 0.4.

在一些實施例中，EC₅₀為約 0.08 或約 0.3。In some embodiments, the_EC50 is about 0.08 or about 0.3.

在一些實施例中，EC₅₀係用人 CD8+ T 細胞及表現 STEAP1 之 LNCaPX1.2KO3-13 細胞在細胞毒殺測定中在 72 小時所判定，且該 EC₅₀為約 0.1 至約 0.8。In some embodiments,_theEC50 is determined in a cytotoxicity assay using human CD8+ T cells and LNCaPX1.2KO3-13 cells expressing STEAP1 at 72 hours and is about 0.1 to about 0.8.

在一些實施例中，EC₅₀為約 0.1 或約 0.7。In some embodiments, the_EC50 is about 0.1 or about 0.7.

在一些實施例中，多特異性抗原結合分子與 STEAP1 單價結合。In some embodiments, the multispecific antigen-binding molecule monovalently binds to STEAP1.

本文所述之本發明的至少另一態樣涉及一種與 STEAP1 結合的抗原結合分子，該抗原結合分子包含：抗 STEAP1 重鏈可變區 (VH)，其包含選自 SEQ ID NO: 7、17 至 25、30 至 34、38 及 68 的 VH 序列之 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含選自 SEQ ID NO: 8、26 至 29、39 及 69 的 VL 序列之 CDR-L1、CDR-L2 及 CDR-L3。At least another aspect of the invention described herein relates to an antigen-binding molecule that binds to STEAP1, the antigen-binding molecule comprising: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3 of a VH sequence selected from SEQ ID NOs: 7, 17 to 25, 30 to 34, 38, and 68; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3 of a VL sequence selected from SEQ ID NOs: 8, 26 to 29, 39, and 69.

本文所述之本發明的至少另一態樣涉及一種與 STEAP1 結合的抗原結合分子，該抗原結合分子包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3；其中 CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)；其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)；其中 Xaa³為 Asp (D) 或 Ser (S)；Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)；Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)；其中 Xaa⁷為 Phe (F) 或 Tyr (Y)；Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。At least another aspect of the invention described herein relates to an antigen binding molecule that binds to STEAP1, the antigen binding molecule comprising: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Asn (N); and Xaa 2 is His (H), Tyr (Y) or Phe (F). or Ser (S); Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (N); and Xaa⁶ is Ser (S) or Tyr (Y); CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，CDR-H1 包含 SEQ ID NO: 10、1 或 9 之胺基酸序列。在一些實施例中，CDR-H2 包含 SEQ ID NO: 2、11、12 或 13 之胺基酸序列。在一些實施例中，CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列。In some embodiments, CDR-H1 comprises the amino acid sequence of SEQ ID NO: 10, 1, or 9. In some embodiments, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 2, 11, 12, or 13. In some embodiments, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 16, 3, 14, or 15.

在一些實施例中，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列；CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列；CDR-H3，其包含 SEQ ID NO: 16 之胺基酸序列；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；以及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。In some embodiments, the antigen binding molecule comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 16; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 1 之胺基酸序列；CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；以及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。In some embodiments, the antigen-binding molecule comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列；CDR-H2，其包含 SEQ ID NO: 11 之胺基酸序列；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；以及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。In some embodiments, the antigen-binding molecule comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列；CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；以及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。In some embodiments, the antigen binding molecule comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列；CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列；CDR-H3，其包含 SEQ ID NO: 14 之胺基酸序列；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；以及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。In some embodiments, the antigen binding molecule comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列；CDR-H2，其包含 SEQ ID NO: 13 之胺基酸序列；CDR-H3，其包含 SEQ ID NO: 15 之胺基酸序列；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；以及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。In some embodiments, the antigen binding molecule comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，抗原結合分子為人源化抗體。In some embodiments, the antigen binding molecule is a humanized antibody.

在一些實施例中，抗原結合分子包含 IgG 骨架區，視情況為 IgG1 骨架區。In some embodiments, the antigen binding molecule comprises an IgG framework region, optionally an IgG1 framework region.

在一些實施例中，抗原結合分子包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列。In some embodiments, the antigen binding molecule comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 7, 17 to 25, 30 to 34, 38 or 68.

在一些實施例中，抗原結合分子包含：抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 8、26 至 29、39 或 69 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the antigen binding molecule comprises: an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 8, 26 to 29, 39 or 69.

在一些實施例中，抗原結合分子包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 68 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 69 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the antigen binding molecule comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 68; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 69.

在一些實施例中，抗原結合分子包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 34 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the antigen binding molecule comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 34; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.

在一些實施例中，抗原結合分子包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 30 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the antigen binding molecule comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 30; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.

在一些實施例中，抗原結合分子包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 31 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the antigen binding molecule comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 31; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.

在一些實施例中，抗原結合分子包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 32 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the antigen binding molecule comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 32; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.

在一些實施例中，抗原結合分子包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 33 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the antigen binding molecule comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 33; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.

在一些實施例中，抗原結合分子包含與 SEQ ID NO: 83 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 重鏈恆定區序列及與 SEQ ID NO: 82 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 輕鏈恆定區序列。In some embodiments, the antigen binding molecule comprises an anti-STEAP1 heavy chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 83 and an anti-STEAP1 light chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 82.

在一些實施例中，抗原結合分子包含與 SEQ ID NO: 85 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 重鏈恆定區序列及與 SEQ ID NO: 84 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 輕鏈恆定區序列。In some embodiments, the antigen binding molecule comprises an anti-STEAP1 heavy chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 85 and an anti-STEAP1 light chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 84.

在一些實施例中，抗原結合分子包含含有 SEQ ID NO: 73 的抗 STEAP1 重鏈及含有 SEQ ID NO: 72 的抗 STEAP1 輕鏈。在一些實施例中，抗原結合分子包含含有 SEQ ID NO: 71 的抗 STEAP1 重鏈及含有 SEQ ID NO: 70 的抗 STEAP1 輕鏈。In some embodiments, the antigen binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 73 and an anti-STEAP1 light chain comprising SEQ ID NO: 72. In some embodiments, the antigen binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 71 and an anti-STEAP1 light chain comprising SEQ ID NO: 70.

在一些實施例中，抗原結合分子為全長抗體或其片段。In some embodiments, the antigen binding molecule is a full-length antibody or a fragment thereof.

在一些實施例中，抗原結合分子為 Fab、Fab'、F(ab')2、Fv、Fd、單鏈 Fv (scFv)、三特異性 (Fab3)、雙特異性 (Fab2)、雙功能抗體 ((VL-VH)2 或 (VH-VL)2)、三功能抗體 (三價)、四抗體 (四價)、微抗體 ((scFV-CH)2)、雙特異性單鏈 Fv (雙-scFv)、IgGδCH2、scFv-Fc 或 (scFv)2-Fc。In some embodiments, the antigen binding molecule is Fab, Fab', F(ab')2, Fv, Fd, single chain Fv (scFv), trispecific (Fab3), bispecific (Fab2), bifunctional antibody ((VL-VH)2 or (VH-VL)2), trifunctional antibody (trivalent), tetraantibody (tetravalent), miniantibody ((scFV-CH)2), bispecific single chain Fv (bi-scFv), IgGδCH2, scFv-Fc or (scFv)2-Fc.

在一些實施例中，抗原結合分子為多特異性抗體，較佳地其中該抗原結合分子為雙特異性抗體或三特異性抗體。In some embodiments, the antigen binding molecule is a multispecific antibody, preferably wherein the antigen binding molecule is a bispecific antibody or a trispecific antibody.

在一些實施例中，抗原結合分子進一步包含與 T 細胞受體結合的抗原結合域。在一些實施例中，T 細胞受體為分化簇 3 (CD3)。In some embodiments, the antigen binding molecule further comprises an antigen binding domain that binds to a T cell receptor. In some embodiments, the T cell receptor is cluster of differentiation 3 (CD3).

在一些實施例中，與 CD3 結合的抗原結合域包含含有 CDR-H1、CDR-H2 及 CDR-H3 的抗 CD3 重鏈可變區 (VH) 及含有 CDR-L1、CDR-L2 及 CDR-L3 的抗 CD3 輕鏈可變區 (VL)，其中 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3 分別包含 SEQ ID NO: 48 至 53 之胺基酸序列。In some embodiments, the antigen-binding domain that binds to CD3 comprises an anti-CD3 heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3, and an anti-CD3 light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences of SEQ ID NOs: 48 to 53, respectively.

在一些實施例中，與 CD3 結合的抗原結合域包含：抗 CD3 重鏈可變區，其包含與 SEQ ID NO: 54 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 CD3 輕鏈可變區，其包含與 SEQ ID NO: 55 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the antigen binding domain that binds to CD3 comprises: an anti-CD3 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 54; and an anti-CD3 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 55.

在一些實施例中，與 CD3 結合的抗原結合域包含含有 CDR-H1、CDR-H2 及 CDR-H3 的抗 CD3 重鏈可變區 (VH) 及含有 CDR-L1、CDR-L2 及 CDR-L3 的抗 CD3 輕鏈可變區 (VL)，其中 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3 分別包含 SEQ ID NO: 40 至 45 之胺基酸序列。In some embodiments, the antigen-binding domain that binds to CD3 comprises an anti-CD3 heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3, and an anti-CD3 light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences of SEQ ID NOs: 40 to 45, respectively.

在一些實施例中，與 CD3 結合的抗原結合域包含：抗 CD3 重鏈可變區，其包含與 SEQ ID NO: 46 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 CD3 輕鏈可變區，其包含與 SEQ ID NO: 47 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the antigen binding domain that binds to CD3 comprises: an anti-CD3 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 46; and an anti-CD3 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 47.

在一些實施例中，抗原結合分子進一步包含與腫瘤相關抗原結合的額外抗原結合域。在一些實施例中，腫瘤相關抗原為在前列腺癌細胞或尤文氏肉瘤上表現的受體。在一些實施例中，腫瘤相關抗原為前列腺特異性膜抗原 (PSMA)、STEAP2、前列腺幹細胞抗原 (PSCA)、上皮細胞黏著分子 (EpCAM)、前列腺特異性抗原 (PSA)、前列腺酸性磷酸酶 (PAP) 或 HBA-71。In some embodiments, the antigen binding molecule further comprises an additional antigen binding domain that binds to a tumor-associated antigen. In some embodiments, the tumor-associated antigen is a receptor expressed on prostate cancer cells or Ewing's sarcoma. In some embodiments, the tumor-associated antigen is prostate-specific membrane antigen (PSMA), STEAP2, prostate stem cell antigen (PSCA), epithelial cell adhesion molecule (EpCAM), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP) or HBA-71.

在一些實施例中，抗原結合分子與人 STEAP1、石蟹獼猴 STEAP1 或其組合結合，較佳地其中該抗原結合分子與 SEQ ID NO: 65 之人 STEAP1 結合。In some embodiments, the antigen binding molecule binds to human STEAP1, red macaque STEAP1, or a combination thereof, preferably wherein the antigen binding molecule binds to human STEAP1 of SEQ ID NO: 65.

在一些實施例中，抗原結合分子與選自 STEAP1 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的至少一個、至少兩個、至少三個、至少四個或至少五個殘基結合，其中殘基位置 101、102、103、195、198、202 及 281 對應於 SEQ ID NO: 65 中所示之位置 101、102、103、195、198、202 及 281。在一些實施例中，抗原結合分子與 STEAP1 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 結合。在一些實施例中，抗原結合分子之重鏈可變區與選自 STEAP1 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的至少一個、至少兩個、至少三個、至少四個或至少五個殘基形成氫鍵。在一些實施例中，抗原結合分子之重鏈可變區的選自 Leu56、Ser73、Asn74、Gly101、Tyr103 及 Tyr107 的至少一個、至少兩個、至少三個、至少四個或至少五個選殘基與選自 STEAP1 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的至少一個、至少兩個、至少三個、至少四個或至少五個殘基形成氫鍵，其中抗原結合分子之重鏈可變區的殘基位置對應於 SEQ ID NO: 18 中所示之位置 56、73、74、101、103 及 107。In some embodiments, the antigen binding molecule binds to at least one, at least two, at least three, at least four, or at least five residues selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202, and Lys281 of STEAP1, wherein residue positions 101, 102, 103, 195, 198, 202, and 281 correspond to positions 101, 102, 103, 195, 198, 202, and 281 as shown in SEQ ID NO: 65. In some embodiments, the antigen binding molecule binds to Ser101, His102, Gln103, Trp195, Gln198, Gln202, and Lys281 of STEAP1. In some embodiments, the heavy chain variable region of the antigen binding molecule forms a hydrogen bond with at least one, at least two, at least three, at least four, or at least five residues selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202, and Lys281 of STEAP1. In some embodiments, at least one, at least two, at least three, at least four or at least five residues selected from Leu56, Ser73, Asn74, Gly101, Tyr103 and Tyr107 of the heavy chain variable region of the antigen binding molecule form hydrogen bonds with at least one, at least two, at least three, at least four or at least five residues selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202 and Lys281 of STEAP1, wherein the residue positions of the heavy chain variable region of the antigen binding molecule correspond to positions 56, 73, 74, 101, 103 and 107 shown in SEQ ID NO: 18.

在一些實施例中，抗原結合分子與選自 STEAP1 之 Gln201、Gln202、Asn203 及 Lys204 的至少一個、至少兩個或至少三個殘基結合，其中殘基位置 201、202、203 及 204 對應於 SEQ ID NO: 65 中所示之位置 201、202、203 及 204。在一些實施例中，抗原結合分子與 STEAP1 之 Gln201、Gln202、Asn203 及 Lys204 結合。在一些實施例中，抗原結合分子之輕鏈可變區與選自 STEAP1 之 Gln201 及 Gln202 的至少一個殘基形成氫鍵。在一些實施例中，抗原結合分子之輕鏈可變區的選自 Tyr53 及 Tyr54 的至少一個殘基與選自 Gln201 及 Gln202 的至少一個殘基形成氫鍵，其中抗原結合分子之輕鏈可變區的殘基位置對應於 SEQ ID NO: 18 中所示之位置 53 及 54。在一些實施例中，抗原結合分子之輕鏈可變區的殘基與選自 STEAP1 之 Asn203 及 Lys204 的至少一個殘基形成凡得瓦交互作用。In some embodiments, the antigen binding molecule binds to at least one, at least two, or at least three residues selected from Gln201, Gln202, Asn203, and Lys204 of STEAP1, wherein residue positions 201, 202, 203, and 204 correspond to positions 201, 202, 203, and 204 as shown in SEQ ID NO: 65. In some embodiments, the antigen binding molecule binds to Gln201, Gln202, Asn203, and Lys204 of STEAP1. In some embodiments, the light chain variable region of the antigen binding molecule forms a hydrogen bond with at least one residue selected from Gln201 and Gln202 of STEAP1. In some embodiments, at least one residue selected from Tyr53 and Tyr54 of the light chain variable region of the antigen binding molecule forms a hydrogen bond with at least one residue selected from Gln201 and Gln202, wherein the positions of the residues in the light chain variable region of the antigen binding molecule correspond to positions 53 and 54 as shown in SEQ ID NO: 18. In some embodiments, the residues in the light chain variable region of the antigen binding molecule form a van der Waals interaction with at least one residue selected from Asn203 and Lys204 of STEAP1.

在一些實施例中，抗原結合分子與 STEAP1 單價結合。In some embodiments, the antigen binding molecule binds monovalently to STEAP1.

本文所述之本發明的至少另一態樣涉及一種抗體，該抗體包含在選自 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的一個或多個殘基處與人 STEAP1 結合的第一抗原結合域，其中殘基位置 101、102、103、195、198、202 及 281 對應於 SEQ ID NO: 65 中所示之位置 101、102、103、195、198、202 及 281。At least another aspect of the invention described herein relates to an antibody comprising a first antigen binding domain that binds to human STEAP1 at one or more residues selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202 and Lys281, wherein residue positions 101, 102, 103, 195, 198, 202 and 281 correspond to positions 101, 102, 103, 195, 198, 202 and 281 as set forth in SEQ ID NO: 65.

在一些實施例中，第一抗原結合域與選自 SEQ ID NO: 65 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的至少一個、至少兩個、至少三個、至少四個或至少五個殘基結合。在一些實施例中，第一抗原結合域與 SEQ ID NO: 65 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 結合。在一些實施例中，第一抗原結合域之重鏈可變區與選自 STEAP1 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的至少一個、至少兩個、至少三個、至少四個或至少五個殘基形成氫鍵。在一些實施例中，第一抗原結合域之重鏈可變區的選自 Leu56、Ser73、Asn74、Gly101、Tyr103 及 Tyr107 的至少一個、至少兩個、至少三個、至少四個或至少五個選殘基與選自 STEAP1 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的至少一個、至少兩個、至少三個、至少四個或至少五個殘基形成氫鍵，其中第一抗原結合域之重鏈可變區的殘基位置對應於 SEQ ID NO: 18 中所示之位置 56、73、74、101、103 及 107。In some embodiments, the first antigen-binding domain binds to at least one, at least two, at least three, at least four, or at least five residues selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202, and Lys281 of SEQ ID NO: 65. In some embodiments, the first antigen-binding domain binds to Ser101, His102, Gln103, Trp195, Gln198, Gln202, and Lys281 of SEQ ID NO: 65. In some embodiments, the heavy chain variable region of the first antigen binding domain forms a hydrogen bond with at least one, at least two, at least three, at least four, or at least five residues selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202, and Lys281 of STEAP1. In some embodiments, at least one, at least two, at least three, at least four or at least five residues selected from Leu56, Ser73, Asn74, Gly101, Tyr103 and Tyr107 of the heavy chain variable region of the first antigen binding domain form hydrogen bonds with at least one, at least two, at least three, at least four or at least five residues selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202 and Lys281 of STEAP1, wherein the residue positions of the heavy chain variable region of the first antigen binding domain correspond to positions 56, 73, 74, 101, 103 and 107 shown in SEQ ID NO: 18.

在一些實施例中，第一抗原結合域包含：抗 STEAP1 重鏈可變區 (VH)，其包含選自 SEQ ID NO: 7、17 至 25、30 至 34、38 及 68 的 VH 序列之 CDR-H1、CDR-H2 及 CDR-H3；以及抗 STEAP1 輕鏈可變區 (VL) 之三個 CDR，其包含選自 SEQ ID NO: 8、26 至 29、39 及 69 的 VL 序列之 CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3 of a VH sequence selected from SEQ ID NOs: 7, 17 to 25, 30 to 34, 38 and 68; and three CDRs of an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3 of a VL sequence selected from SEQ ID NOs: 8, 26 to 29, 39 and 69.

在一些實施例中，第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含含有與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列。In some embodiments, the first antigen binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence comprising at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 7, 17-25, 30-34, 38 or 68.

在一些實施例中，第一抗原結合域包含：抗 STEAP1 輕鏈可變區，其包含含有與 SEQ ID NO: 8、26 至 29、39 或 69 至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the first antigen binding domain comprises: an anti-STEAP1 light chain variable region comprising a VL sequence comprising at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 8, 26 to 29, 39 or 69.

在一些實施例中，抗體進一步包含與 T 細胞受體結合的第二抗原結合域。在一些實施例中，T 細胞受體為分化簇 3 (CD3)。In some embodiments, the antibody further comprises a second antigen binding domain that binds to a T cell receptor. In some embodiments, the T cell receptor is cluster of differentiation 3 (CD3).

在一些實施例中，與 CD3 結合的第二抗原結合域包含含有 CDR-H1、CDR-H2 及 CDR-H3 的抗 CD3 重鏈可變區 (VH) 及含有 CDR-L1、CDR-L2 及 CDR-L3 的抗 CD3 輕鏈可變區 (VL)，其中 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3 分別包含 SEQ ID NO: 48 至 53 之胺基酸序列。In some embodiments, the second antigen-binding domain that binds to CD3 comprises an anti-CD3 heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3, and an anti-CD3 light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences of SEQ ID NOs: 48 to 53, respectively.

在一些實施例中，與 CD3 結合的第二抗原結合域包含：抗 CD3 重鏈可變區，其包含與 SEQ ID NO: 54 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 CD3 輕鏈可變區，其包含與 SEQ ID NO: 55 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the second antigen binding domain that binds to CD3 comprises: an anti-CD3 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 54; and an anti-CD3 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 55.

在一些實施例中，與 CD3 結合的第二抗原結合域包含含有 CDR-H1、CDR-H2 及 CDR-H3 的抗 CD3 重鏈可變區 (VH) 及含有 CDR-L1、CDR-L2 及 CDR-L3 的抗 CD3 輕鏈可變區 (VL)，其中 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3 分別包含 SEQ ID NO: 40 至 45 之胺基酸序列。In some embodiments, the second antigen-binding domain that binds to CD3 comprises an anti-CD3 heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3, and an anti-CD3 light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences of SEQ ID NOs: 40 to 45, respectively.

在一些實施例中，與 CD3 結合的第二抗原結合域包含：抗 CD3 重鏈可變區，其包含與 SEQ ID NO: 46 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 CD3 輕鏈可變區，其包含與 SEQ ID NO: 47 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。In some embodiments, the second antigen binding domain that binds to CD3 comprises: an anti-CD3 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 46; and an anti-CD3 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 47.

在一些實施例中，多特異性抗原結合分子進一步包含與腫瘤相關抗原結合的第三抗原結合域。在一些實施例中，腫瘤相關抗原為在前列腺癌細胞上表現的受體。在一些實施例中，腫瘤相關抗原為前列腺特異性膜抗原 (PSMA)、STEAP2、前列腺幹細胞抗原 (PSCA)、上皮細胞黏著分子 (EpCAM)、前列腺特異性抗原 (PSA) 或前列腺酸性磷酸酶 (PAP)。In some embodiments, the multispecific antigen binding molecule further comprises a third antigen binding domain that binds to a tumor-associated antigen. In some embodiments, the tumor-associated antigen is a receptor expressed on prostate cancer cells. In some embodiments, the tumor-associated antigen is prostate-specific membrane antigen (PSMA), STEAP2, prostate stem cell antigen (PSCA), epithelial cell adhesion molecule (EpCAM), prostate-specific antigen (PSA) or prostatic acid phosphatase (PAP).

本文所述之本發明的至少另一態樣涉及一種或多種經分離核酸，該一種或多種經分離核酸個別或一起編碼本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體。At least another aspect of the invention described herein relates to one or more isolated nucleic acids that individually or collectively encode a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein.

本文所述之本發明的至少另一態樣涉及一種或多種載體，該一種或多種載體個別或一起包含本文所述之經分離核酸。At least another aspect of the invention described herein relates to one or more vectors, which individually or collectively comprise the isolated nucleic acids described herein.

本文所述之本發明的至少另一態樣涉及一種或多種宿主細胞，該一種或多種宿主細胞個別或一起包含本文所述之經分離核酸或本文所述之載體。在一些實施例中，宿主細胞是哺乳動物細胞。在一些實施例中，哺乳動物細胞為中國倉鼠卵巢 (CHO) 細胞。在一些實施例中，宿主細胞是昆蟲細胞。在一些實施例中，宿主細胞為原核細胞。At least another aspect of the invention described herein relates to one or more host cells, which individually or collectively contain an isolated nucleic acid described herein or a vector described herein. In some embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the host cell is an insect cell. In some embodiments, the host cell is a prokaryotic cell.

本文所述之本發明的至少另一態樣涉及一種生產本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體的方法，該方法包含在培養基中培養本文所述之宿主細胞。在一些實施例中，方法進一步包含從宿主細胞或培養基收穫本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體。At least another aspect of the invention described herein relates to a method of producing a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein, the method comprising culturing a host cell described herein in a culture medium. In some embodiments, the method further comprises harvesting a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein from the host cell or the culture medium.

本文所述之本發明的至少另一態樣涉及一種醫藥組成物，該醫藥組成物包含本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體。在一些實施例中，醫藥組成物進一步包含醫藥上可接受之載劑、賦形劑或稀釋劑。At least another aspect of the present invention described herein relates to a pharmaceutical composition comprising a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

在一些實施例中，醫藥組成物用為藥物。在一些實施例中，醫藥組成物用於治療表現 STEAP1 之癌症或延緩其進展。在一些實施例中，醫藥組成物用於治療前列腺癌或尤文氏肉瘤或延緩其進展。In some embodiments, the pharmaceutical composition is used as a medicament. In some embodiments, the pharmaceutical composition is used to treat or slow the progression of a cancer expressing STEAP1. In some embodiments, the pharmaceutical composition is used to treat or slow the progression of prostate cancer or Ewing's sarcoma.

本文所述之本發明的至少另一態樣涉及本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體在治療有需要之個體的表現 STEAP1 之癌症或延緩其進展中的用途。At least another aspect of the invention described herein relates to the use of a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein for treating or delaying the progression of a cancer expressing STEAP1 in a subject in need thereof.

本文所述之本發明的至少另一態樣涉及本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體在抑制或減少表現 STEAP1 之癌症細胞的增生中的用途。At least another aspect of the invention described herein relates to the use of a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein for inhibiting or reducing the proliferation of cancer cells expressing STEAP1.

在一些實施例中，表現 STEAP1 之癌症為實性瘤。In some embodiments, the cancer expressing STEAP1 is a solid tumor.

在一些實施例中，表現 STEAP1 之癌症為前列腺癌或尤文氏肉瘤。In some embodiments, the cancer expressing STEAP1 is prostate cancer or Ewing's sarcoma.

在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係待與額外治療劑或額外治療方案組合使用。In some embodiments, a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein is to be used in combination with an additional therapeutic agent or additional treatment regimen.

在一些實施例中，額外治療劑包含化學治療劑、免疫治療劑、靶向療法、放射療法或其組合。In some embodiments, the additional therapeutic agent comprises chemotherapy, immunotherapy, targeted therapy, radiation therapy, or a combination thereof.

在一些實施例中，額外治療劑包含一線、二線或三線療法。In some embodiments, the additional therapy comprises a first-line, second-line, or third-line therapy.

在一些實施例中，額外治療劑包含手術。In some embodiments, the additional treatment comprises surgery.

在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體與額外治療劑係待同時投予。In some embodiments, a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein and an additional therapeutic agent are to be administered simultaneously.

在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係待經依序投予。In some embodiments, a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein is to be administered sequentially.

在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係待在投予額外治療劑之前先投予。In some embodiments, a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein is administered prior to administration of an additional therapeutic agent.

在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係待在投予額外治療劑之後投予。In some embodiments, a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein is administered after administration of an additional therapeutic agent.

在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係待經全身性投予。In some embodiments, a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein is to be administered systemically.

在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係待經局部性投予。In some embodiments, a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein is to be administered locally.

在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係待藉由腸胃外投予來投予。In some embodiments, a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein is to be administered by parenteral administration.

在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係待經靜脈內或經皮下投予。In some embodiments, a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein is to be administered intravenously or subcutaneously.

在一些實施例中，該個體為人。In some embodiments, the individual is a human.

本文所述之本發明的至少另一態樣涉及本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體在製造藥物中之用途，該藥物用於治療表現 STEAP1 之癌症或延緩其進展，視情況用於治療前列腺癌或尤文氏肉瘤或延緩其進展。At least another aspect of the invention described herein relates to the use of a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein in the manufacture of a medicament for treating or delaying the progression of a cancer expressing STEAP1, in particular for treating or delaying the progression of prostate cancer or Ewing's sarcoma.

本文所述之本發明的至少另一態樣涉及本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體在製造藥物中之用途，該藥物用於抑制或減少表現 STEAP1 之癌症細胞 (視情況前列腺癌細胞或尤文氏肉瘤細胞) 的增生。At least another aspect of the invention described herein relates to the use of a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein in the manufacture of a medicament for inhibiting or reducing the proliferation of cancer cells (prostate cancer cells or Ewing's sarcoma cells, as the case may be) expressing STEAP1.

本文所述之本發明的至少另一態樣涉及一種治療有需要之個體的表現 STEAP1 之癌症或延緩其進展之方法，該方法包含向該個體投予有效量之本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體。At least another aspect of the invention described herein relates to a method of treating or slowing the progression of a cancer expressing STEAP1 in a subject in need thereof, the method comprising administering to the subject an effective amount of a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein.

在一些實施例中，表現 STEAP1 之癌症為實性瘤。在一些實施例中，表現 STEAP1 之癌症為前列腺癌或尤文氏肉瘤。In some embodiments, the cancer expressing STEAP1 is a solid tumor. In some embodiments, the cancer expressing STEAP1 is prostate cancer or Ewing's sarcoma.

在一些實施例中，方法進一步包含向個體投予額外治療劑或額外治療方案。在一些實施例中，額外治療劑包含化學治療劑、免疫治療劑、靶向療法、放射療法或其組合。在一些實施例中，額外治療劑包含一線、二線或三線療法。在一些實施例中，額外治療劑包含手術。In some embodiments, the method further comprises administering an additional therapeutic agent or an additional treatment regimen to the individual. In some embodiments, the additional therapeutic agent comprises chemotherapy, immunotherapy, targeted therapy, radiation therapy, or a combination thereof. In some embodiments, the additional therapeutic agent comprises first-line, second-line, or third-line therapy. In some embodiments, the additional therapeutic agent comprises surgery.

在一些實施例中，額外治療劑與本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係同時投予。In some embodiments, an additional therapeutic agent is administered concurrently with a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein.

在一些實施例中，額外治療劑與本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係依序投予。In some embodiments, an additional therapeutic agent is administered sequentially with a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein.

在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係在投予額外治療劑之前先投予。In some embodiments, a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein is administered prior to administration of an additional therapeutic agent.

在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係在投予額外治療劑之後投予。In some embodiments, a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein is administered after administration of an additional therapeutic agent.

在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係經全身性投予。在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係經局部性投予。在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係藉由腸胃外投予來投予。在一些實施例中，本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體係經靜脈內或經皮下投予。In some embodiments, the multispecific antigen-binding molecules described herein, the antigen-binding molecules described herein, or the antibodies described herein are administered systemically. In some embodiments, the multispecific antigen-binding molecules described herein, the antigen-binding molecules described herein, or the antibodies described herein are administered topically. In some embodiments, the multispecific antigen-binding molecules described herein, the antigen-binding molecules described herein, or the antibodies described herein are administered by parenteral administration. In some embodiments, the multispecific antigen-binding molecules described herein, the antigen-binding molecules described herein, or the antibodies described herein are administered intravenously or subcutaneously.

在一些實施例中，該個體為人。In some embodiments, the individual is a human.

本文所述之本發明的至少另一態樣涉及一種抑制或減少表現 STEAP1 之細胞的增生之方法，該方法包含使該細胞與本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、或本文所述之抗體接觸足以抑制該細胞之增生的時間。At least another aspect of the invention described herein relates to a method of inhibiting or reducing proliferation of a cell expressing STEAP1, the method comprising contacting the cell with a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, or an antibody described herein for a time sufficient to inhibit proliferation of the cell.

在一些實施例中，細胞為前列腺癌細胞或尤文氏肉瘤細胞。在某些實施例中，該方法是活體內方法。在一些實施例中，方法為活體外(in vitro) 或離體(ex vivo) 方法。In some embodiments, the cell is a prostate cancer cell or an Ewing's sarcoma cell. In some embodiments, the method isan in vivo method. In some embodiments, the method is an invitro or exvivo method.

本文所述之本發明的至少另一態樣涉及一種套組，該套組包含本文所述之多特異性抗原結合分子、本文所述之抗原結合分子、本文所述之抗體、本文所述之經分離核酸、本文所述之載體、本文所述之宿主細胞、或本文所述之醫藥組成物，視情況包含一組說明。At least another aspect of the invention described herein relates to a kit comprising a multispecific antigen-binding molecule described herein, an antigen-binding molecule described herein, an antibody described herein, an isolated nucleic acid described herein, a vector described herein, a host cell described herein, or a pharmaceutical composition described herein, optionally comprising a set of instructions.

序列表Sequence Listing

本申請包含序列表，該序列表已經以 XML 格式以電子方式提交，並以引用方式以其全部內容併入本文。該 XML 複本創建於 2023 年 7 月 18 日，命名為「50474-301TW2_Sequence_Listing」，且大小為 170,087 位元組。I.定義This application contains a sequence listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on July 18, 2023, is named "50474-301TW2_Sequence_Listing", and is 170,087 bytes in size.I.Definitions

如本文所用，術語「約」係指本技術領域技術人員易於知曉的各個值的通常誤差範圍。本文提及「約」值或參數包括 (及描述) 針對該值或參數本身的實施例。As used herein, the term "about" refers to the usual error range of each value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter itself.

「親和力」係指分子 (例如抗體) 之單一結合位點與其結合配偶體 (例如抗原) 之間的非共價交互作用總和的強度。除非另有說明，否則如本文中所使用的「結合親和力」，係指反映結合對成員 (例如抗體及抗原) 之間 1:1 交互作用之內在結合親和力。分子 X 對於其配偶體 Y 之親和力通常可藉由平衡解離常數 (K_D) 來表示。可以藉由本領域已知的習知方法測量親和力，包括彼等本文所述之方法。下面描述了用於測量結合親和力的具體的說明性及示例性實施例。"Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (_KD ). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

如本文所用，術語「結合至」係指可測量且可重現之交互作用，諸如標靶與抗原結合分子 (例如，抗體) 之間的結合，其可在異種分子 (包括生物分子) 群體存在下判定標靶的存在。例如，與標靶 (其可為表位) 特異性結合之抗原結合分子為與該標靶結合之親和力、結合性或容易程度及/或持續時間優於與其他標靶結合之親和力、結合性或容易程度及/或持續時間的抗原結合分子。在一個實施例中，抗原結合分子結合不相關的標靶之程度小於該抗原結合分子結合標靶之程度的約 10%，例如藉由表面電漿子共振 (SPR)、放射免疫檢定 (RIA) 或動力學篩析測定 (KinExA®) 所測量。在某些實施例中，與靶標特異性結合的抗原結合分子具有 ≤ 1 μM、≤ 100 nM、≤ 10 nM、≤ 1 nM 或 ≤ 0.1 nM 之平衡解離常數 (K_D)。在某些實施例中，抗原結合分子與不同物種蛋白質中保留的蛋白質上之表位特異性結合。於另一個實施例中，特異性結合可包括但不要求專一結合。As used herein, the term "binds to" refers to a measurable and reproducible interaction, such as binding between a target and an antigen binding molecule (e.g., an antibody), which can be used to determine the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antigen binding molecule that specifically binds to a target (which may be an epitope) is one that binds to the target with greater affinity, binding, ease, and/or duration than to other targets. In one embodiment, the extent to which an antigen binding molecule binds to an unrelated target is less than about 10% of the extent to which the antigen binding molecule binds to the target, such as measured by surface plasmon resonance (SPR), radioimmunoassay (RIA), or kinetic screening assay (KinExA®). In certain embodiments, an antigen binding molecule that specifically binds to a target has an equilibrium dissociation constant (_KD ) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.1 nM. In certain embodiments, an antigen binding molecule specifically binds to an epitope on a protein that is conserved among proteins of different species. In another embodiment, specific binding may include but does not require exclusive binding.

如本文所用，術語「抗原結合分子」係指與標靶表位、抗原、配體或受體特異性結合的分子。抗原結合分子包括但不限於抗體 (例如，單株、多株、重組、人源化及嵌合抗體)、抗體片段或其部分 (例如，Fab 片段，Fab'2、scFv 抗體、SMIP、域抗體、雙抗體、微抗體、scFv-Fc、親合體 (affibody)、奈米抗體、以及抗體之 VH 域及/或 VL 域)、受體、配體、適體、及具有確定結合搭配物的其他分子。「親和力成熟」抗體係指在一個或多個互補決定區 (CDR) 或高度可變區 (HVR) 中具有一種或多種變化之抗體，與不具有此等變化之親代抗體相比，此類變化引起該抗體對抗原之親和力的改善。As used herein, the term "antigen binding molecule" refers to a molecule that specifically binds to a target epitope, antigen, ligand or receptor. Antigen binding molecules include, but are not limited to, antibodies (e.g., monoclonal, polyclonal, recombinant, humanized and chimeric antibodies), antibody fragments or portions thereof (e.g., Fab fragments, Fab'2, scFv antibodies, SMIPs, domain antibodies, bispecific antibodies, miniantibodies, scFv-Fc, affibodies, nanoantibodies, and VH domains and/or VL domains of antibodies), receptors, ligands, aptamers, and other molecules with defined binding partners. "Affinity matured" antibodies refer to antibodies with one or more changes in one or more complementary determining regions (CDRs) or highly variable regions (HVRs), such changes resulting in improved affinity of the antibody for the antigen compared to a parent antibody without such changes.

術語「抗原結合域」係指與標靶表位、抗原、配體或受體特異性結合的化合物或分子的一部分。特徵在於抗原結合域的分子包括但不限於抗體 (例如，單株、多株、重組、人源化及嵌合抗體)、抗體片段或其部分 (例如，Fab 片段，Fab'2、scFv 抗體、SMIP、域抗體、雙抗體、微抗體、scFv-Fc、親合體、奈米抗體及抗體之 VH 域及/或 VL 域)、受體、配體、適體、及具有確定結合搭配物的其他分子。The term "antigen binding domain" refers to a compound or a portion of a molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Molecules characterized by an antigen binding domain include, but are not limited to, antibodies (e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), antibody fragments or portions thereof (e.g., Fab fragments, Fab'2, scFv antibodies, SMIPs, domain antibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies, and VH and/or VL domains of antibodies), receptors, ligands, aptamers, and other molecules with defined binding partners.

本文中的術語「抗體」以最廣義使用且涵蓋各種抗體結構，包括但不限於單株抗體、多株抗體、多特異性抗體 (例如，雙特異性或三特異性抗體) 及抗體片段，只要其等展示出預期抗原結合活性即可。The term "antibody" herein is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific or trispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen-binding activity.

「抗體片段」係指除完整抗體以外的分子，其包含結合完整抗體所結合抗原之完整抗體的一部分。抗體片段之示例包括但不限於 Fv、Fab、Fab’、Fab’-SH、F(ab’)₂；雙抗體 (diabody)；線性抗體；單鏈抗體分子 (例如，scFv 及 scFab)；單域抗體 (dAb)；及從抗體片段所形成之多重特異性抗體。關於某些抗體片段的綜述，參見 Holliger 及 Hudson，Nat. Biotech.23:1126-1136 (2005)。"Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')₂ ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson,Nat. Biotech. 23:1126-1136 (2005).

「單鏈變異片段」 或 「scFv」 為抗體之重鏈 (VH) 及輕鏈 (VL) 的可變域之融合蛋白，其藉由連接子連接。特定而言，連接子通常為 10 個至 25 個胺基酸組成之短多肽，並且通常富含甘胺酸以提高柔韌性，並含有絲胺酸或蘇胺酸以提高溶解性，並且可將 VH 之 N 端與 VL 之 C 端連接，或反之亦然。儘管去除了恆定區並引入了連接子，但是該蛋白仍保留了原始抗體的特異性。關於 scFv 片段的綜述，例如參見 Pluckthün，The Pharmacology of Monoclonal Antibodies，第 113卷，Rosenburg 及 Moore 編輯，Springer-Verlag，New York，第 269-315 頁 (1994)；亦參見 WO 93/16185；及美國專利第 5,571,894 號及第 5,587,458 號。"Single-chain variant fragment" or "scFv" is a fusion protein of the variable domains of the heavy chain (VH) and light chain (VL) of an antibody, connected by a linker. Specifically, the linker is usually a short polypeptide consisting of 10 to 25 amino acids, and is usually rich in glycine to improve flexibility and contains serine or threonine to improve solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, the protein still retains the specificity of the original antibody. For a general review of scFv fragments, see, e.g., Pluckthün,The Pharmacology of Monoclonal Antibodies , Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458.

「單鏈 Fab 片段」或「scFab」是由抗體重鏈可變域 (VH)、抗體重鏈恆定域 1 (CH1)、抗體輕鏈可變域 (VL)、抗體輕鏈恆定域 (CL) 及連接子組成的多肽，其中該抗體域及該連接子在 N 端至 C 端方向具有以下序列之一：a) VH-CH1-連接子-VL-CL、b) VL-CL-連接子-VH-CH1、c) VH-CL-連接子-VL-CH1 或 d) VL-CH1-連接子-VH-CL。特定而言，該連接子為至少 30 個胺基酸且較佳地 32 至 50 個胺基酸組成之多肽。該單鏈 Fab 片段通過 CL 域與 CH1 域之間的天然雙硫鍵達到穩定。此外，這些單鏈 Fab 片段可藉由插入半胱胺酸殘基產生鏈間二硫鍵而得到進一步穩定 (例如，根據 Kabat 編號，在變異重鏈之位置 44 及變異輕鏈之位置 100 處插入)。"Single-chain Fab fragment" or "scFab" is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL. In particular, the linker is a polypeptide composed of at least 30 amino acids and preferably 32 to 50 amino acids. The single-chain Fab fragments are stabilized by the native disulfide bonds between the CL domain and the CH1 domain. In addition, these single-chain Fab fragments can be further stabilized by the insertion of cysteine residues to generate interchain disulfide bonds (e.g., at position 44 of the variant heavy chain and at position 100 of the variant light chain according to Kabat numbering).

術語「cross-Fab 片段」或「xFab 片段」或「交叉 Fab 片段」 是指其中重鏈及輕鏈之可變區或恆定區發生交換的 Fab 片段。cross-Fab 片段包含由輕鏈可變區 (VL) 及重鏈恆定區 1 (CH1) 構成之多肽鏈以及由重鏈可變區 (VH) 及輕鏈恆定區 (CL) 構成之多肽鏈。還可透過將帶電荷或不帶電荷之胺基酸突變引入域界面引導正確 Fab 配對，從而設計不對稱之 Fab 臂。參見例如 WO 2016/172485。The term "cross-Fab fragment" or "xFab fragment" or "cross-Fab fragment" refers to a Fab fragment in which the variable or constant regions of the heavy and light chains are exchanged. The cross-Fab fragment comprises a polypeptide chain consisting of a light chain variable region (VL) and a heavy chain constant region 1 (CH1) and a polypeptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be designed by introducing charged or uncharged amino acid mutations into the domain interface to direct the correct Fab pairing. See, for example, WO 2016/172485.

術語「抗原」表示抗原結合分子 (例如，抗體) 與之結合的蛋白質或非蛋白質分子。抗原可包括蛋白質、蛋白質片段或半抗原。The term "antigen" refers to a protein or non-protein molecule to which an antigen-binding molecule (e.g., an antibody) binds. Antigens may include proteins, protein fragments, or haptens.

術語「表位 (epitope)」表示抗 STEAP1 抗原結合分子 (例如，抗 STEAP1 抗體) 與之結合的抗原上的位點，無論是蛋白性的還是非蛋白性的。表位可由連續的胺基酸延伸形成 (線性表位) 或包含非連續之胺基酸 (構象表位)，例如，由於抗原的折疊，即藉由蛋白抗原的三級折疊而在空間上接近。線性表位通常在蛋白抗原暴露於變性劑後仍與抗 STEAP1 抗原結合分子結合，而構象表位通常在變性劑處理後被破壞。在獨特空間構象中，表位包含至少 3 個、至少 4 個、至少 5 個、至少 6 個、至少 7 個或 8 個至 10 個胺基酸。The term "epitope" refers to a site on an antigen, whether proteinaceous or non-proteinaceous, to which an anti-STEAP1 antigen-binding molecule (e.g., an anti-STEAP1 antibody) binds. An epitope may be formed by a stretch of consecutive amino acids (a linear epitope) or comprise non-consecutive amino acids (a conformational epitope), for example, brought into spatial proximity by folding of the antigen, i.e., by tertiary folding of the protein antigen. A linear epitope typically remains bound to an anti-STEAP1 antigen-binding molecule after exposure of the protein antigen to a denaturing agent, whereas a conformational epitope is typically destroyed after treatment with a denaturing agent. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8 to 10 amino acids in a unique spatial conformation.

篩選與特定表位結合 (亦即與相同表位結合者) 的抗原結合分子 (例如，抗體) 可使用此領域的常規方法進行，例如諸如但不限於丙胺酸掃描、肽印漬 (參見Meth. Mol. Biol.248 (2004) 443-463)、肽裂解分析、表位切除、表位萃取、抗原的化學修飾 (參見Prot. Sci.9 (2000) 487-496)、及交叉阻斷 (參見「Antibodies」，Harlow 及 Lane (Cold Spring Harbor Press, Cold Spring Harb.NY)。Screening for antigen-binding molecules (e.g., antibodies) that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using conventional methods in this field, such as, but not limited to, alanine scanning, peptide blotting (seeMeth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (seeProt. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb. NY).

基於抗原結構的抗體剖析 (ASAP)，亦稱為修飾輔助剖析 (MAP)，允許根據從眾多化學或酶修飾的抗原表面的各抗體結合剖析，將特異性結合 STEAP1 的眾多單株抗體進行分箱 (參見例如，US 2004/0101920)。各分箱中的抗體都與相同表位結合，這個表位可能是獨特的表位，與另一分箱所代表的表位明顯不同或部分重疊。Antibody profiling based on antigen structure (ASAP), also known as modification-assisted profiling (MAP), allows the binning of a number of monoclonal antibodies that specifically bind to STEAP1 based on their binding to a variety of chemically or enzymatically modified antigen surfaces (see, e.g., US 2004/0101920). The antibodies in each bin all bind to the same epitope, which may be a unique epitope, distinct from or partially overlapping with the epitope represented by another bin.

另外，競爭性結合可用來易於確定抗原結合分子 (例如，抗體) 是否與 STEAP1 的相同表位結合，或與參考抗 STEAP1 抗體競爭性結合。舉例而言，與參考抗 STEAP1 抗體「結合在相同表位的抗原結合分子」係指在競爭性測定中阻斷參考抗 STEAP1 抗體與其抗原結合 50% 或更多的抗原結合分子，且反之，參考抗體在競爭測定中阻斷該抗原結合分子與其抗原結合 50% 或更多。又舉例而言，為了確定抗原結合分子是否與參考抗 STEAP1 抗體結合在相同表位，讓參考抗體在飽和條件下與 STEAP1 結合。在去除過量的參考抗 STEAP1 抗體後，評估有關抗 STEAP1 抗原結合分子與 STEAP1 結合的能力。如果在參考抗 STEAP1 抗原結合分子飽和結合後，抗 STEAP1 抗體與能夠與 STEAP1 結合，則可以斷定該抗 STEAP1 抗原結合分子與參考抗 STEAP1 抗體結合的表位不同。但是，如果在參考抗 STEAP1 抗體飽和結合後，所述抗 STEAP1 抗原結合分子不能與 STEAP1 結合，則所述抗 STEAP1 抗原結合分子可能與參考抗 STEAP1 抗體結合的表位相同。為了確認所述抗原結合分子是否與相同的表位結合，或者只是由於立體原因而阻礙了結合，可以使用常規實驗 (例如，使用 ELISA、RIA、表面電漿共振、流式細胞儀或本技術中可獲得的任何其他定量或定性的抗體結合測定進行肽突變及結合分析)。此測定可以分兩次設置進行，亦即以兩種分子為飽和抗體。若在這兩種設置中，只有第一種 (飽和) 抗體能夠與 STEAP1 結合，則可斷定所述抗 STEAP1 抗原結合分子及參考抗 STEAP1 抗體競爭結合至 STEAP1。Additionally, competitive binding can be used to readily determine whether an antigen-binding molecule (e.g., an antibody) binds to the same epitope of STEAP1, or competitively binds to a reference anti-STEAP1 antibody. For example, an "antigen-binding molecule that binds to the same epitope as a reference anti-STEAP1 antibody" refers to an antigen-binding molecule that blocks 50% or more of the binding of the reference anti-STEAP1 antibody to its antigen in a competitive assay, and conversely, the reference antibody blocks 50% or more of the binding of the antigen-binding molecule to its antigen in a competitive assay. For another example, to determine whether an antigen-binding molecule binds to the same epitope as a reference anti-STEAP1 antibody, the reference antibody is allowed to bind to STEAP1 under saturation conditions. After removing excess reference anti-STEAP1 antibody, the ability of the anti-STEAP1 antigen-binding molecule to bind to STEAP1 is assessed. If the anti-STEAP1 antibody is able to bind to STEAP1 after saturation binding of the reference anti-STEAP1 antigen-binding molecule, it can be concluded that the anti-STEAP1 antigen-binding molecule binds to a different epitope than the reference anti-STEAP1 antibody. However, if the anti-STEAP1 antigen-binding molecule is unable to bind to STEAP1 after saturation binding of the reference anti-STEAP1 antibody, the anti-STEAP1 antigen-binding molecule may bind to the same epitope as the reference anti-STEAP1 antibody. To confirm whether the antigen binding molecule binds to the same epitope or is simply blocked for stereological reasons, conventional experiments can be used (e.g., peptide mutagenesis and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art). This assay can be performed in two settings, i.e., with both molecules as saturated antibodies. If in both settings, only the first (saturated) antibody is able to bind to STEAP1, it can be concluded that the anti-STEAP1 antigen binding molecule and the reference anti-STEAP1 antibody compete for binding to STEAP1.

術語「STEAP1」係指來自任何脊椎動物來源之任何 STEAP1，該脊椎動物包括哺乳動物，諸如靈長類動物 (例如，人類或非人靈長類) 及囓齒類動物 (例如，小鼠及大鼠)。該術語涵蓋「全長」STEAP1 及天然發生之 STEAP1 變異體，例如，剪接變異體或對偶基因變異體。STEAP1 為前列腺六段跨膜上皮抗原 (STEAP) 蛋白質家族的成員。STEAP 蛋白質家族包含五個成員：STEAP1、STEAP2、STEAP3、STEAP4 及 STEAP5。已觀察到 STEAP1、STEAP2 及 STEAP4 在不同癌細胞中過度表現，而在正常組織中表現極少。STEAP1 包括例如人 STEAP1 (UniProtKB 參考號：Q9UHE8-1；SEQ ID NO: 65)、石蟹獼猴 STEAP1 (UniProtKB 參考號：A0A2K5X1J3；SEQ ID NO: 66) 及紅毛猩猩 STEAP1 (UniProtKB 參考號：H2PMZ0；SEQ ID NO: 67)。The term "STEAP1" refers to any STEAP1 from any vertebrate source, including mammals, such as primates (e.g., humans or non-human primates) and rodents (e.g., mice and rats). The term encompasses "full-length" STEAP1 and naturally occurring STEAP1 variants, such as splice variants or allelic variants. STEAP1 is a member of the six-segment transmembrane epithelial antigen of the prostate (STEAP) family of proteins. The STEAP protein family includes five members: STEAP1, STEAP2, STEAP3, STEAP4, and STEAP5. STEAP1, STEAP2, and STEAP4 have been observed to be overexpressed in various cancer cells and minimally expressed in normal tissues. STEAP1 includes, for example, human STEAP1 (UniProtKB Reference No.: Q9UHE8-1; SEQ ID NO: 65), red macaque STEAP1 (UniProtKB Reference No.: A0A2K5X1J3; SEQ ID NO: 66), and orangutan STEAP1 (UniProtKB Reference No.: H2PMZ0; SEQ ID NO: 67).

以下為人 STEAP1 的示例性胺基酸序列：MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL (SEQ ID NO: 65)。The following is an exemplary amino acid sequence of human STEAP1: MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL (SEQ ID NO: 65).

以下為石蟹獼猴 (Macaca fascicularis) STEAP1 的示例性胺基酸序列：MESRKDITNEEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLATIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPVVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKMEISSQL  (SEQ ID NO: 66)。The following is an exemplary amino acid sequence ofMacaca fascicularis STEAP1: MESRKDITNEEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLATIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPVVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKMEISSQL (SEQ ID NO: 66).

以下為紅毛猩猩 (Pongo abelii) STEAP1 的示例性胺基酸序列：MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQQTRELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVILPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEISSQL (SEQ ID NO: 67)。The following is an exemplary amino acid sequence of orangutan (Pongo abelii ) STEAP1: MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQQTRELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVILPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEISSQL (SEQ ID NO: 67).

術語「抗 STEAP1 抗原結合分子」或「結合 STEAP1 的抗原結合分子」係指能夠以足夠親和力結合 STEAP1，從而使得該分子可用作靶向 STEAP1 之診斷劑及/或治療劑的任何分子。在一個實施例中，抗 STEAP1 抗原結合分子與無關的非 STEAP1 蛋白質的結合程度小於該抗原結合分子與 STEAP1 的結合程度的約 10%，例如藉由表面電漿子共振 (SPR)、放射免疫檢定 (RIA) 或動力學篩析測定 (KinExA®) 所測量。在某些態樣中，抗 STEAP1 抗原結合分子具有 ≤ 1μM、≤ 100 nM、≤ 10 nM、≤ 1 nM、≤ 0.1 nM、≤ 0.01 nM 或 ≤ 0.001 nM (例如，10^-8M 或更低，例如 10^-8M 至 10^-13M，例如 10^-9M 至 10^-13M) 之解離常數 (K_D)。當抗原結合分子具有 1 μM 或更少之 K_D時，稱該抗原結合分子與 STEAP1「特異性結合」。在某些態樣中，抗 STEAP1 抗原結合分子與 STEAP1 之表位結合，該表位在不同物種之 STEAP 中係保留的。The term "anti-STEAP1 antigen binding molecule" or "antigen binding molecule that binds to STEAP1" refers to any molecule that is capable of binding to STEAP1 with sufficient affinity to render the molecule useful as a diagnostic and/or therapeutic agent targeting STEAP1. In one embodiment, the extent of binding of the anti-STEAP1 antigen binding molecule to an unrelated non-STEAP1 protein is less than about 10% of the extent of binding of the antigen binding molecule to STEAP1, for example as measured by surface plasmon resonance (SPR), radioimmunoassay (RIA), or kinetic screening assay (KinExA®). In certain aspects, the anti-STEAP1 antigen binding molecule has a dissociation constant (K D ) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10^-8 M or lower, such as 10^-8 M to 10^-13 M, such as 10^-9 M to 10^-13 M). When the antigen binding molecule has_{a K Dof} 1 μM or less, the antigen binding molecule is said to "specifically bind" to STEAP1. In certain aspects, the anti-STEAP1 antigen binding molecule binds to an epitope of STEAP1 that is conserved in STEAPs of different species.

術語「抗 STEAP1 抗體」或「與 STEAP1 結合的抗體」係指能夠以足夠親和力結合 STEAP1，從而使得該分子可用作靶向 STEAP1 之診斷劑及/或治療劑之抗體。在一個實施例中，抗 STEAP1 抗體與無關的非 STEAP1 蛋白質的結合程度小於該抗體與 STEAP1 的結合程度的約 10%，例如藉由表面電漿子共振 (SPR)、放射免疫檢定 (RIA) 或動力學篩析測定 (KinExA®) 所測量。在某些態樣中，抗 STEAP1 抗體具有 ≤ 1μM、≤ 100 nM、≤ 10 nM、≤ 1 nM、≤ 0.1 nM、≤ 0.01 nM 或 ≤ 0.001 nM (例如，10^-8M 或更低，例如 10^-8M 至 10^-13M，例如 10^-9M 至 10^-13M) 之解離常數 (K_D)。當抗體具有 1 μM 或更少之 K_D時，稱該抗體與 STEAP1「特異性結合」。在某些態樣中，抗 STEAP1 抗體與 STEAP1 之表位結合，該表位在不同物種之 STEAP 中係保留的。The term "anti-STEAP1 antibody" or "antibody that binds to STEAP1" refers to an antibody that is capable of binding to STEAP1 with sufficient affinity such that the molecule can be used as a diagnostic and/or therapeutic agent targeting STEAP1. In one embodiment, the extent of binding of the anti-STEAP1 antibody to an unrelated, non-STEAP1 protein is less than about 10% of the extent of binding of the antibody to STEAP1, for example as measured by surface plasmon resonance (SPR), radioimmunoassay (RIA), or kinetic screening assay (KinExA®). In certain aspects, the anti-STEAP1 antibody has a dissociation constant (K D ) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10^-8 M or lower, such as 10^-8 M to 10^-13 M, such as 10^-9 M to 10^-13 M). An antibody is said to "specifically_bind " to STEAP1 when it has a K_D of 1 μM or less. In certain aspects, the anti-STEAP1 antibody binds to an epitope of STEAP1 that is conserved in STEAPs of different species.

如本文所用，術語「分化簇 3」或「CD3」涉及來自任何脊椎動物來源的任何天然 CD3，包括哺乳動物，例如靈長類動物 (例如人類) 及囓齒動物 (例如小鼠及大鼠)，除非另有說明，包括例如 CD3ε、CD3γ、CD3α 及 CD3β 鏈。該術語涵蓋「全長」、未處理之 CD3 (例如未處理或未修飾之 CD3ε 或 CD3γ) 以及在細胞處理中得到的任何形式的 CD3。該術語亦涵蓋天然生成之 CD3 變異體，例如，剪接變異體或對偶基因變異體。CD3 包括例如長度為 207 個胺基酸的人類 CD3ε 蛋白 (NCBI RefSeq No. NP_000724) 及長度為 182 個胺基酸的人類 CD3γ 蛋白 (NCBI RefSeq No. NP_000064)。As used herein, the term "cluster of differentiation 3" or "CD3" refers to any native CD3 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated, including, for example, CD3ε, CD3γ, CD3α and CD3β chains. The term encompasses "full-length," unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ) as well as any form of CD3 obtained in cell manipulation. The term also encompasses naturally occurring CD3 variants, such as splice variants or allelic variants. CD3 includes, for example, a human CD3ε protein having a length of 207 amino acids (NCBI RefSeq No. NP_000724) and a human CD3γ protein having a length of 182 amino acids (NCBI RefSeq No. NP_000064).

術語「抗 CD3 抗體」及「結合至 CD3 之抗體」是指能夠以足夠親和力結合 CD3，從而使得該抗體可用作靶向 CD3 之診斷劑及/或治療劑之抗體。在一個實施例中，抗 CD3 抗體與無關的非 CD3 蛋白質的結合程度小於該抗體與 CD3 的結合程度的約 10%，例如藉由表面電漿子共振 (SPR)、放射免疫檢定 (RIA) 或動力學篩析測定 (KinExA®) 所測量。在某些實施例中，與 CD3 結合的抗體之解離常數 (K_D) 為 ≤ 1μM、≤ 100 nM、≤ 10 nM、≤ 1 nM、≤ 0.1 nM、≤ 0.01 nM、或 ≤ 0.001 nM (例如，10^-8M 或更小，例如 10^-8M 至 10^-13M，例如，10^-9M 至 10^-13M)。在某些實施例中，抗 CD3 拮抗劑抗體結合至 CD3 之抗原決定基，其在不同物種之 CD3 是保守性。在一些實施例中，抗 CD3 抗體描述於國際專利申請公開號 WO 2015/095392 中，其全文以引用方式併入本文中。在一些實施例中，抗 CD3 抗體描述於美國專利第 10,174,124 號中，其全文以引用方式併入本文中。在其他實施例中，抗 CD3 抗體描述於國際專利申請號 PCT/US2020/064635 中，其全文以引用方式併入本文中。The terms "anti-CD3 antibody" and "antibody that binds to CD3" refer to an antibody that is capable of binding to CD3 with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent targeting CD3. In one embodiment, the extent of binding of the anti-CD3 antibody to an unrelated non-CD3 protein is less than about 10% of the extent of binding of the antibody to CD3, for example as measured by surface plasmon resonance (SPR), radioimmunoassay (RIA), or kinetic screening assay (KinExA®). In certain embodiments, the dissociation constant (_KD ) of the antibody that binds to CD3 is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g.,^10-8 M or less, such as^10-8 M to^10-13 M, such as^10-9 M to^10-13 M). In certain embodiments, the anti-CD3 antagonist antibody binds to an antigenic determinant of CD3 that is conserved in CD3 of different species. In certain embodiments, the anti-CD3 antibody is described in International Patent Application Publication No. WO 2015/095392, which is incorporated herein by reference in its entirety. In some embodiments, the anti-CD3 antibody is described in U.S. Patent No. 10,174,124, which is incorporated herein by reference in its entirety. In other embodiments, the anti-CD3 antibody is described in International Patent Application No. PCT/US2020/064635, which is incorporated herein by reference in its entirety.

在一些實施例中，抗 CD3 抗體為 40G5c。在一些實施例中，抗 CD3 抗體為 38E4V1.MD1。40G5c 及 38E4V1.MD1 的 CDRs 以及 VH 及 VL 序列之 SEQ ID NO 列於表 1 及表 2 中。In some embodiments, the anti-CD3 antibody is 40G5c. In some embodiments, the anti-CD3 antibody is 38E4V1.MD1. The CDRs and SEQ ID NOs of the VH and VL sequences of 40G5c and 38E4V1.MD1 are listed in Tables 1 and 2.

在一些態樣中，若一種抗體的 1 倍、5 倍、10 倍、20 倍或 100 倍的過量抑制另一抗體的結合至少 50%、至少 75%、至少 90% 或甚至 99% 或更多 (藉由競爭性結合測定測量)，則認為兩個抗體與相同或重疊的表位結合 (參見例如，Junghans 等人，Cancer Res.50 (1990) 1495-1502)。In some aspects, two antibodies are considered to bind to the same or overlapping epitope if a 1-fold, 5-fold, 10-fold, 20-fold or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 50%, at least 75%, at least 90% or even 99% or more (as measured by a competitive binding assay) (see, e.g., Junghans et al.,Cancer Res. 50 (1990) 1495-1502).

在一些態樣中，如果抗原中基本上所有的胺基酸突變降低或消除了一個抗體的結合，也降低或消除了另一抗體的結合，則認為兩個抗體與相同表位結合。如果只有減少或消除一個抗體結合的胺基酸突變的次集合 (subset) 減少或消除另一抗體的結合，則認為兩種抗體具有「重疊表位 (overlapping epitope)」。In some aspects, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies are considered to have "overlapping epitopes" if only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

術語「嵌合」抗體是指其中重鏈及/或輕鏈的一部分源自特定來源或物種，而重鏈及/或輕鏈的其餘部分源自不同來源或物種的抗體。The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and/or light chain is derived from a particular source or species, while the remainder of the heavy chain and/or light chain is derived from a different source or species.

抗體之「類別 (class)」係指為其重鏈所具有的恆定域或恆定區之類型。有五大類抗體：IgA、IgD、IgE、IgG 及 IgM，且該等種類中之若干種可進一步分為亞類 (同型)，例如 IgG₁、IgG₂、IgG₃、IgG₄、IgA₁及 IgA₂。在某些態樣中，該抗體是屬 IgG₁同型。在某些態樣中，該抗體是屬 IgG₁同型，具有 P329G、L234A 及 L235A 突變以減少 Fc 區效應子功能。在其他態樣中，該抗體是屬 IgG₂同型。在某些態樣中，該抗體是屬 IgG₄同型，在鉸鏈區中具有 S228P 突變以改善 IgG₄抗體之穩定性。對應於不同類別之免疫球蛋白的重鏈恆定域分別稱為 α、δ、ε、γ 及 μ。基於其恆定域之胺基酸序列，抗體之輕鏈可被歸類為兩種類型中的一種，稱為卡帕 (κ) 及蘭姆達 (λ)。The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further divided into subclasses (isotypes), such as IgG₁ , IgG₂ , IgG₃ , IgG₄ , IgA₁ , and IgA_2. In some aspects, the antibody is of the IgG₁ isotype. In some aspects, the antibody is of the IgG₁ isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other aspects, the antibody is of the IgG₂ isotype. In certain aspects, the antibody is of the IgG₄ isotype and has an S228P mutation in the hinge region to improve the stability of the IgG₄ antibody. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be classified into one of two types, called kappa (κ) and lambda (λ).

如本文所用，術語「高度可變區」或「HVR」係指抗體可變域中序列高變並決定抗原結合特異性的區域中之各者，例如「互補決定區」(「CDR」)。As used herein, the term "hypervariable region" or "HVR" refers to each of the regions in the antibody variable domain whose sequence is highly variable and determines the antigen binding specificity, such as the "complementary determining region" ("CDR").

通常，抗體包括六個 CDR：三個在 VH 中 (CDR-H1、CDR-H2、CDR-H3)，及三個在 VL 中 (CDR-L1、CDR-L2、CDR-L3)。在本文中，例示性 CDR 包括： (a) 高度可變環存在於胺基酸殘基 26-32 (L1)、50-52 (L2)、91-96 (L3)、26-32 (H1)、53-55 (H2)、及 96-101 (H3) 處 (Chothia 及 Lesk，J. Mol. Biol.196:901-917 (1987))； (b) CDR 存在於胺基酸殘基 24-34 (L1)、50-56 (L2)、89-97 (L3)、31-35b (H1)、50-65 (H2)、及 95-102 (H3)處 (Kabat 等人，Sequences of Proteins of Immunological Interest，第 5 版 Public Health Service，National Institutes of Health，Bethesda, MD (1991))；及 (c) 抗原接觸存在於胺基酸殘基 27c-36 (L1)、46-55 (L2)、89-96 (L3)、30-35b (H1)、47-58 (H2)、及 93-101 (H3) 處 (MacCallum 等人J. Mol. Biol.262: 732-745 (1996))。Typically, antibodies include six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). In this article, exemplary CDRs include: (a) highly variable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk,J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occur at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al.,Sequences of Proteins of Immunological Interest , 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) antigenic contacts are present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).

除非另有說明，否則 CDR 根據 Kabat 等人在上述文獻中所述之方法來確定。本領域之技術人員將理解，也可以根據 Chothia 在上述文獻、McCallum 在上述文獻中所述之方法或任何其他科學上接受之命名系統來確定 CDR 名稱。Unless otherwise indicated, CDRs are identified according to the method described by Kabat et al., supra. Those skilled in the art will appreciate that CDR names may also be identified according to the method described by Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

術語「可變區 (variable region)」或「可變域 (variable domain)」係指參與抗體與抗原結合的抗體重鏈或輕鏈之域。天然抗體之重鏈及輕鏈 (分別為 VH 及 VL) 之可變域通常具有類似的結構，且每個域均包含四個保留性骨架區 (FR) 及三個互補決定區 (CDR) 或高度可變區 (HVR)。(參見，例如，Kindt 等人Kuby Immunology, 6^thed., W.H. Freeman and Co., page 91 (2007)。) 單個 VH 或 VL 域可能足以賦予抗原結合特異性。此外，可以使用 VH 或 VL 域從結合抗原的抗體中分離結合特定抗原的抗體，以分別篩選互補 VL 或 VH 域的文庫。參見，例如，Portolano 等人，J. Immunol.150:880-887 (1993); Clarkson 等人，Nature352:624-628 (1991)。The term "variable region" or "variable domain" refers to the domain of the antibody heavy chain or light chain that is involved in binding the antibody to the antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, and each domain comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs) or highly variable regions (HVRs). (See, e.g., Kindt et al.Kuby Immunology ,^6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains can be used to separate antibodies that bind a specific antigen from antibodies that bind the antigen to screen libraries for complementary VL or VH domains, respectively. See, e.g., Portolano et al.,J. Immunol. 150:880-887 (1993); Clarkson et al.,Nature 352:624-628 (1991).

「衍生自人源的恆定區」或「人恆定區」表示亞類 IgG1、IgG2、IgG3 或 IgG4 的人抗體的恆定重鏈區及/或恆定輕鏈區 κ 或 λ 區。此類恆定區在現有技術中係習知者，且例如描述於以下文獻中：Kabat, E.A. 等人，Sequences of Proteins of Immunological Interest，第 5 版，Public Health Service，National Institutes of Health，Bethesda，MD (1991) (另見例如，Johnson 及 Wu，Nucleic Acids Res.28 (2000) 214-218；Kabat 等人，Proc. Natl. Acad. Sci. USA72 (1975) 2785-2788)。除非本文另有說明，否則恆定區中胺基酸殘基之編號根據 EU 編號系統 (亦稱為 Kabat 之 EU 索引) 進行，如以下文獻所述：Kabat 等人，Sequences of Proteins of Immunological Interest，第 5 版，Public Health Service, National Institutes of Health，Bethesda，MD (1991)，NIH Publication 91-3242。"Constant regions derived from human sources" or "human constant regions" refer to constant heavy chain regions and/or constant light chain regions kappa or lambda regions of human antibodies of subclass IgG1, IgG2, IgG3 or IgG4. Such constant regions are known in the prior art and are described, for example, in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, for example, Johnson and Wu,Nucleic Acids Res. 28 (2000) 214-218; Kabat et al.,Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise indicated herein, the numbering of amino acid residues in the invariant regions is according to the EU numbering system (also known as the EU index of Kabat) as described in Kabat et al.,Sequences of Proteins of Immunological Interest , 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

術語「Fc 區域」或「Fc 域」在本文中可互換使用，並且係指包含恆定區的至少一部分的免疫球蛋白重鏈的 C 端區。該術語包括天然序列 Fc 區域及變異體 Fc 區域。在一個實施例中，人 IgG 重鏈 Fc 區域從 Cys226 或 Pro230 延伸至重鏈之羧基端。然而，Fc 區域的 C 端離胺酸 (Lys447) 可以存在或可以不存在。該術語涵蓋經截短之 Fc 區，諸如具有 C 端截短的那些 (例如，ΔGK 截短，例如，如以下文獻中所述：Hu 等人，Biotechnol. Prog.2017，33: 786–794；及 Jiang 等人，J. Pharm. Sci.2016，105: 2066–2072)。除非本文另有說明，否則 Fc 區域或恆定區中胺基酸殘基之編號根據 EU 編號系統 (也稱為 EU 指數) 進行，如 Kabat 等人所述 (Sequences of Proteins of Immunological Interest, 第 5 版 Public Health Service, National Institutes of Health, Bethesda, MD, 1991) (另見上文)。如本文所使用之 Fc 域之「次單元」，指代形成二聚體 Fc 域之兩個多肽之一，即包含能夠穩定自締合之免疫球蛋白重鏈之 C 端恆定區之多肽。在一個實施例中，IgG Fc 域之次單元包含 IgG CH2 及 IgG CH3 恆定域。The terms "Fc region" or "Fc domain" are used interchangeably herein and refer to the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. The term encompasses truncated Fc regions, such as those with a C-terminal truncation (e.g., ΔGK truncation, e.g., as described in Hu et al.,Biotechnol. Prog . 2017, 33: 786–794; and Jiang et al.,J. Pharm. Sci. 2016, 105: 2066–2072). Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or cognate region is according to the EU numbering system (also known as the EU index) as described by Kabat et al. (Sequences of Proteins of Immunological Interest , 5th edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991) (see also above). As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising a C-terminal constant region that is capable of stabilizing self-associated immunoglobulin heavy chains. In one embodiment, a subunit of an IgG Fc domain comprises IgG CH2 and IgG CH3 constant domains.

「骨架 (framework)」或「FR」係指除互補決定區 (CDR) 或高度可變區 (HVR) 殘基之外的可變域殘基。可變域之 FR 通常由四個 FR 域組成：FR1、FR2、FR3、及 FR4。因此，CDR 及 FR 序列通常以如下順序出現在 VH (或 VL) 中：FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4。"Framework" or "FR" refers to the variable domain residues excluding the complementary determining regions (CDRs) or hypervariable regions (HVRs). The FR of a variable domain is usually composed of four FR domains: FR1, FR2, FR3, and FR4. Therefore, CDR and FR sequences usually appear in the following order in VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

術語「全長抗體」、「完整抗體」及「全抗體」在本文中可互換使用，係指具有與天然抗體結構實質上類似的結構或具有含有如本文中所定義的 Fc 區的重鏈之抗體。The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein.

「人抗體 (human antibody)」為具有胺基酸序列之抗體，該胺基酸序列對應於由人或人體細胞產生或自利用人抗體譜系 (antibody repertoire) 或其他人抗體編碼序列之非人來源衍生之抗體之胺基酸序列。人抗體的該定義特定地排除包含非人抗原結合殘基之人源化抗體。人抗體可使用本領域中已知的各種技術（包括噬菌體顯示庫）來生產。Hoogenboom 及 Winter，J. Mol. Biol.，227:381 (1991)；Marks 等人，J. Mol. Biol.，222:581 (1991)。亦可用於製備人單株抗體之方法描述於：Cole 等人，Monoclonal Antibodies and Cancer Therapy，Alan R. Liss，第 77 頁 (1985)；Boerner 等人，J. Immunol.，147(1):86-95 (1991)。另見 van Dijk 及 van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001)。可藉由將抗原投予轉基因動物來製備人抗體，該轉基因動物已被改造以回應於抗原攻擊而產生此類抗體，但其內源基因座已失去功能，例如，異源小鼠 (參見例如，關於 XENOMOUSE^™技術之美國專利第 6,075,181 號及第 6,150,584 號)。關於藉由人 B 細胞融合瘤技術產生之人抗體，另見例如 Li 等人，Proc. Natl. Acad. Sci. USA，103:3557-3562 (2006)。A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using the human antibody repertoire or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter,J. Mol. Biol. , 227:381 (1991); Marks et al.,J. Mol. Biol. , 222:581 (1991). Methods for preparing human monoclonal antibodies are also described in Cole et al.,Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, p. 77 (1985); Boerner et al.,J. Immunol. , 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol. , 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been engineered to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., a xenogeneic mouse (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE^™ technology). For human antibodies generated by human B cell fusion tumor technology, see, for example, Li et al.,Proc. Natl. Acad. Sci. USA , 103:3557-3562 (2006).

「人共通骨架」是代表一系列人免疫球蛋白 VL 或 VH 骨架序列中最常見的胺基酸殘基的骨架。通常，人免疫球蛋白 VL 或 VH 序列的選擇來自可變域序列的次群組。通常，序列的亞組是如 Kabat 等人在Sequences of Proteins of Immunological Interest(第 5 版，NIH Publication 91-3242，Bethesda MD (1991)，第 1-3 卷) 中所述之亞組。在一個實施例中，對於 VL，亞組是如 Kabat 等人在上述文獻中所述之亞組 κ I。在一個實施例中，對於 VH，亞組為如 Kabat 等人在上述文獻中所述之亞組 III。A "human common framework" is a framework that represents the most common amino acid residues in a series of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences comes from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup as described by Kabat et al. inSequences of Proteins of Immunological Interest (5th edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3). In one embodiment, for VL, the subgroup is subgroup κ I as described by Kabat et al. in the above-mentioned document. In one embodiment, for VH, the subgroup is subgroup III as described by Kabat et al. in the above-mentioned document.

「人源化」抗體係指包含來自非人類 CDR 或 HVR 之胺基酸殘基及來自人類 FR 之胺基酸殘基之嵌合抗體。在某些實施例中，人源化抗體將包括實質上所有至少一個 (且通常兩個) 可變域，其中所有或實質上所有 CDR 或 HVR 對應於非人抗體之其等，及所有或實質上所有 FR 對應對於人抗體之其等。人源化抗體視情況可包含衍生自人抗體之抗體恆定區之至少一部分。抗體 (例如非人抗體) 之「人源化形式 (humanized form)」係指已經歷人源化之抗體。A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs or HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will include substantially all of at least one (and typically two) variable domains, wherein all or substantially all CDRs or HVRs correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

如本文所用的術語「單株抗體」係指獲自實質上同源抗體群體之抗體，即包含群體的個別抗體是相同的及/或結合相同的抗原決定位，除了例如含有天然生成之突變或於單株抗體製劑生產過程中產生的可能的變異體抗體之外，此等變異體通常係以少量存在。與通常包括針對不同決定位 (抗原決定基) 之不同抗體之多株抗體製劑相反，單株抗體製劑之每個單株抗體係針對於抗原上的單一決定位。因此，修飾詞「單株」表示抗體之特徵係獲自實質上同質之抗體群體，且不應解釋為需要藉由任何特定方法產生抗體。例如，意欲根據本發明使用的單株抗體可藉由多種技術來製造，包括但不限於雜交瘤方法、重組 DNA 方法、噬菌體展示方法、及利用包含全部或部分人免疫球蛋白基因座之基因轉殖動物之方法，本文描述此等方法及用於製備單株抗體之其他示例性方法。The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical and/or bind to the same antigenic determinant, except for possible variant antibodies that contain, for example, naturally occurring mutations or that arise during the production of the monoclonal antibody preparation, such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (antigenic determinants), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous antibody population, and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies intended for use in accordance with the present invention may be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals with genes comprising all or part of the human immunoglobulin loci, these methods and other exemplary methods for making monoclonal antibodies are described herein.

如本文所用，術語「單特異性」係指與單一抗原 (例如，STEAP1) 結合的抗原結合分子。如本文所用，術語「多特異性」係指結合與不同抗原 (例如，STEAP1 及 CD3) 結合的抗原結合分子。舉例而言，「多特異性抗體」為對至少兩個不同位點 (即不同抗原上之不同表位或同一抗原上之不同表位) 具有結合特異性的單株抗體。As used herein, the term "monospecific" refers to an antigen binding molecule that binds to a single antigen (e.g., STEAP1). As used herein, the term "multispecific" refers to an antigen binding molecule that binds to different antigens (e.g., STEAP1 and CD3). For example, a "multispecific antibody" is a monoclonal antibody that has binding specificity for at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen).

「天然抗體」係指具有不同結構的天然生成之免疫球蛋白分子。例如，Ig 天然 IgG 抗體為約 150,000 道耳頓、由二條相同的輕鏈及二條相同的重鏈經二硫鍵鍵合所構成之異四聚體糖蛋白。從 N 端至 C 端，每條重鏈具有可變區 (VH)，亦稱為變異重鏈域或重鏈可變域，接著係三個恆定域（CH1、CH2 及 CH3）。類似地，從 N 端至 C 端，每條輕鏈具有可變區 (VL)，亦稱為變異輕鏈域或輕鏈可變域，接著係輕鏈恆定 (CL) 域。基於其恆定域之胺基酸序列，抗體之輕鏈可被歸類為兩種類型中的一種，稱為卡帕 (κ) 及蘭姆達 (λ)。"Native antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, the Ig natural IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also known as a variable light chain domain or a light chain variable domain, followed by a light chain constant (CL) domain. Based on the amino acid sequence of their homeodomains, the light chains of antibodies can be classified into one of two types, called kappa (κ) and lambda (λ).

相對於參照多肽序列所述之「胺基酸序列同一性百分比 (%)」，是指候選序列中胺基酸殘基與參照多肽序列中之胺基酸殘基相同之百分比，在比對序列並引入差異後 (如有必要)，可實現最大的序列同一性百分比，並且不考慮將任何保守取代作為序列同一性之一部分。為確定胺基酸序列同一性百分比之目的而進行的比對可透過本領域中技術範圍內之各種方式實現，例如，使用公開可用的電腦軟體諸如 BLAST、BLAST-2、Clustal W、Megalign (DNASTAR) 軟件或 FASTA 程式套件實現。本領域之技術人員可確定用於比對序列之合適參數，包括在所比較之序列全長上實現最大比對所需之任何算法。可替代地，可使用序列比較電腦程式 ALIGN-2 生成同一性百分比值。ALIGN-2 序列比較電腦程式由建南德克公司開發，並且其源代碼已與用戶文檔一起歸檔在位於美國華盛頓特區 20559 的美國著作權局，其已經注冊 (美國版權註冊號 TXU510087) 並在 WO 2001/007611 中有所描述。"Percentage (%) of amino acid sequence identity" relative to a reference polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing differences (if necessary), the maximum percentage of sequence identity that can be achieved, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage of amino acid sequence identity can be achieved by various means within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program suite. A person skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. Alternatively, percent identity values may be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was developed by ALIGN-2, Inc., and its source code has been filed with user documentation in the U.S. Copyright Office, Washington, D.C. 20559, registered (U.S. Copyright Registration No. TXU510087) and described in WO 2001/007611.

除非另有說明，否則出於本文之目的，使用 FASTA 套件 36.3.8c 版或更高版本的 ggsearch 程式及 BLOSUM50 比較矩陣來生成胺基酸序列同一性百分比值。FASTA 程式包由以下作者開發：Pearson及 Lipman，Proc. Natl. Acad. Sci. USA85:2444-2448 (1988)；PearsonMeth. Enzymol.266:227- 258 (1996)；及 Pearson 等人，Genomics46:24-36 (1997)，並可從以下網址公開存取：www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml 或 www.ebi.ac.uk/Tools/sss/fasta。可替代地，可使用透過 fasta.bioch.virginia.edu/fasta_www2/index.cgi 存取的公用伺服器，使用 ggsearch (global protein:protein) 程式及預設選項 (BLOSUM50; open: -10; ext: -2; Ktup = 2) 比較序列，以確保執行全局而不是局部比對。胺基酸同一性百分比提供於輸出比對標題中。Unless otherwise noted, for the purposes of this article, percent amino acid sequence identity values were generated using the ggsearch program of the FASTA suite, version 36.3.8c or later, and the BLOSUM50 comparison matrix. The FASTA package was developed by the following authors: Pearson and Lipman,Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988); PearsonMeth. Enzymol. 266:227-258 (1996); and Pearson et al.,Genomics 46:24-36 (1997), and is publicly accessible at the following URLs: www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or www.ebi.ac.uk/Tools/sss/fasta. Alternatively, sequences can be compared using a public server accessed through fasta.bioch.virginia.edu/fasta_www2/index.cgi using the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure that a global rather than a local alignment is performed. The percentage of amino acid identity is provided in the output alignment header.

「分離的」抗體是從其自然環境的組分中分離出來之抗體。在一些態樣中，將抗體純化至大於 95% 或 99% 純度，藉由 (例如) 電泳 (例如 SDS-PAGE、等電聚焦 (IEF)、毛細管電泳) 或層析 (例如，離子交換或反相 HPLC) 方法測定。關於評估抗體純度之方法的綜述，參見例如 Flatman 等人，J. Chromatogr. B848:79-87 (2007)。An "isolated" antibody is one that is separated from the components of its natural environment. In some aspects, the antibody is purified to greater than 95% or 99% purity as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al.,J. Chromatogr. B 848:79-87 (2007).

術語「核酸分子」或「多核苷酸」包括任何包含核苷酸聚合物的化合物及/或物質。每個核苷酸由鹼基 (具體而言，嘌呤或嘧啶鹼基 (亦即，胞嘧啶 (C)、鳥嘌呤 (G)、腺嘌呤 (A)、胸腺嘧啶 (T) 或尿嘧啶 (U)))、糖 (亦即，去氧核糖或核糖) 及磷酸基團構成。通常，核酸分子通過鹼基序列進行描述，其中該鹼基代表核酸分子的一級結構 (線性結構)。鹼基序列通常由 5’ 至 3’ 表示。在本文中，術語核酸分子包括：去氧核糖核酸 (DNA)，其包括例如互補 DNA (cDNA) 及基因組 DNA；核糖核酸 (RNA)，特定而言信使 RNA (mRNA)；DNA 或 RNA 的合成形式；以及包含兩個或更多個這些分子的混合聚合物。核酸分子可以是線性或環狀的。此外，術語核酸分子包括有義股及反義股，以及單股及雙股形式。此外，本文所述之核酸分子可包含天然存在或非天然存在之核苷酸。非天然存在之核苷酸的例子包括帶有衍生糖、磷酸鹽連接或化學修飾殘基的經修飾之核苷酸鹼基。核酸分子亦包括適於在活體外及/或活體內例如在宿主或患者體內直接表現本發明之抗體的載體的 DNA 及 RNA 分子。此等 DNA (例如，cDNA) 或 RNA (例如，mRNA) 載體可以是未修飾的或經過修飾的。舉例而言，mRNA 可經過化學修飾以增強 RNA 載體之穩定性及/或編碼分子之表現，從而將 mRNA 注入個體活體內以產生抗體 (參見例如，Stadler 等人，Nat. Med.23(7):815-817，2017；或 EP 2 101 823 B1)。The term "nucleic acid molecule" or "polynucleotide" includes any compound and/or substance comprising a nucleotide polymer. Each nucleotide is composed of a base (specifically, a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U))), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a base sequence, where the base represents the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually represented from 5' to 3'. As used herein, the term nucleic acid molecule includes: deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), specifically messenger RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes sense and antisense strands, as well as single-stranded and double-stranded forms. In addition, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars, phosphate linkages, or chemically modified residues. Nucleic acid molecules also include DNA and RNA molecules that are suitable for vectors for direct expression of antibodies of the present invention in vitro and/or in vivo, such as in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, thereby injecting the mRNA into a living individual to produce antibodies (see, for example, Stadler et al.,Nat. Med. 23(7):815-817, 2017; or EP 2 101 823 B1).

「分離的」核酸係指已經與其天然環境的組分分離的核酸分子。分離的核酸包括通常包含核酸分子之細胞中所含之核酸分子，但是核酸分子存在於染色體外或與自然染色體位置不同之染色體位置。An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from the natural chromosomal location.

「經分離之編碼抗 STEAP1 抗體的核酸」係指編碼抗 STEAP1 抗體重鏈及輕鏈 (或其片段) 之一種或多種核酸分子，包括在單個載體或單獨抗體中之此等核酸分子，並且此等核酸分子存在於宿主細胞中的一個或多個位置。"Isolated nucleic acid encoding an anti-STEAP1 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of an anti-STEAP1 antibody (or fragments thereof), including such nucleic acid molecules in a single vector or a separate antibody, and such nucleic acid molecules are present at one or more locations in a host cell.

「經分離之編碼抗 CD3 抗體的核酸」係指編碼抗 CD3 抗體重鏈及輕鏈 (或其片段) 之一種或多種核酸分子，包括在單個載體或單獨抗體中之此等核酸分子，並且此等核酸分子存在於宿主細胞中的一個或多個位置。"Isolated nucleic acid encoding an anti-CD3 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of an anti-CD3 antibody (or fragments thereof), including such nucleic acid molecules in a single vector or a separate antibody, and such nucleic acid molecules are present at one or more locations in a host cell.

如本文所用，術語「載體」係指能夠繁殖與其連接的另一核酸的核酸分子。該術語包括作為自我複製核酸結構之載體以及併入已引入該宿主細胞的基因體中的載體。某些載體能夠指導與其可操作地連接的核酸的表現。該等載體在本文中稱為「表現載體」。As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell that has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

術語「宿主細胞」、「宿主細胞株」及「宿主細胞培養物」可互換使用，是指已向其中引入外源性核酸的細胞，包括此等細胞的子代細胞。宿主細胞包括「轉形體」及「轉形細胞」，其包括原代轉形細胞及由其衍生的子代細胞，而與傳代次數無關。子代細胞之核酸含量可能與親代細胞不完全相同，但可能含有突變。本文中包括具有與原始轉化細胞中篩選或選擇的功能或生物活性相同的功能或生物活性的突變子代細胞。The terms "host cell," "host cell strain," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The nucleic acid content of the progeny cells may not be exactly the same as that of the parent cell, but may contain mutations. Mutant progeny cells having the same function or biological activity as that screened or selected in the original transformed cell are included herein.

術語「醫藥調配物」係指以下製劑，其形式為允許其中所含之活性成分的生物活性有效，並且不包含對調配物將投予之個體具有不可接受之毒性的其他組分。The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient contained therein to be effective and which contains no other components which are unacceptably toxic to the subject to which the formulation is to be administered.

「醫藥上可接受之載劑」係指醫藥調配物中除對個體無毒之活性成分以外的成分。醫藥上可接受之載劑包括但不限於緩衝劑、賦形劑、穩定劑或防腐劑。"Pharmaceutically acceptable carriers" refer to ingredients in pharmaceutical formulations other than active ingredients that are non-toxic to individuals. Pharmaceutically acceptable carriers include but are not limited to buffers, excipients, stabilizers or preservatives.

如本文所使用，「投予」意指對個體給予化合物 (例如，本發明之抗原結合分子或編碼本發明之抗原結合分子的核酸) 或組成物 (例如，醫藥組成物，例如包括本發明之抗原結合分子的醫藥組成物) 之劑量之方法。例如，本文所述之方法中所用的組成物可藉由例如肌內、靜脈內、皮內、經皮、動脈內、腹膜內、病灶內、顱內、關節內、前列腺內、胸膜內、氣管內、鼻內、玻璃體內、陰道內、直腸內、外用、腫瘤內、腹膜、皮下、結膜下、囊內、黏膜、心包內、臍內、眼內、口服、外用、局部、經吸入、經注射、經輸注、經連續輸注、經局部直接灌注浴靶細胞、經導管、經灌洗、經乳脂或脂質組成物進行投予。投予方法可以根據多種因素而變化（例如，投予之化合物或組成物以及待治療之病狀、疾病或病症的嚴重程度）。As used herein, "administering" refers to a method of giving a dose of a compound (e.g., an antigen-binding molecule of the present invention or a nucleic acid encoding an antigen-binding molecule of the present invention) or a composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising an antigen-binding molecule of the present invention) to a subject. For example, the compositions used in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intracapsularly, intramucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, topically, by inhalation, by injection, by infusion, by continuous infusion, by local direct perfusion of target cells, by catheter, by lavage, by cream or lipid composition. The method of administration can vary according to a variety of factors (e.g., the compound or composition administered and the severity of the condition, disease or disorder to be treated).

如本文中所使用的「治療 (treatment)」 (及其語法變異體，諸如「治療 (treat)」或「治療 (treating)」)，係指試圖改變受治療個體之疾病自然病程的臨床干預，並且可進行預防或在臨床病理過程中執行。期望之治療效果包括但不限於預防疾病之發生或複發、減輕症狀、減輕疾病之任何直接或間接病理後果、預防轉移、降低疾病進展之速度、改善或減輕疾病狀態、緩解或改善預後。在一些實施例中，本發明抗體用於延遲疾病之發展或減緩疾病之進展。As used herein, "treatment" (and grammatical variants such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of a disease in the individual being treated, and can be performed either preventively or during the course of clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or reducing the disease state, relieving or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

如本文所使用，病症或疾病的「延遲進展」意旨延緩、阻礙、減緩、延遲、穩定及/或推遲疾病或病症 (例如細胞增殖性病症，例如癌症) 的發展。此延緩可具有不同時間長度，視所治療之疾病及/或個體之病史而定。如熟習此項技術者顯而易見，充分或顯著延遲可實際上涵蓋預防，使得該個體不發展該疾病。舉例而言，可延緩晚期癌症，諸如癌轉移發展。As used herein, "delaying the progression" of a disorder or disease means delaying, impeding, slowing, retarding, stabilizing and/or postponing the development of a disease or disorder (e.g., a cell proliferative disorder, such as cancer). This delay can be of varying lengths of time, depending on the disease being treated and/or the individual's medical history. As will be apparent to one skilled in the art, a substantial or significant delay can actually encompass prevention, such that the individual does not develop the disease. For example, advanced cancers, such as metastatic development, can be delayed.

「降低或抑制」意指引起總體減少的能力，較佳為 20％ 或更大、更佳為 50% 或更大、最佳為 75%、85%、90%、95% 或更大。在某些實施例中，減少或抑制可係指經抗體 Fc 區媒介的抗原結合分子之效應功能，此類效應功能具體包括補體依賴性細胞毒性 (CDC)、抗體依賴性細胞毒性 (ADCC) 及抗體-依賴性細胞吞噬作用 (ADCP)。"Reducing or inhibiting" means the ability to cause an overall reduction, preferably 20% or greater, more preferably 50% or greater, and most preferably 75%, 85%, 90%, 95% or greater. In certain embodiments, reduction or inhibition may refer to the effector function of the antigen-binding molecule mediated by the antibody Fc region, such effector functions specifically include complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).

術語「癌症」及「癌性」係指或描述哺乳動物中通常以不受調控的細胞生長為特徵的生理狀況。此定義包括良性及惡性癌症。「早期癌症」或「早期腫瘤」意指非侵襲性或轉移性或分類為 0、1 或 2 期癌症之癌症。癌症之實例包括但不限於實性瘤，諸如腦癌、乳癌、大腸直腸癌、子宮內膜癌、腎癌、肝癌、肺癌、黑色素瘤、胰腺癌、前列腺癌、胃癌或甲狀腺癌；血液系統惡性腫瘤，諸如伯基特氏淋巴瘤 (BL)、多發性骨髓瘤、彌漫型大 B 細胞淋巴瘤 (DLBCL)、濾泡性淋巴瘤 (FL)、被套細胞淋巴瘤 (MCL)、急性骨髓性白血病 (AML)、慢性淋巴性白血病 (CLL)、緣帶淋巴瘤 (MZL)、小淋巴球性白血病 (SLL)、淋巴漿細胞淋巴瘤 (LL) 或瓦登斯特隆巨球蛋白血症 (WM)。The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes both benign and malignant cancers. "Early stage cancer" or "early stage tumor" means cancer that is not invasive or metastatic or is classified as stage 0, 1, or 2 cancer. Examples of cancer include, but are not limited to, solid tumors, such as brain cancer, breast cancer, colorectal cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, gastric cancer, or thyroid cancer; hematological malignancies, such as Burkitt's lymphoma (BL), multiple myeloma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), zonal lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), or Waldenstrom's macroglobulinemia (WM).

如本文所用，術語「腫瘤」係指所有贅生性細胞生長及增生，無論惡性或良性，及所有癌前及癌性細胞及組織。術語「癌症」、「癌性」、「細胞增生性疾病」、「增生性疾病」及「腫瘤」在本文中並不互相排斥。As used herein, the term "tumor" refers to all proliferative cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disease", "proliferative disease" and "tumor" are not mutually exclusive herein.

術語「表現 STEAP1 之癌症」係指與同等非癌細胞中 STEAP1 之表現位準相比，癌細胞之特徵在於 STEAP1 過度表現之癌症。表現 STEAP1 之癌症的實例包括但不限於乳癌、膀胱癌、子宮頸癌、大腸直腸癌、尤文氏肉瘤、肺癌、卵巢癌及前列腺癌。The term "STEAP1-expressing cancer" refers to cancers in which the cancer cells are characterized by overexpression of STEAP1 compared to the expression level of STEAP1 in equivalent non-cancerous cells. Examples of STEAP1-expressing cancers include, but are not limited to, breast cancer, bladder cancer, cervical cancer, colorectal cancer, Ewing's sarcoma, lung cancer, ovarian cancer, and prostate cancer.

前列腺癌為男性最常見的癌症之一。前列腺癌類型可包括前列腺腺癌、前列腺肉瘤、前列腺移行細胞癌、前列腺小細胞癌、前列腺神經內分泌瘤及去勢抗性前列腺癌 (CRPC)。前列腺癌亦可基於分子標記進行分類。舉例而言，前列腺癌可分為 ETS 家族轉錄因子 (例如，ERG、ETV1、ETV4 及 FLI1) 重排的癌症以及 ETS 因子呈陰性的癌症。ETS 陽性前列腺癌亦可包括 PI3K 及 p53 傳訊之改變。ETS 陰性前列腺癌可顯示出 SPOP、FOXA1 及 IDH1 中之復現突變；CHD1 之缺失；及 SPINK1 之過度表現。在一些情況下，前列腺癌為轉移性前列腺癌 (例如，轉移性去勢抵抗性前列腺癌)。在其他情況下，前列腺癌為復發性或難治性前列腺癌。Prostate cancer is one of the most common cancers in men. Types of prostate cancer may include prostate adenocarcinoma, prostate sarcoma, prostate transitional cell carcinoma, prostate small cell carcinoma, prostate neuroendocrine tumors, and castration-resistant prostate cancer (CRPC). Prostate cancer may also be classified based on molecular markers. For example, prostate cancer may be divided into cancers that have rearrangements of the ETS family transcription factors (e.g., ERG, ETV1, ETV4, and FLI1) and cancers that are negative for ETS factors. ETS-positive prostate cancer may also include alterations in PI3K and p53 signaling. ETS-negative prostate cancer may show recurrent mutations in SPOP, FOXA1, and IDH1; loss of CHD1; and overexpression of SPINK1. In some cases, the prostate cancer is metastatic prostate cancer (e.g., metastatic castration-resistant prostate cancer). In other cases, the prostate cancer is recurrent or refractory.

尤文氏肉瘤 (亦稱為尤文氏肉瘤或尤文氏腫瘤) 為骨骼及軟組織 (例如，軟骨或神經) 之癌症。存在若干類型之尤文氏肉瘤，諸如骨骼之尤文氏肉瘤、骨外尤文氏肉瘤、周邊原始神經外胚層腫瘤 (pPNET) 及阿斯金腫瘤 (Askin tumor)。尤文氏肉瘤的病因尚不清楚。症狀包括腫瘤部位處之疼痛及腫脹。Ewing sarcoma (also called Ewing's sarcoma or Ewing's tumor) is a cancer of the bones and soft tissues (for example, cartilage or nerves). There are several types of Ewing's sarcoma, such as skeletal Ewing's sarcoma, extraosseous Ewing's sarcoma, peripheral primitive neuroectodermal tumor (pPNET), and Askin tumor. The cause of Ewing's sarcoma is unknown. Symptoms include pain and swelling at the site of the tumor.

如本文所用，術語「腫瘤抗原」可理解為那些在腫瘤細胞上呈遞的抗原。這些抗原可在細胞表面上用細胞外部分呈遞，其通常與分子之跨膜及細胞質部分合併。這些抗原有時只能由腫瘤細胞呈遞，而不能由正常細胞呈遞。腫瘤抗原可僅僅在腫瘤細胞上表現，或者與正常細胞相比可以表示腫瘤特異性突變。在此情況下，它們被稱為腫瘤特異性抗原。更常見的是由腫瘤細胞及正常細胞呈遞的腫瘤抗原，並且它們被稱為腫瘤相關抗原。與正常細胞相比，這些腫瘤相關抗原可過表現，或者由於與正常組織相比腫瘤組織的結構較不緊湊，因此能夠用於腫瘤細胞中的抗體結合。示例性腫瘤抗原包括但不限於前列腺特異性膜抗原 (PSMA)、前列腺幹細胞抗原 (PSCA)、上皮細胞黏著分子 (EpCAM)、前列腺特異性抗原 (PSA)、前列腺酸性磷酸酶 (PAP)、STEAP2 及 HBA-71。在一些情況下，前列腺癌的 TAA 包括前列腺特異性膜抗原 (PSMA)、前列腺幹細胞抗原 (PSCA)、上皮細胞黏著分子 (EpCAM)、前列腺特異性抗原 (PSA) 及前列腺酸性磷酸酶 (PAP)。在一些情況下，尤文氏肉瘤的 TAA 包括 HBA-71 (一種位於腫瘤細胞的細胞表面醣質包被的抗原)。As used herein, the term "tumor antigen" is understood to mean those antigens that are presented on tumor cells. These antigens may be presented on the cell surface with an extracellular portion, which is usually combined with the transmembrane and cytoplasmic portions of the molecule. These antigens are sometimes only presented by tumor cells and not by normal cells. Tumor antigens may be expressed only on tumor cells or may represent tumor-specific mutations compared to normal cells. In this case, they are called tumor-specific antigens. More common are tumor antigens that are presented by tumor cells and normal cells, and they are called tumor-associated antigens. These tumor-associated antigens may be overexpressed compared to normal cells or may be available for antibody binding in tumor cells due to the less compact structure of tumor tissue compared to normal tissue. Exemplary tumor antigens include, but are not limited to, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), epithelial cell adhesion molecule (EpCAM), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), STEAP2, and HBA-71. In some instances, TAAs for prostate cancer include prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), epithelial cell adhesion molecule (EpCAM), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP). In some cases, TAAs for Ewing sarcoma include HBA-71, a cell surface carbohydrate-coated antigen found on tumor cells.

術語「T 細胞受體」或 TCR 係指在 T 細胞上表現的受體。示例性 T 細胞受體包括但不限於 CD3。The term "T cell receptor" or TCR refers to a receptor expressed on T cells. Exemplary T cell receptors include, but are not limited to, CD3.

本發明之雙特異性抗原結合分子、或其組成物 (例如，醫藥組成物) 的「有效量」至少為達到預期治療或預防結果 (諸如特定疾患 (例如，細胞增生性疾病，例如癌症) 的可測量之改善或預防) 所需要的最小量。本文中之有效量可根據諸如以下因素而變化：患者之疾病病況、年齡、性別及體重，以及抗體引發個體發生所需反應之能力。有效量亦為該治療之任意毒性或有害效應被治療有益效應超過的量。對於防治用途而言，有益或所需結果包括諸如以下之結果：消除或降低疾病之風險、減輕疾病之嚴重程度，或延緩疾病發作，疾病包括疾病、其併發症及在疾病發展期間所呈現之中間病理學表型之生物化學、組織學及/或行為症狀。對於治療用途而言，有益或所需結果包括諸如以下之臨床結果：減少由疾病引起之一種或多種症狀、提高患病者之生活品質、降低治療疾病所需之其他藥物的劑量、增強另一藥劑之作用 (諸如經由靶向)、延緩疾病進展及/或延長存活期。就癌症或腫瘤而言，有效量之藥物可具有以下效果：減少癌細胞數；減小腫瘤尺寸；抑制 (亦即，在一定程度上減緩或在理想情況下終止) 癌細胞浸潤入週邊器官中；抑制 (亦即，在一定程度上減緩或在理想情況下終止) 腫瘤轉移；在一定程度上抑制腫瘤生長；及/或在一定程度上減輕與該疾患相關之症狀中的一者或多者。有效量可於一次或多次投予中投予。出於本發明的目的，藥物、化合物或藥物組成物的有效量為足以直接或間接完成預防性或治療性治療的量。如在臨床背景中理解，藥物、化合物或藥物組成物之有效量可與或不與另一藥物、化合物或醫藥組成物聯合而達成。因此，在投予一種或多種治療劑之上下文中可慮及「有效量」，且若單個藥劑與一種或多種其他藥劑聯合而可實現或已實現所需結果，則該單個藥劑可視為以有效量給出。An "effective amount" of the bispecific antigen-binding molecules of the invention, or a composition thereof (e.g., a pharmaceutical composition) is at least the minimum amount required to achieve a desired therapeutic or preventive result, such as a measurable improvement or prevention of a particular disease (e.g., a cell proliferative disease, such as cancer). The effective amount herein may vary depending on factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also an amount in which any toxic or deleterious effects of the treatment are outweighed by the beneficial effects of the treatment. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including the biochemical, histological and/or behavioral symptoms of disease, its complications and intermediate pathological phenotypes presented during the development of disease. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms caused by the disease, improving the quality of life of the patient, reducing the dosage of other drugs required to treat the disease, enhancing the effect of another drug (such as through targeting), delaying the progression of disease and/or prolonging survival. In the case of cancer or tumors, an effective amount of a drug may have the following effects: reducing the number of cancer cells; reducing the size of tumors; inhibiting (i.e., slowing down to some extent or, ideally, stopping) the infiltration of cancer cells into peripheral organs; inhibiting (i.e., slowing down to some extent or, ideally, stopping) tumor metastasis; inhibiting tumor growth to some extent; and/or alleviating to some extent one or more of the symptoms associated with the disease. An effective amount may be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly accomplish a preventive or therapeutic treatment. As understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may be achieved with or without combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if it, in combination with one or more other agents, can achieve or have achieved the desired result.

如本文所用，「一線療法」包含對患有癌症的個體之主要治療。在一些情況下，癌症為原發性癌症。在其他情況下，癌症為轉移性或復發性癌症。在一些情況下，一線治療包含化學療法。在其他情況下，一線治療包含放射療法。技術人員將容易理解,不同的一線治療可適用於不同類型之癌症。As used herein, "first-line therapy" includes the main treatment for an individual with cancer. In some cases, the cancer is a primary cancer. In other cases, the cancer is a metastatic or recurrent cancer. In some cases, the first-line therapy includes chemotherapy. In other cases, the first-line therapy includes radiation therapy. A skilled artisan will readily appreciate that different first-line therapies may be appropriate for different types of cancer.

在一些情況下，額外治療劑包含二線療法、三線療法、四線療法或五線療法。如本文所用，二線療法涵蓋在主要或一線治療停止後所用的治療。三線療法、四線療法或五線療法涵蓋後續治療。如命名慣例所示，三線療法涵蓋主要療法及二線療法業已停止的治療過程。In some cases, the additional treatment comprises a second-line therapy, a third-line therapy, a fourth-line therapy, or a fifth-line therapy. As used herein, second-line therapy encompasses treatment used after the primary or first-line therapy has stopped. Third-line therapy, fourth-line therapy, or fifth-line therapy encompasses subsequent treatment. As indicated by naming convention, third-line therapy encompasses a course of treatment after the primary and second-line therapies have stopped.

如本文所用，術語「受試者」、「患者」或「個體」可互換使用，並且各自係指哺乳動物。哺乳動物包括但不限於馴養的動物 (例如牛、綿羊、貓、狗及馬)、靈長類動物 (例如人及非人類靈長類動物諸如猴)、兔以及囓齒動物 (例如小鼠及大鼠)。在某些態樣中，受試者、患者或個體為人類。II.組成物As used herein, the terms "subject,""patient," or "individual" are used interchangeably and each refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the subject, patient, or individual is a human.II.Compositions

在一個態樣中，本發明部分地基於抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子)。在一些實施例中，抗原結合分子與 STEAP1 結合。在一些實施例中，抗原結合分子為與 STEAP1 及一種或多種額外所關注抗原結合的多特異性抗原結合分子。在某些實施例中，多特異性抗原結合分子與 STEAP1 單價結合。本發明之抗原結合分子可用於例如治療癌症 (例如，表現 STEAP1 之癌症) 或延緩其進展。與STEAP1結合的示例性抗原結合分子與STEAP1結合的抗原結合分子In one aspect, the present invention is based in part on antigen binding molecules (e.g., monospecific and/or multispecific antigen binding molecules). In some embodiments, the antigen binding molecule binds to STEAP1. In some embodiments, the antigen binding molecule is a multispecific antigen binding molecule that binds to STEAP1 and one or more additional antigens of interest. In certain embodiments, the multispecific antigen binding molecule monovalently bindsto STEAP1. The antigen binding molecules of the present invention can be used, for example, to treat cancer (e.g., a cancer expressing STEAP1) or slow its progression.Exemplary antigen binding molecules that bindtoSTEAP1Antigen binding molecules that bind toSTEAP1

在一些實施例中，本發明提供與 STEAP1 結合的經分離之抗原結合分子。在一些實施例中，本發明之抗原結合分子包含如表 1 (Kabat) 或表 2 (Chothia) 中所示之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。在一些情況下，抗原結合分子包含如表 1 中所示之 VH 及/或 VL。表1SEQ. ID. NO 列表CDR (Kabat)VH1H2H3L1L2L3VHVLSTEAP1-4412345678STEAP1-44.NGS.HC19113456178STEAP1-44.NGS.HC29123456188STEAP1-44.NGS.HC391214456198STEAP1-44.NGS.HC4101315456208STEAP1-44.NGS.HC510216456218huAb44.v11234562226huAb44.v21234562227huAb44.v31234562228huAb44.v41234562229huAb44.v51234562326huAb44.v61234562327huAb44.v71234562328huAb44.v81234562329huAb44.v91234562426huAb44.v101234562427huAb44.v111234562428huAb44.v121234562429huAb44.v131234562526huAb44.v141234562527huAb44.v151234562528huAb44.v161234562529huAb44.v6.0191134563027huAb44.v6.0291234563127huAb44.v6.03912144563227huAb44.v6.041013154563327huAb44.v6.05102164563427huAb44.v6.05 EKKE102164566869huAb44 共通353637456383938E4v1.MD1404142434445464740G5c4849505152535455rbAb34049091929394959697rbAb33499899100101102103104105表2SEQ. ID. NO 列表CDR (Chothia)H1H2H3L1L2L3STEAP1-4456573456STEAP1-44.NGS.HC158603456STEAP1-44.NGS.HC258613456STEAP1-44.NGS.HC3586114456STEAP1-44.NGS.HC4596215456STEAP1-44.NGS.HC5595716456huAb44.v156573456huAb44.v256573456huAb44.v356573456huAb44.v456573456huAb44.v556573456huAb44.v656573456huAb44.v756573456huAb44.v856573456huAb44.v956573456huAb44.v1056573456huAb44.v1156573456huAb44.v1256573456huAb44.v1356573456huAb44.v1456573456huAb44.v1556573456huAb44.v1656573456huAb44.v6.0158603456huAb44.v6.0258613456huAb44.v6.03586114456huAb44.v6.04596215456huAb44.v6.05595716456huAb44 共通63643745638E4v1.MD186874243444540G5c888950515253rbAb340410610792939495rbAb3349108109100101102103In some embodiments, the present invention provides an isolated antigen-binding molecule that binds to STEAP1. In some embodiments, the antigen-binding molecule of the present invention comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs as shown in Table 1 (Kabat) or Table 2 (Chothia). In some cases, the antigen-binding molecule comprises a VH and/or VL as shown in Table 1.Table1 SEQ. ID. NO List CDR (Kabat) V H1 H2 H3 L1 L2 L3 VH V L STEAP1-44 1 2 3 4 5 6 7 8 STEAP1-44.NGS.HC1 9 11 3 4 5 6 17 8 STEAP1-44.NGS.HC2 9 12 3 4 5 6 18 8 STEAP1-44.NGS.HC3 9 12 14 4 5 6 19 8 STEAP1-44.NGS.HC4 10 13 15 4 5 6 20 8 STEAP1-44.NGS.HC5 10 2 16 4 5 6 twenty one 8 huAb44.v1 1 2 3 4 5 6 twenty two 26 huAb44.v2 1 2 3 4 5 6 twenty two 27 huAb44.v3 1 2 3 4 5 6 twenty two 28 huAb44.v4 1 2 3 4 5 6 twenty two 29 huAb44.v5 1 2 3 4 5 6 twenty three 26 huAb44.v6 1 2 3 4 5 6 twenty three 27 huAb44.v7 1 2 3 4 5 6 twenty three 28 huAb44.v8 1 2 3 4 5 6 twenty three 29 huAb44.v9 1 2 3 4 5 6 twenty four 26 huAb44.v10 1 2 3 4 5 6 twenty four 27 huAb44.v11 1 2 3 4 5 6 twenty four 28 huAb44.v12 1 2 3 4 5 6 twenty four 29 huAb44.v13 1 2 3 4 5 6 25 26 huAb44.v14 1 2 3 4 5 6 25 27 huAb44.v15 1 2 3 4 5 6 25 28 huAb44.v16 1 2 3 4 5 6 25 29 huAb44.v6.01 9 11 3 4 5 6 30 27 huAb44.v6.02 9 12 3 4 5 6 31 27 huAb44.v6.03 9 12 14 4 5 6 32 27 huAb44.v6.04 10 13 15 4 5 6 33 27 huAb44.v6.05 10 2 16 4 5 6 34 27 huAb44.v6.05 EKKE 10 2 16 4 5 6 68 69 huAb44 Common 35 36 37 4 5 6 38 39 38E4v1.MD1 40 41 42 43 44 45 46 47 40G5c 48 49 50 51 52 53 54 55 rbAb3404 90 91 92 93 94 95 96 97 rbAb3349 98 99 100 101 102 103 104 105Table2 SEQ. ID. NO List CDR (Chothia) H1 H2 H3 L1 L2 L3 STEAP1-44 56 57 3 4 5 6 STEAP1-44.NGS.HC1 58 60 3 4 5 6 STEAP1-44.NGS.HC2 58 61 3 4 5 6 STEAP1-44.NGS.HC3 58 61 14 4 5 6 STEAP1-44.NGS.HC4 59 62 15 4 5 6 STEAP1-44.NGS.HC5 59 57 16 4 5 6 huAb44.v1 56 57 3 4 5 6 huAb44.v2 56 57 3 4 5 6 huAb44.v3 56 57 3 4 5 6 huAb44.v4 56 57 3 4 5 6 huAb44.v5 56 57 3 4 5 6 huAb44.v6 56 57 3 4 5 6 huAb44.v7 56 57 3 4 5 6 huAb44.v8 56 57 3 4 5 6 huAb44.v9 56 57 3 4 5 6 huAb44.v10 56 57 3 4 5 6 huAb44.v11 56 57 3 4 5 6 huAb44.v12 56 57 3 4 5 6 huAb44.v13 56 57 3 4 5 6 huAb44.v14 56 57 3 4 5 6 huAb44.v15 56 57 3 4 5 6 huAb44.v16 56 57 3 4 5 6 huAb44.v6.01 58 60 3 4 5 6 huAb44.v6.02 58 61 3 4 5 6 huAb44.v6.03 58 61 14 4 5 6 huAb44.v6.04 59 62 15 4 5 6 huAb44.v6.05 59 57 16 4 5 6 huAb44 Common 63 64 37 4 5 6 38E4v1.MD1 86 87 42 43 44 45 40G5c 88 89 50 51 52 53 rbAb3404 106 107 92 93 94 95 rbAb3349 108 109 100 101 102 103

在一些實施例中，與 STEAP1 結合的抗原結合分子包含：抗 STEAP1 重鏈可變區 (VH)，其包含選自 SEQ ID NO: 7、17 至 25、30 至 34 及 38 的 VH 序列之 CDR-H1、CDR-H2 及/或 CDR-H3、基本上由其組成、或由其組成；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含選自 SEQ ID NO: 8、26 至 29、39 及 69 的 VL 序列之 CDR-L1、CDR-L2 及/或 CDR-L3、基本上由其組成、或由其組成。在一些情況下，至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR 選自含有 SEQ ID NO: 7、17 至 25、30 至 34、38 及 68 之 VH 序列及選自 SEQ ID NO: 8、26 至 29、39 及 69 之 VL 序列的 CDR。在一些情況下，六個 CDR 係根據 Kabat 編號定義 (參見表 1)。在其他情況下，六個 CDR 係根據 Chothia 編號定義 (參見表 2)。在額外情況下，六個 CDR 係根據 EU 編號定義。In some embodiments, the antigen binding molecule that binds to STEAP1 comprises: an anti-STEAP1 heavy chain variable region (VH) comprising, consisting essentially of, or consisting of CDR-H1, CDR-H2 and/or CDR-H3 of a VH sequence selected from SEQ ID NOs: 7, 17 to 25, 30 to 34, and 38; and/or an anti-STEAP1 light chain variable region (VL) comprising, consisting essentially of, or consisting of CDR-L1, CDR-L2 and/or CDR-L3 of a VL sequence selected from SEQ ID NOs: 8, 26 to 29, 39, and 69. In some cases, at least one, at least two, at least three, at least four, at least five, or all six CDRs are selected from a CDR comprising a VH sequence of SEQ ID NOs: 7, 17 to 25, 30 to 34, 38, and 68 and a VL sequence selected from SEQ ID NOs: 8, 26 to 29, 39, and 69. In some cases, the six CDRs are defined according to Kabat numbering (see Table 1). In other cases, the six CDRs are defined according to Chothia numbering (see Table 2). In additional cases, the six CDRs are defined according to EU numbering.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3；其中： CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)； 其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)； 其中 Xaa³為 Asp (D) 或 Ser (S)； Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)； Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen-binding molecule that binds to STEAP1 comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein: CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Ser (S); Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (N); and Xaa⁶ is Ser (S) or Tyr (Y); CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen binding molecule comprises at least two, at least three, at least four, at least five, or all six of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3；其中： CDR-H1 包含 SEQ ID NO: 10、1 或 9 之胺基酸序列； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)； 其中 Xaa³為 Asp (D) 或 Ser (S)； Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)； Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen-binding molecule that binds to STEAP1 comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein: CDR-H1 comprises the amino acid sequence of SEQ ID NO: 10, 1 or 9; CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Ser (S); Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (N); and Xaa⁶ is Ser (S) or Tyr (Y); CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen binding molecule comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3； 其中 CDR-H1 包含 SEQ ID NO: 10、1 或 9 之胺基酸序列； CDR-H2 包含 SEQ ID NO: 2、11、12 或 13 之胺基酸序列； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen-binding molecule that binds to STEAP1 comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 10, 1 or 9; CDR-H2 comprises the amino acid sequence of SEQ ID NO: 2, 11, 12 or 13; CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen-binding molecule comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3； 其中 CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)； 其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 SEQ ID NO: 2、11、12 或 13 之胺基酸序列； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen binding molecule that binds to STEAP1 comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises the amino acid sequence of SEQ ID NO: 2, 11, 12 or 13; CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen-binding molecule comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3； 其中 CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)； 其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 SEQ ID NO: 2、11、12 或 13 之胺基酸序列； CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen binding molecule that binds to STEAP1 comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises the amino acid sequence of SEQ ID NO: 2, 11, 12 or 13; CDR-H3 comprises the amino acid sequence of SEQ ID NO: 16, 3, 14 or 15; CDR-L1 comprises SEQ ID NO: 35; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen-binding molecule comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3； 其中 CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)； 其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)； 其中 Xaa³為 Asp (D) 或 Ser (S)； Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)； Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)； CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen-binding molecule that binds to STEAP1 comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Ser (S); Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (N); and Xaa⁶ is Ser (S) or Tyr (Y); CDR-H3 comprises the amino acid sequence of SEQ ID NO: 16, 3, 14 or 15; CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen-binding molecule comprises at least two, at least three, at least four, at least five or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含 抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3； 其中 CDR-H1 包含 SEQ ID NO: 10、1 或 9 之胺基酸序列； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)； 其中 Xaa³為 Asp (D) 或 Ser (S)； Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)； Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)； CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen-binding molecule that binds to STEAP1 comprises an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 10, 1 or 9; CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Ser (S); Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (S); (N); and Xaa⁶ is Ser (S) or Tyr (Y); CDR-H3 comprises the amino acid sequence of SEQ ID NO: 16, 3, 14 or 15; CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen-binding molecule comprises at least two, at least three, at least four, at least five or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3；其中 CDR-H1 包含 SEQ ID NO: 10、1 或 9 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2 包含 SEQ ID NO: 2、11、12 或 13 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen binding molecule that binds to STEAP1 comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 10, 1 or 9; CDR-H2 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 2, 11, 12 or 13; CDR-H3 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 16, 3, 14 or 15; CDR-L1 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 4 In some cases, the anti-STEAP1 antigen-binding molecule comprises at least two, at least three, at least four, at least five, or all six of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

在一些情況下，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 1 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the antigen binding molecule comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 11 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the antigen binding molecule comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 11; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the antigen binding molecule comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 12; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 14 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the antigen binding molecule comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 12; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 14; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 13 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 15 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the antigen binding molecule comprises: a CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 10; a CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 13; a CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 15; a CDR-L1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 4; a CDR-L2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 5; and a CDR-L3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 6.

在一些情況下，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 16 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the antigen binding molecule comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 10; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3；其中 CDR-H1 包含 GFTFSXaa¹⁰Xaa¹¹(SEQ ID NO: 63)； 其中 Xaa¹⁰為 Asn (N) 或 Asp (D)；且 Xaa¹¹為 Tyr (Y)、Phe (F) 或 His (H)； CDR-H2 包含 Xaa¹²YDGXaa¹³Xaa¹⁴(SEQ ID NO: 64)； 其中 Xaa¹²為 Asp (D) 或 Ser (S)； Xaa¹³為 Gly (G)、Asp (D) 或 Leu (L)；且 Xaa¹⁴為 Ser (S)、Asp (D) 或 Asn (N)； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen-binding molecule that binds to STEAP1 comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises GFTFSXaa¹⁰ Xaa¹¹ (SEQ ID NO: 63); wherein Xaa¹⁰ is Asn (N) or Asp (D); and Xaa¹¹ is Tyr (Y), Phe (F) or His (H); CDR-H2 comprises Xaa¹² YDGXaa¹³ Xaa¹⁴ (SEQ ID NO: 64); wherein Xaa¹² is Asp (D) or Ser (S); Xaa¹³ is Gly (G), Asp (D) or Leu (L); and Xaa¹⁴ is Ser (S), Asp (D) or Asn (N); CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen binding molecule comprises at least two, at least three, at least four, at least five, or all six of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含 抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3；其中 CDR-H1 包含 SEQ ID NO: 56、58 或 59 之胺基酸序列； CDR-H2 包含 Xaa¹²YDGXaa¹³Xaa¹⁴(SEQ ID NO: 64)； 其中 Xaa¹²為 Asp (D) 或 Ser (S)； Xaa¹³為 Gly (G)、Asp (D) 或 Leu (L)；且 Xaa¹⁴為 Ser (S)、Asp (D) 或 Asn (N)； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen-binding molecule that binds to STEAP1 comprises an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 56, 58 or 59; CDR-H2 comprises Xaa¹² YDGXaa¹³ Xaa¹⁴ (SEQ ID NO: 64); wherein Xaa¹² is Asp (D) or Ser (S); Xaa¹³ is Gly (G), Asp (D) or Leu (L); and Xaa¹⁴ is Ser (S), Asp (D) or Asn (N); CDR-H3 comprising RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen-binding molecule comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含 抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3；其中 CDR-H1 包含 SEQ ID NO: 56、58 或 59 之胺基酸序列； CDR-H2 包含 SEQ ID NO: 57、60、61 或 62 之胺基酸序列； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen binding molecule that binds to STEAP1 comprises an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises an amino acid sequence of SEQ ID NO: 56, 58 or 59; CDR-H2 comprises an amino acid sequence of SEQ ID NO: 57, 60, 61 or 62; CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen-binding molecule comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含 抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3；其中 CDR-H1 包含 GFTFSXaa¹⁰Xaa¹¹(SEQ ID NO: 63)； 其中 Xaa¹⁰為 Asn (N) 或 Asp (D)；且 Xaa¹¹為 Tyr (Y)、Phe (F) 或 His (H)； CDR-H2 包含 SEQ ID NO: 57、60、61 或 62 之胺基酸序列； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen binding molecule that binds to STEAP1 comprises an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises GFTFSXaa¹⁰ Xaa¹¹ (SEQ ID NO: 63); wherein Xaa¹⁰ is Asn (N) or Asp (D); and Xaa¹¹ is Tyr (Y), Phe (F) or His (H); CDR-H2 comprises the amino acid sequence of SEQ ID NO: 57, 60, 61 or 62; CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen-binding molecule comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含 抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3；其中 CDR-H1 包含 GFTFSXaa¹⁰Xaa¹¹(SEQ ID NO: 63)； 其中 Xaa¹⁰為 Asn (N) 或 Asp (D)；且 Xaa¹¹為 Tyr (Y)、Phe (F) 或 His (H)； CDR-H2 包含 SEQ ID NO: 57、60、61 或 62 之胺基酸序列； CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen binding molecule that binds to STEAP1 comprises an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises GFTFSXaa¹⁰ Xaa¹¹ (SEQ ID NO: 63); wherein Xaa¹⁰ is Asn (N) or Asp (D); and Xaa¹¹ is Tyr (Y), Phe (F) or His (H); CDR-H2 comprises the amino acid sequence of SEQ ID NO: 57, 60, 61 or 62; CDR-H3 comprises the amino acid sequence of SEQ ID NO: 16, 3, 14 or 15; CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen-binding molecule comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含 抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3；其中 CDR-H1 包含 GFTFSXaa¹⁰Xaa¹¹(SEQ ID NO: 63)； 其中 Xaa¹⁰為 Asn (N) 或 Asp (D)；且 Xaa¹¹為 Tyr (Y)、Phe (F) 或 His (H)； CDR-H2 包含 Xaa¹²YDGXaa¹³Xaa¹⁴(SEQ ID NO: 64)； 其中 Xaa¹²為 Asp (D) 或 Ser (S)； Xaa¹³為 Gly (G)、Asp (D) 或 Leu (L)；且 Xaa¹⁴為 Ser (S)、Asp (D) 或 Asn (N)； CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen binding molecule that binds to STEAP1 comprises an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises GFTFSXaa¹⁰ Xaa¹¹ (SEQ ID NO: 63); wherein Xaa¹⁰ is Asn (N) or Asp (D); and Xaa¹¹ is Tyr (Y), Phe (F) or His (H); CDR-H2 comprises Xaa¹² YDGXaa¹³ Xaa¹⁴ (SEQ ID NO: 64); wherein Xaa¹² is Asp (D) or Ser (S); Xaa and^{Xaa 13} is Gly (G), Asp (D) or Leu (L); and Xaa¹⁴ is Ser (S), Asp (D) or Asn (N); CDR-H3 comprises the amino acid sequence of SEQ ID NO: 16, 3, 14 or 15; CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen-binding molecule comprises at least two, at least three, at least four, at least five or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含 抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3；其中 CDR-H1 包含 SEQ ID NO: 56、58 或 59 之胺基酸序列； CDR-H2 包含 Xaa¹²YDGXaa¹³Xaa¹⁴(SEQ ID NO: 64)； 其中 Xaa¹²為 Asp (D) 或 Ser (S)； Xaa¹³為 Gly (G)、Asp (D) 或 Leu (L)；且 Xaa¹⁴為 Ser (S)、Asp (D) 或 Asn (N)； CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen-binding molecule that binds to STEAP1 comprises an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 56, 58 or 59; CDR-H2 comprises Xaa¹² YDGXaa¹³ Xaa¹⁴ (SEQ ID NO: 64); wherein Xaa¹² is Asp (D) or Ser (S); Xaa¹³ is Gly (G), Asp (D) or Leu (L); and Xaa¹⁴ is Ser (S), Asp (D) or Asn (N); CDR-H3 comprising an amino acid sequence of SEQ ID NO: 16, 3, 14 or 15; CDR-L1 comprising an amino acid sequence of SEQ ID NO: 4; CDR-L2 comprising an amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprising an amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen-binding molecule comprises at least two, at least three, at least four, at least five or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3.

在一些實施例中，與 STEAP1 結合的抗原結合分子包含 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及/或 CDR-L3；其中 CDR-H1 包含 SEQ ID NO: 56、58 或 59 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2 包含 SEQ ID NO: 57、60、61 或 62 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由組成；CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由組成；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，抗 STEAP1 抗原結合分子包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。In some embodiments, the antigen binding molecule that binds to STEAP1 comprises a heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3; wherein CDR-H1 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 56, 58 or 59; CDR-H2 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 57, 60, 61 or 62; CDR-H3 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 16, 3, 14 or 15; CDR-L1 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 4 ; CDR-L2 comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6. In some cases, the anti-STEAP1 antigen-binding molecule comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

在一些情況下，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 56 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 57 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the antigen binding molecule comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 56; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 57; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 58 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 60 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the antigen binding molecule comprises: a CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 58; a CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 60; a CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 3; a CDR-L1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 4; a CDR-L2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 5; and a CDR-L3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 6.

在一些情況下，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 58 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 61 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the antigen binding molecule comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 58; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 61; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 58 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 61 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 14 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the antigen binding molecule comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 58; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 61; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 14; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 59 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 62 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 15 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the antigen binding molecule comprises: a CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 59; a CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 62; a CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 15; a CDR-L1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 4; a CDR-L2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 5; and a CDR-L3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 6.

在一些情況下，抗原結合分子包含：CDR-H1，其包含 SEQ ID NO: 59 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 57 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 16 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the antigen binding molecule comprises: a CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 59; a CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 57; a CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 16; a CDR-L1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 4; a CDR-L2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 5; and a CDR-L3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 6.

本發明之抗原結合分子可為人源化抗體。在一些情況下，本發明之抗原結合分子包含衍生自 IgG 骨架區之恆定區。在一些情況下，IgG 骨架區為 IgG₁、IgG₂或 IgG₄骨架區。在一些情況下，IgG 骨架區為 IgG₁骨架區。The antigen-binding molecules of the present invention may be humanized antibodies. In some cases, the antigen-binding molecules of the present invention comprise a constant region derived from an IgG framework region. In some cases, the IgG framework region is an IgG₁ , IgG₂ or IgG₄ framework region. In some cases, the IgG framework region is an IgG₁ framework region.

在一些實施例中，抗原結合分子包含重鏈可變區，該重鏈可變區包含以下項中之一個或多個 (例如，1 個、2 個、3 個或全部 4 個)：與 SEQ ID NO: 110 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的骨架區 (FR)-H1 序列；與 SEQ ID NO: 118 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-H2；與 SEQ ID NO: 112 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-H3；及/或與 SEQ ID NO: 113 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-H4。在一些態樣中，抗原結合分子包含重鏈可變區，該重鏈可變區包含與 SEQ ID NO: 118 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-H2。在一些實施例中，抗原結合分子包含重鏈可變區，該重鏈可變區包含具有 SEQ ID NO: 118 之胺基酸序列的 FR-H2。在其他實施例中，抗原結合分子包含重鏈可變區，該重鏈可變區包含具有 SEQ ID NO: 120 之胺基酸序列的 FR-H2。In some embodiments, the antigen binding molecule comprises a heavy chain variable region comprising one or more (e.g., 1, 2, 3, or all 4) of the following: a framework region (FR)-H1 sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 110; a FR-H2 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118; a FR-H2 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 112; and/or a FR-H4 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 113. In some aspects, the antigen binding molecule comprises a heavy chain variable region comprising a FR-H2 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118. In some embodiments, the antigen binding molecule comprises a heavy chain variable region comprising FR-H2 having an amino acid sequence of SEQ ID NO: 118. In other embodiments, the antigen binding molecule comprises a heavy chain variable region comprising FR-H2 having an amino acid sequence of SEQ ID NO: 120.

在一些實施例中，抗原結合分子包含重鏈可變區，該重鏈可變區包含以下項中之一個或多個 (例如，1 個、2 個、3 個或全部 4 個)：與 SEQ ID NO: 110 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-H1 序列；與 SEQ ID NO: 111 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-H2；與 SEQ ID NO: 112 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-H3；及/或與 SEQ ID NO: 113 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-H4。In some embodiments, the antigen binding molecule comprises a heavy chain variable region comprising one or more (e.g., 1, 2, 3, or all 4) of the following: a FR-H1 sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 110; a FR-H2 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 111; a FR-H2 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 112; 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 113; and/or a FR-H4 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 113.

在一些實施例中，抗原結合分子包含輕鏈可變區，該輕鏈可變區包含以下項中之一個或多個 (例如，1 個、2 個、3 個或全部 4 個)：與 SEQ ID NO: 114 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-L1；與 SEQ ID NO: 119 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-L2；與 SEQ ID NO: 116 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-L3；及/或與 SEQ ID NO: 117 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-L4。在一些態樣中，抗原結合分子包含重鏈可變區，該重鏈可變區包含與 SEQ ID NO: 119 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-L2。在一些實施例中，抗原結合分子包含重鏈可變區，該重鏈可變區包含具有 SEQ ID NO: 119 之胺基酸序列的 FR-L2。在其他實施例中，抗原結合分子包含重鏈可變區，該重鏈可變區包含具有 SEQ ID NO: 121 之胺基酸序列的 FR-L2。In some embodiments, the antigen binding molecule comprises a light chain variable region comprising one or more (e.g., 1, 2, 3, or all 4) of the following: a FR-L1 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 114; a FR-L2 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 119; a FR-L2 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 116. and/or a FR-L4 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 117. In some aspects, the antigen binding molecule comprises a heavy chain variable region comprising a FR-L2 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the antigen binding molecule comprises a heavy chain variable region comprising a FR-L2 having an amino acid sequence of SEQ ID NO: 119. In other embodiments, the antigen binding molecule comprises a heavy chain variable region comprising a FR-L2 having an amino acid sequence of SEQ ID NO: 121.

在一些實施例中，抗原結合分子包含輕鏈可變區，該輕鏈可變區包含以下項中之一個或多個 (例如，1 個、2 個、3 個或全部 4 個)：與 SEQ ID NO: 114 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-L1；與 SEQ ID NO: 115 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-L2；與 SEQ ID NO: 116 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-L3；及/或與 SEQ ID NO: 117 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-L4。In some embodiments, the antigen binding molecule comprises a light chain variable region comprising one or more (e.g., 1, 2, 3, or all 4) of the following: a FR-L1 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 114; a FR-L2 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 115; a FR-L2 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 116. 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 117; and/or an FR-L4 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 117.

舉例而言，在一些情況下，抗原結合分子包含：FR-H1，其包含 SEQ ID NO: 110 之胺基酸序列、基本上由其組成、或由其組成；FR-H2，其包含 SEQ ID NO: 111 之胺基酸序列、基本上由其組成、或由其組成；FR-H3，其包含 SEQ ID NO: 112 之胺基酸序列、基本上由其組成、或由其組成；FR-H4，其包含 SEQ ID NO: 113 之胺基酸序列、基本上由其組成、或由其組成；FR-L1，其包含 SEQ ID NO: 114 之胺基酸序列、基本上由其組成、或由其組成；FR-L2，其包含 SEQ ID NO: 115 之胺基酸序列、基本上由其組成、或由其組成；FR-L3，其包含 SEQ ID NO: 116 之胺基酸序列、基本上由其組成、或由其組成；及 FR-L4，其包含 SEQ ID NO: 117 之胺基酸序列、基本上由其組成、或由其組成。For example, in some cases, the antigen binding molecule comprises: FR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 110; FR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 111; FR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 112; FR-H4 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 113; FR-L1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 114; FR-L2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 115; FR-L3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 116; , consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 117; and FR-L4 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 117.

在一些實施例中，抗原結合分子包含重鏈可變區，該重鏈可變區包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 VH 序列。在一些情況下，重鏈可變區包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 具有至少 90% 序列同一性的 VH 序列。在一些情況下，重鏈可變區包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 具有至少 95% 序列同一性的 VH 序列。在一些情況下，重鏈可變區包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 具有至少 98% 序列同一性的 VH 序列。在一些情況下，重鏈可變區包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 具有至少 99% 序列同一性的 VH 序列。在一些情況下，重鏈可變區包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 具有 100% 序列同一性的 VH 序列。在某些態樣中，具有至少 90%、91%、92%、93%、94%、95%、96%、97%、98% 或 99% 的同一性的 VH 序列含有相對於 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 的取代 (例如，保留取代)、插入或缺失，但是包含該序列的抗 STEAP1 抗體保留與 STEAP1 結合之能力。在某些態樣中，在 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 中，共有 1 至 10 個胺基酸已被取代、插入及/或缺失。在某些方面，取代、插入或缺失發生在 CDR 以外的區域 (即，在 FR 中)。In some embodiments, the antigen binding molecule comprises a heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7, 17-25, 30-34, 38 or 68. In some cases, the heavy chain variable region comprises a VH sequence having at least 90% sequence identity to SEQ ID NO: 7, 17-25, 30-34, 38 or 68. In some cases, the heavy chain variable region comprises a VH sequence having at least 95% sequence identity to SEQ ID NO: 7, 17-25, 30-34, 38 or 68. In some cases, the heavy chain variable region comprises a VH sequence having at least 98% sequence identity to SEQ ID NO: 7, 17-25, 30-34, 38, or 68. In some cases, the heavy chain variable region comprises a VH sequence having at least 99% sequence identity to SEQ ID NO: 7, 17-25, 30-34, 38, or 68. In some cases, the heavy chain variable region comprises a VH sequence having 100% sequence identity to SEQ ID NO: 7, 17-25, 30-34, 38, or 68. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains a substitution (e.g., a retention substitution), insertion or deletion relative to SEQ ID NO: 7, 17-25, 30-34, 38 or 68, but an anti-STEAP1 antibody comprising the sequence retains the ability to bind to STEAP1. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 7, 17-25, 30-34, 38 or 68. In certain aspects, the substitution, insertion or deletion occurs in a region outside of a CDR (i.e., in a FR).

在一些實施例中，抗原結合分子包含輕鏈可變區，該輕鏈可變區包含與 SEQ ID NO: 8、26 至 29、39 或 69 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 VL 序列。在一些情況下，輕鏈可變區包含與 SEQ ID NO: 8、26 至 29、39 或 69 具有至少 90% 序列同一性的 VL 序列。在一些情況下，輕鏈可變區包含與 SEQ ID NO: 8、26 至 29、39 或 69 具有至少 95% 序列同一性的 VL 序列。在一些情況下，輕鏈可變區包含與 SEQ ID NO: 8、26 至 29、39 或 69 具有至少 98% 序列同一性的 VL 序列。在一些情況下，輕鏈可變區包含與 SEQ ID NO: 8、26 至 29、39 或 69 具有至少 99% 序列同一性的 VL 序列。在一些情況下，輕鏈可變區包含與 SEQ ID NO: 8、26 至 29、39 或 69 具有 100% 序列同一性的 VL 序列。在某些態樣中，具有至少 90%、91%、92%、93%、94%、95%、96%、97%、98% 或 99% 的同一性的 VL 序列含有相對於 SEQ ID NO: 8、26 至 29、39 或 69 的取代 (例如，保留取代)、插入或缺失，但是包含該序列的抗 STEAP1 抗體保留與 STEAP1 結合之能力。在某些態樣中，在 SEQ ID NO: 8、26 至 29、39 或 69 中，共有 1 至 10 個胺基酸已被取代、插入及/或缺失。在某些方面，取代、插入或缺失發生在 CDR 以外的區域 (即，在 FR 中)。In some embodiments, the antigen binding molecule comprises a light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8, 26-29, 39, or 69. In some cases, the light chain variable region comprises a VL sequence having at least 90% sequence identity to SEQ ID NO: 8, 26-29, 39, or 69. In some cases, the light chain variable region comprises a VL sequence having at least 95% sequence identity to SEQ ID NO: 8, 26-29, 39, or 69. In some cases, the light chain variable region comprises a VL sequence having at least 98% sequence identity to SEQ ID NO: 8, 26-29, 39, or 69. In some cases, the light chain variable region comprises a VL sequence having at least 99% sequence identity to SEQ ID NO: 8, 26-29, 39, or 69. In some cases, the light chain variable region comprises a VL sequence having 100% sequence identity to SEQ ID NO: 8, 26-29, 39, or 69. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains a substitution (e.g., a retention substitution), insertion or deletion relative to SEQ ID NO: 8, 26 to 29, 39 or 69, but an anti-STEAP1 antibody comprising the sequence retains the ability to bind to STEAP1. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 8, 26 to 29, 39 or 69. In certain aspects, the substitution, insertion or deletion occurs in a region outside of a CDR (i.e., in a FR).

在一些實施例中，前述實施例中任一項之抗原結合分子的特徵在於 VH 及 VL 區，其中該 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 11 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 58 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 60 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，VH 區包含與 SEQ ID NO: 30 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 30 具有至少 95% 序列同一性、與 SEQ ID NO: 30 具有至少 96% 序列同一性、與 SEQ ID NO: 30 具有至少 97% 序列同一性、與 SEQ ID NO: 30 具有至少 98% 序列同一性、與 SEQ ID NO: 30 具有至少 99% 序列同一性、或與 SEQ ID NO: 30 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，VL 區包含與 SEQ ID NO: 27 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 27 具有至少 95% 序列同一性、與 SEQ ID NO: 27 具有至少 96% 序列同一性、與 SEQ ID NO: 27 具有至少 97% 序列同一性、與 SEQ ID NO: 27 具有至少 98% 序列同一性、與 SEQ ID NO: 27 具有至少 99% 序列同一性、或與 SEQ ID NO: 27 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the antigen binding molecule of any of the preceding embodiments is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9; (b) CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 11; or (c) CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3. In some cases, the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 58; (b) CDR-H2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 60; or (c) CDR-H3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 30 (e.g., at least 95% sequence identity to SEQ ID NO: 30, at least 96% sequence identity to SEQ ID NO: 30, at least 97% sequence identity to SEQ ID NO: 30, at least 98% sequence identity to SEQ ID NO: 30, at least 99% sequence identity to SEQ ID NO: 30, or 100% sequence identity to SEQ ID NO: 30). In some embodiments, the VL region comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5; or (c) CDR-L3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 27 (e.g., at least 95% sequence identity to SEQ ID NO: 27, at least 96% sequence identity to SEQ ID NO: 27, at least 97% sequence identity to SEQ ID NO: 27, at least 98% sequence identity to SEQ ID NO: 27, at least 99% sequence identity to SEQ ID NO: 27, or 100% sequence identity to SEQ ID NO: 27).

在一些實施例中，前述實施例中任一項之抗原結合分子的特徵在於 VH 及 VL 區，其中該 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 58 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 61 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，VH 區包含與 SEQ ID NO: 31 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 31 具有至少 95% 序列同一性、與 SEQ ID NO: 31 具有至少 96% 序列同一性、與 SEQ ID NO: 31 具有至少 97% 序列同一性、與 SEQ ID NO: 31 具有至少 98% 序列同一性、與 SEQ ID NO: 31 具有至少 99% 序列同一性、或與 SEQ ID NO: 31 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，VL 區包含與 SEQ ID NO: 27 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 27 具有至少 95% 序列同一性、與 SEQ ID NO: 27 具有至少 96% 序列同一性、與 SEQ ID NO: 27 具有至少 97% 序列同一性、與 SEQ ID NO: 27 具有至少 98% 序列同一性、與 SEQ ID NO: 27 具有至少 99% 序列同一性、或與 SEQ ID NO: 27 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the antigen binding molecule of any of the preceding embodiments is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9; (b) CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 12; or (c) CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3. In some cases, the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 58; (b) CDR-H2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 61; or (c) CDR-H3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 31 (e.g., at least 95% sequence identity to SEQ ID NO: 31, at least 96% sequence identity to SEQ ID NO: 31, at least 97% sequence identity to SEQ ID NO: 31, at least 98% sequence identity to SEQ ID NO: 31, at least 99% sequence identity to SEQ ID NO: 31, or 100% sequence identity to SEQ ID NO: 31). In some embodiments, the VL region comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5; or (c) CDR-L3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 27 (e.g., at least 95% sequence identity to SEQ ID NO: 27, at least 96% sequence identity to SEQ ID NO: 27, at least 97% sequence identity to SEQ ID NO: 27, at least 98% sequence identity to SEQ ID NO: 27, at least 99% sequence identity to SEQ ID NO: 27, or 100% sequence identity to SEQ ID NO: 27).

在一些實施例中，前述實施例中任一項之抗原結合分子的特徵在於 VH 及 VL 區，其中該 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 14 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 58 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 61 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 14 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，VH 區包含與 SEQ ID NO: 32 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 32 具有至少 95% 序列同一性、與 SEQ ID NO: 32 具有至少 96% 序列同一性、與 SEQ ID NO: 32 具有至少 97% 序列同一性、與 SEQ ID NO: 32 具有至少 98% 序列同一性、與 SEQ ID NO: 32 具有至少 99% 序列同一性、或與 SEQ ID NO: 32 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，VL 區包含與 SEQ ID NO: 27 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 27 具有至少 95% 序列同一性、與 SEQ ID NO: 27 具有至少 96% 序列同一性、與 SEQ ID NO: 27 具有至少 97% 序列同一性、與 SEQ ID NO: 27 具有至少 98% 序列同一性、與 SEQ ID NO: 27 具有至少 99% 序列同一性、或與 SEQ ID NO: 27 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the antigen binding molecule of any of the preceding embodiments is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9; (b) CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 12; or (c) CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 14. In some cases, the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 58; (b) CDR-H2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 61; or (c) CDR-H3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 32 (e.g., at least 95% sequence identity to SEQ ID NO: 32, at least 96% sequence identity to SEQ ID NO: 32, at least 97% sequence identity to SEQ ID NO: 32, at least 98% sequence identity to SEQ ID NO: 32, at least 99% sequence identity to SEQ ID NO: 32, or 100% sequence identity to SEQ ID NO: 32). In some embodiments, the VL region comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5; or (c) CDR-L3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 27 (e.g., at least 95% sequence identity to SEQ ID NO: 27, at least 96% sequence identity to SEQ ID NO: 27, at least 97% sequence identity to SEQ ID NO: 27, at least 98% sequence identity to SEQ ID NO: 27, at least 99% sequence identity to SEQ ID NO: 27, or 100% sequence identity to SEQ ID NO: 27).

在一些實施例中，前述實施例中任一項之抗原結合分子的特徵在於 VH 及 VL 區，其中該 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 13 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 15 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 59 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 62 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 15 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，VH 區包含與 SEQ ID NO: 33 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 33 具有至少 95% 序列同一性、與 SEQ ID NO: 33 具有至少 96% 序列同一性、與 SEQ ID NO: 33 具有至少 97% 序列同一性、與 SEQ ID NO: 33 具有至少 98% 序列同一性、與 SEQ ID NO: 33 具有至少 99% 序列同一性、或與 SEQ ID NO: 33 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，VL 區包含與 SEQ ID NO: 27 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 27 具有至少 95% 序列同一性、與 SEQ ID NO: 27 具有至少 96% 序列同一性、與 SEQ ID NO: 27 具有至少 97% 序列同一性、與 SEQ ID NO: 27 具有至少 98% 序列同一性、與 SEQ ID NO: 27 具有至少 99% 序列同一性、或與 SEQ ID NO: 27 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the antigen binding molecule of any of the preceding embodiments is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 10; (b) CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 13; or (c) CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 15. In some cases, the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 59; (b) CDR-H2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 62; or (c) CDR-H3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the VH region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 33 (e.g., at least 95% sequence identity to SEQ ID NO: 33, at least 96% sequence identity to SEQ ID NO: 33, at least 97% sequence identity to SEQ ID NO: 33, at least 98% sequence identity to SEQ ID NO: 33, at least 99% sequence identity to SEQ ID NO: 33, or 100% sequence identity to SEQ ID NO: 33). In some embodiments, the VL region comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5; or (c) CDR-L3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 27 (e.g., at least 95% sequence identity to SEQ ID NO: 27, at least 96% sequence identity to SEQ ID NO: 27, at least 97% sequence identity to SEQ ID NO: 27, at least 98% sequence identity to SEQ ID NO: 27, at least 99% sequence identity to SEQ ID NO: 27, or 100% sequence identity to SEQ ID NO: 27).

在一些實施例中，前述實施例中任一項之抗原結合分子的特徵在於 VH 及 VL 區，其中該 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 16 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 59 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 57 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 16 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，VH 區包含與 SEQ ID NO: 34 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 34 具有至少 95% 序列同一性、與 SEQ ID NO: 34 具有至少 96% 序列同一性、與 SEQ ID NO: 34 具有至少 97% 序列同一性、與 SEQ ID NO: 34 具有至少 98% 序列同一性、與 SEQ ID NO: 34 具有至少 99% 序列同一性、或與 SEQ ID NO: 34 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，VL 區包含與 SEQ ID NO: 27 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 27 具有至少 95% 序列同一性、與 SEQ ID NO: 27 具有至少 96% 序列同一性、與 SEQ ID NO: 27 具有至少 97% 序列同一性、與 SEQ ID NO: 27 具有至少 98% 序列同一性、與 SEQ ID NO: 27 具有至少 99% 序列同一性、或與 SEQ ID NO: 27 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the antigen binding molecule of any of the preceding embodiments is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 10; (b) CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2; or (c) CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16. In some cases, the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 59; (b) CDR-H2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 57; or (c) CDR-H3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 34 (e.g., at least 95% sequence identity to SEQ ID NO: 34, at least 96% sequence identity to SEQ ID NO: 34, at least 97% sequence identity to SEQ ID NO: 34, at least 98% sequence identity to SEQ ID NO: 34, at least 99% sequence identity to SEQ ID NO: 34, or 100% sequence identity to SEQ ID NO: 34). In some embodiments, the VL region comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5; or (c) CDR-L3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 27 (e.g., at least 95% sequence identity to SEQ ID NO: 27, at least 96% sequence identity to SEQ ID NO: 27, at least 97% sequence identity to SEQ ID NO: 27, at least 98% sequence identity to SEQ ID NO: 27, at least 99% sequence identity to SEQ ID NO: 27, or 100% sequence identity to SEQ ID NO: 27).

在一些實施例中，VH 區包含與 SEQ ID NO: 68 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 68 具有至少 95% 序列同一性、與 SEQ ID NO: 68 具有至少 96% 序列同一性、與 SEQ ID NO: 68 具有至少 97% 序列同一性、與 SEQ ID NO: 68 具有至少 98% 序列同一性、與 SEQ ID NO: 68 具有至少 99% 序列同一性、或與 SEQ ID NO: 68 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，VL 區包含與 SEQ ID NO: 69 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 69 具有至少 95% 序列同一性、與 SEQ ID NO: 69 具有至少 96% 序列同一性、與 SEQ ID NO: 69 具有至少 97% 序列同一性、與 SEQ ID NO: 69 具有至少 98% 序列同一性、與 SEQ ID NO: 69 具有至少 99% 序列同一性、或與 SEQ ID NO: 69 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the VH region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 68 (e.g., at least 95% sequence identity to SEQ ID NO: 68, at least 96% sequence identity to SEQ ID NO: 68, at least 97% sequence identity to SEQ ID NO: 68, at least 98% sequence identity to SEQ ID NO: 68, at least 99% sequence identity to SEQ ID NO: 68, or 100% sequence identity to SEQ ID NO: 68). In some embodiments, the VL region comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5; or (c) CDR-L3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 69 (e.g., at least 95% sequence identity to SEQ ID NO: 69, at least 96% sequence identity to SEQ ID NO: 69, at least 97% sequence identity to SEQ ID NO: 69, at least 98% sequence identity to SEQ ID NO: 69, at least 99% sequence identity to SEQ ID NO: 69, or 100% sequence identity to SEQ ID NO: 69).

在一些實施例中，前述實施例中任一項之抗原結合分子的特徵在於 VH 及 VL 區，其中該 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 35 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 36 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 37 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 63 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 64 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 37 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，VH 區包含與 SEQ ID NO: 38 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 38 具有至少 95% 序列同一性、與 SEQ ID NO: 38 具有至少 96% 序列同一性、與 SEQ ID NO: 38 具有至少 97% 序列同一性、與 SEQ ID NO: 38 具有至少 98% 序列同一性、與 SEQ ID NO: 38 具有至少 99% 序列同一性、或與 SEQ ID NO: 38 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，VL 區包含與 SEQ ID NO: 39 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 39 具有至少 95% 序列同一性、與 SEQ ID NO: 39 具有至少 96% 序列同一性、與 SEQ ID NO: 39 具有至少 97% 序列同一性、與 SEQ ID NO: 39 具有至少 98% 序列同一性、與 SEQ ID NO: 39 具有至少 99% 序列同一性、或與 SEQ ID NO: 39 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the antigen binding molecule of any of the preceding embodiments is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 35; (b) CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 36; or (c) CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 37. In some cases, the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 63; (b) CDR-H2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 64; or (c) CDR-H3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 38 (e.g., at least 95% sequence identity to SEQ ID NO: 38, at least 96% sequence identity to SEQ ID NO: 38, at least 97% sequence identity to SEQ ID NO: 38, at least 98% sequence identity to SEQ ID NO: 38, at least 99% sequence identity to SEQ ID NO: 38, or 100% sequence identity to SEQ ID NO: 38). In some embodiments, the VL region comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5; or (c) CDR-L3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL region comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 39 (e.g., at least 95% sequence identity to SEQ ID NO: 39, at least 96% sequence identity to SEQ ID NO: 39, at least 97% sequence identity to SEQ ID NO: 39, at least 98% sequence identity to SEQ ID NO: 39, at least 99% sequence identity to SEQ ID NO: 39, or 100% sequence identity to SEQ ID NO: 39).

在一些實施例中，抗原結合分子與 STEAP1 上之表位結合，該表位包含選自 STEAP1 之 Ser101、His102、Gln103 及 Lys281 的一個或多個胺基酸殘基。在一些實施例中，表位包含 STEAP1 之 ECL1 的胺基酸殘基 Ser101。在一些實施例中，表位包含 STEAP1 之 ECL1 的胺基酸殘基 His102。在一些實施例中，表位包含 STEAP1 之 ECL1 的胺基酸殘基 Gln103。在一些實施例中，表位包含 STEAP1 之 ECL3 的胺基酸殘基 Lys281。在一些實施例中，表位包含 STEAP1 之 ECL1 的 Ser101、His102、Gln103 及 STEAP1 之 ECL3 的 Lys281。在一些實施例中，表位進一步包含選自 STEAP1 之 ECL2 的 Trp195、Gln198、Gln201、Gln202、Asn203 及 Lys204 的一個或多個額外胺基酸殘基。在一些實施例中，表位不包含 STEAP1 之胺基酸殘基 Gln104。在一些實施例中，表位不包含 STEAP1 之胺基酸殘基 Tyr107。在一些實施例中，表位不包含 STEAP1 之胺基酸殘基 Asn194。在一些實施例中，表位不包含 STEAP1 之胺基酸殘基 Glu205。在一些實施例中，表位不包含 STEAP1 之胺基酸殘基 Ala207。In some embodiments, the antigen binding molecule binds to an epitope on STEAP1, the epitope comprising one or more amino acid residues selected from Ser101, His102, Gln103, and Lys281 of STEAP1. In some embodiments, the epitope comprises the amino acid residue Ser101 of ECL1 of STEAP1. In some embodiments, the epitope comprises the amino acid residue His102 of ECL1 of STEAP1. In some embodiments, the epitope comprises the amino acid residue Gln103 of ECL1 of STEAP1. In some embodiments, the epitope comprises the amino acid residue Lys281 of ECL3 of STEAP1. In some embodiments, the epitope comprises Ser101, His102, Gln103 of ECL1 of STEAP1 and Lys281 of ECL3 of STEAP1. In some embodiments, the epitope further comprises one or more additional amino acid residues selected from Trp195, Gln198, Gln201, Gln202, Asn203 and Lys204 of ECL2 of STEAP1. In some embodiments, the epitope does not comprise the amino acid residue Gln104 of STEAP1. In some embodiments, the epitope does not comprise the amino acid residue Tyr107 of STEAP1. In some embodiments, the epitope does not comprise the amino acid residue Asn194 of STEAP1. In some embodiments, the epitope does not comprise the amino acid residue Glu205 of STEAP1. In some embodiments, the epitope does not comprise the amino acid residue Ala207 of STEAP1.

在一些實施例中，抗原結合分子以約 100 nM 或更低之 K_D結合人 STEAP1，如藉由動力學篩析測定 (KinExA®) 所測量。在一些實施例中，抗原結合分子以在約 10 pM 至約 100 nM 之間的 K_D結合人 STEAP1。在一些實施例中，抗原結合分子以在約 100 pM 至約 50 nM 之間的 K_D結合人 STEAP1。在一些實施例中，抗原結合分子以在約 1 nM 至約 30 nM 之間的 K_D結合人 STEAP1。在一些實施例中，抗原結合分子以約 100 nM 或更低之 K_D結合石蟹獼猴 STEAP1，如藉由動力學篩析測定 (KinExA®) 所測量。在一些實施例中，抗原結合分子以在約 10 pM 至約 100 nM 之間的 K_D結合石蟹獼猴 STEAP1。在一些實施例中，抗原結合分子以在約 100 pM 至約 50 nM 之間的 K_D結合石蟹獼猴 STEAP1。在一些實施例中，抗原結合分子以在約 1 nM 至約 30 nM 之間的 K_D結合石蟹獼猴 STEAP1。多特異性抗原結合分子In some embodiments, the antigen binding molecule binds to human STEAP1 with a_K of about 100 nM or less, as measured by a kinetic screening assay (KinExA®). In some embodiments, the antigen binding molecule binds to human STEAP1 with a_K between about 10 pM to about 100 nM. In some embodiments, the antigen binding molecule binds to human STEAP1 with a_K between about 100 pM to about 50 nM. In some embodiments, the antigen binding molecule binds to human STEAP1 with a_K between about 1 nM to about 30 nM. In some embodiments, the antigen binding molecule binds to stone crab macaque STEAP1 with a_K of about 100 nM or less, as measured by a kinetic screening assay (KinExA®). In some embodiments, the antigen binding molecule binds to red macaque STEAP1 with a_KD between about 10 pM and about 100 nM. In some embodiments, the antigen binding molecule binds to red macaque STEAP1 with a_KD between about 100 pM and about 50 nM. In some embodiments, the antigen binding molecule binds to red macaque STEAP1 with a_KD between about 1 nM and about 30 nM.Multispecific Antigen Binding Molecules

在一些實施例中，本發明提供經分離之多特異性抗原結合分子，該等經分離之多特異性抗原結合分子包含如上所述之與 STEAP1 結合的第一抗原結合域。在一些情況下，多特異性抗原結合分子與 STEAP1 單價結合。在一些情況下，多特異性抗原結合分子進一步包含與 T 細胞受體結合的第二結合域。在額外的情況下，多特異性抗原結合分子包含於額外抗原 (例如，腫瘤相關抗原) 結合的第三結合域。In some embodiments, the present invention provides isolated multispecific antigen-binding molecules comprising a first antigen-binding domain that binds to STEAP1 as described above. In some cases, the multispecific antigen-binding molecule monovalently binds to STEAP1. In some cases, the multispecific antigen-binding molecule further comprises a second binding domain that binds to a T cell receptor. In additional cases, the multispecific antigen-binding molecule comprises a third binding domain that binds to an additional antigen (e.g., a tumor-associated antigen).

在一些實施例中，本發明之多特異性抗原結合分子為雙特異性抗原結合分子 (例如，雙特異性抗體)，該雙特異性抗原結合分子包含與 STEAP1 結合的第一抗原結合域及與 T 細胞受體結合的第二抗原結合域。示例性 T 細胞受體包括分化簇 3 (CD3)。在一些情況下，第二抗原結合域包含與 CD3 結合的抗體或其片段。與 CD3 結合的示例性抗原結合域包括但不限於 40G5c 及 38E4v1.MD1。與 CD3 結合的額外的抗原結合域 (例如，UCHT1.v9、38E4v11、30E4c、SP34v52 等) 描述於美國專利第 10,174,124 號、WO2015/095392、WO2016/205520 及國際專利申請第 PCT/US2020/064635 號中，其各自藉由引用的方式而以其整體併入本文。In some embodiments, the multispecific antigen-binding molecules of the present invention are bispecific antigen-binding molecules (e.g., bispecific antibodies) comprising a first antigen-binding domain that binds to STEAP1 and a second antigen-binding domain that binds to a T cell receptor. Exemplary T cell receptors include cluster of differentiation 3 (CD3). In some cases, the second antigen-binding domain comprises an antibody or fragment thereof that binds to CD3. Exemplary antigen-binding domains that bind to CD3 include, but are not limited to, 40G5c and 38E4v1.MD1. Additional antigen binding domains that bind to CD3 (e.g., UCHT1.v9, 38E4v11, 30E4c, SP34v52, etc.) are described in U.S. Patent No. 10,174,124, WO2015/095392, WO2016/205520, and International Patent Application No. PCT/US2020/064635, each of which is incorporated herein by reference in its entirety.

在一些實施例中，第二抗原結合域與 CD3 上之表位結合，該表位包含 CD3 之胺基酸殘基 Glu6。在一些實施例中，表位進一步包含選自 CD3 之 Gln1、Asp2 及 Met7 的一個或多個額外胺基酸殘基。在一些實施例中，表位包含 CD3 之胺基酸殘基 Gln1、Asp2 及 Glu6。在一些實施例中，表位包含 CD3 之胺基酸殘基 Gln1、Asp2、Glu6 及 Met7。在一些實施例中，表位不包含 CD3 之胺基酸殘基 Glu5。在一些實施例中，表位不包含 CD3 之胺基酸殘基 Gly3 及 Glu5。在一些實施例中，表位由 CD3 之胺基酸殘基 Gln1、Asp2、Glu6 及 Met7 組成。在一些實施例中，第二抗原結合域能夠與人 CD3 多肽或石蟹獼猴 CD3 多肽結合。在一些實施例中，人 CD3 多肽或石蟹獼猴 CD3 多肽分別為人 CD3e 多肽或石蟹獼猴 CD3e 多肽。在一些實施例中，人 CD3 多肽或石蟹獼猴 CD3 多肽分別為人 CD3y 多肽或石蟹獼猴 CD3y 多肽。在一些實施例中，第二抗原結合域以約 100 nM 或更低之 K_D結合人 CD3e 多肽。在一些實施例中，第二抗原結合域以在約 10 pM 至約 100 nM 之間的 K_D結合人 CD3e。在一些實施例中，第二抗原結合域以在約 100 pM 至約 50 nM 之間的 K_D結合人 CD3e。在一些實施例中，第二抗原結合域以在約 1 nM 至約 10 nM 之間的 K_D結合人 CD3e。In some embodiments, the second antigen binding domain binds to an epitope on CD3 that comprises the amino acid residue Glu6 of CD3. In some embodiments, the epitope further comprises one or more additional amino acid residues selected from Gln1, Asp2, and Met7 of CD3. In some embodiments, the epitope comprises the amino acid residues Gln1, Asp2, and Glu6 of CD3. In some embodiments, the epitope comprises the amino acid residues Gln1, Asp2, Glu6, and Met7 of CD3. In some embodiments, the epitope does not comprise the amino acid residue Glu5 of CD3. In some embodiments, the epitope does not comprise the amino acid residues Gly3 and Glu5 of CD3. In some embodiments, the epitope consists of the amino acid residues Gln1, Asp2, Glu6 and Met7 of CD3. In some embodiments, the second antigen binding domain is capable of binding to a human CD3 polypeptide or a red macaque CD3 polypeptide. In some embodiments, the human CD3 polypeptide or the red macaque CD3 polypeptide is a human CD3e polypeptide or a red macaque CD3e polypeptide, respectively. In some embodiments, the human CD3 polypeptide or the red macaque CD3 polypeptide is a human CD3y polypeptide or a red macaque CD3y polypeptide, respectively. In some embodiments, the second antigen binding domain binds to a human CD3e polypeptide with a_KD of about 100 nM or less. In some embodiments, the second antigen binding domain binds to human CD3e with a_KD between about 10 pM and about 100 nM. In some embodiments, the second antigen binding domain binds human CD3 epsilon with a_KD between about 100 pM to about 50 nM. In some embodiments, the second antigen binding domain binds human CD3 epsilon with a_KD between about 1 nM to about 10 nM.

在一些實施例中，第二抗原結合域包含 38E4v1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域包含至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR，其中 CDR-H1 包含 SEQ ID NO: 40 之胺基酸序列、基本上由其組成、或由其組成，CDR-H2 包含 SEQ ID NO: 41 之胺基酸序列、基本上由其組成、或由其組成，CDR-H3 包含 SEQ ID NO: 42 之胺基酸序列、基本上由其組成、或由其組成，CDR-L1 包含 SEQ ID NO: 43 之胺基酸序列、基本上由其組成、或由其組成，CDR-L2 包含 SEQ ID NO: 44，或 CDR-L3 包含 SEQ ID NO: 45 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，第二抗原結合域包含與 SEQ ID NO: 46 具有至少 80% (例如，90%、91%、92%、93%、94%、95%、96%、97%、98% 或 99%) 序列同一性的 VH 序列。在一些情況下，第二抗原結合域包含與 SEQ ID NO: 47 具有至少 80% (例如，90%、91%、92%、93%、94%、95%、96%、97%、98% 或 99%) 序列同一性的 VL 序列。在某些態樣中，具有至少 90%、91%、92%、93%、94%、95%、96%、97%、98% 或 99% 同一性的 VH 或 VL 序列含有相對於 SEQ ID NO: 46 或 47 的取代 (例如，保留取代)、插入或缺失，但是包含該序列的多特異性抗體保留與 CD3 結合之能力。在某些態樣中，在 SEQ ID NO: 46 或 47 中，共有 1 至 10 個胺基酸被取代、插入及/或缺失。在某些方面，取代、插入或缺失發生在 CDR 以外的區域 (即，在 FR 中)。In some embodiments, the second antigen-binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of 38E4v1.MD1. In some cases, the second antigen-binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs, wherein CDR-H1 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 40, CDR-H2 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 41, CDR-H3 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 42, CDR-L1 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 43, CDR-L2 comprises SEQ ID NO: 44, or CDR-L3 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 45. In some cases, the second antigen binding domain comprises a VH sequence having at least 80% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 46. In some cases, the second antigen binding domain comprises a VL sequence having at least 80% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 47. In certain aspects, a VH or VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains a substitution (e.g., a retention substitution), insertion or deletion relative to SEQ ID NO: 46 or 47, but the multispecific antibody comprising the sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO: 46 or 47. In certain aspects, the substitution, insertion or deletion occurs in a region outside of the CDR (i.e., in the FR).

在一些實施例中，第二抗原結合域包含 40G5c 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域包含至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR，其中 CDR-H1 包含 SEQ ID NO: 48 之胺基酸序列、基本上由其組成、或由其組成，CDR-H2 包含 SEQ ID NO: 49 之胺基酸序列、基本上由其組成、或由其組成，CDR-H3 包含 SEQ ID NO: 50 之胺基酸序列、基本上由其組成、或由其組成，CDR-L1 包含 SEQ ID NO: 51 之胺基酸序列、基本上由其組成、或由其組成，CDR-L2 包含 SEQ ID NO: 52，或 CDR-L3 包含 SEQ ID NO: 53 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，第二抗原結合域包含與 SEQ ID NO: 54 具有至少 80% (例如，90%、91%、92%、93%、94%、95%、96%、97%、98% 或 99%) 序列同一性的 VH 序列。在一些情況下，第二抗原結合域包含與 SEQ ID NO: 55 具有至少 80% (例如，90%、91%、92%、93%、94%、95%、96%、97%、98% 或 99%) 序列同一性的 VL 序列。在某些態樣中，具有至少 90%、91%、92%、93%、94%、95%、96%、97%、98% 或 99% 同一性的 VH 或 VL 序列含有相對於 SEQ ID NO: 54 或 55 的取代 (例如，保留取代)、插入或缺失，但是包含該序列的多特異性抗體保留與 CD3 結合之能力。在某些態樣中，在 SEQ ID NO: 54 或 55 中，共有 1 至 10 個胺基酸被取代、插入及/或缺失。在某些方面，取代、插入或缺失發生在 CDR 以外的區域 (即，在 FR 中)。In some embodiments, the second antigen-binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of 40G5c. In some cases, the second antigen-binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs, wherein CDR-H1 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 48, CDR-H2 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 49, CDR-H3 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 50, CDR-L1 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 51, CDR-L2 comprises SEQ ID NO: 52, or CDR-L3 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 53. In some cases, the second antigen binding domain comprises a VH sequence having at least 80% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 54. In some cases, the second antigen binding domain comprises a VL sequence having at least 80% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 55. In certain aspects, a VH or VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains a substitution (e.g., a retention substitution), insertion or deletion relative to SEQ ID NO: 54 or 55, but the multispecific antibody comprising the sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO: 54 or 55. In certain aspects, the substitution, insertion or deletion occurs in a region outside of the CDR (i.e., in the FR).

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含選自 SEQ ID NO: 7、17 至 25、30 至 34、38 及 68 的 VH 序列之 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含選自 SEQ ID NO: 8、26 至 29、39 及 69 的 VL 序列之 CDR-L1、CDR-L2 及/或 CDR-L3；以及 (B) 第二抗原結合域，該第二抗原結合域與 T 細胞受體結合。在一些情況下，至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR 選自含有 SEQ ID NO: 7、17 至 25、30 至 34、38 及 68 之 VH 序列及選自 SEQ ID NO: 8、26 至 29、39 及 69 之 VL 序列的 CDR。在一些情況下，至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR 選自 40G5c 或 38E4v1.MD1 的 CDR。在一些情況下，六個 CDR 係根據 Kabat 編號定義。在其他情況下，六個 CDR 是根據 Chothia 編號定義。在額外情況下，六個 CDR 係根據 EU 編號定義。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3 of a VH sequence selected from SEQ ID NOs: 7, 17 to 25, 30 to 34, 38 and 68; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3 of a VL sequence selected from SEQ ID NOs: 8, 26 to 29, 39 and 69; and (B) a second antigen-binding domain that binds to a T cell receptor. In some cases, at least one, at least two, at least three, at least four, at least five, or all six CDRs are selected from the CDRs of a VH sequence comprising SEQ ID NOs: 7, 17 to 25, 30 to 34, 38, and 68 and a VL sequence selected from SEQ ID NOs: 8, 26 to 29, 39, and 69. In some cases, at least one, at least two, at least three, at least four, at least five, or all six CDRs are selected from the CDRs of 40G5c or 38E4v1.MD1. In some cases, the six CDRs are defined according to Kabat numbering. In other cases, the six CDRs are defined according to Chothia numbering. In additional cases, the six CDRs are defined according to EU numbering.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)； 其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)； 其中 Xaa³為 Asp (D) 或 Ser (S)； Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)； Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)；且 CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4v1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Ser (S); Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (N); and Xaa⁶ is Ser (S) or Tyr (Y); and CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises SEQ ID NO: 6 and (B) a second antigen-binding domain that binds to a T cell receptor. In some cases, the first antigen-binding domain comprises at least two, at least three, at least four, at least five, or all six CDRs of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen-binding domain binds to CD3. In some cases, the second antigen-binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4v1.MD1.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 SEQ ID NO: 10、1 或 9 之胺基酸序列； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)； 其中 Xaa³為 Asp (D) 或 Ser (S)； Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)； Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4v1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 10, 1 or 9; CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Ser (S); Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (N); and Xaa⁶ is Ser (S) or Tyr (Y); CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) a second antigen binding domain that binds to a T cell receptor. In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4v1.MD1.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 SEQ ID NO: 10、1 或 9 之胺基酸序列； CDR-H2 包含 SEQ ID NO: 2、11、12 或 13 之胺基酸序列； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4v1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises an amino acid sequence of SEQ ID NO: 10, 1 or 9; CDR-H2 comprises an amino acid sequence of SEQ ID NO: 2, 11, 12 or 13; CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) a second antigen binding domain that binds to a T cell receptor. In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen-binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4v1.MD1.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)； 其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 SEQ ID NO: 2、11、12 或 13 之胺基酸序列； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4V1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises the amino acid sequence of SEQ ID NO: 2, 11, 12 or 13; CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) a second antigen binding domain that binds to a T cell receptor. In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4V1.MD1.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)； 其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 SEQ ID NO: 2、11、12 或 13 之胺基酸序列； CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4V1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises an amino acid sequence of SEQ ID NO: 2, 11, 12 or 13; CDR-H3 comprises SEQ ID NO: 16, 3, 14 or 15; CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) a second antigen binding domain that binds to a T cell receptor. In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five, or all six CDRs of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4V1.MD1.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)； 其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)； 其中 Xaa³為 Asp (D) 或 Ser (S)； Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)； Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)； CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4V1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Ser (S); Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (N); and Xaa⁶ is Ser (S) or Tyr (Y); CDR-H3 comprises the amino acid sequence of SEQ ID NO: 16, 3, 14 or 15; CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) a second antigen binding domain that binds to a T cell receptor. In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five, or all six CDRs of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4V1.MD1.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 SEQ ID NO: 10、1 或 9 之胺基酸序列； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)； 其中 Xaa³為 Asp (D) 或 Ser (S)； Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)； Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)； CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4V1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 10, 1 or 9; CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Ser (S); Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (N); and Xaa⁶ is Ser (S) or Tyr (Y); CDR-H3 comprises the amino acid sequence of SEQ ID NO: 16, 3, 14 or 15; CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) a second antigen binding domain that binds to a T cell receptor. In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen-binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4V1.MD1.

在一些實施例中，多特異性抗原結合分子包含 (A) 第一抗原結合域，其與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3；其中 CDR-H1 包含 SEQ ID NO: 10、1 或 9 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2 包含 SEQ ID NO: 2、11、12 或 13 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成；以及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4V1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3; wherein CDR-H1 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 10, 1, or 9; CDR-H2 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 2, 11, 12, or 13; and CDR-H3 comprises SEQ ID NO: 16, 3, 14, or 15. In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five, or all six of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen-binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4V1.MD1.

在一些情況下，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 1 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the first antigen-binding domain comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 11 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the first antigen-binding domain comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 11; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the first antigen-binding domain comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 12; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 14 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the first antigen-binding domain comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 12; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 14; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 13 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 15 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the first antigen-binding domain comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 10; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 13; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 15; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 16 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the first antigen-binding domain comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 10; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 GFTFSXaa¹⁰Xaa¹¹(SEQ ID NO: 63)； 其中 Xaa¹⁰為 Asn (N) 或 Asp (D)；且 Xaa¹¹為 Tyr (Y)、Phe (F) 或 His (H)； CDR-H2 包含 Xaa¹²YDGXaa¹³Xaa¹⁴(SEQ ID NO: 64)； 其中 Xaa¹²為 Asp (D) 或 Ser (S)； Xaa¹³為 Gly (G)、Asp (D) 或 Leu (L)；且 Xaa¹⁴為 Ser (S)、Asp (D) 或 Asn (N)； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4V1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3; wherein CDR-H1 comprises GFTFSXaa¹⁰ Xaa¹¹ (SEQ ID NO: 63); wherein Xaa¹⁰ is Asn (N) or Asp (D); and Xaa¹¹ is Tyr (Y), Phe (F), or His (H); CDR-H2 comprises Xaa¹² YDGXaa¹³ Xaa¹⁴ (SEQ ID NO: 64); wherein Xaa wherein^{Xaa 12} is Asp (D) or Ser (S); Xaa¹³ is Gly (G), Asp (D) or Leu (L); and Xaa¹⁴ is Ser (S), Asp (D) or Asn (N); CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) a second antigen-binding domain that binds to T In some cases, the first antigen-binding domain comprises at least two, at least three, at least four, at least five, or all six CDRs of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen-binding domain binds to CD3. In some cases, the second antigen-binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4V1.MD1.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 SEQ ID NO: 56、58 或 59 之胺基酸序列； CDR-H2 包含 Xaa¹²YDGXaa¹³Xaa¹⁴(SEQ ID NO: 64)； 其中 Xaa¹²為 Asp (D) 或 Ser (S)； Xaa¹³為 Gly (G)、Asp (D) 或 Leu (L)；且 Xaa¹⁴為 Ser (S)、Asp (D) 或 Asn (N)； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4V1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 56, 58 or 59; CDR-H2 comprises Xaa¹² YDGXaa¹³ Xaa¹⁴ (SEQ ID NO: 64); wherein Xaa¹² is Asp (D) or Ser (S); Xaa¹³ is Gly (G), Asp (D) or Leu (L); and Xaa¹⁴ is Ser (S), Asp (D) or Asn (N); CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) a second antigen binding domain that binds to a T cell receptor. In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4V1.MD1.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 SEQ ID NO: 56、58 或 59 之胺基酸序列； CDR-H2 包含 SEQ ID NO: 57、60、61 或 62 之胺基酸序列； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4V1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises an amino acid sequence of SEQ ID NO: 56, 58 or 59; CDR-H2 comprises an amino acid sequence of SEQ ID NO: 57, 60, 61 or 62; CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) a second antigen binding domain that binds to a T cell receptor. In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen-binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4V1.MD1.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 GFTFSXaa¹⁰Xaa¹¹(SEQ ID NO: 63)； 其中 Xaa¹⁰為 Asn (N) 或 Asp (D)；且 Xaa¹¹為 Tyr (Y)、Phe (F) 或 His (H)； CDR-H2 包含 SEQ ID NO: 57、60、61 或 62 之胺基酸序列； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4V1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises GFTFSXaa¹⁰ Xaa¹¹ (SEQ ID NO: 63); wherein Xaa¹⁰ is Asn (N) or Asp (D); and Xaa¹¹ is Tyr (Y), Phe (F) or His (H); CDR-H2 comprises the amino acid sequence of SEQ ID NO: 57, 60, 61 or 62; CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) a second antigen binding domain that binds to a T cell receptor. In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4V1.MD1.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 GFTFSXaa¹⁰Xaa¹¹(SEQ ID NO: 63)； 其中 Xaa¹⁰為 Asn (N) 或 Asp (D)；且 Xaa¹¹為 Tyr (Y)、Phe (F) 或 His (H)； CDR-H2 包含 SEQ ID NO: 57、60、61 或 62 之胺基酸序列； CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4V1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises GFTFSXaa¹⁰ Xaa¹¹ (SEQ ID NO: 63); wherein Xaa¹⁰ is Asn (N) or Asp (D); and Xaa¹¹ is Tyr (Y), Phe (F) or His (H); CDR-H2 comprises the amino acid sequence of SEQ ID NO: 57, 60, 61 or 62; CDR-H3 comprises SEQ ID NO: In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five, or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen-binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4V1.MD1.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 GFTFSXaa¹⁰Xaa¹¹(SEQ ID NO: 63)； 其中 Xaa¹⁰為 Asn (N) 或 Asp (D)；且 Xaa¹¹為 Tyr (Y)、Phe (F) 或 His (H)； CDR-H2 包含 Xaa¹²YDGXaa¹³Xaa¹⁴(SEQ ID NO: 64)； 其中 Xaa¹²為 Asp (D) 或 Ser (S)； Xaa¹³為 Gly (G)、Asp (D) 或 Leu (L)；且 Xaa¹⁴為 Ser (S)、Asp (D) 或 Asn (N)； CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4V1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3; wherein CDR-H1 comprises GFTFSXaa¹⁰ Xaa¹¹ (SEQ ID NO: 63); wherein Xaa¹⁰ is Asn (N) or Asp (D); and Xaa¹¹ is Tyr (Y), Phe (F), or His (H); CDR-H2 comprises Xaa¹² YDGXaa¹³ Xaa¹⁴ (SEQ ID NO: 64); wherein Xaa¹² is Asp (D) or Ser (S); Xaa¹³ is Gly (G), Asp (D) or Leu (L); and Xaa¹⁴ is Ser (S), Asp (D) or Asn (N); CDR-H3 comprises the amino acid sequence of SEQ ID NO: 16, 3, 14 or 15; CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) a second antigen binding domain that binds to a T cell receptor. In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five, or all six CDRs of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4V1.MD1.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 SEQ ID NO: 56、58 或 59 之胺基酸序列； CDR-H2 包含 Xaa¹²YDGXaa¹³Xaa¹⁴(SEQ ID NO: 64)； 其中 Xaa¹²為 Asp (D) 或 Ser (S)； Xaa¹³為 Gly (G)、Asp (D) 或 Leu (L)；且 Xaa¹⁴為 Ser (S)、Asp (D) 或 Asn (N)； CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4V1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 56, 58 or 59; CDR-H2 comprises Xaa¹² YDGXaa¹³ Xaa¹⁴ (SEQ ID NO: 64); wherein Xaa¹² is Asp (D) or Ser (S); Xaa¹³ is Gly (G), Asp (D) or Leu (L); and Xaa¹⁴ is Ser (S), Asp (D) or Asn (N); CDR-H3 comprises the amino acid sequence of SEQ ID NO: 16, 3, 14 or 15; CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) a second antigen binding domain that binds to a T cell receptor. In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five or all six CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen-binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4V1.MD1.

在一些實施例中，多特異性抗原結合分子包含 (A) 第一抗原結合域，其與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3；其中 CDR-H1 包含 SEQ ID NO: 56、58 或 59 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2 包含 SEQ ID NO: 57、60、61 或 62 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成；以及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域包含至少兩個、至少三個、至少四個、至少五個或全部六個 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含- 40G5c 或 38E4V1.MD1 之至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個 CDR。In some embodiments, the multispecific antigen-binding molecule comprises (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3; wherein CDR-H1 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 56, 58, or 59; CDR-H2 comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 57, 60, 61, or 62; and CDR-H3 comprises SEQ ID NO: 16, 13, 14, or 15. In some cases, the first antigen binding domain comprises at least two, at least three, at least four, at least five, or all six of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen-binding domain comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs of -40G5c or 38E4V1.MD1.

在一些情況下，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 56 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 57 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the first antigen-binding domain comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 56; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 57; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 58 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 60 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the first antigen-binding domain comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 58; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 60; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 58 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 61 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the first antigen-binding domain comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 58; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 61; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 58 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 61 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 14 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the first antigen-binding domain comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 58; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 61; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 14; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 59 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 62 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 15 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the first antigen-binding domain comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 59; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 62; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 15; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

在一些情況下，第一抗原結合域包含：CDR-H1，其包含 SEQ ID NO: 59 之胺基酸序列、基本上由其組成、或由其組成；CDR-H2，其包含 SEQ ID NO: 57 之胺基酸序列、基本上由其組成、或由其組成；CDR-H3，其包含 SEQ ID NO: 16 之胺基酸序列、基本上由其組成、或由其組成；CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；及 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。In some cases, the first antigen-binding domain comprises: CDR-H1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 59; CDR-H2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 57; CDR-H3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16; CDR-L1, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6.

本發明之多特異性抗原結合分子可為人源化抗體。在一些情況下，第一抗原結合域及/或第二抗原結合域各自獨立地包含衍生自 IgG 骨架區的恆定區。在一些情況下，IgG 骨架區為 IgG₁、IgG₂或 IgG₄骨架區。在一些情況下，IgG 骨架區為 IgG₁骨架區。The multispecific antigen-binding molecules of the present invention may be humanized antibodies. In some cases, the first antigen-binding domain and/or the second antigen-binding domain each independently comprises a constant region derived from an IgG framework region. In some cases, the IgG framework region is an IgG₁ , IgG₂ or IgG₄ framework region. In some cases, the IgG framework region is an IgG₁ framework region.

在一些實施例中，多特異性抗原結合分子包含 (A) 第一抗原結合域，該第一抗原結合域包含重鏈可變區，該重鏈可變區包含以下項中之一個或多個 (例如，1 個、2 個、3 個或全部 4 個)：與 SEQ ID NO: 110 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-H1 序列；與 SEQ ID NO: 111 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-H2；與 SEQ ID NO: 112 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-H3；及/或與 SEQ ID NO: 113 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-H4。In some embodiments, the multispecific antigen-binding molecule comprises (A) a first antigen-binding domain comprising a heavy chain variable region comprising one or more (e.g., 1, 2, 3, or all 4) of the following: a FR-H1 sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 110; a FR-H2 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 111; a FR-H3 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 112; 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 113; and/or a FR-H4 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 113.

在一些實施例中，多特異性抗原結合分子包含 (A) 第一抗原結合域，該第一抗原結合域包含輕鏈可變區，該輕鏈可變區包含以下項中之一個或多個 (例如，1 個、2 個、3 個或全部 4 個)：與 SEQ ID NO: 114 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-L1；與 SEQ ID NO: 115 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-L2；與 SEQ ID NO: 116 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-L3；及/或與 SEQ ID NO: 117 之胺基酸序列具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 FR-L4。In some embodiments, the multispecific antigen-binding molecule comprises (A) a first antigen-binding domain comprising a light chain variable region comprising one or more (e.g., 1, 2, 3, or all 4) of the following: a FR-L1 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 114; a FR-L2 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 115; a FR-L2 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 116; 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 117; and/or an FR-L4 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 117.

舉例而言，在一些實施例中，多特異性抗原結合分子包含 (A) 第一抗原結合域，該第一抗原結合域包含：FR-H1，其包含 SEQ ID NO: 110 之胺基酸序列、基本上由其組成、或由其組成；FR-H2，其包含 SEQ ID NO: 111 之胺基酸序列、基本上由其組成、或由其組成；FR-H3，其包含 SEQ ID NO: 112 之胺基酸序列、基本上由其組成、或由其組成；FR-H4，其包含 SEQ ID NO: 113 之胺基酸序列、基本上由其組成、或由其組成；FR-L1，其包含 SEQ ID NO: 114 之胺基酸序列、基本上由其組成、或由其組成；FR-L2，其包含 SEQ ID NO: 115 之胺基酸序列、基本上由其組成、或由其組成；FR-L3，其包含 SEQ ID NO: 116 之胺基酸序列、基本上由其組成、或由其組成；及 FR-L4，其包含 SEQ ID NO: 117 之胺基酸序列、基本上由其組成、或由其組成。For example, in some embodiments, a multispecific antigen-binding molecule comprises (A) a first antigen-binding domain comprising: FR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 110; FR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 111; FR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 112; FR-H4 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 113; FR-L1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 114; FR-L2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 115; , consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 116; FR-L4, comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 117.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，其包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 VH 序列；以及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，第一抗原結合域之 VH 序列包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 的至少 90% 序列同一性。在一些情況下，第一抗原結合域之 VH 序列包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 的至少 95% 序列同一性。在一些情況下，第一抗原結合域之 VH 序列包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 的至少 98% 序列同一性。在一些情況下，第一抗原結合域之 VH 序列包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 的至少 99% 序列同一性。在一些情況下，第一抗原結合域之 VH 序列包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 的 100% 序列同一性。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含與 SEQ ID NO: 46 或 54 具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 VH 序列。在一些情況下，第二抗原結合域之 VH 序列包含與 SEQ ID NO: 46 或 54 的至少 95% 序列同一性。在一些情況下，第二抗原結合域之 VH 序列包含與 SEQ ID NO: 46 或 54 的至少 99% 序列同一性。在一些情況下，第二抗原結合域之 VH 序列包含與 SEQ ID NO: 46 或 54 的 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain comprising a VH sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7, 17-25, 30-34, 38, or 68; and (B) a second antigen-binding domain that binds to a T cell receptor. In some cases, the VH sequence of the first antigen-binding domain comprises at least 90% sequence identity to SEQ ID NO: 7, 17-25, 30-34, 38, or 68. In some cases, the VH sequence of the first antigen binding domain comprises at least 95% sequence identity to SEQ ID NO: 7, 17-25, 30-34, 38, or 68. In some cases, the VH sequence of the first antigen binding domain comprises at least 98% sequence identity to SEQ ID NO: 7, 17-25, 30-34, 38, or 68. In some cases, the VH sequence of the first antigen binding domain comprises at least 99% sequence identity to SEQ ID NO: 7, 17-25, 30-34, 38, or 68. In some cases, the VH sequence of the first antigen binding domain comprises 100% sequence identity to SEQ ID NO: 7, 17-25, 30-34, 38, or 68. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen binding domain comprises a VH sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 46 or 54. In some cases, the VH sequence of the second antigen binding domain comprises at least 95% sequence identity to SEQ ID NO: 46 or 54. In some cases, the VH sequence of the second antigen binding domain comprises at least 99% sequence identity to SEQ ID NO: 46 or 54. In some cases, the VH sequence of the second antigen binding domain comprises 100% sequence identity to SEQ ID NO: 46 or 54.

在一些實施例中，多特異性抗原結合分子包含：(A) 第一抗原結合域，其包含與 SEQ ID NO: 8、26 至 29、39 或 69 具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 VL 序列；以及 (B) 第二抗原結合域，其與 T 細胞受體結合。在一些情況下，VL 序列包含與 SEQ ID NO: 8、26 至 29、39 或 69 的至少 90% 序列同一性。在一些情況下，VL 序列包含與 SEQ ID NO: 8、26 至 29、39 或 69 的至少 95% 序列同一性。在一些情況下，VL 序列包含與 SEQ ID NO: 8、26 至 29、39 或 69 的至少 98% 序列同一性。在一些情況下，VL 序列包含與 SEQ ID NO: 8、26 至 29、39 或 69 的至少 99% 序列同一性。在一些情況下，VL 序列包含與 SEQ ID NO: 8、26 至 29、39 或 69 的 100% 序列同一性。在一些情況下，第二抗原結合域與 CD3 結合。在一些情況下，第二抗原結合域包含與 SEQ ID NO: 47 或 55 具有至少 80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99% 或 100% 序列同一性的 VL 序列。在一些情況下，第二抗原結合域之 VL 序列包含與 SEQ ID NO: 47 或 55 的至少 95% 序列同一性。在一些情況下，第二抗原結合域之 VL 序列包含與 SEQ ID NO: 47 或 55 的至少 99% 序列同一性。在一些情況下，第二抗原結合域之 VL 序列包含與 SEQ ID NO: 47 或 55 的 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain comprising a VL sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8, 26 to 29, 39, or 69; and (B) a second antigen-binding domain that binds to a T cell receptor. In some cases, the VL sequence comprises at least 90% sequence identity to SEQ ID NO: 8, 26 to 29, 39, or 69. In some cases, the VL sequence comprises at least 95% sequence identity to SEQ ID NO: 8, 26 to 29, 39, or 69. In some cases, the VL sequence comprises at least 98% sequence identity to SEQ ID NO: 8, 26-29, 39, or 69. In some cases, the VL sequence comprises at least 99% sequence identity to SEQ ID NO: 8, 26-29, 39, or 69. In some cases, the VL sequence comprises 100% sequence identity to SEQ ID NO: 8, 26-29, 39, or 69. In some cases, the second antigen binding domain binds to CD3. In some cases, the second antigen binding domain comprises a VL sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 47 or 55. In some cases, the VL sequence of the second antigen binding domain comprises at least 95% sequence identity to SEQ ID NO: 47 or 55. In some cases, the VL sequence of the second antigen binding domain comprises at least 99% sequence identity to SEQ ID NO: 47 or 55. In some cases, the VL sequence of the second antigen binding domain comprises 100% sequence identity to SEQ ID NO: 47 or 55.

在一些實施例中，本發明之多特異性抗原結合分子包含第一抗原結合域，該第一抗原結合域與 STEAP1 結合並包含至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個如表 1 或 2 中所示之 CDR。在一些情況下，第一抗原結合域包含如表 1 中所示之 VH 及/或 VL。在一些情況下，多特異性抗原結合分子包含與 T 細胞受體 (例如，CD3) 結合的第二抗原結合域。在一些情況下，與 CD3 結合的第二抗原結合域包含至少一個、至少兩個、至少三個、至少四個、至少五個或全部六個如表 1 或 2 中所示之 CDR。在一些情況下，與 CD3 結合的第二抗原結合域包含如表 1 中所示之 VH 及/或 VL。In some embodiments, the multispecific antigen-binding molecule of the present invention comprises a first antigen-binding domain that binds to STEAP1 and comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs as shown in Table 1 or 2. In some cases, the first antigen-binding domain comprises a VH and/or VL as shown in Table 1. In some cases, the multispecific antigen-binding molecule comprises a second antigen-binding domain that binds to a T cell receptor (e.g., CD3). In some cases, the second antigen-binding domain that binds to CD3 comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs as shown in Table 1 or 2. In some cases, the second antigen-binding domain that binds to CD3 comprises a VH and/or VL as shown in Table 1.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含與 STEAP1 結合的第一抗原結合域及與 T 細胞受體 (例如，CD3) 結合的第二抗原結合域。在一些實施例中，第一抗原結合域的特徵在於 VH 及 VL 區，其中 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 11 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，第一抗原結合域之 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 58 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 60 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第一抗原結合域之 VH 區包含與 SEQ ID NO: 30 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 30 具有至少 95% 序列同一性、與 SEQ ID NO: 30 具有至少 96% 序列同一性、與 SEQ ID NO: 30 具有至少 97% 序列同一性、與 SEQ ID NO: 30 具有至少 98% 序列同一性、與 SEQ ID NO: 30 具有至少 99% 序列同一性、或與 SEQ ID NO: 30 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第一抗原結合域之 VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第一抗原結合域之 VL 區包含與 SEQ ID NO: 27 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 27 具有至少 95% 序列同一性、與 SEQ ID NO: 27 具有至少 96% 序列同一性、與 SEQ ID NO: 27 具有至少 97% 序列同一性、與 SEQ ID NO: 27 具有至少 98% 序列同一性、與 SEQ ID NO: 27 具有至少 99% 序列同一性、或與 SEQ ID NO: 27 具有 100% 序列同一性) 的胺基酸序列。在一些情況下，第二抗原結合域的特徵在於 VH 及 VL 區，其中 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 40 或 48 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 41 或 49 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 42 或 50 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第二抗原結合域之 VH 區包含與 SEQ ID NO: 46 或 54 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 46 或 54 具有至少 95% 序列同一性、與 SEQ ID NO: 46 或54 具有至少 96% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 97% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 98% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 99% 序列同一性、或與 SEQ ID NO: 46 或 54 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第二抗原結合域之 VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 43 或 51 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 44 或 52 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 45 或 53 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第二抗原結合域之 VL 區包含與 SEQ ID NO: 47 或 55 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 47 或 55 具有至少 95% 序列同一性、與 SEQ ID NO: 47 或55 具有至少 96% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 97% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 98% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 99% 序列同一性、或與 SEQ ID NO: 47 或 55 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises a first antigen-binding domain that binds to STEAP1 and a second antigen-binding domain that binds to a T cell receptor (e.g., CD3). In some embodiments, the first antigen-binding domain is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 9; (b) CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 11; or (c) CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 3. In some cases, the VH region of the first antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 58; (b) CDR-H2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 60; or (c) CDR-H3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH region of the first antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 30 (e.g., at least 95% sequence identity to SEQ ID NO: 30, at least 96% sequence identity to SEQ ID NO: 30, at least 97% sequence identity to SEQ ID NO: 30, at least 98% sequence identity to SEQ ID NO: 30, at least 99% sequence identity to SEQ ID NO: 30, or 100% sequence identity to SEQ ID NO: 30). In some embodiments, the VL region of the first antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5; or (c) CDR-L3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL region of the first antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 27 (e.g., at least 95% sequence identity to SEQ ID NO: 27, at least 96% sequence identity to SEQ ID NO: 27, at least 97% sequence identity to SEQ ID NO: 27, at least 98% sequence identity to SEQ ID NO: 27, at least 99% sequence identity to SEQ ID NO: 27, or 100% sequence identity to SEQ ID NO: 27). In some cases, the second antigen-binding domain is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 40 or 48; (b) CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 41 or 49; or (c) CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 42 or 50. In some embodiments, the VH region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 46 or 54 (e.g., at least 95% sequence identity to SEQ ID NO: 46 or 54, at least 96% sequence identity to SEQ ID NO: 46 or 54, at least 97% sequence identity to SEQ ID NO: 46 or 54, at least 98% sequence identity to SEQ ID NO: 46 or 54, at least 99% sequence identity to SEQ ID NO: 46 or 54, or 100% sequence identity to SEQ ID NO: 46 or 54). In some embodiments, the VL region of the second antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 43 or 51; (b) CDR-L2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 44 or 52; or (c) CDR-L3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 45 or 53. In some embodiments, the VL region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 47 or 55 (e.g., at least 95% sequence identity to SEQ ID NO: 47 or 55, at least 96% sequence identity to SEQ ID NO: 47 or 55, at least 97% sequence identity to SEQ ID NO: 47 or 55, at least 98% sequence identity to SEQ ID NO: 47 or 55, at least 99% sequence identity to SEQ ID NO: 47 or 55, or 100% sequence identity to SEQ ID NO: 47 or 55).

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含與 STEAP1 結合的第一抗原結合域及與 T 細胞受體 (例如，CD3) 結合的第二抗原結合域。在一些實施例中，第一抗原結合域的特徵在於 VH 及 VL 區，其中 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，第一抗原結合域之 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 58 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 61 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第一抗原結合域之 VH 區包含與 SEQ ID NO: 31 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 31 具有至少 95% 序列同一性、與 SEQ ID NO: 31 具有至少 96% 序列同一性、與 SEQ ID NO: 31 具有至少 97% 序列同一性、與 SEQ ID NO: 31 具有至少 98% 序列同一性、與 SEQ ID NO: 31 具有至少 99% 序列同一性、或與 SEQ ID NO: 31 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第一抗原結合域之 VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第一抗原結合域之 VL 區包含與 SEQ ID NO: 27 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 27 具有至少 95% 序列同一性、與 SEQ ID NO: 27 具有至少 96% 序列同一性、與 SEQ ID NO: 27 具有至少 97% 序列同一性、與 SEQ ID NO: 27 具有至少 98% 序列同一性、與 SEQ ID NO: 27 具有至少 99% 序列同一性、或與 SEQ ID NO: 27 具有 100% 序列同一性) 的胺基酸序列。在一些情況下，第二抗原結合域的特徵在於 VH 及 VL 區，其中 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 40 或 48 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 41 或 49 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 42 或 50 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第二抗原結合域之 VH 區包含與 SEQ ID NO: 46 或 54 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 46 或 54 具有至少 95% 序列同一性、與 SEQ ID NO: 46 或54 具有至少 96% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 97% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 98% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 99% 序列同一性、或與 SEQ ID NO: 46 或 54 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第二抗原結合域之 VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 43 或 51 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 44 或 52 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 45 或 53 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第二抗原結合域之 VL 區包含與 SEQ ID NO: 47 或 55 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 47 或 55 具有至少 95% 序列同一性、與 SEQ ID NO: 47 或55 具有至少 96% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 97% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 98% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 99% 序列同一性、或與 SEQ ID NO: 47 或 55 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises a first antigen-binding domain that binds to STEAP1 and a second antigen-binding domain that binds to a T cell receptor (e.g., CD3). In some embodiments, the first antigen-binding domain is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 9; (b) CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 12; or (c) CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 3. In some cases, the VH region of the first antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 58; (b) CDR-H2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 61; or (c) CDR-H3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH region of the first antigen-binding domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 31 (e.g., at least 95% sequence identity to SEQ ID NO: 31, at least 96% sequence identity to SEQ ID NO: 31, at least 97% sequence identity to SEQ ID NO: 31, at least 98% sequence identity to SEQ ID NO: 31, at least 99% sequence identity to SEQ ID NO: 31, or 100% sequence identity to SEQ ID NO: 31). In some embodiments, the VL region of the first antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5; or (c) CDR-L3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL region of the first antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 27 (e.g., at least 95% sequence identity to SEQ ID NO: 27, at least 96% sequence identity to SEQ ID NO: 27, at least 97% sequence identity to SEQ ID NO: 27, at least 98% sequence identity to SEQ ID NO: 27, at least 99% sequence identity to SEQ ID NO: 27, or 100% sequence identity to SEQ ID NO: 27). In some cases, the second antigen-binding domain is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 40 or 48; (b) CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 41 or 49; or (c) CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 42 or 50. In some embodiments, the VH region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 46 or 54 (e.g., at least 95% sequence identity to SEQ ID NO: 46 or 54, at least 96% sequence identity to SEQ ID NO: 46 or 54, at least 97% sequence identity to SEQ ID NO: 46 or 54, at least 98% sequence identity to SEQ ID NO: 46 or 54, at least 99% sequence identity to SEQ ID NO: 46 or 54, or 100% sequence identity to SEQ ID NO: 46 or 54). In some embodiments, the VL region of the second antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 43 or 51; (b) CDR-L2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 44 or 52; or (c) CDR-L3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 45 or 53. In some embodiments, the VL region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 47 or 55 (e.g., at least 95% sequence identity to SEQ ID NO: 47 or 55, at least 96% sequence identity to SEQ ID NO: 47 or 55, at least 97% sequence identity to SEQ ID NO: 47 or 55, at least 98% sequence identity to SEQ ID NO: 47 or 55, at least 99% sequence identity to SEQ ID NO: 47 or 55, or 100% sequence identity to SEQ ID NO: 47 or 55).

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含與 STEAP1 結合的第一抗原結合域及與 T 細胞受體 (例如，CD3) 結合的第二抗原結合域。在一些實施例中，第一抗原結合域的特徵在於 VH 及 VL 區，其中 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 14 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，第一抗原結合域之 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 58 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 61 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 14 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第一抗原結合域之 VH 區包含與 SEQ ID NO: 32 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 32 具有至少 95% 序列同一性、與 SEQ ID NO: 32 具有至少 96% 序列同一性、與 SEQ ID NO: 32 具有至少 97% 序列同一性、與 SEQ ID NO: 32 具有至少 98% 序列同一性、與 SEQ ID NO: 32 具有至少 99% 序列同一性、或與 SEQ ID NO: 32 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第一抗原結合域之 VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第一抗原結合域之 VL 區包含與 SEQ ID NO: 27 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 27 具有至少 95% 序列同一性、與 SEQ ID NO: 27 具有至少 96% 序列同一性、與 SEQ ID NO: 27 具有至少 97% 序列同一性、與 SEQ ID NO: 27 具有至少 98% 序列同一性、與 SEQ ID NO: 27 具有至少 99% 序列同一性、或與 SEQ ID NO: 27 具有 100% 序列同一性) 的胺基酸序列。在一些情況下，第二抗原結合域的特徵在於 VH 及 VL 區，其中 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 40 或 48 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 41 或 49 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 42 或 50 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第二抗原結合域之 VH 區包含與 SEQ ID NO: 46 或 54 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 46 或 54 具有至少 95% 序列同一性、與 SEQ ID NO: 46 或54 具有至少 96% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 97% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 98% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 99% 序列同一性、或與 SEQ ID NO: 46 或 54 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第二抗原結合域之 VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 43 或 51 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 44 或 52 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 45 或 53 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第二抗原結合域之 VL 區包含與 SEQ ID NO: 47 或 55 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 47 或 55 具有至少 95% 序列同一性、與 SEQ ID NO: 47 或55 具有至少 96% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 97% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 98% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 99% 序列同一性、或與 SEQ ID NO: 47 或 55 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises a first antigen-binding domain that binds to STEAP1 and a second antigen-binding domain that binds to a T cell receptor (e.g., CD3). In some embodiments, the first antigen-binding domain is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 9; (b) CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 12; or (c) CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 14. In some cases, the VH region of the first antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 58; (b) CDR-H2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 61; or (c) CDR-H3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH region of the first antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 32 (e.g., at least 95% sequence identity to SEQ ID NO: 32, at least 96% sequence identity to SEQ ID NO: 32, at least 97% sequence identity to SEQ ID NO: 32, at least 98% sequence identity to SEQ ID NO: 32, at least 99% sequence identity to SEQ ID NO: 32, or 100% sequence identity to SEQ ID NO: 32). In some embodiments, the VL region of the first antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5; or (c) CDR-L3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL region of the first antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 27 (e.g., at least 95% sequence identity to SEQ ID NO: 27, at least 96% sequence identity to SEQ ID NO: 27, at least 97% sequence identity to SEQ ID NO: 27, at least 98% sequence identity to SEQ ID NO: 27, at least 99% sequence identity to SEQ ID NO: 27, or 100% sequence identity to SEQ ID NO: 27). In some cases, the second antigen-binding domain is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 40 or 48; (b) CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 41 or 49; or (c) CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 42 or 50. In some embodiments, the VH region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 46 or 54 (e.g., at least 95% sequence identity to SEQ ID NO: 46 or 54, at least 96% sequence identity to SEQ ID NO: 46 or 54, at least 97% sequence identity to SEQ ID NO: 46 or 54, at least 98% sequence identity to SEQ ID NO: 46 or 54, at least 99% sequence identity to SEQ ID NO: 46 or 54, or 100% sequence identity to SEQ ID NO: 46 or 54). In some embodiments, the VL region of the second antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 43 or 51; (b) CDR-L2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 44 or 52; or (c) CDR-L3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 45 or 53. In some embodiments, the VL region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 47 or 55 (e.g., at least 95% sequence identity to SEQ ID NO: 47 or 55, at least 96% sequence identity to SEQ ID NO: 47 or 55, at least 97% sequence identity to SEQ ID NO: 47 or 55, at least 98% sequence identity to SEQ ID NO: 47 or 55, at least 99% sequence identity to SEQ ID NO: 47 or 55, or 100% sequence identity to SEQ ID NO: 47 or 55).

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含與 STEAP1 結合的第一抗原結合域及與 T 細胞受體 (例如，CD3) 結合的第二抗原結合域。在一些實施例中，第一抗原結合域的特徵在於 VH 及 VL 區，其中 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 13 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 15 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，第一抗原結合域之 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 59 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 62 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 15 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第一抗原結合域之 VH 區包含與 SEQ ID NO: 33 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 33 具有至少 95% 序列同一性、與 SEQ ID NO: 33 具有至少 96% 序列同一性、與 SEQ ID NO: 33 具有至少 97% 序列同一性、與 SEQ ID NO: 33 具有至少 98% 序列同一性、與 SEQ ID NO: 33 具有至少 99% 序列同一性、或與 SEQ ID NO: 33 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第一抗原結合域之 VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第一抗原結合域之 VL 區包含與 SEQ ID NO: 27 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 27 具有至少 95% 序列同一性、與 SEQ ID NO: 27 具有至少 96% 序列同一性、與 SEQ ID NO: 27 具有至少 97% 序列同一性、與 SEQ ID NO: 27 具有至少 98% 序列同一性、與 SEQ ID NO: 27 具有至少 99% 序列同一性、或與 SEQ ID NO: 27 具有 100% 序列同一性) 的胺基酸序列。在一些情況下，第二抗原結合域的特徵在於 VH 及 VL 區，其中 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 40 或 48 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 41 或 49 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 42 或 50 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第二抗原結合域之 VH 區包含與 SEQ ID NO: 46 或 54 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 46 或 54 具有至少 95% 序列同一性、與 SEQ ID NO: 46 或54 具有至少 96% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 97% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 98% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 99% 序列同一性、或與 SEQ ID NO: 46 或 54 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第二抗原結合域之 VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 43 或 51 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 44 或 52 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 45 或 53 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第二抗原結合域之 VL 區包含與 SEQ ID NO: 47 或 55 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 47 或 55 具有至少 95% 序列同一性、與 SEQ ID NO: 47 或55 具有至少 96% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 97% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 98% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 99% 序列同一性、或與 SEQ ID NO: 47 或 55 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises a first antigen-binding domain that binds to STEAP1 and a second antigen-binding domain that binds to a T cell receptor (e.g., CD3). In some embodiments, the first antigen-binding domain is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 10; (b) CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 13; or (c) CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 15. In some cases, the VH region of the first antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 59; (b) CDR-H2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 62; or (c) CDR-H3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the VH region of the first antigen binding domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 33 (e.g., at least 95% sequence identity to SEQ ID NO: 33, at least 96% sequence identity to SEQ ID NO: 33, at least 97% sequence identity to SEQ ID NO: 33, at least 98% sequence identity to SEQ ID NO: 33, at least 99% sequence identity to SEQ ID NO: 33, or 100% sequence identity to SEQ ID NO: 33). In some embodiments, the VL region of the first antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5; or (c) CDR-L3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL region of the first antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 27 (e.g., at least 95% sequence identity to SEQ ID NO: 27, at least 96% sequence identity to SEQ ID NO: 27, at least 97% sequence identity to SEQ ID NO: 27, at least 98% sequence identity to SEQ ID NO: 27, at least 99% sequence identity to SEQ ID NO: 27, or 100% sequence identity to SEQ ID NO: 27). In some cases, the second antigen-binding domain is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 40 or 48; (b) CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 41 or 49; or (c) CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 42 or 50. In some embodiments, the VH region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 46 or 54 (e.g., at least 95% sequence identity to SEQ ID NO: 46 or 54, at least 96% sequence identity to SEQ ID NO: 46 or 54, at least 97% sequence identity to SEQ ID NO: 46 or 54, at least 98% sequence identity to SEQ ID NO: 46 or 54, at least 99% sequence identity to SEQ ID NO: 46 or 54, or 100% sequence identity to SEQ ID NO: 46 or 54). In some embodiments, the VL region of the second antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 43 or 51; (b) CDR-L2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 44 or 52; or (c) CDR-L3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 45 or 53. In some embodiments, the VL region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 47 or 55 (e.g., at least 95% sequence identity to SEQ ID NO: 47 or 55, at least 96% sequence identity to SEQ ID NO: 47 or 55, at least 97% sequence identity to SEQ ID NO: 47 or 55, at least 98% sequence identity to SEQ ID NO: 47 or 55, at least 99% sequence identity to SEQ ID NO: 47 or 55, or 100% sequence identity to SEQ ID NO: 47 or 55).

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含與 STEAP1 結合的第一抗原結合域及與 T 細胞受體 (例如，CD3) 結合的第二抗原結合域。在一些實施例中，第一抗原結合域的特徵在於 VH 及 VL 區，其中 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 16 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，第一抗原結合域之 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 59 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 57 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 16 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第一抗原結合域之 VH 區包含與 SEQ ID NO: 34 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 34 具有至少 95% 序列同一性、與 SEQ ID NO: 34 具有至少 96% 序列同一性、與 SEQ ID NO: 34 具有至少 97% 序列同一性、與 SEQ ID NO: 34 具有至少 98% 序列同一性、與 SEQ ID NO: 34 具有至少 99% 序列同一性、或與 SEQ ID NO: 34 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第一抗原結合域之 VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第一抗原結合域之 VL 區包含與 SEQ ID NO: 27 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 27 具有至少 95% 序列同一性、與 SEQ ID NO: 27 具有至少 96% 序列同一性、與 SEQ ID NO: 27 具有至少 97% 序列同一性、與 SEQ ID NO: 27 具有至少 98% 序列同一性、與 SEQ ID NO: 27 具有至少 99% 序列同一性、或與 SEQ ID NO: 27 具有 100% 序列同一性) 的胺基酸序列。在一些情況下，第二抗原結合域的特徵在於 VH 及 VL 區，其中 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 40 或 48 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 41 或 49 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 42 或 50 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第二抗原結合域之 VH 區包含與 SEQ ID NO: 46 或 54 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 46 或 54 具有至少 95% 序列同一性、與 SEQ ID NO: 46 或54 具有至少 96% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 97% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 98% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 99% 序列同一性、或與 SEQ ID NO: 46 或 54 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第二抗原結合域之 VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 43 或 51 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 44 或 52 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 45 或 53 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第二抗原結合域之 VL 區包含與 SEQ ID NO: 47 或 55 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 47 或 55 具有至少 95% 序列同一性、與 SEQ ID NO: 47 或55 具有至少 96% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 97% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 98% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 99% 序列同一性、或與 SEQ ID NO: 47 或 55 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises a first antigen-binding domain that binds to STEAP1 and a second antigen-binding domain that binds to a T cell receptor (e.g., CD3). In some embodiments, the first antigen-binding domain is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 10; (b) CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 2; or (c) CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 16. In some cases, the VH region of the first antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 59; (b) CDR-H2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 57; or (c) CDR-H3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH region of the first antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 34 (e.g., at least 95% sequence identity to SEQ ID NO: 34, at least 96% sequence identity to SEQ ID NO: 34, at least 97% sequence identity to SEQ ID NO: 34, at least 98% sequence identity to SEQ ID NO: 34, at least 99% sequence identity to SEQ ID NO: 34, or 100% sequence identity to SEQ ID NO: 34). In some embodiments, the VL region of the first antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5; or (c) CDR-L3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL region of the first antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 27 (e.g., at least 95% sequence identity to SEQ ID NO: 27, at least 96% sequence identity to SEQ ID NO: 27, at least 97% sequence identity to SEQ ID NO: 27, at least 98% sequence identity to SEQ ID NO: 27, at least 99% sequence identity to SEQ ID NO: 27, or 100% sequence identity to SEQ ID NO: 27). In some cases, the second antigen-binding domain is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 40 or 48; (b) CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 41 or 49; or (c) CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 42 or 50. In some embodiments, the VH region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 46 or 54 (e.g., at least 95% sequence identity to SEQ ID NO: 46 or 54, at least 96% sequence identity to SEQ ID NO: 46 or 54, at least 97% sequence identity to SEQ ID NO: 46 or 54, at least 98% sequence identity to SEQ ID NO: 46 or 54, at least 99% sequence identity to SEQ ID NO: 46 or 54, or 100% sequence identity to SEQ ID NO: 46 or 54). In some embodiments, the VL region of the second antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 43 or 51; (b) CDR-L2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 44 or 52; or (c) CDR-L3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 45 or 53. In some embodiments, the VL region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 47 or 55 (e.g., at least 95% sequence identity to SEQ ID NO: 47 or 55, at least 96% sequence identity to SEQ ID NO: 47 or 55, at least 97% sequence identity to SEQ ID NO: 47 or 55, at least 98% sequence identity to SEQ ID NO: 47 or 55, at least 99% sequence identity to SEQ ID NO: 47 or 55, or 100% sequence identity to SEQ ID NO: 47 or 55).

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含與 STEAP1 結合的第一抗原結合域及與 T 細胞受體 (例如，CD3) 結合的第二抗原結合域。在一些實施例中，第一抗原結合域的特徵在於 VH 及 VL 區，其中 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 35 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 36 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 37 之胺基酸序列、基本上由其組成、或由其組成。在一些情況下，第一抗原結合域之 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 63 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 64 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 37 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第一抗原結合域之 VH 區包含與 SEQ ID NO: 38 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 38 具有至少 95% 序列同一性、與 SEQ ID NO: 38 具有至少 96% 序列同一性、與 SEQ ID NO: 38 具有至少 97% 序列同一性、與 SEQ ID NO: 38 具有至少 98% 序列同一性、與 SEQ ID NO: 38 具有至少 99% 序列同一性、或與 SEQ ID NO: 38 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第一抗原結合域之 VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第一抗原結合域之 VL 區包含與 SEQ ID NO: 39 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 39 具有至少 95% 序列同一性、與 SEQ ID NO: 39 具有至少 96% 序列同一性、與 SEQ ID NO: 39 具有至少 97% 序列同一性、與 SEQ ID NO: 39 具有至少 98% 序列同一性、與 SEQ ID NO: 39 具有至少 99% 序列同一性、或與 SEQ ID NO: 39 具有 100% 序列同一性) 的胺基酸序列。在一些情況下，第二抗原結合域的特徵在於 VH 及 VL 區，其中 VH 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-H1，其包含 SEQ ID NO: 40 或 48 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-H2，其包含 SEQ ID NO: 41 或 49 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-H3，其包含 SEQ ID NO: 42 或 50 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第二抗原結合域之 VH 區包含與 SEQ ID NO: 46 或 54 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 46 或 54 具有至少 95% 序列同一性、與 SEQ ID NO: 46 或54 具有至少 96% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 97% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 98% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 99% 序列同一性、或與 SEQ ID NO: 46 或 54 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第二抗原結合域之 VL 區包含以下 CDR 中之一個、兩個或全部三個：(a) CDR-L1，其包含 SEQ ID NO: 43 或 51 之胺基酸序列、基本上由其組成、或由其組成；(b) CDR-L2，其包含 SEQ ID NO: 44 或 52 之胺基酸序列、基本上由其組成、或由其組成；或 (c) CDR-L3，其包含 SEQ ID NO: 45 或 53 之胺基酸序列、基本上由其組成、或由其組成。在一些實施例中，第二抗原結合域之 VL 區包含與 SEQ ID NO: 47 或 55 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 47 或 55 具有至少 95% 序列同一性、與 SEQ ID NO: 47 或55 具有至少 96% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 97% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 98% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 99% 序列同一性、或與 SEQ ID NO: 47 或 55 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises a first antigen-binding domain that binds to STEAP1 and a second antigen-binding domain that binds to a T cell receptor (e.g., CD3). In some embodiments, the first antigen-binding domain is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 35; (b) CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 36; or (c) CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 37. In some cases, the VH region of the first antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 63; (b) CDR-H2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 64; or (c) CDR-H3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH region of the first antigen binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 38 (e.g., at least 95% sequence identity to SEQ ID NO: 38, at least 96% sequence identity to SEQ ID NO: 38, at least 97% sequence identity to SEQ ID NO: 38, at least 98% sequence identity to SEQ ID NO: 38, at least 99% sequence identity to SEQ ID NO: 38, or 100% sequence identity to SEQ ID NO: 38). In some embodiments, the VL region of the first antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5; or (c) CDR-L3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL region of the first antigen binding domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 39 (e.g., at least 95% sequence identity to SEQ ID NO: 39, at least 96% sequence identity to SEQ ID NO: 39, at least 97% sequence identity to SEQ ID NO: 39, at least 98% sequence identity to SEQ ID NO: 39, at least 99% sequence identity to SEQ ID NO: 39, or 100% sequence identity to SEQ ID NO: 39). In some cases, the second antigen-binding domain is characterized by VH and VL regions, wherein the VH region comprises one, two, or all three of the following CDRs: (a) CDR-H1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 40 or 48; (b) CDR-H2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 41 or 49; or (c) CDR-H3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 42 or 50. In some embodiments, the VH region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 46 or 54 (e.g., at least 95% sequence identity to SEQ ID NO: 46 or 54, at least 96% sequence identity to SEQ ID NO: 46 or 54, at least 97% sequence identity to SEQ ID NO: 46 or 54, at least 98% sequence identity to SEQ ID NO: 46 or 54, at least 99% sequence identity to SEQ ID NO: 46 or 54, or 100% sequence identity to SEQ ID NO: 46 or 54). In some embodiments, the VL region of the second antigen-binding domain comprises one, two, or all three of the following CDRs: (a) CDR-L1 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 43 or 51; (b) CDR-L2 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 44 or 52; or (c) CDR-L3 comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 45 or 53. In some embodiments, the VL region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 47 or 55 (e.g., at least 95% sequence identity to SEQ ID NO: 47 or 55, at least 96% sequence identity to SEQ ID NO: 47 or 55, at least 97% sequence identity to SEQ ID NO: 47 or 55, at least 98% sequence identity to SEQ ID NO: 47 or 55, at least 99% sequence identity to SEQ ID NO: 47 or 55, or 100% sequence identity to SEQ ID NO: 47 or 55).

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含與 STEAP1 結合的第一抗原結合域及與 T 細胞受體 (例如，CD3) 結合的第二抗原結合域。在一些實施例中，第一抗原結合域之 VH 區包含與 SEQ ID NO: 68 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 68 具有至少 95% 序列同一性、與 SEQ ID NO: 68 具有至少 96% 序列同一性、與 SEQ ID NO: 68 具有至少 97% 序列同一性、與 SEQ ID NO: 68 具有至少 98% 序列同一性、與 SEQ ID NO: 68 具有至少 99% 序列同一性、或與 SEQ ID NO: 68 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第一抗原結合域之 VL 區包含與 SEQ ID NO: 69 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 69 具有至少 95% 序列同一性、與 SEQ ID NO: 69 具有至少 96% 序列同一性、與 SEQ ID NO: 69 具有至少 97% 序列同一性、與 SEQ ID NO: 69 具有至少 98% 序列同一性、與 SEQ ID NO: 69 具有至少 99% 序列同一性、或與 SEQ ID NO: 69 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第二抗原結合域之 VH 區包含與 SEQ ID NO: 46 或 54 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 46 或 54 具有至少 95% 序列同一性、與 SEQ ID NO: 46 或54 具有至少 96% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 97% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 98% 序列同一性、與 SEQ ID NO: 46 或 54 具有至少 99% 序列同一性、或與 SEQ ID NO: 46 或 54 具有 100% 序列同一性) 的胺基酸序列。在一些實施例中，第二抗原結合域之 VL 區包含與 SEQ ID NO: 47 或 55 具有至少 90% 序列同一性 (例如，與 SEQ ID NO: 47 或 55 具有至少 95% 序列同一性、與 SEQ ID NO: 47 或55 具有至少 96% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 97% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 98% 序列同一性、與 SEQ ID NO: 47 或 55 具有至少 99% 序列同一性、或與 SEQ ID NO: 47 或 55 具有 100% 序列同一性) 的胺基酸序列。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises a first antigen-binding domain that binds to STEAP1 and a second antigen-binding domain that binds to a T cell receptor (e.g., CD3). In some embodiments, the VH region of the first antigen-binding domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 68 (e.g., at least 95% sequence identity to SEQ ID NO: 68, at least 96% sequence identity to SEQ ID NO: 68, at least 97% sequence identity to SEQ ID NO: 68, at least 98% sequence identity to SEQ ID NO: 68, at least 99% sequence identity to SEQ ID NO: 68, or 100% sequence identity to SEQ ID NO: 68). In some embodiments, the VL region of the first antigen binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 69 (e.g., at least 95% sequence identity to SEQ ID NO: 69, at least 96% sequence identity to SEQ ID NO: 69, at least 97% sequence identity to SEQ ID NO: 69, at least 98% sequence identity to SEQ ID NO: 69, at least 99% sequence identity to SEQ ID NO: 69, or 100% sequence identity to SEQ ID NO: 69). In some embodiments, the VH region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 46 or 54 (e.g., at least 95% sequence identity to SEQ ID NO: 46 or 54, at least 96% sequence identity to SEQ ID NO: 46 or 54, at least 97% sequence identity to SEQ ID NO: 46 or 54, at least 98% sequence identity to SEQ ID NO: 46 or 54, at least 99% sequence identity to SEQ ID NO: 46 or 54, or 100% sequence identity to SEQ ID NO: 46 or 54). In some embodiments, the VL region of the second antigen-binding domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 47 or 55 (e.g., at least 95% sequence identity to SEQ ID NO: 47 or 55, at least 96% sequence identity to SEQ ID NO: 47 or 55, at least 97% sequence identity to SEQ ID NO: 47 or 55, at least 98% sequence identity to SEQ ID NO: 47 or 55, at least 99% sequence identity to SEQ ID NO: 47 or 55, or 100% sequence identity to SEQ ID NO: 47 or 55).

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 STEAP1 重鏈恆定區，其與 SEQ ID NO: 83 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 STEAP1 輕鏈恆定區，其與 SEQ ID NO: 82 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises: an anti-STEAP1 heavy chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 83; and an anti-STEAP1 light chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 STEAP1 重鏈恆定區，其與 SEQ ID NO: 85 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 STEAP1 輕鏈恆定區，其與 SEQ ID NO: 84 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises: an anti-STEAP1 heavy chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 85; and an anti-STEAP1 light chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 84.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 CD3 重鏈恆定區，其與 SEQ ID NO: 83 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 CD3 輕鏈恆定區，其與 SEQ ID NO: 82 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises: an anti-CD3 heavy chain constant region that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 83; and an anti-CD3 light chain constant region that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 CD3 重鏈恆定區，其與 SEQ ID NO: 85 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 CD3 輕鏈恆定區，其與 SEQ ID NO: 84 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises: an anti-CD3 heavy chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 85; and an anti-CD3 light chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 84.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 STEAP1 重鏈，其與 SEQ ID NO: 71 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 STEAP1 輕鏈，其與 SEQ ID NO: 70 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises: an anti-STEAP1 heavy chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71; and an anti-STEAP1 light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 STEAP1 重鏈其與 SEQ ID NO: 73 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 STEAP1 輕鏈，其與 SEQ ID NO: 72 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any preceding embodiment comprises: an anti-STEAP1 heavy chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73; and an anti-STEAP1 light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 STEAP1 重鏈其與 SEQ ID NO: 75 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 STEAP1 輕鏈，其與 SEQ ID NO: 74 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any preceding embodiment comprises: an anti-STEAP1 heavy chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75; and an anti-STEAP1 light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 STEAP1 重鏈其與 SEQ ID NO: 77 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 STEAP1 輕鏈，其與 SEQ ID NO: 76 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises: an anti-STEAP1 heavy chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77; and an anti-STEAP1 light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 STEAP1 重鏈恆定區，其與 SEQ ID NO: 122 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 STEAP1 輕鏈恆定區，其與 SEQ ID NO: 123 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises: an anti-STEAP1 heavy chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 122; and an anti-STEAP1 light chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 123.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 STEAP1 重鏈恆定區，其與 SEQ ID NO: 126 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 STEAP1 輕鏈恆定區，其與 SEQ ID NO: 127 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises: an anti-STEAP1 heavy chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 126; and an anti-STEAP1 light chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 127.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 STEAP1 重鏈恆定區，其與 SEQ ID NO: 130 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 STEAP1 輕鏈恆定區，其與 SEQ ID NO: 131 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises: an anti-STEAP1 heavy chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 130; and an anti-STEAP1 light chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 131.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 STEAP1 重鏈恆定區，其與 SEQ ID NO: 134 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 STEAP1 輕鏈恆定區，其與 SEQ ID NO: 135 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises: an anti-STEAP1 heavy chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 134; and an anti-STEAP1 light chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 135.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 STEAP1 重鏈恆定區，其與 SEQ ID NO: 138 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 STEAP1 輕鏈恆定區，其與 SEQ ID NO: 139 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises: an anti-STEAP1 heavy chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 138; and an anti-STEAP1 light chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 139.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 STEAP1 重鏈恆定區，其與 SEQ ID NO: 142 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 STEAP1 輕鏈恆定區，其與 SEQ ID NO: 143 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any of the preceding embodiments comprises: an anti-STEAP1 heavy chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 142; and an anti-STEAP1 light chain constant region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 143.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 CD3 重鏈其與 SEQ ID NO: 79 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 CD3 輕鏈，其與 SEQ ID NO: 78 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any preceding embodiment comprises: an anti-CD3 heavy chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79; and an anti-CD3 light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 78.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 CD3 重鏈其與 SEQ ID NO: 81 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 CD3 輕鏈，其與 SEQ ID NO: 80 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any preceding embodiment comprises: an anti-CD3 heavy chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 81; and an anti-CD3 light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 80.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 CD3 重鏈其與 SEQ ID NO: 124 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 CD3 輕鏈，其與 SEQ ID NO: 125 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any preceding embodiment comprises: an anti-CD3 heavy chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 124; and an anti-CD3 light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 125.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 CD3 重鏈其與 SEQ ID NO: 128 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 CD3 輕鏈，其與 SEQ ID NO: 129 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any preceding embodiment comprises: an anti-CD3 heavy chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 128; and an anti-CD3 light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 129.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 CD3 重鏈其與 SEQ ID NO: 132 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 CD3 輕鏈，其與 SEQ ID NO: 133 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any preceding embodiment comprises: an anti-CD3 heavy chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 132; and an anti-CD3 light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 133.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 CD3 重鏈其與 SEQ ID NO: 136 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 CD3 輕鏈，其與 SEQ ID NO: 137 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any preceding embodiment comprises: an anti-CD3 heavy chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; and an anti-CD3 light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 137.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 CD3 重鏈其與 SEQ ID NO: 140 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 CD3 輕鏈，其與 SEQ ID NO: 141 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any preceding embodiment comprises: an anti-CD3 heavy chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 140; and an anti-CD3 light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 141.

在一些實施例中，任何前述實施例之多特異性抗原結合分子包含：抗 CD3 重鏈其與 SEQ ID NO: 144 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性；及抗 CD3 輕鏈，其與 SEQ ID NO: 145 具有至少 90%、至少 91%、至少 92%、至少 93%、至少 94%、至少 95%、至少 96%、至少 97%、至少 98%、至少 99% 或 100% 序列同一性。In some embodiments, the multispecific antigen-binding molecule of any preceding embodiment comprises: an anti-CD3 heavy chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 144; and an anti-CD3 light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 145.

在一些實施例中，多特異性抗原結合分子包含含有 SEQ ID NO: 122 的抗 STEAP1 重鏈、含有 SEQ ID NO: 123 的抗 STEAP1 輕鏈、含有 SEQ ID NO: 124 的抗 CD3 重鏈及含有 SEQ ID NO: 125 的抗 CD3 輕鏈。In some embodiments, the multispecific antigen-binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 122, an anti-STEAP1 light chain comprising SEQ ID NO: 123, an anti-CD3 heavy chain comprising SEQ ID NO: 124, and an anti-CD3 light chain comprising SEQ ID NO: 125.

在一些實施例中，多特異性抗原結合分子包含含有 SEQ ID NO: 126 的抗 STEAP1 重鏈、含有 SEQ ID NO: 127 的抗 STEAP1 輕鏈、含有 SEQ ID NO: 128 的抗 CD3 重鏈及含有 SEQ ID NO: 129 的抗 CD3 輕鏈。In some embodiments, the multispecific antigen-binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 126, an anti-STEAP1 light chain comprising SEQ ID NO: 127, an anti-CD3 heavy chain comprising SEQ ID NO: 128, and an anti-CD3 light chain comprising SEQ ID NO: 129.

在一些實施例中，多特異性抗原結合分子包含含有 SEQ ID NO: 130 的抗 STEAP1 重鏈、含有 SEQ ID NO: 131 的抗 STEAP1 輕鏈、含有 SEQ ID NO: 132 的抗 CD3 重鏈及含有 SEQ ID NO: 133 的抗 CD3 輕鏈。In some embodiments, the multispecific antigen-binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 130, an anti-STEAP1 light chain comprising SEQ ID NO: 131, an anti-CD3 heavy chain comprising SEQ ID NO: 132, and an anti-CD3 light chain comprising SEQ ID NO: 133.

在一些實施例中，多特異性抗原結合分子包含含有 SEQ ID NO: 134 的抗 STEAP1 重鏈、含有 SEQ ID NO: 135 的抗 STEAP1 輕鏈、含有 SEQ ID NO: 136 的抗 CD3 重鏈及含有 SEQ ID NO: 137 的抗 CD3 輕鏈。In some embodiments, the multispecific antigen-binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 134, an anti-STEAP1 light chain comprising SEQ ID NO: 135, an anti-CD3 heavy chain comprising SEQ ID NO: 136, and an anti-CD3 light chain comprising SEQ ID NO: 137.

在一些實施例中，多特異性抗原結合分子包含含有 SEQ ID NO: 138 的抗 STEAP1 重鏈、含有 SEQ ID NO: 139 的抗 STEAP1 輕鏈、含有 SEQ ID NO: 140 的抗 CD3 重鏈及含有 SEQ ID NO: 141 的抗 CD3 輕鏈。In some embodiments, the multispecific antigen-binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 138, an anti-STEAP1 light chain comprising SEQ ID NO: 139, an anti-CD3 heavy chain comprising SEQ ID NO: 140, and an anti-CD3 light chain comprising SEQ ID NO: 141.

在一些實施例中，多特異性抗原結合分子包含含有 SEQ ID NO: 142 的抗 STEAP1 重鏈、含有 SEQ ID NO: 143 的抗 STEAP1 輕鏈、含有 SEQ ID NO: 144 的抗 CD3 重鏈及含有 SEQ ID NO: 145 的抗 CD3 輕鏈。In some embodiments, the multispecific antigen-binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 142, an anti-STEAP1 light chain comprising SEQ ID NO: 143, an anti-CD3 heavy chain comprising SEQ ID NO: 144, and an anti-CD3 light chain comprising SEQ ID NO: 145.

在一些實施例中，多特異性抗原結合分子進一步包含第三抗原結合域。第三抗原結合域可為在細胞之表面上表現的抗原。抗原可在從實性瘤獲得的細胞之表面上表現。抗原可在前列腺癌、尤文氏肉瘤、肺癌、大腸直腸癌、乳癌、膀胱癌、卵巢癌或子宮頸癌細胞之表面上表現。In some embodiments, the multispecific antigen binding molecule further comprises a third antigen binding domain. The third antigen binding domain may be an antigen expressed on the surface of a cell. The antigen may be expressed on the surface of a cell obtained from a solid tumor. The antigen may be expressed on the surface of a prostate cancer, Ewing's sarcoma, lung cancer, colorectal cancer, breast cancer, bladder cancer, ovarian cancer, or cervical cancer cell.

在一些實施例中，抗原為腫瘤相關抗原 (TAA)。TAA 可為在前列腺癌、尤文氏肉瘤、肺癌、大腸直腸癌、乳癌、膀胱癌、卵巢癌或子宮頸癌的細胞之表面上表現的受體。在一些情況下，TAA 為在前列腺癌細胞之表面上表現的受體。在一些情況下，TAA 為在尤文氏肉瘤細胞上表現的受體。示例性腫瘤相關抗原包括但不限於前列腺特異性膜抗原 (PSMA)、前列腺幹細胞抗原 (PSCA)、上皮細胞黏著分子 (EpCAM)、前列腺特異性抗原 (PSA)、前列腺酸性磷酸酶 (PAP)、STEAP2 及 HBA-71。In some embodiments, the antigen is a tumor-associated antigen (TAA). TAA can be a receptor expressed on the surface of cells of prostate cancer, Ewing's sarcoma, lung cancer, colorectal cancer, breast cancer, bladder cancer, ovarian cancer, or cervical cancer. In some cases, TAA is a receptor expressed on the surface of prostate cancer cells. In some cases, TAA is a receptor expressed on Ewing's sarcoma cells. Exemplary tumor-associated antigens include, but are not limited to, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), epithelial cell adhesion molecule (EpCAM), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), STEAP2, and HBA-71.

在一些情況下，多特異性抗原結合分子進一步包含與腫瘤相關抗原結合的第三抗原結合域。在一些情況下，第三抗原結合域與前列腺癌、尤文氏肉瘤、肺癌、大腸直腸癌、乳癌、膀胱癌、卵巢癌或子宮頸癌之 TAA 結合。在一些情況下，第三抗原結合域與前列腺特異性膜抗原 (PSMA)、前列腺幹細胞抗原 (PSCA)、上皮細胞黏著分子 (EpCAM)、前列腺特異性抗原 (PSA) 或前列腺酸性磷酸酶 (PAP)、STEAP2 或 HBA-71 結合。在一些情況下，第三抗原結合域與 PSMA 結合。在一些情況下，第三抗原結合域與 PSCA 結合。在一些情況下，第三抗原結合域與 EpCAM 結合。在一些情況下，第三抗原結合域與 PSA 結合。在一些情況下，第三抗原結合域與 PAP 結合。在一些情況下，第三抗原結合域與 STEAP2 結合。在一些情況下，第三抗原結合域與 HBA-71 結合。In some cases, the multispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to a tumor-associated antigen. In some cases, the third antigen-binding domain binds to a TAA of prostate cancer, Ewing's sarcoma, lung cancer, colorectal cancer, breast cancer, bladder cancer, ovarian cancer, or cervical cancer. In some cases, the third antigen-binding domain binds to prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), epithelial cell adhesion molecule (EpCAM), prostate-specific antigen (PSA) or prostatic acid phosphatase (PAP), STEAP2, or HBA-71. In some cases, the third antigen-binding domain binds to PSMA. In some cases, the third antigen-binding domain binds to PSCA. In some cases, the third antigen-binding domain binds to EpCAM. In some cases, the third antigen binding domain binds to PSA. In some cases, the third antigen binding domain binds to PAP. In some cases, the third antigen binding domain binds to STEAP2. In some cases, the third antigen binding domain binds to HBA-71.

在一些實施例中，多特異性抗原結合分子以小於 100 nM、小於 75 nM、小於 50 nM、小於 40 nM、小於 30 nM、小於 20 nM、小於 10 nM、小於 1 nM 或更低之 K_D與人 STEAP1 結合。In some embodiments, the multispecific antigen-binding molecule binds to human STEAP1 with a_KD of less than 100 nM, less than 75 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 1 nM, or less.

在一些實施例中，多特異性抗原結合分子以約 1 nM 至約 100 nM、約 1 nM 至約 75 nM、約 1 nM 至約 50 nM、約 1 nM 至約 40 nM、約 1 nM 至約 30 nM、約 1 nM 至約 20 nM、約 1 nM 至約 10 nM、約 1 nM 至約 5 nM、約 5 nM 至約 100 nM、約 5 nM 至約 75 nM、約 5 nM 至約 50 nM、約 5 nM 至約 40 nM、約 5 nM 至約 30 nM、約 5 nM 至約 20 nM、約 5 nM 至約 10 nM、約 10 nM 至約 100 nM、約 10 nM 至約 75 nM、約 10 nM 至約 50 nM、約 10 nM 至約 40 nM、約 10 nM 至約 30 nM、約 10 nM 至約 20 nM、約 15 nM 至約 100 nM、約 15 nM 至約 75 nM、約 15 nM 至約 50 nM、約 15 nM 至約 40 nM、約 15 nM 至約 30 nM、約 15 nM 至約 20 nM、約 20 nM 至約 100 nM、約 20 nM 至約 75 nM、約 20 nM 至約 50 nM、約 20 nM 至約 40 nM、約 20 nM 至約 30 nM、約 25 nM 至約 100 nM、約 25 nM 至約 75 nM、約 25 nM 至約 50 nM、約 25 nM 至約 40 nM、約 25 nM 至約 30 nM、約 30 nM 至約 100 nM、約 30 nM 至約 75 nM、約 30 nM 至約 50 nM、約 30 nM 至約 40 nM、約 35 nM 至約 100 nM、約 35 nM 至約 75 nM、約 35 nM 至約 50 nM、約 35 nM 至約 40 nM、約 40 nM 至約 100 nM、約 40 nM 至約 75 nM、約 40 nM 至約 50 nM、約 45 nM 至約 100 nM、約 45 nM 至約 75 nM、約 45 nM 至約 50 nM、約 50 nM 至約 100 nM、約 50 nM 至約 75 nM、 約 70 nM 至約 100 nM、約 70 nM 至約 75 nM、約 80 nM 至約 100 nM、或約 90 nM 至約 100 nM 之 K_D與人 STEAP1 結合。在一些實施例中，多特異性抗原結合分子以約 1 nM、約 2 nM、約 3 nM、約 4 nM、約 5 nM、約 6 nM、約 7 nM、約 8 nM、約 9 nM、約 10 nM、約 11 nM、約 12 nM、約 13 nM、約 14 nM、約 15 nM、約 16 nM、約 17 nM、約 18 nM、約 19 nM、約 20 nM、約 21 nM、約 22 nM、約 23 nM、約 24 nM、約 25 nM、約 26 nM、約 27 nM、約 28 nM、約 29 nM、約 30 nM、約 31 nM、約 32 nM、約 33 nM、約 34 nM、約 35 nM、約 36 nM、約 37 nM、約 38 nM、約 39 nM、約 40 nM、約 41 nM、約 42 nM、約 43 nM、約 44 nM、約 45 nM、約 46 nM、約 47 nM、約 48 nM、約 49 nM、或約 50 nM 之 K_D與人 STEAP1 結合。在一些實施例中，多特異性抗原結合分子以約 29 nM 之 K_D與人 STEAP1 結合。在一些實施例中，K_D係使用動力學篩析測定 (KinExA®) 所確定，如實例 8 中所述。In some embodiments, the multispecific antigen-binding molecule is present in an amount of about 1 nM to about 100 nM, about 1 nM to about 75 nM, about 1 nM to about 50 nM, about 1 nM to about 40 nM, about 1 nM to about 30 nM, about 1 nM to about 20 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, about 5 nM to about 100 nM, about 5 nM to about 75 nM, about 5 nM to about 50 nM, about 5 nM to about 40 nM, about 5 nM to about 30 nM, about 5 nM to about 20 nM, about 5 nM to about 10 nM, about 10 nM to about 100 nM, about 10 nM to about 75 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM, about 10 nM to about 20 nM, about 15 nM to about 100 nM, about 15 nM to about 75 nM, about 15 nM to about 50 nM, about 15 nM to about 40 nM, about 15 nM to about 30 nM, about 15 nM to about 20 nM, about 20 nM to about 100 nM, about 20 nM to about 75 nM, about 20 nM to about 50 nM, about 20 nM to about 40 nM, about 20 nM to about 30 nM, about 25 nM to about 100 nM, about 25 nM to about 75 nM, about 25 nM to about 50 nM, about 25 nM to about 40 nM, about 25 nM to about 30 nM, about 30 nM to about 100 nM nM, about 30 nM to about 75 nM, about 30 nM to about 50 nM, about 30 nM to about 40 nM, about 35 nM to about 100 nM, about 35 nM to about 75 nM, about 35 nM to about 50 nM, about 35 nM to about 40 nM, about 40 nM to about 100 nM, about 40 nM to about 75 nM, about 40 nM to about 50 nM, about 45 nM to about 100 nM, about 45 nM to about 75 nM, about 45 nM to about 50 nM, about 50 nM to about 100 nM, about 50 nM to about 75 nM, about 70 nM to about 100 nM, about 70 nM to about 75 nM, about 80 nM to about 100 nM, or about 90 nM. Binds to human STEAP1 with a_KD of approximately 100 nM. In some embodiments, the multispecific antigen-binding molecule is about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19 nM, about 20 nM, about 21 nM, about 22 nM, about 23 nM, about 24 nM, about 25 nM, about 26 nM, about 27 nM, about 28 nM, about 29 nM, about 30 nM, about 31 nM, about 32 nM, about 33 nM, about 34 nM, about 35 nM, about 36 nM, about 37 nM, about 38 nM, about 39 nM, about 40 nM, about In some embodiments, the multispecific_{antigen-binding molecule} binds to human STEAP1 with a_K of about 29 nM. In some embodiments, the_K is determined using a kinetic screening assay (KinExA®), as described in Example 8.

在一些實施例中，多特異性抗原結合分子以小於 100 nM、小於 75 nM、小於 50 nM、小於 40 nM、小於 30 nM、小於 20 nM、小於 10 nM、小於 1 nM 或更低之 K_D與石蟹獼猴 STEAP1 結合。In some embodiments, the multispecific antigen-binding molecule binds to C. macaque_STEAP1 with a KD of less than 100 nM, less than 75 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 1 nM, or less.

在一些實施例中，多特異性抗原結合分子以約 1 nM 至約 100 nM、約 1 nM 至約 75 nM、約 1 nM 至約 50 nM、約 1 nM 至約 40 nM、約 1 nM 至約 30 nM、約 1 nM 至約 20 nM、約 1 nM 至約 10 nM、約 1 nM 至約 5 nM、約 5 nM 至約 100 nM、約 5 nM 至約 75 nM、約 5 nM 至約 50 nM、約 5 nM 至約 40 nM、約 5 nM 至約 30 nM、約 5 nM 至約 20 nM、約 5 nM 至約 10 nM、約 10 nM 至約 100 nM、約 10 nM 至約 75 nM、約 10 nM 至約 50 nM、約 10 nM 至約 40 nM、約 10 nM 至約 30 nM、約 10 nM 至約 20 nM、約 15 nM 至約 100 nM、約 15 nM 至約 75 nM、約 15 nM 至約 50 nM、約 15 nM 至約 40 nM、約 15 nM 至約 30 nM、約 15 nM 至約 20 nM、約 20 nM 至約 100 nM、約 20 nM 至約 75 nM、約 20 nM 至約 50 nM、約 20 nM 至約 40 nM、約 20 nM 至約 30 nM、約 25 nM 至約 100 nM、約 25 nM 至約 75 nM、約 25 nM 至約 50 nM、約 25 nM 至約 40 nM、約 25 nM 至約 30 nM、約 30 nM 至約 100 nM、約 30 nM 至約 75 nM、約 30 nM 至約 50 nM、約 30 nM 至約 40 nM、約 35 nM 至約 100 nM、約 35 nM 至約 75 nM、約 35 nM 至約 50 nM、約 35 nM 至約 40 nM、約 40 nM 至約 100 nM、約 40 nM 至約 75 nM、約 40 nM 至約 50 nM、約 45 nM 至約 100 nM、約 45 nM 至約 75 nM、約 45 nM 至約 50 nM、約 50 nM 至約 100 nM、約 50 nM 至約 75 nM、約 70 nM 至約 100 nM、約 70 nM 至約 75 nM、約 80 nM 至約 100 nM、或約 90 nM 至約 100 nM 之 K_D與石蟹獼猴 STEAP1 結合。在一些實施例中，多特異性抗原結合分子以 1 nM、約 2 nM、約 3 nM、約 4 nM、約 5 nM、約 6 nM、約 7 nM、約 8 nM、約 9 nM、約 10 nM、約 11 nM、約 12 nM、約 13 nM、約 14 nM、約 15 nM、約 16 nM、約 17 nM、約 18 nM、約 19 nM、約 20 nM、約 21 nM、約 22 nM、約 23 nM、約 24 nM、或約 25 nM 之 K_D與石蟹獼猴 STEAP1 結合。在一些實施例中，多特異性抗原結合分子以約 14 nM 之 K_D與石蟹獼猴 STEAP1 結合。在一些實施例中，K_D係使用動力學篩析測定 (KinExA®) 所確定，如實例 8 中所述。In some embodiments, the multispecific antigen-binding molecule is present in an amount of about 1 nM to about 100 nM, about 1 nM to about 75 nM, about 1 nM to about 50 nM, about 1 nM to about 40 nM, about 1 nM to about 30 nM, about 1 nM to about 20 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, about 5 nM to about 100 nM, about 5 nM to about 75 nM, about 5 nM to about 50 nM, about 5 nM to about 40 nM, about 5 nM to about 30 nM, about 5 nM to about 20 nM, about 5 nM to about 10 nM, about 10 nM to about 100 nM, about 10 nM to about 75 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM, about 10 nM to about 20 nM, about 15 nM to about 100 nM, about 15 nM to about 75 nM, about 15 nM to about 50 nM, about 15 nM to about 40 nM, about 15 nM to about 30 nM, about 15 nM to about 20 nM, about 20 nM to about 100 nM, about 20 nM to about 75 nM, about 20 nM to about 50 nM, about 20 nM to about 40 nM, about 20 nM to about 30 nM, about 25 nM to about 100 nM, about 25 nM to about 75 nM, about 25 nM to about 50 nM, about 25 nM to about 40 nM, about 25 nM to about 30 nM, about 30 nM to about 100 nM nM, about 30 nM to about 75 nM, about 30 nM to about 50 nM, about 30 nM to about 40 nM, about 35 nM to about 100 nM, about 35 nM to about 75 nM, about 35 nM to about 50 nM, about 35 nM to about 40 nM, about 40 nM to about 100 nM, about 40 nM to about 75 nM, about 40 nM to about 50 nM, about 45 nM to about 100 nM, about 45 nM to about 75 nM, about 45 nM to about 50 nM, about 50 nM to about 100 nM, about 50 nM to about 75 nM, about 70 nM to about 100 nM, about 70 nM to about 75 nM, about 80 nM to about 100 nM, or about 90 nM. In some embodiments, the multispecific antigen-binding molecule binds to stone macaque STEAP1 with a_KD of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19 nM, about 20 nM, about 21 nM, about 22 nM, about 23 nM, about 24 nM, or about 25 nM. In some embodiments, the multispecific antigen-binding molecule binds to stone macaque STEAP1 with_{a KDof} about 14 nM. In some embodiments,_KD is determined using a kinetic screening assay (KinExA®), as described in Example 8.

在一些實施例中，多特異性抗原結合分子具有高達或約 15、20、25 或 30 μg/mL 之 C_max。在一些情況下，多特異性抗原結合分子具有約 11、11.5、12、12.1、12.6、13、13.2、13.5、15、15.3、18、20、23.4、25、27.9、29.1 或 30 µg/mL 之 C_max。In some embodiments, the multispecific antigen-binding molecule has a_Cmax of up to or about 15, 20, 25, or 30 μg/mL. In some cases, the multispecific antigen-binding molecule has a_Cmax of about 11, 11.5, 12, 12.1, 12.6, 13, 13.2, 13.5, 15, 15.3, 18, 20, 23.4, 25, 27.9, 29.1, or 30 μg/mL.

在一些實施例中，多特異性抗原結合分子具有約 10 µg/mL 至約 30 µg/mL (例如，約 10 µg/mL 至約 30 µg/mL、約 10 µg/mL 至約 28 µg/mL、約 10 µg/mL 至約 26 µg/mL、約 10 µg/mL 至約 24 µg/mL、約 10 µg/mL 至約 22 µg/mL、約 10 µg/mL 至約 20 µg/mL、約 10 µg/mL 至約 18 µg/mL、約 10 µg/mL 至約 16 µg/mL、約 10 µg/mL 至約 14 µg/mL、約 10 µg/mL 至約 12 µg/mL、約 12 µg/mL 至約 30 µg/mL、約 12 µg/mL 至約 28 µg/mL、約 12 µg/mL 至約 26 µg/mL、約 12 µg/mL 至約 24 µg/mL、約 12 µg/mL 至約 22 µg/mL、約 12 µg/mL 至約 20 µg/mL、約 12 µg/mL 至約 18 µg/mL、約 12 µg/mL 至約 16 µg/mL、約 12 µg/mL 至約 14 µg/mL、約 14 µg/mL 至約 30 µg/mL、約 14 µg/mL 至約 28 µg/mL、約 14 µg/mL 至約 26 µg/mL、約 14 µg/mL 至約 24 µg/mL、約 14 µg/mL 至約 22 µg/mL、約 14 µg/mL 至約 20 µg/mL、約 14 µg/mL 至約 18 µg/mL、約 14 µg/mL 至約 16 µg/mL、約 16 µg/mL 至約 30 µg/mL、約 16 µg/mL 至約 28 µg/mL、約 16 µg/mL 至約 26 µg/mL、約 16 µg/mL 至約 24 µg/mL、約 16 µg/mL 至約 22 µg/mL、約 16 µg/mL 至約 20 µg/mL、約 16 µg/mL 至約 18 µg/mL、約 18 µg/mL 至約 30 µg/mL、約 18 µg/mL 至約 28 µg/mL、約 18 µg/mL 至約 26 µg/mL、約 18 µg/mL 至約 24 µg/mL、約 18 µg/mL 至約 22 µg/mL、約 18 µg/mL 至約 20 µg/mL、約 20 µg/mL 至約 30 µg/mL、約 20 µg/mL 至約 28 µg/mL、約 20 µg/mL 至約 26 µg/mL、約 20 µg/mL 至約 24 µg/mL、約 20 µg/mL 至約 22 µg/mL、約 22 µg/mL 至約 30 µg/mL、約 22 µg/mL 至約 28 µg/mL、約 22 µg/mL 至約 26 µg/mL、約 22 µg/mL 至約 24 µg/mL、約 24 µg/mL 至約 30 µg/mL、約 24 µg/mL 至約 28 µg/mL、約 22 µg/mL 至約 26 µg/mL、約 22 µg/mL 至約 24 µg/mL、約 26 µg/mL 至約 30 µg/mL、約 26 µg/mL 至約 28 µg/mL、或約 28 µg/mL 至約 30 µg/mL) 之 C_max。在一些實施例中，多特異性抗原結合分子具有約 20 µg/mL 至約 28 µg/mL (例如，約 20 µg/mL、約 21 µg/mL、約 22 µg/mL、約 23 µg/mL、約 24 µg/mL、約 25 µg/mL、約 26 µg/mL、約 27 µg/mL、或約 28 µg/mL) 之 C_max。在一些實施例中，多特異性抗原結合分子具有約 24 µg/mL 之 C_max。在一些實施例中，C_max可以如實例 9 (在雌性 SCID 小鼠中) 中所述來確定。在一些實施例中，C_max可以如實例 10 (在石蟹獼猴中) 中所述來確定。In some embodiments, the multispecific antigen-binding molecule has an OD of about 10 μg/mL to about 30 μg/mL (e.g., about 10 μg/mL to about 30 μg/mL, about 10 μg/mL to about 28 μg/mL, about 10 μg/mL to about 26 μg/mL, about 10 μg/mL to about 24 μg/mL, about 10 μg/mL to about 22 μg/mL, about 10 μg/mL to about 20 μg/mL, about 10 μg/mL to about 18 μg/mL, about 10 μg/mL to about 16 μg/mL, about 10 μg/mL to about 14 μg/mL, about 10 μg/mL to about 12 μg/mL, about 12 μg/mL to about 30 μg/mL, about 12 μg/mL to about 28 μg/mL, about 12 μg/mL to about 26 µg/mL, about 12 µg/mL to about 24 µg/mL, about 12 µg/mL to about 22 µg/mL, about 12 µg/mL to about 20 µg/mL, about 12 µg/mL to about 18 µg/mL, about 12 µg/mL to about 16 µg/mL, about 12 µg/mL to about 14 µg/mL, about 14 µg/mL to about 30 µg/mL, about 14 µg/mL to about 28 µg/mL, about 14 µg/mL to about 26 µg/mL, about 14 µg/mL to about 24 µg/mL, about 14 µg/mL to about 22 µg/mL, about 14 µg/mL to about 20 µg/mL, about 14 µg/mL to about 18 µg/mL, about 14 µg/mL to about 16 µg/mL, about 16 16 µg/mL to about 28 µg/mL, about 16 µg/mL to about 26 µg/mL, about 16 µg/mL to about 24 µg/mL, about 16 µg/mL to about 22 µg/mL, about 16 µg/mL to about 20 µg/mL, about 16 µg/mL to about 18 µg/mL, about 18 µg/mL to about 30 µg/mL, about 18 µg/mL to about 28 µg/mL, about 18 µg/mL to about 26 µg/mL, about 18 µg/mL to about 24 µg/mL, about 18 µg/mL to about 22 µg/mL, about 18 µg/mL to about 20 µg/mL, about 20 µg/mL to about 30 µg/mL, about 20 µg/mL to about 28 about 22 µg/mL to about 24 µg/mL, about 22 µg/mL to about 26 µg/mL, about 22 µg/mL to about 24 µg/mL, about 24 µg/mL to about 30 µg/mL, about 26 µg/mL to about 28 µg/mL, about 22 µg/mL to about 26 µg/mL, about 22 µg/mL to about 24 µg/mL, about 24 µg/mL to about 30 µg/mL, about 24 µg/mL to about 28 µg/mL, about 22 µg/mL to about 26 µg/mL, about 22 µg/mL to about 24 µg/mL, about 26 µg/mL to about 30 µg/mL, about 26 µg/mL to about 28 µg/mL, or about 28 µg/mL to about 30 µg/mL₎ . In some embodiments, the multispecific antigen-binding molecule has a Cmax of about 20 μg/mL to about 28 μg/mL (e.g., about 20 μg/mL, about 21 μg/mL, about 22 μg/mL, about 23 μg/mL, about 24 μg/mL, about 25 μg/mL, about 26 μg/mL, about 27 μg/mL, or about 28 μg/mL). In some embodiments, the multispecific antigen-binding molecule has_aCmax of about 24 μg/mL. In some embodiments,_Cmax can be determined as described in Example 9 (in female SCID mice). In some embodiments,_Cmax can be determined as described in Example 10 (in stone crab macaques).

在一些實施例中，多特異性抗原結合分子具有約 0.6、0.56、0.5、0.45、0.4、0.35、0.3、0.25、0.2、0.15、0.1、0.09、0.05 或更低之 EC₅₀。在一些情況下，多特異性抗原結合分子具有約 0.5 或更低之 EC₅₀。在一些情況下，多特異性抗原結合分子具有約 0.4 或更低之 EC₅₀。在一些情況下，多特異性抗原結合分子具有約 0.3 或更低之 EC₅₀。在一些情況下，多特異性抗原結合分子具有約 0.2 或更低之 EC₅₀。在一些情況下，多特異性抗原結合分子具有約 0.1 或更低之 EC₅₀。In some embodiments, the multispecific antigen-binding molecule has an EC₅₀ of about 0.6, 0.56, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.09, 0.05 or less. In some cases, the multispecific antigen-binding molecule has an EC₅₀ of about 0.5 or less. In some cases, the multispecific antigen-binding molecule has an EC₅₀ of about 0.4 or less. In some cases, the multispecific antigen-binding molecule has an EC₅₀ of about 0.3 or less. In some cases, the multispecific antigen-binding molecule has an EC₅₀ of about 0.2 or less. In some cases, the multispecific antigen-binding molecule has an EC₅₀ of about 0.1 or less.

在一些實施例中，多特異性抗原結合分子具有約 0.05 至約 0.8 (例如，約 0.05 至約 0.8、約 0.05 至約 0.75、約 0.05 至約 0.70、約 0.05 至約 0.65、約 0.05 至約 0.6、約 0.05 至約 0.55、約 0.05 至約 0.5、約 0.05 至約 0.45、約 0.05 至約 0.4、約 0.05 至約 0.35、約 0.05 至約 0.3、約 0.05 至約 0.25、約 0.05 至約 0.2、約 0.05 至約 0.15、約 0.05 至約 0.1、約 0.05 至約 0.09、約 0.05 至約 0.08、約 0.05 至約 0.07、約 0.05 至約 0.06、約 0.06 至約 0.8、約 0.06 至約 0.75、約 0.06 至約 0.70、約 0.06 至約 0.65、約 0.06 至約 0.6、約 0.06 至約 0.55、約 0.06 至約 0.5、約 0.06 至約 0.45、約 0.06 至約 0.4、約 0.06 至約 0.35、約 0.06 至約 0.3、約 0.06 至約 0.25、約 0.06 至約 0.2、約 0.06 至約 0.15、約 0.06 至約 0.1、約 0.06 至約 0.09、約 0.06 至約 0.08、約 0.06 至約 0.07、約 0.07 至約 0.8、約 0.07 至約 0.75、約 0.07 至約 0.70、約 0.07 至約 0.65、約 0.07 至約 0.6、約 0.07 至約 0.55、約 0.07 至約 0.5、約 0.07 至約 0.45、約 0.07 至約 0.4、約 0.07 至約 0.35、約 0.07 至約 0.3、約 0.07 至約 0.25、約 0.07 至約 0.2、約 0.07 至約 0.15、約 0.07 至約 0.1、約 0.07 至約 0.09、約 0.07 至約 0.08、約 0.08 至約 0.8、約 0.08 至約 0.75、約 0.08 至約 0.70、約 0.08 至約 0.65、約 0.08 至約 0.6、約 0.08 至約 0.55、約 0.08 至約 0.5、約 0.08 至約 0.45、約 0.08 至約 0.4、約 0.08 至約 0.35、約 0.08 至約 0.3、約 0.08 至約 0.25、約 0.08 至約 0.2、約 0.08 至約 0.15、約 0.08 至約 0.1、約 0.08 至約 0.09、約 0.09 至約 0.8、約 0.09 至約 0.75、約 0.09 至約 0.70、約 0.09 至約 0.65、約 0.09 至約 0.6、約 0.09 至約 0.55、約 0.09 至約 0.5、約 0.09 至約 0.45、約 0.09 至約 0.4、約 0.09 至約 0.35、約 0.09 至約 0.3、約 0.09 至約 0.25、約 0.09 至約 0.2、約 0.09 至約 0.15、約 0.09 至約 0.1、約 0.1 至約 0.8、約 0.1 至約 0.75、約 0.1 至約 0.70、約 0.1 至約 0.65、約 0.1 至約 0.6、約 0.1 至約 0.55、約 0.1 至約 0.5、約 0.1 至約 0.45、約 0.1 至約 0.4、約 0.1 至約 0.35、約 0.1 至約 0.3、約 0.1 至約 0.25、約 0.1 至約 0.2、約 0.1 至約 0.15、約 0.15 至約 0.8、約 0.15 至約 0.75、約 0.15 至約 0.70、約 0.15 至約 0.65、約 0.15 至約 0.6、約 0.15 至約 0.55、約 0.15 至約 0.5、約 0.15 至約 0.45、約 0.15 至約 0.4、約 0.15 至約 0.35、約 0.15 至約 0.3、約 0.15 至約 0.25、約 0.15 至約 0.2、約 0.2 至約 0.8、約 0.2 至約 0.75、約 0.2 至約 0.70、約 0.2 至約 0.65、約 0.2 至約 0.6、約 0.2 至約 0.55、約 0.2 至約 0.5、約 0.2 至約 0.45、約 0.2 至約 0.4、約 0.2 至約 0.35、約 0.2 至約 0.3、約 0.2 至約 0.25、約 0.25 至約 0.8、約 0.25 至約 0.75、約 0.25 至約 0.70、約 0.25 至約 0.65、約 0.25 至約 0.6、約 0.25 至約 0.55、約 0.25 至約 0.5、約 0.25 至約 0.45、約 0.25 至約 0.4、約 0.25 至約 0.35、約 0.25 至約 0.3、約 0.3 至約 0.8、約 0.3 至約 0.75、約 0.3 至約 0.70、約 0.3 至約 0.65、約 0.3 至約 0.6、約 0.3 至約 0.55、約 0.3 至約 0.5、約 0.3 至約 0.45、約 0.3 至約 0.4、約 0.3 至約 0.35、約 0.35 至約 0.8、約 0.35 至約 0.75、約 0.35 至約 0.70、約 0.35 至約 0.65、約 0.35 至約 0.6、約 0.35 至約 0.55、約 0.35 至約 0.5、約 0.35 至約 0.45、約 0.35 至約 0.4、約 0.4 至約 0.8、約 0.4 至約 0.75、約 0.4 至約 0.70、約 0.4 至約 0.65、約 0.4 至約 0.6、約 0.4 至約 0.55、約 0.4 至約 0.5、約 0.4 至約 0.45、約 0.45 至約 0.8、約 0.45 至約 0.75、約 0.45 至約 0.70、約 0.45 至約 0.65、約 0.45 至約 0.6、約 0.45 至約 0.55、約 0.45 至約 0.5、約 0.5 至約 0.8、約 0.5 至約 0.75、約 0.5 至約 0.70、約 0.5 至約 0.65、約 0.5 至約 0.6、約 0.5 至約 0.55、約 0.55 至約 0.8、約 0.55 至約 0.75、約 0.55 至約 0.70、約 0.55 至約 0.65、約 0.55 至約 0.6、約 0.6 至約 0.8、約 0.6 至約 0.75、約 0.6 至約 0.70、約 0.6 至約 0.65、約 0.65 至約 0.8、約 0.65 至約 0.75、約 0.65 至約 0.70、約 0.7 至約 0.8、約 0.7 至約 0.75、或約 0.75 至約 0.8) 之 EC₅₀。In some embodiments, the multispecific antigen-binding molecule has a molecular weight of about 0.05 to about 0.8 (e.g., about 0.05 to about 0.8, about 0.05 to about 0.75, about 0.05 to about 0.70, about 0.05 to about 0.65, about 0.05 to about 0.6, about 0.05 to about 0.55, about 0.05 to about 0.5, about 0.05 to about 0.45, about 0.05 to about 0.4, about 0.05 to about 0.35, about 0.05 to about 0.3, about 0.05 to about 0.25, about 0.05 to about 0.2, about 0.05 to about 0.15, about 0.05 to about 0.1, about 0.05 to about 0.09, about 0.05 to about 0.08, about 0.05 to about 0.07, about 0.05 to about 0.06, about 0.06 to about 0.8, about 0.06 to about 0.75, about 0.06 to about 0.70, about 0.06 to about 0.65, about 0.06 to about 0.6, about 0.06 to about 0.55, about 0.06 to about 0.5, about 0.06 to about 0.45, about 0.06 to about 0.4, about 0.06 to about 0.35, about 0.06 to about 0.3, about 0.06 to about 0.25, about 0.06 to about 0.2, about 0.06 to about 0.15, about 0.06 to about 0.1, about 0.06 to about 0.09, about 0.06 to about 0.08, about 0.06 to about 0.07, about 0.07 to about 0.8, about 0.07 to about 0.75, about 0.07 to about 0.70, about 0.07 to about 0.65, about 0.07 to about 0.6, about 0.07 to about 0.55, about 0.07 to about 0.5, about 0.07 to about 0.45, about 0.07 to about 0.4, about 0.07 to about 0.35, about 0.07 to about 0.3, about 0.07 to about 0.25, about 0.07 to about 0.2, about 0.07 to about 0.15, about 0.07 to about 0.1, about 0.07 to about 0.09, about 0.07 to about 0.08, about 0.08 to about 0.8, about 0.08 to about 0.75, about 0.08 to about 0.70, about 0.08 to about 0.65, about 0.08 to about 0.6, about 0.08 to about 0.55, about 0.08 to about 0.5, about 0.08 to about 0.45, about 0.08 to about 0.4, about 0.08 to about 0.35, about 0.08 to about 0.3, about 0.08 to about 0.25, about 0.08 to about 0.2, about 0.08 to about 0.15, about 0.08 to about 0.1, about 0.08 to about 0.09, about 0.09 to about 0.8, about 0.09 to about 0.75, about 0.09 to about 0.70, about 0.09 to about 0.65, about 0.09 to about 0.6, about 0.09 to about 0.55, about 0.09 to about 0.5, about 0.09 to about 0.45, about 0.09 to about 0.4, about 0.09 to about 0.35, about 0.09 to about 0.3, about 0.09 to about 0.25, about 0.09 to about 0.2, about 0.09 to about 0.15, about 0.09 to about 0.1, about 0.1 to about 0.8, about 0.1 to about 0.75, about 0.1 to about 0.70, about 0.1 to about 0.65, about 0.1 to about 0.6, about 0.1 to about 0.55, about 0.1 to about 0.5, about 0.1 to about 0.45, about 0.1 to about 0.4, about 0.1 to about 0.35, about 0.1 to about 0.3, about 0.1 to about 0.25, about 0.1 to about 0.2, about 0.1 to about 0.15, about 0.15 to about 0.8, about 0.15 to about 0.75, about 0.15 to about 0.70, about 0.15 to about 0.65, about 0.15 to about 0.6, about 0.15 to about 0.55, about 0.15 to about 0.5, about 0.15 to about 0.45, about 0.15 to about 0.4, about 0.15 to about 0.35, about 0.15 to about 0.3, about 0.15 to about 0.25, about 0.15 to about 0.2, about 0.2 to about 0.8, about 0.2 to about 0.75, about 0.2 to about 0.70, about 0.2 to about 0.65, about 0.2 to about 0.6, about 0.2 to about 0.55, about 0.2 to about 0.5, about 0.2 to about 0.45, about 0.2 to about 0.4, about 0.2 to about 0.35, about 0.2 to about 0.3, about 0.2 to about 0.25, about 0.25 to about 0.8, about 0.25 to about 0.75, about 0.25 to about 0.70, about 0.25 to about 0.65, about 0.25 to about 0.6, about 0.25 to about 0.55, about 0.25 to about 0.5, about 0.25 to about 0.45, about 0.25 to about 0.4, about 0.25 to about 0.35, about 0.25 to about 0.3, about 0.3 to about 0.8, about 0.3 to about 0.75, about 0.3 to about 0.70, about 0.3 to about 0.65, about 0.3 to about 0.6, about 0.3 to about 0.55, about 0.3 to about 0.5, about 0.3 to about 0.45, about 0.3 to about 0.4, about 0.3 to about 0.35, about 0.35 to about 0.8, about 0.35 to about 0.75, about 0.35 to about 0.70, about 0.35 to about 0.65, about 0.35 to about 0.6, about 0.35 to about 0.55, about 0.35 to about 0.5, about 0.35 to about 0.45, about 0.35 to about 0.4, about 0.4 to about 0.8, about 0.4 to about 0.75, about 0.4 to about 0.70, about 0.4 to about 0.65, about 0.4 to about 0.6, about 0.4 to about 0.55, about 0.4 to about 0.5, about 0.4 to about 0.45, about 0.45 to about 0.8, about 0.45 to about 0.75, about 0.45 to about 0.70, about 0.45 to about 0.65, about 0.45 to about 0.6, about 0.45 to about 0.55, about 0.45 to about 0.5, about 0.5 to about 0.8, about 0.5 to about 0.75, about 0.45 to about 0.70, about 0.5 to about 0.65, about 0.45 to about 0.6, about 0.45 to about 0.55, about 0.45 to about 0.5, about 0.5 to about 0.8, about 0.5 to about 0.75, about 0.5 to about 0.70, about 0.5 to about 0.65, about 0.5 to about 0.6, about 0.5 to about 0.55, about 0.5 to about 0.8, about 0.55 to about 0.75, about 0.55 to about 0.70, about 0.55 to about 0.65, about 0.55 to about 0.6, about 0.6 to about 0.8, about 0.6 to about 0.75, about 0.6 to about 0.70, about 0.6 to about 0.65, about 0.65 to about 0.8, about 0.65 to about 0.75, about 0.65 to about 0.70, about 0.7 to about_0.8 , about 0.7 to about 0.75, or about 0.75 to about 0.8).

在一些實施例中，EC₅₀係用人 CD8+ T 細胞及表現 STEAP1 之 LNCaP-X1.2 細胞在細胞毒殺測定中在 72 小時所判定。在一些實施例中，EC₅₀為約 0.05 至約 0.4 (例如，約 0.05、約 0.06、約 0.07、約 0.08、約 0.09、約 0.1、約 0.15、約 0.2、約 0.25、約 0.3、約 0.35、或約 0.4)。在一些實施例中，EC₅₀為約 0.08 或約 0.3。在一些實施例中，EC₅₀為約 0.08。在一些實施例中，EC₅₀為約 0.3。In some embodiments, the EC₅₀ is determined at 72 hours in a cytotoxicity assay using human CD8+ T cells and LNCaP-X1.2 cells expressing STEAP1. In some embodiments, the EC₅₀ is about 0.05 to about 0.4 (e.g., about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, or about 0.4). In some embodiments, the EC₅₀ is about 0.08 or about 0.3. In some embodiments, the EC₅₀ is about 0.08. In some embodiments, the EC₅₀ is about 0.3.

在一些實施例中，EC₅₀係用人 CD8+ T 細胞及表現 STEAP1 之 LNCaPX1.2KO3-13 細胞在細胞毒殺測定中在 72 小時所判定。在一些實施例中，EC₅₀為約 0.1 至約 0.8 (例如，約 0.1、約 0.15、約 0.2、約 0.25、約 0.3、約 0.35、約 0.4、約 0.45、約 0.5、約 0.55、約 0.6、約 0.65、約 0.7、約 0.75、或約 0.8)。在一些實施例中，EC₅₀為約 0.1 或約 0.7。在一些實施例中，EC₅₀為約 0.1。在一些實施例中，EC₅₀為約 0.7。In some embodiments, the EC₅₀ is determined in a cytotoxicity assay using human CD8+ T cells and LNCaPX1.2KO3-13 cells expressing STEAP1 at 72 hours. In some embodiments, the EC₅₀ is about 0.1 to about 0.8 (e.g., about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, or about 0.8). In some embodiments, the EC₅₀ is about 0.1 or about 0.7. In some embodiments, the EC₅₀ is about 0.1. In some embodiments, the EC₅₀ is about 0.7.

在一些實施例中，多特異性抗原結合分子與 STEAP1 單價結合。在其他實施例中，多特異性抗原結合分子與 STEAP1 多價 (例如，二價) 結合。與特定STEAP1表位結合的抗體In some embodiments, the multispecific antigen-binding molecule binds monovalently to STEAP1. In other embodiments, the multispecific antigen-binding molecule binds multivalently (eg, bivalently) to STEAP1. Antibodies that bindto specificSTEAP1epitopes

在某些實施例中，本發明之抗體包含與 STEAP1 蛋白結合的第一抗原結合域。在一些情況下，第一抗原結合域與人 STEAP1 蛋白結合。在其他情況下，第一抗原結合域與靈長類 STEAP1 蛋白 (例如，來自石蟹獼猴或Pongo abelii的 STEAP1蛋白) 結合。In certain embodiments, the antibodies of the present invention comprise a first antigen binding domain that binds to a STEAP1 protein. In some cases, the first antigen binding domain binds to a human STEAP1 protein. In other cases, the first antigen binding domain binds to a primate STEAP1 protein (eg, a STEAP1 protein from a macaque orPongo abelii ).

在一些實施例中，抗體包含第一抗原結合域，該第一抗原結合域在選自 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的一個或多個殘基處與人 STEAP1 結合，其中殘基位置 101、102、103、195、198、202 及 281 對應於 SEQ ID NO: 65 中所示之位置 101、102、103、195、198、202 及 281。在一些情況下，第一抗原結合域與選自 SEQ ID NO: 65 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的至少一個殘基結合。In some embodiments, the antibody comprises a first antigen-binding domain that binds to human STEAP1 at one or more residues selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202, and Lys281, wherein residue positions 101, 102, 103, 195, 198, 202, and 281 correspond to positions 101, 102, 103, 195, 198, 202, and 281 as shown in SEQ ID NO: 65. In some cases, the first antigen-binding domain binds to at least one residue selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202, and Lys281 of SEQ ID NO: 65.

在一些情況下，來自第一抗原結合域的殘基與選自 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的至少一個殘基形成氫鍵。在一些情況下，第一抗原結合域之 VH 的至少一個選自 Leu56、Ser73、Asn74、Gly101、Tyr103 及 Tyr107 的殘基與選自 STEAP1 之 Ser101、His102、Gln103、Trp195、Gln198、Gln202 及 Lys281 的至少一個殘基形成氫鍵，其中殘基位置 56、73、 74、101、103 及 107 對應於 SEQ ID NO: 18 中所示之位置 56、73、74、101、103 及 107。在一些情況下，第一抗原結合域之 VL 的至少一個選自 Tyr35 或 Tyr54 的殘基與選自 Gln202 或 Gln201 的至少一個殘基形成氫鍵，其中殘基位置 35 及 54 對應於 SEQ ID NO: 8 中所示之位置 35 及 54。在一些情況下，人 STEAP1 之殘基 Asn203 及/或 Lys204 與一個或多個 VL CDR 殘基形成凡得瓦交互作用。在一些情況下，形成氫鍵的殘基如表 3 中所示。表3第一抗原結合域人STEAP1VHTyr103Trp195Gly101Gln202Tyr107Gln198Leu56Lys281Ser73His102Asn74Ser101Asn74Gln103VLTyr35Gln202Tyr54Gln201In some cases, the residue from the first antigen binding domain forms a hydrogen bond with at least one residue selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202 and Lys281. In some cases, at least one residue selected from Leu56, Ser73, Asn74, Gly101, Tyr103 and Tyr107 of the VH of the first antigen binding domain forms a hydrogen bond with at least one residue selected from Ser101, His102, Gln103, Trp195, Gln198, Gln202 and Lys281 of STEAP1, wherein residue positions 56, 73, 74, 101, 103 and 107 correspond to positions 56, 73, 74, 101, 103 and 107 shown in SEQ ID NO: 18. In some cases, at least one residue selected from Tyr35 or Tyr54 of the VL of the first antigen binding domain forms a hydrogen bond with at least one residue selected from Gln202 or Gln201, wherein residue positions 35 and 54 correspond to positions 35 and 54 as shown in SEQ ID NO: 8. In some cases, residues Asn203 and/or Lys204 of human STEAP1 form van der Waals interactions with one or more VL CDR residues. In some cases, the residues that form hydrogen bonds are shown in Table 3.Table3First antigen binding domainHumanSTEAP1 VH Tyr103 Trp195 Gly101 Gln202 Tyr107 Gln198 Leu56 Lys281 Ser73 His102 Asn74 Ser101 Asn74 Gln103 V L Tyr35 Gln202 Tyr54 Gln201

在一些實施例中，本發明之抗體為上文 A 部分「與 STEAP1 結合的抗原結合分子」中描述的單特異性抗體。在一些情況下，抗體包含：VH 區，該 VH 區包含選自 SEQ ID NO: 7、17 至 25、30 至 34、38 及 68 的 VH 序列之 CDR-H1、CDR-H2 及 CDR-H3；以及 VL 區，該 VL 區包含選自 SEQ ID NO: 8、26 至 29、39 及 69 的 VL 序列之 CDR-L1、CDR-L2 及 CDR-L3。在一些情況下，抗體包含六個 CDR，其中： CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)； 其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)； 其中 Xaa³為 Asp (D) 或 Ser (S)； Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)； Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。 在一些情況下，抗體包含六個 CDR，其中： CDR-H1 包含 GFTFSXaa¹⁰Xaa¹¹(SEQ ID NO: 63)； 其中 Xaa¹⁰為 Asn (N) 或 Asp (D)；且 Xaa¹¹為 Tyr (Y)、Phe (F) 或 His (H)； CDR-H2 包含 Xaa¹²YDGXaa¹³Xaa¹⁴(SEQ ID NO: 64)； 其中 Xaa¹²為 Asp (D) 或 Ser (S)； Xaa¹³為 Gly (G)、Asp (D) 或 Leu (L)；且 Xaa¹⁴為 Ser (S)、Asp (D) 或 Asn (N)； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。In some embodiments, the antibody of the present invention is a monospecific antibody described in Section A "Antigen Binding Molecules that Bind to STEAP1". In some cases, the antibody comprises: a VH region comprising CDR-H1, CDR-H2 and CDR-H3 of a VH sequence selected from SEQ ID NOs: 7, 17 to 25, 30 to 34, 38 and 68; and a VL region comprising CDR-L1, CDR-L2 and CDR-L3 of a VL sequence selected from SEQ ID NOs: 8, 26 to 29, 39 and 69. In some cases, the antibody comprises six CDRs, wherein: CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Ser (S); Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (N); and Xaa⁶ is Ser (S) or Tyr (Y); CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the antibody comprises six CDRs, wherein: CDR-H1 comprises GFTFSXaa¹⁰ Xaa¹¹ (SEQ ID NO: 63); wherein Xaa¹⁰ is Asn (N) or Asp (D); and Xaa¹¹ is Tyr (Y), Phe (F) or His (H); CDR-H2 comprises Xaa¹² YDGXaa¹³ Xaa¹⁴ (SEQ ID NO: 64); wherein Xaa¹² is Asp (D) or Ser (S); Xaa¹³ is Gly (G), Asp (D) or Leu (L); and Xaa¹⁴ is Ser (S), Asp (D) or Asn (N); CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6.

在一些實施例中，本發明之抗體為上文 B 節「多特異性抗原結合分子」中描述的多特異性抗體。在一些情況下，抗體包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含選自 SEQ ID NO: 7、17 至 25、30 至 34、38 及 68 的 VH 序列之 CDR-H1、CDR-H2 及/或 CDR-H3；及/或抗 STEAP1 輕鏈可變區 (VL)，其包含選自 SEQ ID NO: 8、26 至 29、39 及 69 的 VL 序列之 CDR-L1、CDR-L2 及/或 CDR-L3；以及 (B) 第二抗原結合域，該第二抗原結合域與 T 細胞受體 (例如，CD3) 結合。In some embodiments, the antibody of the present invention is a multispecific antibody described in Section B "Multispecific Antigen Binding Molecules" above. In some cases, the antibody comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and/or CDR-H3 of a VH sequence selected from SEQ ID NOs: 7, 17 to 25, 30 to 34, 38 and 68; and/or an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and/or CDR-L3 of a VL sequence selected from SEQ ID NOs: 8, 26 to 29, 39 and 69; and (B) a second antigen-binding domain that binds to a T cell receptor (e.g., CD3).

在一些情況下，抗體包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)； 其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)； 其中 Xaa³為 Asp (D) 或 Ser (S)； Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)； Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)；且 CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，該第二抗原結合域與 T 細胞受體 (例如，CD3) 結合。In some cases, the antibody comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Ser (S); Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (N); and Xaa⁶ is Ser (S) or Tyr (Y); and CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) A second antigen binding domain that binds to a T cell receptor (eg, CD3).

在一些實施例中，抗體包含：(A) 第一抗原結合域，該第一抗原結合域與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 GFTFSXaa¹⁰Xaa¹¹(SEQ ID NO: 63)； 其中 Xaa¹⁰為 Asn (N) 或 Asp (D)；且 Xaa¹¹為 Tyr (Y)、Phe (F) 或 His (H)； CDR-H2 包含 Xaa¹²YDGXaa¹³Xaa¹⁴(SEQ ID NO: 64)； 其中 Xaa¹²為 Asp (D) 或 Ser (S)； Xaa¹³為 Gly (G)、Asp (D) 或 Leu (L)；且 Xaa¹⁴為 Ser (S)、Asp (D) 或 Asn (N)； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；及/或 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，該第二抗原結合域與 T 細胞受體 (例如，CD3) 結合。融合第一抗原結合域及第二抗原結合域的肽連接子In some embodiments, the antibody comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises GFTFSXaa¹⁰ Xaa¹¹ (SEQ ID NO: 63); wherein Xaa¹⁰ is Asn (N) or Asp (D); and Xaa¹¹ is Tyr (Y), Phe (F) or His (H); CDR-H2 comprises Xaa¹² YDGXaa¹³ Xaa¹⁴ (SEQ ID NO: 64); wherein Xaa¹² is Asp (D) or Ser (D); (S); Xaa¹³ is Gly (G), Asp (D) or Leu (L); and Xaa¹⁴ is Ser (S), Asp (D) or Asn (N); CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and/or CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) a second antigen binding domain that binds to a T cell receptor (e.g., CD3) Binding.A peptide linker that fuses the first antigen binding domain and the second antigen binding domain

在一些實施例中，本發明之多特異性抗原結合分子 (例如，雙特異性或三特異性抗原結合分子) 的特徵在於其中第一抗原結合域之 C 端經由肽連接子融合至第二抗原結合域之 N 端的結構。肽連接子的長度可為 5 至 20 個胺基酸 (例如，長度為 5 至 10 個、10 至 15 個、或 15 至 20 個，例如長度為 5、6、7、8、9、10、11、12、13、14、15、16、17、18、19 或 20 個胺基酸)。在一些實施例中，肽連接子包含可變重鏈鉸鏈區 (例如，DKTHT) 之天然胺基酸序列。在一些實施例中，肽連接子包含 (Gly₄Ser)_n連接子 (或 (G₄S)_n連接子)，其中 n 為 1 至 10、2 至 10、3 至 10、4 至 10、5 至 10、6 至 10、2 至 6、2 至 6、3 至 6、或 4 至 6。在一些情況下，n 為 1、2、3、4、5、6、7、8、9 或 10。在一些實施例中，肽連接子包含 G₄SG₂連接子。在一些實施例中，肽連接子包含 G₄SG₂連接子及鉸鏈區 (例如，DKTHT)。在一些實施例中，肽連接子包含複數個甘胺酸、丙胺酸或其組合。Fc結構域In some embodiments, the multispecific antigen-binding molecules (e.g., bispecific or trispecific antigen-binding molecules) of the present invention are characterized by a structure in which the C-terminus of the first antigen-binding domain is fused to the N-terminus of the second antigen-binding domain via a peptide linker. The peptide linker can be 5 to 20 amino acids in length (e.g., 5 to 10, 10 to 15, or 15 to 20, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length). In some embodiments, the peptide linker comprises the natural amino acid sequence of the variable heavy chain hinge region (e.g., DKTHT). In some embodiments, the peptide linker comprises a (Gly₄ Ser)_n linker (or a (G₄ S)_n linker), wherein n is 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 2 to 6, 2 to 6, 3 to 6, or 4 to 6. In some cases, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the peptide linker comprises a G₄ SG₂ linker. In some embodiments, the peptide linker comprises a G₄ SG₂ linker and a hinge region (e.g., DKTHT). In some embodiments, the peptide linker comprises a plurality of glycine, alanine, or a combination thereof.Fcdomain

本發明之抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 可以以 Fc 域為特徵。Fc 域可以為 IgG Fc 域 (例如，IgG₁、IgG₂或 IgG₄Fc 域)。舉例而言，Fc 域可為人 Fc 域。在一些實施例中，Fc 域包含降低與 Fc 受體之結合及/或效應功能之一個或多個胺基酸取代。舉例而言，在一些實施例中，降低與 Fc 受體之結合及/或效應功能之一個或多個胺基酸取代係位於選自 L234、L235 及 P329 的組的一個或多個位置處 (例如，其中第一 Fc 次單元及第二 Fc 次單元各自包含 L234A、L235A 及 P329G 之胺基酸取代)。Fc 受體可以為例如 Fcγ 受體。因此，本發明之抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 可以經組態降低抗體依賴性細胞媒介的細胞毒性 (ADCC)。The antigen binding molecules (e.g., monospecific and/or multispecific antigen binding molecules such as bispecific or trispecific antigen binding molecules) of the present invention can be characterized by an Fc domain. The Fc domain can be an IgG Fc domain (e.g., an IgG₁ , IgG₂ or IgG₄ Fc domain). For example, the Fc domain can be a human Fc domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function. For example, in some embodiments, one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function are located at one or more positions selected from the group consisting of L234, L235, and P329 (e.g., wherein the first Fc subunit and the second Fc subunit each comprise amino acid substitutions of L234A, L235A, and P329G). The Fc receptor can be, for example, an Fcγ receptor. Thus, the antigen-binding molecules of the present invention (e.g., monospecific and/or multispecific antigen-binding molecules such as bispecific or trispecific antigen-binding molecules) can be configured to reduce antibody-dependent cell-mediated cytotoxicity (ADCC).

在一些情況下，Fc 域包含經組態用於促進第一 Fc 次單元與第二 Fc 次單元締合的修飾。雙特異性抗體的「杵臼 (Knob-in-hole)」工程可用於產生包含杵 (Knob) 的第一臂以及包含第一臂之杵可結合於其中的臼 (hole) 的第二臂。在一個實施例中，本發明之多特異性抗原結合分子的杵可為抗 STEAP1 臂。替代性地，本發明之多特異性抗原結合分子的杵可為抗 CD3 臂。在一個實施例中，本發明之多特異性抗原結合分子的臼可為抗 STEAP1 臂。替代性地，本發明之多特異性抗原結合分子的臼可為抗 CD3 臂。多特異性抗原結合分子諸如雙特異性抗體亦可使用免疫球蛋白交叉 (immunoglobulin crossover) (亦稱為 Fab 域交換或 CrossMab 型式) 技術進行工程化 (參見例如，WO2009/080253；Schaefer 等人,Proc. Natl. Acad. Sci. USA，108:11187-11192 (2011))。多特異性抗體亦可透過以下方法進行製備：用於製備抗體 Fc-異型二聚體分子的工程靜電轉向效應 (WO 2009/089004A1)；交聯兩個或更多個抗體或片段 (參見例如，美國專利第 4,676,980 號；及 Brennan 等人, Science, 229: 81 (1985))；或藉由使用白胺酸拉鏈產生雙特異性抗體 (參見例如，Kostelny 等人,J. Immunol., 148(5):1547-1553 (1992))。In some cases, the Fc domain comprises a modification configured to promote association of the first Fc subunit with the second Fc subunit. "Knob-in-hole" engineering of bispecific antibodies can be used to generate a first arm comprising a knob and a second arm comprising a hole into which the knob of the first arm can bind. In one embodiment, the knob of the multispecific antigen-binding molecule of the present invention may be an anti-STEAP1 arm. Alternatively, the knob of the multispecific antigen-binding molecule of the present invention may be an anti-CD3 arm. In one embodiment, the hole of the multispecific antigen-binding molecule of the present invention may be an anti-STEAP1 arm. Alternatively, the hole of the multispecific antigen-binding molecule of the present invention may be an anti-CD3 arm. Multispecific antigen-binding molecules such as bispecific antibodies can also be engineered using immunoglobulin crossover (also known as Fab domain exchange or CrossMab format) technology (see, e.g., WO 2009/080253; Schaefer et al.,Proc. Natl. Acad. Sci. USA , 108:11187-11192 (2011)). Multispecific antibodies can also be prepared by the following methods: engineering electrostatic switching for preparing antibody Fc-heterodimer molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980; and Brennan et al., Science , 229: 81 (1985)); or by using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al.,J. Immunol. , 148(5):1547-1553 (1992)).

第二 Fc 次單元的 CH3 域中之胺基酸殘基可以被具有較大側鏈體積之胺基酸殘基替換，從而在該第二 Fc 次單元的 CH3 域內產生隆凸 (例如，杵)，該隆凸可定位於第一 Fc 次單元的 CH3 域內的腔窩 (例如，臼) 中，並且第一 Fc 次單元的 CH3 域中之胺基酸殘基可以被具有較小側鏈體積之胺基酸殘基替換，從而在該第一 Fc 次單元的 CH3 域內產生腔窩 (例如，臼)，該腔窩可定位於第二 Fc 次單元的 CH3 域內的隆凸 (例如，杵) 內。在一些實施例中，第二 Fc 次單元的 CH3 域包含 T366 之胺基酸取代，並且第一 Fc 次單元的 CH3 域包含 T366、L368 及/或 Y407 (全部為 EU 編號) 中之一個、兩個或全部三個處之胺基酸取代。在一些實施例中，第二 Fc 次單元的 CH3 域包含 T366W 之胺基酸取代，並且第一 Fc 次單元的 CH3 域包含 T366S、L368A 及/或 Y407V (全部為 EU 編號) 中之一個、兩個或全部三個處之胺基酸取代。Amino acid residues in the CH3 domain of the second Fc subunit may be replaced with amino acid residues having a larger side chain volume, thereby generating a protuberance (e.g., a knob) in the CH3 domain of the second Fc subunit, which may be positioned in a cavity (e.g., a hole) in the CH3 domain of the first Fc subunit, and amino acid residues in the CH3 domain of the first Fc subunit may be replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity (e.g., a hole) in the CH3 domain of the first Fc subunit, which may be positioned in the protuberance (e.g., a knob) in the CH3 domain of the second Fc subunit. In some embodiments, the CH3 domain of the second Fc subunit comprises an amino acid substitution of T366, and the CH3 domain of the first Fc subunit comprises an amino acid substitution of one, two or all three of T366, L368 and/or Y407 (all EU numbering). In some embodiments, the CH3 domain of the second Fc subunit comprises an amino acid substitution of T366W, and the CH3 domain of the first Fc subunit comprises an amino acid substitution of one, two or all three of T366S, L368A and/or Y407V (all EU numbering).

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 N297G (EU 編號) 及 T366W (EU 編號) 取代的抗 STEAP1 臂，以及重鏈中含有 N297G (EU 編號)、T366S (EU 編號)、L368A (EU 編號) 及 Y407V (EU 編號) 取代的抗 CD3 臂。在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 N297G (EU 編號) 及 T366W (EU 編號) 取代的抗 STEAP1 臂，以及重鏈中含有 N297G (EU 編號)、T366S (EU 編號)、L368A (EU 編號) 及 Y407V (EU 編號) 取代的抗 CD3 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising N297G (EU numbering) and T366W (EU numbering) substitutions in the heavy chain, and an anti-CD3 arm comprising N297G (EU numbering), T366S (EU numbering), L368A (EU numbering) and Y407V (EU numbering) substitutions in the heavy chain. In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising N297G (EU numbering) and T366W (EU numbering) substitutions in the heavy chain, and an anti-CD3 arm comprising N297G (EU numbering), T366S (EU numbering), L368A (EU numbering) and Y407V (EU numbering) substitutions in the heavy chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：a) 與第一抗原結合的第一重鏈/輕鏈對，該第一重鏈/輕鏈對包含第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1)，及 b) 與第二抗原結合的第二重鏈/輕鏈對，該第二重鏈/輕鏈對包含第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2)，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域(CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，且 L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基；H2 的 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，且 L2 的 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，H1 之 VH 域中的 Q39 (Kabat編號) 處之胺基酸被替換為帶正電荷的殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，且 L1 之 VL 域中的 Q38 (Kabat編號) 處之胺基酸被替換為帶負電荷的殘基；並且 H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，且 L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基。在一些實施例中，帶正電荷的殘基選自 R 及 K，並且帶負電荷的殘基選自 D 及 E。在一些實施例中，帶正電荷的殘基為 R。在其他實施例中，帶正電荷的殘基為 K。在一些實施例中，帶負電荷的殘基為 D。在其他實施例中，帶負電荷的殘基為 E。在一些實施例中，第一抗原為 STEAP1 且第二抗原為 CD3。在其他實施例中，第一抗原為 CD3 並且第二抗原為 STEAP1。In some embodiments, the multispecific antigen-binding molecules described herein comprise: a) a first heavy chain/light chain pair that binds to a first antigen, the first heavy chain/light chain pair comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and b) a second heavy chain/light chain pair that binds to a second antigen, the second heavy chain/light chain pair comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) S183 (EU numbering) in the CH1 domain of H1 (i) an amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced with a positively charged residue, an amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced with a negatively charged residue, an amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced with a negatively charged residue, and an amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced with a positively charged residue; an amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced with a positively charged residue, and an amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced with a negatively charged residue; or (ii) S183 (EU numbering) in the CH1 domain of H1 is replaced with a negatively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced with a positively charged residue, the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced with a positively charged residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced with a negatively charged residue; and the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced with a negatively charged residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced with a positively charged residue. In some embodiments, the positively charged residue is selected from R and K, and the negatively charged residue is selected from D and E. In some embodiments, the positively charged residue is R. In other embodiments, the positively charged residue is K. In some embodiments, the negatively charged residue is D. In other embodiments, the negatively charged residue is E. In some embodiments, the first antigen is STEAP1 and the second antigen is CD3. In other embodiments, the first antigen is CD3 and the second antigen is STEAP1.

舉例而言，在一些實施例中，本文所述之多特異性抗原結合分子包含：含有第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1) 的抗 STEAP1 臂，及含有第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2) 的抗 CD3 臂，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域 (CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 R 或 K 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 D 或 E 殘基，且 L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基；並且 H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基，且 L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 D 或 E 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 R 或 K 殘基，且 L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基；並且 H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基，且 L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基。For example, in some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and an anti-CD3 arm comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each of H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each of L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by an R or K residue, and the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a D or E residue. residue in the CL domain of L1, the amino acid at V133 (EU numbering) is replaced by a D or E residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by an R or K residue; and the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by an R or K residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by a D or E residue; or (ii) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by a D or E residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by an R or K residue, and the amino acid at L1 the amino acid at V133 (EU numbering) in the CL domain of H2 is replaced by an R or K residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a D or E residue; and the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by a D or E residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by an R or K residue.

在一些實施例中，本文所述之多特異性抗原結合分子包含：含有第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1) 的抗 STEAP1 臂，及含有第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2) 的抗 CD3 臂，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域 (CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 K 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 E 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 E 殘基，且 L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 K 殘基；並且 H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 K 殘基，且 L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 E 殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 E 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 K 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 K 殘基，且 L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 E 殘基；並且 H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 E 殘基，且 L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 K 殘基。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and an anti-CD3 arm comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each of H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each of L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced with a K residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced with an E residue, and the amino acid at Q39 (Kabat numbering) in the CL domain of L1 is replaced with an E residue. the amino acid at V133 (EU numbering) is replaced by an E residue and the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a K residue; and the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by a K residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by an E residue; or (ii) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by an E residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a K residue, and the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced by a K residue. residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by an E residue; and the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by an E residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by a K residue.

舉例而言，在其他實施例中，本文所述之多特異性抗原結合分子包含：含有第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1) 的抗 CD3 臂，及含有第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2) 的抗 STEAP1 臂，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域 (CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 R 或 K 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 D 或 E 殘基，且 L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基；並且 H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基，且 L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 D 或 E 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 R 或 K 殘基，且 L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基；並且 H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基，且 L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基。For example, in other embodiments, the multispecific antigen-binding molecule described herein comprises: an anti-CD3 arm comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and an anti-STEAP1 arm comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each of H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each of L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by an R or K residue, and the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a D or E residue. residue in the CL domain of L1, the amino acid at V133 (EU numbering) is replaced by a D or E residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by an R or K residue; and the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by an R or K residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by a D or E residue; or (ii) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by a D or E residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by an R or K residue, and the amino acid at L1 the amino acid at V133 (EU numbering) in the CL domain of H2 is replaced by an R or K residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a D or E residue; and the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by a D or E residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by an R or K residue.

在一些實施例中，本文所述之多特異性抗原結合分子包含：含有第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1) 的抗 CD3 臂，及含有第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2) 的抗 STEAP1 臂，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域 (CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 K 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 E 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 E 殘基，且 L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 K 殘基；並且 H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 K 殘基，且 L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 E 殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 E 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 K 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 K 殘基，且 L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 E 殘基；並且 H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 E 殘基，且 L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 K 殘基。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-CD3 arm comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and an anti-STEAP1 arm comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each of H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each of L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced with a K residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced with an E residue, and the amino acid at Q39 (Kabat numbering) in the CL domain of L1 is replaced with an E residue. the amino acid at V133 (EU numbering) is replaced by an E residue and the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a K residue; and the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by a K residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by an E residue; or (ii) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by an E residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a K residue, and the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced by a K residue. residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by an E residue; and the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by an E residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by a K residue.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39K (Kabat 編號) 及 S183E (EU 編號) 取代且輕鏈含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 STEAP1 臂；以及重鏈中含有 Q39E (Kabat 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 取代的抗 CD3 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising Q39K (Kabat numbering) and S183E (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain; and an anti-CD3 arm comprising Q39E (Kabat numbering) substitution in the heavy chain and Q38K (Kabat numbering) substitution in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39K (Kabat 編號) 及 S183E (EU 編號) 取代且輕鏈含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 CD3 臂；以及重鏈中含有 Q39E (Kabat 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 取代的抗 STEAP1 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-CD3 arm comprising Q39K (Kabat numbering) and S183E (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain; and an anti-STEAP1 arm comprising Q39E (Kabat numbering) substitution in the heavy chain and Q38K (Kabat numbering) substitution in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39E (Kabat 編號) 及 S183K (EU 編號) 取代且輕鏈含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 STEAP1 臂；以及重鏈中含有 Q39K (Kabat 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 取代的抗 CD3 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising Q39E (Kabat numbering) and S183K (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain; and an anti-CD3 arm comprising Q39K (Kabat numbering) substitution in the heavy chain and Q38E (Kabat numbering) substitution in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39E (Kabat 編號) 及 S183K (EU 編號) 取代且輕鏈含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 CD3 臂；以及重鏈中含有 Q39K (Kabat 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 取代的抗 STEAP1 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-CD3 arm comprising Q39E (Kabat numbering) and S183K (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain; and an anti-STEAP1 arm comprising Q39K (Kabat numbering) substitution in the heavy chain and Q38E (Kabat numbering) substitution in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：a) 與第一抗原結合的第一重鏈/輕鏈對，該第一重鏈/輕鏈對包含第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1)，及 b) 與第二抗原結合的第二重鏈/輕鏈對，該第二重鏈/輕鏈對包含第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2)，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域(CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶正電荷殘基，H2 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，H2 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基。在一些實施例中，帶正電荷的殘基選自 R 及 K，並且帶負電荷的殘基選自 D 及 E。在一些實施例中，帶正電荷的殘基為 R。在其他實施例中，帶正電荷的殘基為 K。在一些實施例中，帶負電荷的殘基為 D。在其他實施例中，帶負電荷的殘基為 E。在一些實施例中，第一抗原為 STEAP1 且第二抗原為 CD3。在其他實施例中，第一抗原為 CD3 並且第二抗原為 STEAP1。In some embodiments, the multispecific antigen-binding molecules described herein comprise: a) a first heavy chain/light chain pair that binds to a first antigen, the first heavy chain/light chain pair comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and b) a second heavy chain/light chain pair that binds to a second antigen, the second heavy chain/light chain pair comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) S183 (EU numbering) in the CH1 domain of H1 The amino acid at S183 (EU numbering) in the CH1 domain of H2 was replaced with a negatively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 was replaced with a negatively charged residue, the amino acid at V133 (EU numbering) in the CL domain of L1 was replaced with a negatively charged residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 was replaced with a positively charged residue, the amino acid at S183 (EU numbering) in the CH1 domain of H2 was replaced with a negatively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 was replaced with a positively charged residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L2 was replaced with a positively charged residue. is replaced by a negatively charged residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced by a positively charged residue; or (ii) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by a negatively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a positively charged residue, the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced by a positively charged residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a negatively charged residue, and S183 (EU numbering) in the CH1 domain of H2 is replaced by a negatively charged residue. In some embodiments, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced with a positively charged residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced with a positively charged residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced with a negatively charged residue. In some embodiments, the positively charged residue is selected from R and K, and the negatively charged residue is selected from D and E. In some embodiments, the positively charged residue is R. In other embodiments, the positively charged residue is K. In some embodiments, the negatively charged residue is D. In other embodiments, the negatively charged residue is E. In some embodiments, the first antigen is STEAP1 and the second antigen is CD3. In other embodiments, the first antigen is CD3 and the second antigen is STEAP1.

舉例而言，在一些實施例中，本文所述之多特異性抗原結合分子包含：含有第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1) 的抗 STEAP1 臂，及含有第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2) 的抗 CD3 臂，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域 (CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 R 或 K 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 D 或 E 殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被 R 或 K 殘基替換，H2 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 D 或 E 殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 R 或 K 殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 D 或 E 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 R 或 K 殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基，H2 之 CH1域中的 S183 (EU 編號) 處之胺基酸被替換為 R 或 K 殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 D 或 E 殘基。For example, in some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and an anti-CD3 arm comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each of H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each of L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by an R or K residue, and the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a D or E residue. the amino acid at S183 (EU numbering) in the CH1 domain of H2 is replaced by a D or E residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by an R or K residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by a D or E residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced by an R or K residue; or (ii) the CH1 domain of H1 is replaced by a D or E residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by an R or K residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by a D or E residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced by an R or K residue; The amino acid at S183 (EU numbering) in the CH1 domain of H2 is replaced by an R or K residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by an R or K residue, the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced by an R or K residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a D or E residue, the amino acid at S183 (EU numbering) in the CH1 domain of H2 is replaced by an R or K residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by a D or E residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by an R or K residue. The amino acid at 1 is replaced by an R or K residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced by a D or E residue.

舉例而言，在一些實施例中，本文所述之多特異性抗原結合分子包含：含有第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1) 的抗 STEAP1 臂，及含有第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2) 的抗 CD3 臂，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域 (CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 K 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 E 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 E 殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 K 殘基，H2 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 E 殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 K 殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 E 殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 K 殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 E 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 K 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 K 殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 E 殘基，H2 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 K 殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 E 殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 K 殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 E 殘基。For example, in some embodiments, the multispecific antigen-binding molecule described herein comprises: an anti-STEAP1 arm comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and an anti-CD3 arm comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each of H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each of L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by a K residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by an E residue, and the amino acid at L1 is replaced by a the amino acid at V133 (EU numbering) in the CL domain is replaced by an E residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a K residue, the amino acid at S183 (EU numbering) in the CH1 domain of H2 is replaced by an E residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by a K residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by an E residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced by a K residue; or (ii) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by an E residue residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 was replaced by a K residue, the amino acid at V133 (EU numbering) in the CL domain of L1 was replaced by a K residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 was replaced by an E residue, the amino acid at S183 (EU numbering) in the CH1 domain of H2 was replaced by a K residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 was replaced by an E residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L2 was replaced by a K residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 was replaced by a K residue. The amino acid at the site is replaced by an E residue.

在其他實例中，在一些實施例中，本文所述之多特異性抗原結合分子包含：含有第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1) 的抗 CD3 臂，及含有第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2) 的抗 STEAP1 臂，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域 (CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 R 或 K 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 D 或 E 殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被 R 或 K 殘基替換，H2 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 D 或 E 殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 R 或 K 殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 D 或 E 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 R 或 K 殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基，H2 之 CH1域中的 S183 (EU 編號) 處之胺基酸被替換為 R 或 K 殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 D 或 E 殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 R 或 K 殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 D 或 E 殘基。In other examples, in some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-CD3 arm comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and an anti-STEAP1 arm comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each of H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each of L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by an R or K residue, and the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a D or E residue. the amino acid at S183 (EU numbering) in the CH1 domain of H2 is replaced by a D or E residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by an R or K residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by a D or E residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced by an R or K residue; or (ii) the CH1 domain of H1 is replaced by a D or E residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by an R or K residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by a D or E residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced by an R or K residue; The amino acid at S183 (EU numbering) in the CH1 domain of H2 is replaced by an R or K residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by an R or K residue, the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced by an R or K residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a D or E residue, the amino acid at S183 (EU numbering) in the CH1 domain of H2 is replaced by an R or K residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by a D or E residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by an R or K residue. The amino acid at 1 is replaced by an R or K residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced by a D or E residue.

舉例而言，在一些實施例中，本文所述之多特異性抗原結合分子包含：含有第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1) 的抗 CD3 臂，及含有第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2) 的抗 STEAP1 臂，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域 (CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 K 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 E 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 E 殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 K 殘基，H2 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 E 殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 K 殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 E 殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 K 殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 E 殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 K 殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 K 殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 E 殘基，H2 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為 K 殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為 E 殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為 K 殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為 E 殘基。For example, in some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-CD3 arm comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and an anti-STEAP1 arm comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each of H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each of L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by a K residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by an E residue, and the amino acid at L1 is replaced by a the amino acid at V133 (EU numbering) in the CL domain is replaced by an E residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a K residue, the amino acid at S183 (EU numbering) in the CH1 domain of H2 is replaced by an E residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by a K residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced by an E residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced by a K residue; or (ii) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by an E residue residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 was replaced by a K residue, the amino acid at V133 (EU numbering) in the CL domain of L1 was replaced by a K residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 was replaced by an E residue, the amino acid at S183 (EU numbering) in the CH1 domain of H2 was replaced by a K residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 was replaced by an E residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L2 was replaced by a K residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 was replaced by a K residue. The amino acid at the site is replaced by an E residue.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39K (Kabat 編號) 及 S183E (EU 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 STEAP1 臂；以及重鏈中含有 Q39E (Kabat 編號) 及 S183K (EU 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 CD3 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising Q39K (Kabat numbering) and S183E (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain; and an anti-CD3 arm comprising Q39E (Kabat numbering) and S183K (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39K (Kabat 編號) 及 S183E (EU 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 CD3 臂；以及重鏈中含有 Q39E (Kabat 編號) 及 S183K (EU 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 STEAP1 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-CD3 arm comprising Q39K (Kabat numbering) and S183E (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain; and an anti-STEAP1 arm comprising Q39E (Kabat numbering) and S183K (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39E (Kabat 編號) 及 S183K (EU 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 STEAP1 臂；以及重鏈中含有 Q39K (Kabat 編號) 及 S183E (EU 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 CD3 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising Q39E (Kabat numbering) and S183K (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain; and an anti-CD3 arm comprising Q39K (Kabat numbering) and S183E (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39E (Kabat 編號) 及 S183K (EU 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 CD3 臂；以及重鏈中含有 Q39K (Kabat 編號) 及 S183E (EU 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 STEAP1 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-CD3 arm comprising Q39E (Kabat numbering) and S183K (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain; and an anti-STEAP1 arm comprising Q39K (Kabat numbering) and S183E (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈恆定區中含有 S183K (EU 編號)、N297G (EU 編號) 及 T366W (EU 編號) 取代且輕鏈恆定區中含有 V133E (EU 編號) 取代的抗 STEAP1 臂，以及重鏈恆定區中含有 S183E (EU 編號)、N297G (EU 編號)、T366S (EU 編號) L368A (EU 編號) 及 Y407V (EU 編號) 取代且輕鏈恆定區中含有 V133K (EU 編號) 取代的抗 CD3 臂。在一些實施例中，本文所述之多特異性抗原結合分子包含：含有 S183E (EU 編號)、N297G (EU 編號)、T366S (EU 編號)、L368A (EU 編號) 及 Y407V (EU 編號) 取代且輕鏈恆定區中含有 V133K (EU 編號) 取代的抗 STEAP1 臂，以及重鏈恆定區中含有 S183K (EU 編號)、N297G (EU 編號) 及 T366W (EU 編號) 取代且輕鏈恆定區中含有 V133E (EU 編號) 取代的抗 CD3 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising S183K (EU numbering), N297G (EU numbering), and T366W (EU numbering) substitutions in the heavy chain constant region and V133E (EU numbering) substitution in the light chain constant region, and an anti-CD3 arm comprising S183E (EU numbering), N297G (EU numbering), T366S (EU numbering), L368A (EU numbering), and Y407V (EU numbering) substitutions in the heavy chain constant region and V133K (EU numbering) substitution in the light chain constant region. In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising S183E (EU numbering), N297G (EU numbering), T366S (EU numbering), L368A (EU numbering) and Y407V (EU numbering) substitutions and a V133K (EU numbering) substitution in the light chain constant region, and an anti-CD3 arm comprising S183K (EU numbering), N297G (EU numbering) and T366W (EU numbering) substitutions in the heavy chain constant region and a V133E (EU numbering) substitution in the light chain constant region.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39E (Kabat 編號)、S183K (EU 編號)、N297G (EU 編號) 及 T366W (EU 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 STEAP1 臂，以及重鏈中含有 Q39K (Kabat 編號)、S183E (EU 編號)、N297G (EU 編號)、T366S (EU 編號)、L368A (EU 編號) 及 Y407V (EU 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 CD3 臂。在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39K (Kabat 編號)、S183E (EU 編號)、N297G (EU 編號)、T366S (EU 編號)、L368A (EU 編號) 及 Y407V (EU 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 STEAP1 臂，以及重鏈中含有 Q39E (Kabat 編號)、S183K (EU 編號)、N297G (EU 編號) 及 T366W (EU 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 CD3 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising Q39E (Kabat numbering), S183K (EU numbering), N297G (EU numbering), and T366W (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain, and an anti-CD3 arm comprising Q39K (Kabat numbering), S183E (EU numbering), N297G (EU numbering), T366S (EU numbering), L368A (EU numbering), and Y407V (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain. In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising Q39K (Kabat numbering), S183E (EU numbering), N297G (EU numbering), T366S (EU numbering), L368A (EU numbering), and Y407V (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain, and an anti-CD3 arm comprising Q39E (Kabat numbering), S183K (EU numbering), N297G (EU numbering), and T366W (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain.

明確考慮到在本文列舉的任何情況下中使用的本文所述之抗原結合分子可單獨或組合地具有下文第 1 節至第 6 節中描述的任何特徵。1.抗體親和力It is expressly contemplated that the antigen binding molecules described herein for use in any of the situations listed herein may have any of the characteristics described in Sections 1 to 6 below, either alone or in combination.1.Antibody Affinity

在某些情況下，本發明之抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 具有 ≤ 1μM、≤ 100 nM、≤ 10 nM、≤ 1 nM、≤ 0.1 nM、≤ 0.01 nM、或 ≤ 0.001 nM (例如，10^-8M 或更低，例如 10^-8M 至 10^-13M，例如 10^-9M 至 10^-13M) 之平衡解離常數 (K_D)。In certain cases, the antigen-binding molecules of the invention (e.g., monospecific and/or multispecific antigen-binding molecules such as bispecific or trispecific antigen-binding molecules) have an equilibrium dissociation constant (KD) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM,_≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g.,^10-8 M or lower, e.g.,^10-8 M to^10-13 M, e.g.,^10-9 M to^10-13 M).

在一個實例中，K_D藉由放射性標記的抗原結合測定 (RIA) 進行測量。在一種情況下，使用所關注之抗原結合分子及其抗原之 Fab 版執行 RIA。例如，藉由在連續系列未標記的抗原存在下用最小濃度的 (¹²⁵I) 標記的抗原平衡 Fab，然後用抗 Fab 抗體塗覆的板捕獲結合的抗原，來測量 Fab 對抗原的溶液結合親和力 (參見例如 Chen 等人，J. Mol. Biol.293:865-881(1999))。為了建立測定條件，將 MICROTITER® 多孔盤 (Thermo Scientific) 以含 5 μg/ml 捕獲抗 Fab 抗體 (Cappel Labs) 的 50 mM 碳酸鈉 (pH 9.6) 包被過夜，然後於室溫 (約 23℃) 以含 2% (w/v) 的牛血清白蛋白的 PBS 封閉二至五小時。在非吸附板 (Nunc #269620) 中，將 100 pM 或 26 pM [¹²⁵I]-抗原與所關注 Fab 的系列稀釋液混合 (例如，與 Presta 等人在Cancer Res.57: 4593-4599 (1997) 中所述之抗 VEGF 抗體 Fab-12 的評估結果一致)。然後將所關注 Fab 過夜孵育；但是，可繼續孵育更長時間 (例如約 65 小時)，以確保達到平衡。此後，將混合物轉移至捕獲板上，在室溫下進行孵育 (例如，孵育 1 小時)。然後移除溶液，且用含 0.1% 聚山梨糖醇酯 20 (TWEEN-20®) 之 PBS 將板洗滌八次。當盤乾燥後，添加 150 μl/孔之閃爍劑 (MICROSCINT-20™; Packard)，並將盤在 TOPCOUNT™ 伽瑪計數器 (Packard) 上計數十分鐘。選擇提供小於或等於最大結合濃度的 20% 的各種 Fab 的濃度以用於競爭性結合測定中。In one example,_KD is measured by a radiolabeled antigen binding assay (RIA). In one case, the RIA is performed using a Fab version of the antigen-binding molecule of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with a minimal concentration of (^125I )-labeled antigen in the presence of a serial series of unlabeled antigens and then capturing the bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al.,J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with 5 μg/ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked for two to five hours at room temperature (approximately 23°C) with 2% (w/v) bovine serum albumin in PBS. In nonadsorbent plates (Nunc #269620), 100 pM or 26 pM [^125I ]-antigen was mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 described by Presta et al.,Cancer Res. 57: 4593-4599 (1997)). The Fab of interest is then incubated overnight; however, incubation may be continued for longer periods of time (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (e.g., for 1 hour). The solution is then removed and the plate is washed eight times with PBS containing 0.1% polysorbate 20 (TWEEN-20®). When the plate is dry, 150 μl/well of scintillator (MICROSCINT-20™; Packard) is added and the plate is counted for ten minutes on a TOPCOUNT™ Gamma Counter (Packard). Concentrations of each Fab that provide less than or equal to 20% of the maximum binding concentration are selected for use in competitive binding assays.

根據另一實例，K_D係使用 BIACORE® 表面電漿子共振測定測得。例如，使用 BIACORE®-2000 或 BIACORE®-3000 (BIACORE®, Inc., Piscataway, NJ) 的測定，是以固定的抗原 CM5 晶片在約 10 個反應單位 (RU) 於 25℃ 下進行。在一種情況下，根據供應商的說明，用N-乙基-N'-(3-二甲基胺基丙基)-碳二亞胺鹽酸鹽 (EDC) 及N-羥基琥珀醯亞胺 (NHS) 活化羧甲基化葡聚醣生物感測器晶片 (CM5, BIACORE®, Inc.)。將抗原用 10 mM 醋酸鈉 (pH 4.8) 稀釋至 5 μg/ml (約 0.2 μM)，然後以 5 μl/分鐘的流速注入，以獲得大約 10 反應單位 (RU) 的偶合蛋白。注入抗原後，注入 1 M 乙醇胺以封閉未反應的基團。對於動力學測量，將 Fab 之兩倍連續稀釋液 (0.78 nM 至 500 nM) 在 25℃ 以約 25 μl/min 之流速注入含 0.05% 聚山梨醇酯 20 (TWEEN-20™) 界面活性劑 (PBST) 的 PBS 中。藉由同時擬合結合及解離感測圖，使用簡單的一對一 Langmuir 結合模型 (BIACORE® 評估軟體版本 3.2) 計算結合速率 (k_on) 及解離速率 (k_off)。平衡解離常數 (K_D) 藉由 k_off/k_on比率計算得出。參見例如：Chen 等人，J. Mol. Biol.293:865-881 (1999)。若藉由上述表面電漿子共振測定法測得的結合率 (on-rate) 超過 10⁶M^-1s^-1，則可以藉由使用螢光淬滅技術確定結合率，該技術可測量 25℃ PBS (pH 7.2) 中的 20 nM 抗抗原抗體 (Fab 形式) 在存在濃度升高的抗原的情況下螢光發射強度的增加或減少 (激發波長 = 295 nm；發射波長 = 340 nm，帶通 16 nm)，該抗原濃度可藉由分光光度計諸如停流分光光度計 (Aviv Instruments) 或帶有攪拌比色皿的 8000 系列 SLM-AMINCO™ 分光光度計 (ThermoSpectronic) 測得。According to another example,_KD is measured using a BIACORE® surface plasmon resonance assay. For example, the assay using a BIACORE®-2000 or BIACORE®-3000 (BIACORE®, Inc., Piscataway, NJ) is performed with an immobilized antigen CM5 chip at approximately 10 reaction units (RU) at 25°C. In one case, a carboxymethylated dextran biosensor chip (CM5, BIACORE®, Inc.) is activated withN -ethyl-N' -(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN -hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5 μg/ml (approximately 0.2 μM) in 10 mM sodium acetate (pH 4.8) and injected at a flow rate of 5 μl/min to obtain approximately 10 reaction units (RU) of coupled protein. After the injection of antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C at a flow rate of approximately 25 μl/min. The association rate (k_on ) and dissociation rate (k_off ) were calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software Version 3.2). The equilibrium dissociation constant (K_D ) was calculated from the ratio of k_off /k_on . See, e.g., Chen et al.,J. Mol. Biol. 293:865-881 (1999). If the on-rate measured by the surface plasmon resonance assay described above exceeds 10⁶ M^-1 s^-1 , the on-rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen (excitation wavelength = 295 nm; emission wavelength = 340 nm, bandpass 16 nm) as measured by a spectrophotometer such as a stopped-flow spectrophotometer (Aviv Instruments) or a Series 8000 SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

根據另一情況，K_D係藉由動力學篩析測定 (KinExA®) 來測量，其可藉由 Sapidyne Instruments 來執行。動力學篩析測定 (KinExA®) 測量溶液中之平衡結合親和力及動力學。平衡解離常數K_D及締合常數k_a係藉由實驗確定，而解離速率k_d係基於方程式k_d= K_D×k_a來計算。在一個實施例中，K_D係用人 STEAP1 來測量。在另一實施例中，K_D係用石蟹獼猴 STEAP1 來測量。在一個實施例中，本文所述之抗 STEAP1/CD3 TDB 對人 STEAP1 之 K_D為 0.01 nM 至 1 nM、或 1 nM 至 10 nM、或 10 nM 至 100 nM、或 100 nM 至 1,000 nM。在另一實施例中，本文所述之抗 STEAP1/CD3 TDB 對石蟹獼猴 STEAP1 之 K_D為 0.01 nM 至 1 nM、或 1 nM 至 10 nM、或 10 nM 至 100 nM、或 100 nM 至 1,000 nM。在一個實施例中，本文所述之抗 STEAP1/CD3 TDB 對人 STEAP1 之k_a為 100 M^-1S^-1至 1,000 M^-1S^-1、或 1,000 M^-1S^-1至 10,000 M^-1S^-1、或 10,000 M^-1S^-1至 100,000 M^-1S^-1、或 100,000 M^-1S^-1至 1,000,000 M^-1S^-1。在另一實施例中，本文所述之抗 STEAP1/CD3 TDB 對石蟹獼猴 STEAP1 之k_a為 100 M^-1S^-1至 1,000 M^-1S^-1、或 1,000 M^-1S^-1至 10,000 M^-1S^-1、或 10,000 M^-1S^-1至 100,000 M^-1S^-1、或 100,000 M^-1S^-1至 1,000,000 M^-1S^-1。在一個實施例中，本文所述之抗 STEAP1/CD3 TDB 對人 STEAP1 之k_d為 0.0001 S^-1至 0.001 S^-1、0.001 S^-1至 0.01 S^-1、或 0.01 S^-1至 0.1 S^-1、或 0.1 S^-1至 1 S^-1。在另一實施例中，本文所述之抗 STEAP1/CD3 TDB 對石蟹獼猴 STEAP1 之k_d為 0.0001 S^-1至 0.001 S^-1、0.001 S^-1至 0.01 S^-1、或 0.01 S^-1至 0.1 S^-1、或 0.1 S^-1至 1 S^-1。2.抗體片段及多特異性抗體According to another aspect,_KD is measured by a kinetic screening assay (KinExA®), which can be performed by Sapidyne Instruments. The kinetic screening assay (KinExA®) measures equilibrium binding affinity and kinetics in solution. The equilibrium dissociation_constantKD_and the association constantka are_determined experimentally, and the dissociation ratekd is_calculated based on the equationkd =_{KD× ka}. In one embodiment,_KD is measured using human STEAP1. In another embodiment,_KD is measured using macaque monkey STEAP1. In one embodiment, the anti-STEAP1/CD3 TDB described herein has a_KD of 0.01 nM to 1 nM, or 1 nM to 10 nM, or 10 nM to 100 nM, or 100 nM to 1,000 nM for human STEAP1. In another embodiment, the anti-STEAP1/CD3 TDB described herein has a_KD of 0.01 nM to 1 nM, or 1 nM to 10 nM, or 10 nM to 100 nM, or 100 nM to 1,000 nM for stone crab macaque STEAP1. In one embodiment, the anti-STEAP1/CD3 TDB described herein has_aka of 100 M^-1 S^-1 to 1,000 M^-1 S^-1 , or 1,000 M^-1 S^-1 to 10,000 M^-1 S^- 1, or 10,000 M-¹ S^-1 to 100,000 M^-1 S^-1 , or 100,000 M^-1 S-¹ to 1,000,000 M^-1 S^-1 for human STEAP1. In another embodiment, the anti-STEAP1/CD3 TDB described herein hasa k_a of 100 M^-1 S^-1 to 1,000 M^-1 S^-1 , or 1,000 M^-1 S^-1 to 10,000 M^-1 S^-1 , or 10,000 M^-1 S^-1 to 100,000 M^-1 S^-1 , or 100,000 M^-1 S-¹ to 1,000,000 M^-1 S^-1 . In one embodiment, the anti-STEAP1/CD3 TDB described herein hasa k_d of 0.0001 S^-1 to 0.001 S^-1 , 0.001 S^-1 to 0.01 S^-1 , or 0.01 S^-1 to 0.1 S^-1 , or 0.1 S^-1 to 1 S^-1 . In another embodiment, the anti-STEAP1/CD3 TDB described herein has_akd of 0.0001 S^-1 to 0.001 S^-1 , 0.001 S^-1 to 0.01 S^-1 , or 0.01 S^-1 to 0.1 S^-1 , or 0.1 S^-1 to 1 S^-1 .2.Antibody fragments and multispecific antibodies

在某些情況下，本文所提供之抗原結合分子包括一種或多種抗體片段。抗體片段包括但不限於 Fab、Fab'、Fab'-SH、F(ab')₂,、Fv、Fd、單鏈 Fv (scFv)、單鏈 Fab 片段 (scFab)、三特異性 (Fab₃)、雙特異性 (Fab₂)、雙功能抗體 ((V_L-V_H)₂或 (V_H-V_L)₂)、三功能抗體 (三價)、四功能抗體 (四價)、微抗體 ((scF_V-C_H)₂)、雙特異性單鏈 Fv (Bis-scFv)、IgGδCH2、scFv-Fc、或 (scFv)₂-Fc 以及下文所述之其他片段。關於某些抗體片段的綜述，參閱 Hudson 等人,Nat. Med.9:129-134 (2003)。關於 scFv 片段的綜述，參見例如 Pluckthün，The Pharmacology of Monoclonal Antibodies，第 113卷，Rosenburg 及 Moore 編，Springer-Verlag，New York，第 269-315 頁 (1994)；亦可參見 WO 93/16185；及美國專利第 5,571,894 號及第 5,587,458 號。關於包含補救受體結合抗原決定位殘基且具有增加的活體內半衰期之 Fab 及 F(ab')₂片段的論述，參見美國專利號 5,869,046。In certain instances, the antigen-binding molecules provided herein include one or more antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')₂ , Fv, Fd, single-chain Fv (scFv), single-chain Fab fragment (scFab), trispecific (Fab₃ ), bispecific (Fab₂ ), bifunctional antibodies ((_VL -_VH )₂ or (_VH -_VL )₂ ), trifunctional antibodies (trivalent), tetrafunctional antibodies (tetravalent), miniantibodies ((_scFV -_CH )₂ ), bispecific single-chain Fv (Bis-scFv), IgGδCH2, scFv-Fc, or (scFv)₂ -Fc, and other fragments described below. For a review of certain antibody fragments, see Hudson et al.,Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthün,The Pharmacology of Monoclonal Antibodies , Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a review of Fab and F(ab')₂ fragments that contain antigen-binding residues that rescue receptor binding and have increased in vivo half-life, see U.S. Patent No. 5,869,046.

在一些情況下，抗體片段為單鏈可變片段 (scFv)。In some cases, the antibody fragment is a single-chain variable fragment (scFv).

在一些情況下，抗體片段為單鏈 Fab 片段。In some cases, the antibody fragment is a single-chain Fab fragment.

在某些情況下，本文所提供之抗原結合分子包括多特異性抗原結合分子。在某些態樣中，本文所提供之多特異性抗原結合分子為多特異性抗體，例如雙特異性抗體或三特異性抗體。在某些態樣中，多特異性抗體為雙特異性抗體。在某些態樣中，多特異性抗體具有三種或更多種結合特異性。在某些態樣中，結合特異性中之一者針對 STEAP1，而其他特異性則針對任何其他抗原 (例如，CD3)。多特異性 (例如，雙特異性或三特異性) 抗體亦可用於將細胞毒性劑或細胞定位於表現 STEAP1 之細胞。多特異性抗體可製成全長抗體或抗體片段。In some cases, the antigen binding molecules provided herein include multispecific antigen binding molecules. In some aspects, the multispecific antigen binding molecules provided herein are multispecific antibodies, such as bispecific antibodies or trispecific antibodies. In some aspects, the multispecific antibodies are bispecific antibodies. In some aspects, the multispecific antibodies have three or more binding specificities. In some aspects, one of the binding specificities is for STEAP1, while the other specificities are for any other antigen (e.g., CD3). Multispecific (e.g., bispecific or trispecific) antibodies can also be used to localize cytotoxic agents or cells to cells expressing STEAP1. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

用於多特異性抗體之各種分子形式為業內已知且包括於本文中 (參見例如，Spiess 等人，Mol. Immunol.67 (2015) 95-106；及 Brinkmann 等人，MAbs9 (2017) 182-212)。Various molecular formats for multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al.,Mol. Immunol. 67 (2015) 95-106; and Brinkmann et al.,MAbs 9 (2017) 182-212).

在一些實施例中，本文所述之多特異性抗體為雙特異性抗體，該雙特異性抗體被設計為同時結合至標靶細胞 (例如，腫瘤細胞) 上之表面抗原以及 T 細胞受體 (TCR) 複合體之活化不變組分 (例如，CD3)，用於重定向 T 細胞以毒殺標靶細胞。在某些態樣中，本文所提供之抗體為多特異性抗體，特定而言雙特異性抗體，其中結合特異性中之一者係針對 STEAP1，且另一者係針對 CD3。In some embodiments, the multispecific antibodies described herein are bispecific antibodies that are designed to simultaneously bind to a surface antigen on a target cell (e.g., a tumor cell) and an activation-invariant component of the T cell receptor (TCR) complex (e.g., CD3) for redirecting T cells to kill target cells. In certain aspects, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies, wherein one of the binding specificities is for STEAP1 and the other is for CD3.

可用於此目的之雙特異性抗體形式的實例包括但不限於所謂的「BiTE」(雙特異性 T 細胞銜接體) 分子，其中兩個 scFv 分子藉由柔性連接體融合 (參見例如，WO 2004/106381、WO 2005/061547、WO 2007/042261 及 WO 2008/119567；Nagorsen 及 Bäuerle，Exp. Cell Res.317，1255-1260 (2011))；雙功能抗體 (Holliger 等人，Prot. Eng.9，299-305 (1996)) 及其衍生物，諸如串聯雙功能抗體 (「TandAb」；Kipriyanov 等人，J. Mol. Biol.293, 41-56 (1999))；「DART」(雙親和力重靶向) 分子，其基於雙功能抗體形式，但具有 C 端雙硫鍵以供額外穩定 (Johnson 等人，J. Mol. Biol.399, 436-449 (2010))；以及所謂的三功能單抗，其係完整的小鼠/大鼠 IgG 雜合分子 (參見 Seimetz 等人的以下綜述：Cancer Treat.Rev.36，458-467 (2010))。本文所包括之特定 T 細胞雙特異性抗體形式描述於：WO 2013/026833；WO 2013/026839；WO 2016/020309；及 Bacac 等人Oncoimmunology5(8):e1203498 (2016) 中。Examples of bispecific antibody formats that can be used for this purpose include, but are not limited to, so-called "BiTE" (bispecific T-cell engager) molecules in which two scFv molecules are fused via a flexible linker (see, e.g., WO 2004/106381, WO 2005/061547, WO 2007/042261, and WO 2008/119567; Nagorsen and Bäuerle,Exp. Cell Res. 317, 1255-1260 (2011)); bifunctional antibodies (Holliger et al.,Prot. Eng. 9, 299-305 (1996)) and their derivatives, such as tandem bifunctional antibodies ("TandAb"; Kipriyanov et al.,J. Mol. Biol. 293, 294). 41-56 (1999)); "DART" (dual affinity retargeting) molecules, which are based on the bifunctional antibody format but have a C-terminal disulfide bond for additional stabilization (Johnson et al.,J. Mol. Biol. 399, 436-449 (2010)); and so-called trifunctional mAbs, which are complete mouse/rat IgG hybrid molecules (see the following review by Seimetz et al.:Cancer Treat. Rev. 36, 458-467 (2010)). Specific T cell bispecific antibody formats included herein are described in: WO 2013/026833; WO 2013/026839; WO 2016/020309; and Bacac et al.Oncoimmunology 5(8):e1203498 (2016).

額外的多特異性抗體形式包括雙抗體，該等雙抗體為具有兩個抗原結合位點的抗體片段 (其可為二價或雙特異性的)。參見例如，EP 404,097；WO 1993/01161；Hudson 等人，Nat. Med.9:129-134 (2003)；及 Hollinger 等人，Proc. Natl. Acad. Sci. USA90: 6444-6448 (1993)。Hudson 等人 (Nat. Med.9: 129-134，2003) 中亦描述了三功能抗體(Triabodies)及四功能抗體(tetrabodies)。Additional multispecific antibody formats include diabodies, which are antibody fragments (which may be bivalent or bispecific) with two antigen binding sites. See, e.g., EP 404,097; WO 1993/01161; Hudson et al.,Nat. Med. 9:129-134 (2003); and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al. (Nat. Med. 9:129-134, 2003).

多特異性抗體亦可提供為不對稱形式，其包含一個或多個具有相同抗原特異性之結合臂中交叉的域，即藉由交換 VH/VL 之 (參見例如，WO 2009/080252 及 WO 2015/150447)、CH1/CL 之 (參見例如，WO 2009/080253) 或完整的 Fab 臂 (參見例如，WO 2009/080251、WO 2016/016299，亦見 Schaefer 等人，Proc. Natl. Acad. Sci. USA108: 1187-1191 (2011)，以及 Klein 等人，MAbs8:1010-20 (2016)) 實現。在一個態樣中，多特異性抗體包含 cross-Fab 片段。Multispecific antibodies can also be provided in an asymmetric format comprising cross-domains in one or more binding arms with the same antigenic specificity, i.e., by exchanging VH/VL (see, e.g., WO 2009/080252 and WO 2015/150447), CH1/CL (see, e.g., WO 2009/080253) or complete Fab arms (see, e.g., WO 2009/080251, WO 2016/016299, see also Schaefer et al.,Proc. Natl. Acad. Sci. USA 108: 1187-1191 (2011), and Klein et al.,MAbs 8: 1010-20 (2016)). In one aspect, the multispecific antibody comprises a cross-Fab fragment.

本文亦包括具有三個或更多個抗原結合位點的經工程化改造之抗體，包括例如「章魚抗體」(Octopus antibodies) 或 DVD-Ig (參見例如，WO 2001/77342 及 WO 2008/024715)。具有三個或更多個抗原結合位點之多特異性抗體的其他實例可參見 WO 2010/115589、WO 2010/112193、WO 2010/136172、WO 2010/145792 及 WO 2013/026831 中。雙特異性抗體或其抗原結合片段亦包括「雙重作用 FAb」或「DAF」，其包含與 STEAP1 以及另一種不同抗原 (例如，CD3) 結合之抗原結合位點 (參見例如，US 2008/0069820 及 WO 2015/095539)。Also included herein are engineered antibodies with three or more antigen binding sites, including, for example, "Octopus antibodies" or DVD-Ig (see, for example, WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792 and WO 2013/026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual-acting FAbs" or "DAFs," which comprise antigen-binding sites that bind to STEAP1 and another different antigen (e.g., CD3) (see, e.g., US 2008/0069820 and WO 2015/095539).

用於製備多特異性抗體之技術包括但不限於重組共表現兩個具有不同特異性之免疫球蛋白重鏈-輕鏈對 (參見 Milstein 及 Cuello，Nature305:537 (1983)) 及「杵臼」(knob-in-hole) 工程化改造 (參見例如，美國專利第 5,731,168 號，以及 Atwell 等人，J. Mol. Biol. 270:26 (1997))。多特異性抗體亦可藉由以下方法進行製備：用於製備抗體 Fc-異二聚體分子的工程改造靜電轉向效應 (參見例如，WO 2009/089004)；交聯兩個或更多個抗體或片段 (參見例如，美國專利第 4,676,980 號；以及 Brennan 等人，Science，229:81 (1985))；使用白胺酸拉鏈產生雙特異性抗體 (參見例如，Kostelny 等人，J. Immunol.，148(5):1547-1553 (1992)；及 WO 2011/034605)；使用常用輕鏈技術規避輕鏈錯配問題 (參見例如，WO 98/50431)；使用「雙功能抗體」技術製備雙特異性抗體片段 (參見例如，Hollinger 等人，Proc. Natl. Acad. Sci. USA，90:6444-6448 (1993))；以及使用單鏈 Fv (sFv) 二聚體 (參見例如，Gruber 等人，J. Immunol.，152:5368 (1994))；以及按照例如 Tutt 等人於J. Immunol.147:60 (1991) 中所述製備三特異性抗體。3.嵌合及人源化抗體Techniques for preparing multispecific antibodies include, but are not limited to, recombining immunoglobulin heavy chain-light chain pairs that co-express two different specificities (see Milstein and Cuello,Nature 305:537 (1983)) and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be prepared by the following methods: engineering electrostatic switching effects for preparing antibody Fc-heterodimer molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980; and Brennan et al.,Science , 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al.,J. Immunol. , 148(5):1547-1553 (1992); and WO 2011/034605); using common light chain technology to circumvent the light chain mispairing problem (see, e.g., WO 98/50431); using "bifunctional antibody" technology to prepare bispecific antibody fragments (see, e.g., Hollinger et al.,Proc. Natl. Acad. Sci. USA , 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al.,J. Immunol. , 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., by Tutt et al.,J. Immunol. 147:60 (1991).3.Chimeric and humanized antibodies

在某些情況下，本文所提供之抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子，諸如雙特異性或三特異性抗原結合分子) 為嵌合抗體。某些嵌合抗體描述於例如，美國專利號 4,816,567；及 Morrison 等人，Proc. Natl. Acad. Sci. USA，81:6851-6855，1984。在一個實例中，嵌合抗體包含非人可變區 (例如，來源於小鼠、大鼠、倉鼠、兔或非人類靈長類動物如猴的可變區) 及人恆定區。在又一個實例中，嵌合抗體為「類別轉換」抗體，其中類或子類相比於其親代抗體已發生變更。嵌合抗體包括其抗原結合片段。In certain instances, the antigen binding molecules provided herein (e.g., monospecific and/or multispecific antigen binding molecules, such as bispecific or trispecific antigen binding molecules) are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA , 81:6851-6855, 1984. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed compared to its parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

在某些實例中，嵌合抗體為人源化抗體。通常，非人抗體為人源化抗體以降低對人的免疫原性，同時保留親代非人抗體之特異性及親和力。通常，人源化抗體包含一個或多個可變域，其中 HVR 如 CDR (或其部分) 來源於非人抗體，並且 FR (或其部分) 來源於人抗體序列。人源化抗體視情況將包含人恆定區之至少一部分。在一些情況下，人源化抗體中的一些 FR 殘基經來自非人抗體 (例如，衍生 CDR/HVR 殘基之抗體) 之對應殘基取代，以例如恢復或改善抗體特異性或親和力。In some instances, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized antibodies to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies comprise one or more variable domains, wherein HVR such as CDR (or a portion thereof) is derived from a non-human antibody, and FR (or a portion thereof) is derived from a human antibody sequence. Humanized antibodies will optionally comprise at least a portion of a human constant region. In some cases, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies from which CDR/HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

人源化抗體及其製備方法綜述於例如 Almagro 及 Fransson，Front. Biosci.13:1619-1633 (2008) 中，且進一步描述於例如：Riechmann 等人，Nature332:323-329 (1988)；Queen 等人，Proc. Natl. Acad. Sci. USA86:10029-10033 (1989)；美國專利第 5, 821,337 號、第 7,527,791 號、第 6,982,321 號 及 第 7,087,409 號；Kashmiri等人，Methods36:25-34 (2005) (具體描述了決定區 (SDR) 接枝)；Padlan，Mol. Immunol.28:489-498 (1991) (描述了「表面重塑」)；Dall'Acqua 等人，Methods36:43-60 (2005) (描述了「FR 改組」)；Osbourn 等人，Methods36:61-68 (2005)；及 Klimka 等人，Br. J. Cancer，83:252-260 (2000) (描述了 FR 改組的「導向選擇」法)。Humanized antibodies and methods for their preparation are generally described in, for example, Almagro and Fransson,Front. Biosci. 13:1619-1633 (2008), and further described in, for example, Riechmann et al.,Nature 332:323-329 (1988); Queen et al.,Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiriet al. ,Methods 36:25-34 (2005) (specifically describing SDR grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing "surface remodeling");Dall'Acqua et al.,Methods 36:43-60 (2005) (describing "FR shuffling"); Osbourn et al.,Methods 36:61-68 (2005); and Klimka et al.,Br. J. Cancer , 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

可以用於人源化的人骨架區包括但不限於：使用「最佳匹配」方法選擇的骨架區域 (參見例如，Sims 等人J. Immunol.151:2296 (1993))；來源於輕鏈或重鏈可變區的特定亞組的人抗體的共通序列的骨架區域 (參見例如，Carter 等人Proc. Natl. Acad. Sci. USA，89:4285 (1992)；以及 Presta 等人J. Immunol.，151:2623 (1993))；人成熟的 (體細胞突變) 骨架區域或人種系骨架區域 (參見例如，Almagro 及 Fransson，Front. Biosci.13:1619-1633 (2008))；以及來源於篩選 FR 文庫的骨架區域 (參見例如，Baca 等人，J. Biol. Chem.272:10678-10684 (1997)；以及 Rosok 等人，J. Biol. Chem.271:22611-22618 (1996))。4.人抗體Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best match" method (see, e.g., Sims et al.J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of a particular subset of light chain or heavy chain variable regions of human antibodies (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA , 89:4285 (1992); and Presta et al.J. Immunol. , 151:2623 (1993)); human mature (somatic cell mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson,Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al.,J. Biol. Chem. 272:10678-10684 (1997); and Rosok et al.,J. Biol. Chem. 271:22611-22618 (1996).4.Human antibodies

在某些情況下，本文所提供之抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子，諸如雙特異性或三特異性抗原結合分子) 為人類抗體。可使用此領域中所公知的各種技術生產人抗體。人抗體一般性描述於：van Dijk 及 van de Winkel，Curr. Opin. Pharmacol.5: 368-74 (2001)；及 Lonberg，Curr. Opin. Immunol.20:450-459 (2008)。In certain cases, the antigen binding molecules provided herein (e.g., monospecific and/or multispecific antigen binding molecules, such as bispecific or trispecific antigen binding molecules) are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in: van Dijk and van de Winkel,Curr. Opin. Pharmacol. 5: 368-74 (2001); and Lonberg,Curr. Opin. Immunol. 20: 450-459 (2008).

可透過對轉基因動物投予免疫原來製備人抗體，該轉基因動物已被修飾以回應於抗原攻擊而產生完整的人抗體或具有人可變區的完整抗體。此等動物通常包含全部或部分人免疫球蛋白基因座，其取代內源性免疫球蛋白基因座，或存在於染色體外或隨機整合到動物的染色體中。在此等轉基因小鼠中，內源性免疫球蛋白基因座通常已被滅活。有關從轉基因動物中獲得人抗體的方法的綜述，參見 Lonberg，Nat. Biotech.23:1117-1125 (2005)。另見例如，美國專利第 6,075,181 號及第 6,150,584 號 (描述了 XENOMOUSE^™技術)；美國專利第 5,770,429 號 (描述了 HUMAB® 技術)；美國專利第 7,041,870 號 (描述了 K-M MOUSE® 技術)；及美國專利申請公開號 US 2007/0061900 (描述了 VELOCIMOUSE® 技術)。由此等動物產生的來源於完整抗體的人可變區可被進一步修飾，例如透過與不同的人恆定區結合來修飾。Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic challenge. These animals typically contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or are present extrachromosomally or randomly integrated into the chromosomes of the animal. In these transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg,Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE^™ technology); U.S. Patent No. 5,770,429 (describing HUMAB® technology); U.S. Patent No. 7,041,870 (describing KM MOUSE® technology); and U.S. Patent Application Publication No. US 2007/0061900 (describing VELOCIMOUSE® technology). The human variable regions from intact antibodies generated by these animals can be further modified, for example, by combining with different human constant regions.

人抗體也可透過基於融合瘤的方法進行製備。用於生產人單株抗體的人骨髓瘤及小鼠-人异源骨髓瘤細胞株已有描述。(參見例如：KozborJ. Immunol.，133: 3001 (1984)；Brodeur 等人，Monoclonal Antibody Production Techniques and Applications，pp. 51-63 (Marcel Dekker，Inc.，New York，1987)；及 Boerner 等人，J. Immunol.，147: 86 (1991)。) 透過人 B 細胞融合瘤技術產生的人抗體也描述於 Li 等人，Proc. Natl. Acad. Sci. USA，103:3557-3562 (2006)。其他方法包括描述於例如以下文獻中的那些：美國專利號 7,189,826 (描述了由融合瘤細胞株生產單株人 IgM 抗體)，及 Ni，Xiandai Mianyixue，26(4):265-268 (2006) (描述了人-人融合瘤)。人融合瘤技術 (Trioma 技術) 也描述於以下文獻中：Vollmers 和 Brandlein，Histology and Histopathology，20(3):927-937 (2005)；及 Vollmers 和 Brandlein，Methods and Findings in Experimental and Clinical Pharmacology，27(3):185-91 (2005)。Human antibodies can also be prepared by fusion tumor-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, KozborJ. Immunol. , 133: 3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al.,J. Immunol ., 147: 86 (1991).) Human antibodies produced by human B cell fusion tumor technology are also described in Li et al.,Proc. Natl. Acad. Sci. USA , 103: 3557-3562 (2006). Other methods include those described in, for example, U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies by hybridoma cell lines), and Ni,Xiandai Mianyixue , 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in the following references: Vollmers and Brandlein,Histology and Histopathology , 20(3):927-937 (2005); and Vollmers and Brandlein,Methods and Findings in Experimental and Clinical Pharmacology , 27(3):185-91 (2005).

人抗體也可以藉由分離選自人源性噬菌體展示庫的 Fv 選殖株可變域序列來產生。然後可以將此等可變域序列與所需的人恆定域結合。下文描述了從抗體文庫中選擇人類抗體的技術。5.來源於文庫之抗體Human antibodies can also be generated by isolating variable domain sequences of Fv clones selected from human phage display libraries. These variable domain sequences can then be combined with the desired human constant domains. The following describes techniques for selecting human antibodies from antibody libraries.5.Antibodies from libraries

在某些實施例中，本文提供的抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子，諸如雙特異性或三特異性抗原結合分子) 可包括衍生自文庫的抗體。用於本發明之抗體可透過篩選組合文庫中具有所需之一種或多種活性的抗體來分離。例如，用於篩選組合文庫的方法綜述於例如 Lerner 等人Nature Reviews16:498-508 (2016) 中。例如，此領域中所公知的多種方法用於產生噬菌體展示庫並篩選此等庫中具有所需之結合特性的抗體。此等方法綜述於例如以下文獻中：Frenzel 等人，mAbs8:1177-1194 (2016)；Bazan 等人，Human Vaccines and Immunotherapeutics8:1817-1828 (2012)；及 Zhao 等人，Critical Reviews in Biotechnology36:276-289 (2016)；以及 Hoogenboom 等人，Methods in Molecular Biology178:1-37 (O'Brien 等人主編，Human Press，Totowa，NJ，2001)；以及 Marks 及 Bradbury 的Methods in Molecular Biology248:161-175 (Lo 主編，Human Press，Totowa，NJ，2003)。In certain embodiments, the antigen binding molecules provided herein (e.g., monospecific and/or multispecific antigen binding molecules, such as bispecific or trispecific antigen binding molecules) may include antibodies derived from a library. Antibodies used in the present invention can be isolated by screening combinatorial libraries for antibodies having one or more desired activities. For example, methods for screening combinatorial libraries are summarized in, for example, Lerner et al.Nature Reviews 16:498-508 (2016). For example, a variety of methods known in the art are used to generate phage display libraries and screen such libraries for antibodies having desired binding properties. Such methods are summarized in, for example, Frenzel et al.,mAbs 8:1177-1194 (2016); Bazan et al.,Human Vaccines and Immunotherapeutics 8:1817-1828 (2012); and Zhao et al.,Critical Reviews in Biotechnology 36:276-289 (2016); and Hoogenboom et al.,Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001); and Marks and Bradbury,Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

在某些噬菌體展示方法中，藉由聚合酶鏈反應 (PCR) 分別選殖 VH 及 VL 基因庫，並在噬菌體庫中隨機重組，然後可按照 Winter 等人於Ann. Rev. Immunol.12: 433-455 (1994) 中所述之方法篩選抗原結合噬菌體。噬菌體通常以單鏈 Fv (scFv) 片段或 Fab 片段展示抗體片段。來自免疫源的文庫無需構建融合瘤即可向免疫原提供高親和性抗體。替代性地，可以在不進行任何免疫作用的情況下選殖天然譜系 (例如，來自人) 以向各種非自身以及自身抗原提供抗體的單一來源，如 Griffiths 等人EMBO Journal12: 725-734 (1993) 中所述。最後，還可以透過選殖幹細胞中未重排的 V 基因片段，並使用包含隨機序列的 PCR 引子來編碼高變異性 CDR3 區域並在活體外完成重排，由此合成天然庫，如 Hoogenboom 及 Winter 在J. Mol. Biol.，227: 381-388 (1992) 中所述。描述人抗體噬菌體庫的專利公開包括例如：美國專利第 5,750,373 號、第 7,985,840 號、第 7,785,903 號及第 8,679,490 號；以及美國專利公開號 2005/0079574、2007/0117126、2007/0237764 及 2007/0292936。In certain phage display methods, VH and VL gene libraries are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, and then the antigen-binding phage can be screened according to the method described by Winter et al. inAnn. Rev. Immunol. 12: 433-455 (1994). Phage usually display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources can provide high-affinity antibodies to the immunogen without the need to construct fusion tumors. Alternatively, natural repertoires (e.g., from humans) can be cloned without any immunization to provide a single source of antibodies to a variety of non-self and self antigens, as described by Griffiths et al.EMBO Journal 12: 725-734 (1993). Finally, natural libraries can also be synthesized by cloning unrearranged V gene segments in stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and complete rearrangement invitro , as described by Hoogenboom and Winter inJ. Mol. Biol. , 227: 381-388 (1992). Patents describing human antibody phage libraries include, for example, U.S. Patent Nos. 5,750,373, 7,985,840, 7,785,903, and 8,679,490; and U.S. Patent Publication Nos. 2005/0079574, 2007/0117126, 2007/0237764, and 2007/0292936.

用於篩選組合文庫中具有所需活性之抗體的此領域中所公知方法的其他實例包括核醣體及 mRNA 展示以及用於細菌、哺乳動物細胞、昆蟲細胞或酵母細胞上的抗體展示及選擇的方法。酵母表面展示方法綜述於例如以下文獻中：Scholler 等人，Methods Mol. Biol.503:135-56 (2012)；及 Cherf 等人的Methods Mol. Biol.1319:155-175 (2015)；以及 Zhao 等人，Methods Mol. Biol.889:73-84 (2012)。核醣體展示方法描述於例如以下文獻中：He 等人，Nucleic Acids Research25:5132-5134 (1997)；及 Hanes 等人，Proc. Natl. Acad. Sci. USA94:4937-4942 (1997)。Other examples of methods known in the art for screening combinatorial libraries for antibodies with desired activity include ribosomal and mRNA display and methods for antibody display and selection on bacteria, mammalian cells, insect cells, or yeast cells. Yeast surface display methods are summarized in, for example, Scholler et al.,Methods Mol. Biol. 503:135-56 (2012); and Cherf et al.,Methods Mol. Biol. 1319:155-175 (2015); and Zhao et al.,Methods Mol. Biol. 889:73-84 (2012). Ribosome display methods are described, for example, in He et al.,Nucleic Acids Research 25:5132-5134 (1997); and Hanes et al.,Proc. Natl. Acad. Sci. USA 94:4937-4942 (1997).

從人抗體庫分離的抗體或抗體片段在本文中被視作人抗體或人抗體片段。6.抗原結合分子變異體Antibodies or antibody fragments isolated from human antibody libraries are referred to herein as human antibodies or human antibody fragments.6.Antigen Binding Molecule Variants

在某些情況下，考慮到本發明之抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 之胺基酸序列變異體。舉例而言，可能需要改良抗原結合分子之結合親和力及/或其他生物特性。抗原結合分子之胺基酸序列變異體可藉由將適合的修飾引入編碼抗原結合分子的核苷酸序列中或藉由肽合成來製備。此類修飾包括例如抗原結合分子之胺基酸序列中的殘基的缺失及/或插入及/或取代。可實施缺失、插入及取代之任意組合以得到最終構建體，前提條件是最終構建體具有所需之特徵，例如，抗原結合特徵。In some cases, amino acid sequence variants of the antigen binding molecules of the present invention (e.g., monospecific and/or multispecific antigen binding molecules such as bispecific or trispecific antigen binding molecules) are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antigen binding molecule. Amino acid sequence variants of antigen binding molecules can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antigen binding molecule or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of the antigen binding molecule. Any combination of deletions, insertions and substitutions may be implemented to obtain the final construct, provided that the final construct has the desired characteristics, for example, antigen binding characteristics.

在一些實施例中，本發明之抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 在 VH/VL 區及/或 CH1/CL 區中包含一個或多個修飾以促進正確的重鏈/輕鏈配對。在一些實施例中，本發明之抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 在 Fc 區中包含一個或多個修飾以促進抗原結合分子之兩個臂的異源二聚化。VH/VL 區、CH1/CL 區及/或 FC 區中的此類修飾描述於國際專利公開號 WO 2016/172485 中，其全文以引用方式併入本文中。a)取代、插入及刪除變異體In some embodiments, the antigen-binding molecules of the present invention (e.g., monospecific and/or multispecific antigen-binding molecules such as bispecific or trispecific antigen-binding molecules) comprise one or more modifications in the VH/VL region and/or CH1/CL region to promote correct heavy chain/light chain pairing. In some embodiments, the antigen-binding molecules of the present invention (e.g., monospecific and/or multispecific antigen-binding molecules such as bispecific or trispecific antigen-binding molecules) comprise one or more modifications in the Fc region to promote heterodimerization of the two arms of the antigen-binding molecule. Such modifications in the VH/VL region, CH1/CL region and/or FC region are described in International Patent Publication No. WO 2016/172485, which is incorporated herein by reference in its entirety.a)Substitution, Insertion and Deletion Variants

在某些情況下，提供具有一個或多個胺基酸取代的抗原結合域變異體。取代誘變的目標位點包括 CDR 及 FR。保留取代列於表 4 之「優選取代」標題下。表 4 中之「例示性取代」標題下提供了更多實質性變更，並且下文將參考胺基酸側鏈類別進行進一步描述。可將胺基酸取代引入所關注抗原結合分子中，並篩選具有所需活性之產物，舉例而言，保留/改善的抗原結合特徵、降低的免疫原性或改善的抗體依賴性細胞媒介的細胞毒性 (ADCC) 或補體依賴性細胞毒性 (CDC)。表4.例示性及優選胺基酸取代原始殘基例示性取代較佳取代Ala (A)Val；Leu；IleValArg (R)Lys；Gln；AsnLysAsn (N)Gln；His；Asp；Lys；ArgGlnAsp (D)Glu；AsnGluCys (C)Ser；AlaSerGln (Q)Asn；GluAsnGlu (E)Asp；GlnAspGly (G)AlaAlaHis (H)Asn；Gln；Lys；ArgArgIle (I)Leu；Val；Met；Ala；Phe；正白胺酸LeuLeu (L)正白胺酸；Ile；Val；Met；Ala；PheIleLys (K)Arg；Gln；AsnArgMet (M)Leu；Phe；IleLeuPhe (F)Trp；Leu；Val；Ile；Ala；TyrTyrPro (P)AlaAlaSer (S)ThrThrThr (T)Val；SerSerTrp (W)Tyr；PheTyrTyr (Y)Trp；Phe；Thr；SerPheVal (V)Ile；Leu；Met；Phe；Ala；正白胺酸Leu胺基酸可根據常見的側鏈特性進行分組： (1) 疏水性：正白胺酸，Met，Ala，Val，Leu，Ile； (2) 中性親水性：Cys、Ser、Thr、Asn、Gln； (3) 酸性：Asp，Glu； (4) 鹼性：His，Lys，Arg; (5) 影響鏈取向之殘基：Gly、Pro；或 (6) 芳香族：Trp，Tyr，Phe。In certain instances, antigen binding domain variants are provided having one or more amino acid substitutions. Target sites for substitution-induced mutagenesis include CDRs and FRs. Conservative substitutions are listed in Table 4 under the heading "Preferred Substitutions." More substantial changes are provided under the heading "Exemplary Substitutions" in Table 4 and are further described below with reference to amino acid side chain categories. Amino acid substitutions can be introduced into the antigen binding molecules of interest and the products screened for desired activity, for example, retained/improved antigen binding characteristics, reduced immunogenicity, or improved antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).Table4. Exemplary and preferred amino acid substitutionsOriginalResidueExemplarysubstitutionsBetterreplacement Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp; Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn；Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; nor-leucine Leu Leu (L) nor-leucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; nor-leucine Leu Amino acids can be grouped according to common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; or (6) aromatic: Trp, Tyr, Phe.

非保守取代需要將這些類別中之一類的成員交換為另一類的成員。Non-conservative substitutions entail exchanging a member of one of these classes for a member of another class.

一種類型的取代變異體涉及取代一個或多個親本抗原結合分子 (例如，人源化或人抗體) 之高度可變區殘基。一般而言，所選擇用於進一步研究的所產生之變異體與親代抗原結合分子相比將具有某些生物特性之修飾 (例如，改善) (例如，提高的親和力及/或、降低的免疫原性) 及/或將實質上保持親本抗原結合分子之某些生物特性。例示性取代變異體是親和力成熟的抗體，其可以方便地產生，例如，使用基於噬菌體展示的親和性成熟技術，例如本文所述的那些。簡言之，一個或多個 CDR/HVR 殘基發生突變，並且變異體抗體在噬菌體上展示並篩選出特定的生物活性 (例如，結合親和力)。One type of substitutional variant involves replacing one or more highly variable region residues of a parent antigen-binding molecule (e.g., a humanized or human antibody). In general, the resulting variants selected for further study will have modifications (e.g., improvements) of certain biological properties compared to the parent antigen-binding molecule (e.g., increased affinity and/or, reduced immunogenicity) and/or will substantially retain certain biological properties of the parent antigen-binding molecule. Exemplary substitutional variants are affinity-matured antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display, such as those described herein. In brief, one or more CDR/HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).

可以在 CDR/HVR 中進行更改 (例如，取代)，以改善抗體親和力。此類修改可以在 CDR/HVR「熱點」中進行，亦即由密碼子編碼的殘基在體細胞成熟過程中經歷高頻率突變 (參見例如，Chowdhury，Methods Mol. Biol.207:179-196 (2008)) 及/或與抗原接觸的殘基，並測試所得變異體 VH 或 VL 之結合親和力。藉由構建二級文庫且自其中重新選擇以實現親和力成熟已描述於例如 Hoogenboom 等人Methods Mol. Biol.178:1-37 (O'Brien 等人編，Human Press，Totowa，NJ (2001)) 中。在親和力成熟之某些實例中，藉由多種方法（例如，易錯 PCR、鏈改組或寡核苷酸定點突變）將多樣性引入選擇用於成熟的變異基因中。然後創建第二文庫。然後篩選該文庫，以識別具有所需之親和性的任何抗體變異體。引入多樣性的另一種方法涉及 CDR/HVR 定向方法，其中將若干 CDR/HVR 殘基 (例如，每次 4 至 6 個殘基) 隨機化。可例如使用丙胺酸掃描誘變或建模以特異性識別參與抗原結合的 CDR/HVR 殘基。特別地，CDR-H3 及 CDR-L3 經常成為靶點。Changes (e.g., substitutions) can be made in CDR/HVR to improve antibody affinity. Such modifications can be made in CDR/HVR "hotspots," i.e., residues encoded by codons that undergo high frequency mutation during somatic maturation (see, e.g., Chowdhury,Methods Mol. Biol. 207:179-196 (2008)) and/or residues that contact the antigen, and the resulting variant VH or VL tested for binding affinity. Affinity maturation by constructing secondary libraries and reselecting therefrom has been described, e.g., in Hoogenboom et al., Methods Mol. Biol. 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ (2001)). In certain examples of affinity maturation, diversity is introduced into the variant genes selected for maturation by a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide directed mutagenesis). A second library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR/HVR directed approach, in which a number of CDR/HVR residues (e.g., 4 to 6 residues at a time) are randomized. Alanine scanning mutagenesis or modeling can be used, for example, to specifically identify CDR/HVR residues involved in antigen binding. In particular, CDR-H3 and CDR-L3 are often targeted.

在某些情況下，一個或多個 CDR/HVR 內可存在取代、插入或缺失，只要此類變化不實質上降低抗原結合分子結合抗原之能力即可。舉例而言，可在 CDR/HVR 中實施基本上不降低結合親和力的保留修改 (例如，本文所提供之保留取代)。此等修改可例如在 CDR/HVR 中之抗原接觸殘基之外。在上文提供的變異體 VH 及 VL 序列的某些情況下，每個 CDR/HVR 保持不變或含有不超過一個、兩個或三個胺基酸取代。In some cases, substitutions, insertions or deletions may be present within one or more CDR/HVRs, as long as such changes do not substantially reduce the ability of the antigen-binding molecule to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be implemented in the CDR/HVRs. Such modifications may, for example, be outside of antigen-contacting residues in the CDR/HVRs. In some cases of the variant VH and VL sequences provided above, each CDR/HVR remains unchanged or contains no more than one, two or three amino acid substitutions.

如 Cunningham 及 WellsScience, 244:1081-1085 (1989) 所述，用於鑑別可能誘變的抗原結合分子殘基或區域的一種有用的方法稱為「丙胺酸掃描誘變」。在該方法中，鑑別殘基或標靶殘基組 (例如，帶電荷的殘基，諸如 Arg、Asp、His、Lys 及 Glu)，並用中性或帶負電荷之胺基酸 (例如，丙胺酸或聚丙胺酸) 取代以確定抗原結合分子與抗原之交互作用是否受到影響。可在胺基酸位置引入更多取代，表明對初始取代具有良好的功能敏感性。替代性地或另外地，抗原-抗原結合分子之晶體結構複合以鑑別抗原結合分子與抗原之間的接觸點。此等接觸殘基及鄰近殘基可靶向或消除為取代的候選物。可篩選變異體以確定它們是否含有所需之特性。As described in Cunningham and WellsScience , 244:1081-1085 (1989), a useful method for identifying residues or regions of antigen-binding molecules that may be induced is called "alanine scanning induction". In this method, residues or target residue groups (e.g., charged residues such as Arg, Asp, His, Lys and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antigen-binding molecule and the antigen is affected. More substitutions can be introduced at the amino acid position, indicating good functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antigen binding molecule is complexed to identify the contact points between the antigen-binding molecule and the antigen. These contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

胺基酸序列插入包括胺基及/或羧基末端融合體之長度，從一個殘基到包含一百個或更多殘基之多肽，以及單個或多個胺基酸殘基的序列內插入。末端插入的實例包括具有 N 端甲硫胺醯基殘基的抗原結合分子。抗原結合分子之其他插入變異體包括與抗原結合分子的 N 端或 C 端融合的酶 (例如，對於 ADEPT) 或提高抗原結合分子血清半衰期之多肽。b)醣基化變異體Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antigen binding molecules with an N-terminal methionyl residue. Other insertion variants of the antigen binding molecule include enzymes fused to the N-terminus or C-terminus of the antigen binding molecule (e.g., for ADEPT) or polypeptides that increase the serum half-life of the antigen binding molecule.b)Glycosylation variants

在某些情況下，可改變本發明之抗體以增加或減少抗體醣基化之程度。本發明之抗體中添加或缺失糖基化位點可藉由改變胺基酸序列以使得產生或去除一個或多個醣基化位點而方便地實現。In certain cases, the antibodies of the invention may be altered to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites in the antibodies of the invention may be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

當抗體包含 Fc 區域時，可改變與其相連的碳水化合物。由哺乳動物細胞產生的天然抗體通常包含分支的雙觸角寡醣，該寡醣通常藉由 N-鍵結附接至 Fc 區之 CH2 域的 Asn297。參見例如 Wright 等人，TIBTECH15:26-32 (1997)。寡醣可包括各種碳水化合物，例如甘露醣、N-乙醯基葡醣胺 (GlcNAc)、半乳醣及唾液酸以及在雙觸角寡醣結構之「莖」中附接至 GlcNAc 的岩藻醣。在一些情況下，可對本發明之抗體中的寡醣進行修飾，以產生具有某些改善之特性的變異體。When the antibody comprises an Fc region, the carbohydrates attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise branched bitactinic oligosaccharides that are typically attached to Asn297 of the CH2 domain of the Fc region by an N-bond. See, e.g., Wright et al.,TIBTECH 15:26-32 (1997). Oligosaccharides may include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the bitactinic oligosaccharide structure. In some cases, the oligosaccharides in the antibodies of the present invention may be modified to produce variants with certain improved properties.

在一個情況下，提供具有缺少岩藻醣的碳水化合物結構接附 (直接或間接地) 至 Fc 區的抗體變異體。例如，此等抗體中的岩藻醣含量可為 1% 至 80%、1% 至 65%、5% 至 65% 或 20% 至 40%。藉由計算 Asn297 糖鏈中岩藻糖的平均含量來測定岩藻糖相對於藉由 MALDI-TOF 質譜測得的連接至 Asn 297 的所有糖結構 (例如複雜型、雜合型及高甘露糖型) 的總和之含量，例如 WO 2008/077546 中所述。Asn297 係指位於 Fc 區域位置 297 附近之天冬醯胺酸殘基 (Fc 區域殘基的 EU 編號)；但是，Asn297 也可以位於位置 297 上游或下游大約 ±3 個胺基酸處，即由於抗體之微小序列變化而介於位置 294 及 300 之間。此類岩藻醣基化變異體可具有改善的 ADCC 功能。參見例如，美國專利公開號 US 2003/0157108 及 US 2004/0093621。與「去岩藻醣基化」或「岩藻醣缺乏」抗體變異體相關的出版物示例包括：US 2003/0157108；WO 2000/61739；WO 2001/29246；US 2003/0115614；US 2002/0164328；US 2004/0093621；US 2004/0132140；US 2004/0110704；US 2004/0110282；US 2004/0109865；WO 2003/085119；WO 2003/084570；WO 2005/035586；WO 2005/035778；WO2005/053742；WO2002/031140；Okazaki 等人，J. Mol. Biol.336:1239-1249 (2004)；Yamane-Ohnuki 等人，Biotech. Bioeng.87: 614 (2004)。能夠產生去岩藻醣基化抗體的細胞株的實例包括缺乏蛋白岩藻醣基化之 Lec13 CHO 細胞 (Ripka 等人Arch. Biochem. Biophys.249:533-545 (1986)；美國專利申請號 US 2003/0157108 A1；及 Adams 等人 WO 2004/056312 A1，尤其是在實例 11 中)，及剔除細胞株，諸如剔除 α-1,6-岩藻醣基化轉移酶基因FUT8的 CHO 細胞 (參見例如，Yamane-Ohnuki 等人，Biotech. Bioeng.87: 614 (2004)；Kanda, Y. 等人，Biotechnol. Bioeng, 94(4):680-688 (2006)；及 WO2003/085107)。在一些實例中，本文所述之任何抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子 (例如，抗體)) 可以為無岩藻醣基化的。In one embodiment, antibody variants are provided that have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the fucose content in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The content of fucose relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose) attached to Asn 297 as measured by MALDI-TOF mass spectrometry is determined by calculating the average content of fucose in the Asn297 sugar chain, such as described in WO 2008/077546. Asn297 refers to the asparagine residue located near position 297 of the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300 due to minor sequence variations of the antibody. Such fucosylated variants may have improved ADCC function. See, e.g., U.S. Patent Publication Nos. US 2003/0157108 and US 2004/0093621. Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al.,J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al.,Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003/0157108 A1; and Adams et al. WO 2004/056312 A1, particularly in Example 11), and knockout cell lines, such as CHO cells knocked out for the α-1,6-fucosyltransferase geneFUT8 (see, e.g., Yamane-Ohnuki et al.,Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al.,Biotechnol. Bioeng , 94(4):680-688). (2006); and WO2003/085107). In some embodiments, any antigen-binding molecule described herein (e.g., monospecific and/or multispecific antigen-binding molecules such as bispecific or trispecific antigen-binding molecules (e.g., antibodies)) may be afucosylated.

進一步提供具有二分支寡醣的抗體變異體，例如，其中接附至抗體之 Fc 區的雙觸角寡醣被 GlcNAc 平分。此類抗體變異體可具有減少的岩藻醣基化及/或改善的 ADCC 功能。此等抗體變異體的實例描述於例：WO 2003/011878；美國第 6,602,684 號專利；及 US 2005/0123546。亦提供了在寡醣上具有至少一個連接至 Fc 區域之半乳糖殘基的抗體變異體。此等抗體變異體可具有改善的 CDC 功能。此等抗體變異體描述於例如 WO 1997/30087、WO 1998/58964 及 WO 1999/22764 中。c)   Fc區域變異體Further provided are antibody variants having bi-branched oligosaccharides, for example, wherein the bi-antennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described in, for example: WO 2003/011878; U.S. Patent No. 6,602,684; and US 2005/0123546. Antibody variants having at least one galactose residue attached to the Fc region on the oligosaccharide are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087, WO 1998/58964 and WO 1999/22764.c) Fcregion variants

在某些情況下，可在本發明之抗體的 Fc 區中引入一個或多個胺基酸修飾，從而產生 Fc 區變異體。Fc 區域變體可包含人 Fc 區域序列 (例如，人 IgG1、IgG2、IgG3 或 IgG4 Fc 區域)，其在一個或多個胺基酸位置包含胺基酸修飾 (例如，取代)。In certain cases, one or more amino acid modifications may be introduced into the Fc region of an antibody of the invention, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

在某些情況下，本發明涉及具有一些但不是全部效應功能的抗體變異體，這使其成為應用 (其中抗體的活體內半衰期很重要而某些效應功能 (諸如補體及 ADCC) 為不必要或有害的) 的理想候選藥物。可實施活體外及/或活體內細胞毒性測定，以確認 CDC 及/或 ADCC 活性之下降/耗竭。舉例而言，可實施 Fc 受體 (FcR) 結合測定以確保抗體缺乏 FcγR 結合 (因此可能缺乏 ADCC 活性)，但保留 FcRn 結合能力。介導 ADCC 的主要細胞 NK 細胞僅表現 FcγRIII，而單核細胞表現 FcγRI、FcγRII 及 FcγRIII。FcR 在造血細胞上之表現匯總於 Ravetch 及 Kinet 的論文 (Annu. Rev. Immunol.9:457-492 (1991)) 之第 464 頁的表 3 中。用於評定所關注分子之 ADCC 活性的或活體外測定的非限制性實例描述於美國專利第 5,500,362 號中 (參見例如，Hellstrom, I. 等人，Proc. Natl. Acad. Sci. USA83:7059-7063 (1986))；及 Hellstrom, I 等人，Proc. Natl. Acad. Sci. USA82:1499-1502 (1985)；美國專利第 5,821,337 號 (參見 Bruggemann, M. 等人，J. Exp. Med.166:1351-1361 (1987))。替代性地，可採用非放射性測定 (參見例如，用於流式細胞分析技術的 ACTI™ 非放射性細胞毒性測定 (CellTechnology, Inc. Mountain View, CA；及 CYTOTOX 96® 非放射性細胞毒性測定 (Promega, Madison, WI)))。用於此等測定的有用的效應細胞包括外周血單核細胞 (PBMC) 及自然殺手 (NK) 細胞。替代性地或另外地，可在例如 Clynes 等人在Proc. Natl. Acad. Sci. USA95:652-656 (1998) 中揭示的動物模型中在活體內評定所關注分子之 ADCC 活性。還可實施 C1q 結合測定以確認該抗體無法結合 C1q 並因此缺乏 CDC 活性。參見例如 WO 2006/029879 及 WO 2005/100402 中的 C1q 及 C3c 結合 ELISA。為了評定補體活化，可以進行 CDC 測定 (參見例如，Gazzano-Santoro 等人，J. Immunol. Methods202:163 (1996)；Cragg 等人，Blood.101:1045-1052 (2003)；及 Cragg 等人，Blood.103:2738-2743 (2004))。FcRn 結合及活體內清除率/半衰期測定亦可使用此領域中己知的方法進行 (參見例如，Petkova 等人，Int'l. Immunol.18(12):1759-1769 (2006))。In certain instances, the invention relates to antibody variants that possess some but not all effector functions, making them ideal drug candidates for applications where the in vivo half-life of the antibody is important and certain effector functions (such as complement and ADCC) are unnecessary or detrimental.In vitro and/orin vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. The primary cells that mediate ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of the paper by Ravetch and Kinet (Annu. Rev. Immunol. 9:457-492 (1991)). Non-limiting examples of in vitro or in vitro assays for assessing ADCC activity of molecules of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al.,Proc. Natl. Acad. Sci. USA 83:7059-7063 (1986)); and Hellstrom, I et al.,Proc. Natl. Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al.,J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be employed (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA; and CYTOTOX 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3c binding ELISAs. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996); Cragg et al.,Blood. 101:1045-1052 (2003); and Cragg et al.,Blood. 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova et al.,Int'l. Immunol. 18(12):1759-1769 (2006)).

效用功能下降的抗體包括一個或多個 Fc 區域殘基 238、265、269、270、297、327 及 329 被取代之抗體 (美國專利號 6,737,056 及 8,219,149)。此等 Fc 突變體包括在胺基酸位置 265、269、270、297 及 327 中的兩個或更多個位置具有取代的 Fc 突變體，包括所謂的「DANA」 Fc 突變體，其中殘基 265 及 297 被丙胺酸取代（美國專利第 7,332,581 號及第 8,219,149 號）。Antibodies with reduced efficacy include those in which one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (U.S. Patent Nos. 6,737,056 and 8,219,149). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (U.S. Patent Nos. 7,332,581 and 8,219,149).

描述了某些與 FcR 之結合得到改善或減弱的抗體變異體。參見例如，美國專利第 6,737,056 號；第 WO 2004/056312 號及Shields 等人，J. Biol. Chem.9(2): 6591-6604 (2001)。Certain antibody variants with improved or reduced binding to FcRs are described. See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312 and Shields et al.,J. Biol. Chem. 9(2): 6591-6604 (2001).

在某些情況下，抗體變異體包含具有一個或多個胺基酸取代的 Fc 區，這些取代改善了 ADCC，例如 Fc 區的位置 298、333 及/或 334 (殘基的 EU 編號) 處之取代。In certain instances, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333 and/or 334 (EU numbering of residues) of the Fc region.

在某些方面，抗體變異體包含具有一個或多個胺基酸取代的 Fc 區域，這些取代減弱了 FcγR 結合，例如 Fc 區域的位置 234 及 235 (殘基的 EU 編號) 處之取代。在一個態樣中，取代為 L234A 及 L235A (LALA)。在某些態樣中，抗體變異體進一步包含 Fc 區中之 D265A 及/或 P329G，其來源於人 IgG₁Fc 區。在一個態樣中，取代為 Fc 區域中的 L234A、L235A 及 P329G (LALA-PG)，其來源於人 IgG₁Fc 區域。例如參見 WO 2012/130831。在另一態樣中，取代為 Fc 區域中的 L234A、L235A 及 D265A (LALA-DA)，其來源於人 IgG₁Fc 區域。In certain aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcγR binding, such as substitutions at positions 234 and 235 (EU numbering of residues) of the Fc region. In one aspect, the substitutions are L234A and L235A (LALA). In certain aspects, the antibody variant further comprises D265A and/or P329G in the Fc region, which are derived from a human IgG₁ Fc region. In one aspect, the substitutions are L234A, L235A, and P329G (LALA-PG) in the Fc region, which are derived from a human IgG₁ Fc region. See, for example, WO 2012/130831. In another aspect, the substitutions are L234A, L235A and D265A (LALA-DA) in the Fc region, which is derived from the human IgG₁ Fc region.

在一些情況下，在 Fc 區中進行修改，得到修改 (亦即，改善或減少) 之 C1q 結合及/或 CDC，例如，美國專利第 6,194,551 號、WO 99/51642 及 Idusogie 等人J. Immunol.164: 4178-4184 (2000) 中所述。In some cases, modifications are made in the Fc region resulting in modified (ie, improved or reduced) C1q binding and/or CDC, as described, for example, in U.S. Patent No. 6,194,551, WO 99/51642, and Idusogie et al.J. Immunol. 164: 4178-4184 (2000).

具有更長半衰期並改善了與新生兒 Fc 受體 (FcRn) (其負責將母體 IgG 轉移給胎兒，見 Guyer 等人J. Immunol.117: 587 (1976) 及 Kim 等人J. Immunol.24: 249 (1994)) 之結合的抗體描述於 US2005/0014934 (Hinton 等人) 中。那些抗體包含其中具有一個或多個取代之 Fc 區域，其改善了 Fc 區域與 FcRn 之結合。此類 Fc 變異體包括在一個或多個 Fc 區域殘基上發生取代之 Fc 變異體：238、252、254、256、265、272、286、303、305、307、311、312、317、340、356、360、362、376、378、380、382、413、424 或 434，例如 Fc 區殘基 434 之取代 (參見例如，美國專利第 7,371,826 號；Dall'Acqua, W.F. 等人，J. Biol. Chem.281:23514-23524(2006))。Antibodies with longer half-lives and improved binding to the neonatal Fc receptor (FcRn) (which is responsible for the transfer of maternal IgG to the fetus, see Guyer et al. J. Immunol. 117: 587 (1976) and Kim et al. J. Immunol. 24: 249 (1994)) are described in US2005/0014934 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, such as substitution of Fc region residue 434 (see, e.g., U.S. Pat. No. 7,371,826; Dall'Acqua, WF et al.,J. Biol. Chem. 281:23514-23524 (2006)).

藉由定點誘變已經識別出對小鼠 Fc-小鼠 FcRn 交互作用至關重要之 Fc 區殘基 (參見例如，Dall'Acqua 等人，J. Immunol.169:5171-5180 (2002))。殘基 I253、H310、H433、N434 及 H435 (殘基的 EU 編號) 參與交互作用 (Medesan 等人，Eur. J. Immunol.26:2533 (1996)；Firan 等人，Int. Immunol.13:993 (2001)；Kim 等人，Eur. J. Immunol.24:542 (1994))。已發現殘基 I253、H310 及 H435 對於人 Fc 與小鼠 FcRn 之交互作用至關重要 (Kim, J.K. 等人，Eur. J. Immunol.29:2819 (1999))。對人 Fc-人 FcRn 複合體的研究表明，殘基 I253、S254、H435 及 Y436 對於交互作用至關重要 (Firan 等人，Int. Immunol.13:993 (2001)；Shields 等人，J. Biol. Chem.276:6591-6604 (2001))。在 Yeung 等人 (J. Immunol.182:7667-7671 (2009)) 中，已經報導並研究了殘基 248 至 259 及 301 至 317 及 376 至 382 及 424 至 437 的各種突變體。Fc region residues that are critical for mouse Fc-mouse FcRn interactions have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua et al.,J. Immunol. 169:5171-5180 (2002)). Residues I253, H310, H433, N434, and H435 (EU numbering of residues) are involved in the interaction (Medesan et al.,Eur. J. Immunol. 26:2533 (1996); Firan et al.,Int. Immunol. 13:993 (2001); Kim et al.,Eur. J. Immunol. 24:542 (1994)). Residues I253, H310, and H435 have been found to be critical for the interaction of human Fc with mouse FcRn (Kim, JK et al.,Eur. J. Immunol. 29:2819 (1999)). Studies on the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are critical for the interaction (Firan et al.,Int. Immunol. 13:993 (2001); Shields et al.,J. Biol. Chem. 276:6591-6604 (2001)). In Yeung et al. (J. Immunol. 182:7667-7671 (2009)), various mutants at residues 248 to 259, 301 to 317, 376 to 382, and 424 to 437 have been reported and studied.

在某些方面，抗體變異體包含具有一個或多個胺基酸取代的 Fc 區域，這些取代減少 FcRn 結合，例如 Fc 區域之位置 253、及/或 310、及/或 435 (殘基的 EU 編號) 處之取代。在某些態樣中，抗體變異體包含 Fc 區域，該 Fc 區域具有在位置 253、310 及 435 處之胺基酸取代。在一個態樣中，取代為 Fc 區域中之 I253A、H310A 及 H435A，其來源於人 IgG1 Fc 區域。參見例如，Grevys 等人，J. Immunol.194: 5497-5508 (2015)。In certain aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, such as substitutions at positions 253, and/or 310, and/or 435 (EU numbering of residues) of the Fc region. In certain aspects, the antibody variant comprises an Fc region having amino acid substitutions at positions 253, 310, and 435. In one aspect, the substitutions are I253A, H310A, and H435A in the Fc region, which are derived from a human IgG1 Fc region. See, e.g., Grevys et al.,J. Immunol. 194: 5497-5508 (2015).

在某些方面，抗體變異體包含具有一個或多個胺基酸取代的 Fc 區域，這些取代減少 FcRn 結合，例如 Fc 區域之位置 310、及/或 433、及/或 436 (殘基的 EU 編號) 處之取代。在某些態樣中，抗體變異體包含 Fc 區域，該 Fc 區域具有在位置 310、433 及 436 處之胺基酸取代。在一個態樣中，取代為 Fc 區域中之 H310A、H433A 及 Y436A，其來源於人 IgG1 Fc 區域。參見例如 WO 2014/177460。In certain aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, such as substitutions at positions 310, and/or 433, and/or 436 (EU numbering of residues) of the Fc region. In certain aspects, the antibody variant comprises an Fc region having amino acid substitutions at positions 310, 433, and 436. In one aspect, the substitutions are H310A, H433A, and Y436A in the Fc region, which are derived from a human IgG1 Fc region. See, e.g., WO 2014/177460.

在某些態樣中，抗體變異體包含具有一個或多個胺基酸取代的 Fc 區域，這些取代增加 FcRn 結合，例如 Fc 區域之位置 252、及/或 254、及/或 256 (殘基的 EU 編號) 處之取代。在某些態樣中，抗體變異體包含 Fc 區域，該 Fc 區域具有在位置 252、254 及 256 處之胺基酸取代。在一個態樣中，取代為 Fc 區域中之 M252Y、S254T 及 T256E，其來源於人 IgG₁Fc 區域。亦參見 Duncan 及 Winter，Nature322:738-40 (1988)；美國專利第 5,648,260 號；美國專利第 5,624,821 號；及 WO 94/29351，其中涉及 Fc 區變異體的其他實例。d)半胱胺酸工程化抗體變異體In certain aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, such as substitutions at positions 252, and/or 254, and/or 256 (EU numbering of residues) of the Fc region. In certain aspects, the antibody variant comprises an Fc region having amino acid substitutions at positions 252, 254, and 256. In one aspect, the substitutions are M252Y, S254T, and T256E in the Fc region, which are derived from a human IgG₁ Fc region. See also Duncan and Winter,Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 for other examples of Fc region variants.d)Cysteine Engineered Antibody Variants

在某些情況下，可能希望創建胱胺酸工程化抗體，例如「thioMAb」，其中抗體之一個或多個殘基被半胱胺酸殘基取代。在特定實例中，取代殘基出現在抗體之可進入的位點。透過用半胱胺酸取代那些殘基，反應性硫醇基團由此被定位在抗體之可進入的位點，並可用於使抗體與其他部分 (例如藥物部分或連接子-藥物部分) 結合，以形成免疫結合物，如本文進一步所述。在某些情況下，以下任何一個或多個殘基可被半胱胺酸取代：輕鏈的 V205 (Kabat 編號)；重鏈的 A118 (EU 編號)；及重鏈 Fc 區的 S400 (EU 編號)。可例如美國專利第 7,521,541 號中所述產生經半胱胺酸工程化改造之抗體，該美國專利全文以引用方式併入本文中。e)抗體衍生物In certain instances, it may be desirable to create a cystine engineered antibody, e.g., a "thioMAb," in which one or more residues of the antibody are replaced with cysteine residues. In particular instances, the replacement residues occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties (e.g., a drug moiety or a linker-drug moiety) to form an immunoconjugate, as further described herein. In certain instances, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the Fc region of the heavy chain. Cysteine engineered antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541, which is incorporated herein by reference in its entirety.e)Antibody Derivatives

在某些情況下，可進一步修飾本文所提供之抗原結合分子，以使其含有此領域中已知且容易獲得的額外非蛋白質部分。適用於抗體之衍生化的部分包括但不限於水溶性聚合物。水溶性聚合物之非限制性實例包括但不限於聚乙二醇 (PEG)、乙二醇/丙二醇共聚物、羧甲基纖維素、葡聚醣、聚乙烯醇、聚乙烯基吡咯啶酮、聚-1,3-二氧戊環、聚-1,3,6-三㗁𠮿、乙烯/馬來酸酐共聚物、聚胺基酸 (均聚物或隨機共聚物) 以及葡聚醣或聚(n-乙烯基吡咯啶酮)聚乙二醇、丙二醇均聚物、聚環氧丙烷/環氧乙烷共聚物、聚氧乙烯化多元醇 (例如甘油)、聚乙烯醇及其混合物。聚乙二醇丙醛由於其水中之穩定性而可能在製造中具有優勢。該聚合物可具有任何分子量，且可聚支鏈或無支鏈。連接至抗體的聚合物之數量可以變化，並且如果連接的聚合物超過一種，則它們可以為相同或不同之分子。通常，用於衍生化的聚合物之數量及/或類型可基於以下考慮因素來確定，這些考慮因素包括但不限於待改善之抗體的特定性質或功能、抗體衍生物是否將用於指定條件下的治療中等。In some cases, the antigen binding molecules provided herein may be further modified to contain additional non-protein moieties known in the art and readily available. Suitable derivatized moieties for antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-triazine, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the amount and/or type of polymer used for derivatization may be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under specified conditions, etc.

在另一情況下，提供可藉由暴露於輻射而選擇性加熱之抗原結合分子及非蛋白質部分的結合體。在一種情況下，非蛋白質部分為碳奈米管 (Kam 等人，Proc. Natl. Acad. Sci. USA102:11600-11605 (2005))。輻射可具有任何波長，並且包括但不限於不損害普通細胞但將非蛋白質性部分加熱至接近抗原結合分子-非蛋白質性部分的細胞被毒殺之溫度的波長。f)免疫結合物In another aspect, a combination of an antigen binding molecule and a non-protein portion is provided that can be selectively heated by exposure to radiation. In one aspect, the non-protein portion is a carbon nanotube (Kam et al.,Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, a wavelength that does not damage normal cells but heats the non-protein portion to a temperature close to that at which the cells of the antigen binding molecule-non-protein portion are killed.f)Immunoconjugate

本發明亦提供包含抗原結合分子的免疫結合物，該抗原結合分子結合至一種或多種細胞毒性劑，諸如化學治療劑或藥物、生長抑制劑、毒素 (例如，蛋白毒素、來源於細菌、真菌、植物或動物之酶活性毒素、或其片段) 或放射性同位素。The invention also provides immunoconjugates comprising an antigen binding molecule conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins derived from bacteria, fungi, plants or animals, or fragments thereof), or radioactive isotopes.

在一種情況下，免疫結合物為抗體-藥物結合物 (ADC)，其中抗體結合至一種或多種藥物，該一種或多種藥物包括但不限於美登木素生物鹼 (參見美國專利第 5,208,020 號及第 5,416,064 號及歐洲專利 EP 0 425 235 B1)；澳瑞他汀諸如單甲基澳瑞他汀藥物部分 DE 及 DF (MMAE 及 MMAF) (參見美國專利第 5,635,483 號、第 5,780,588 號及第 7,498,298 號)；尾海兔素；加利車黴素 (calicheamicin) 或其衍生物 (參見美國專利第 5,712,374 號、第 5,714,586 號、第 5,739,116 號、第 5,767,285 號、第 5,770,701 號、第 5,770,710 號、第 5,773,001 號及第 5,877,296 號；Hinman 等人,Cancer Res.53:3336-3342 (1993)；及 Lode 等人，Cancer Res.58:2925-2928 (1998))；蒽環類藥物，諸如道諾黴素或多柔比星 (參見 Kratz 等人，Current Med. Chem.13:477-523 (2006)；Jeffrey 等人，Bioorganic & Med. Chem. Letters16:358-362 (2006)；Torgov 等人，Bioconj. Chem.16:717-721 (2005)；Nagy 等人，Proc. Natl. Acad. Sci. USA97:829-834 (2000)；Dubowchik 等人，Bioorg. & Med. Chem. Letters12:1529-1532 (2002)；King 等人，J. Med. Chem.45:4336-4343 (2002)；及美國專利第 6,630,579 號)；胺甲蝶呤；長春地辛；紫杉烷類，諸如多西他賽、紫杉醇、拉洛紫杉醇、特賽紫杉醇及奧他紫杉醇；單端孢黴烯；及 CC1065。In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020 and 5,416,064 and European Patent EP 0 425 235 B1); auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588 and 7,498,298); urocystin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586 and 7,498,298); , 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al.,Cancer Res. 53:3336-3342 (1993); and Lode et al.,Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunorubicin or doxorubicin (see Kratz et al.,Current Med. Chem. 13:477-523 (2006); Jeffrey et al.,Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al.,Bioconj. Chem. 16:717-721 (2005); Nagy et al.,Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al.,J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, lalotaxel, tasitaxel, and ortataxel; trichothecenes; and CC1065.

在另一情況下，免疫結合物包含與酶活性毒素或其片段結合之抗原結合分子，該酶活性毒素或其片段包括但不限於白喉 A 鏈、白喉毒素之非結合活性片段、外毒素 A 鏈 (來源於銅綠假單胞菌 (Pseudomonas aeruginosa))、蓖麻毒蛋白 A 鏈、相思子毒素 A 鏈、莫迪素 A 鏈 (modeccin A chain)、α-八疊球菌、油桐 (Aleurites fordii) 蛋白、香石竹毒蛋白、美洲商陸 (Phytolaca americana) 蛋白 (PAPI、PAPII 及 PAP-S)、苦瓜 (Momordica charantia) 抑制劑、瀉果素 (curcin)、巴豆毒素 (crotin)、肥皂草 (Sapaonaria officinalis) 抑制劑、白樹毒素 (gelonin)、米托菌素 (mitogellin)、局限曲菌素 (restrictocin)、酚黴素 (phenomycin)、伊諾黴素 (enomycin) 及新月毒素。In another aspect, the immunoconjugate comprises an antigen binding molecule bound to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, α-octadecene,Aleurites fordii proteins, caryophyllein proteins,Phytolaca americana proteins (PAPI, PAPII and PAP-S),Momordica charantia inhibitor, curcin, crotin,Sapaonaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin and crescent toxin.

在另一情況下，免疫結合物包含結合至放射性原子以形成放射性結合物的如本文所述之抗原結合分子。多種放射性同位素可用於產生放射性結合物。實例包括 At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²及 Lu 之放射性同位素。當放射性結合物用於檢測時，它可能包含用於閃爍顯像研究之放射性原子，例如 tc99m 或 I¹²³，或用於核磁共振 (NMR) 成像 (亦稱為磁共振成像，MRI) 之自旋標記物，諸如碘-123、碘-131、銦-111、氟-19、碳-13、氮-15、氧-17、釓、錳或鐵。In another embodiment, the immunoconjugate comprises an antigen binding molecule as described herein bound to a radioactive atom to form a radioactive conjugate. A variety of radioactive isotopes can be used to produce radioactive conjugates. Examples include radioactive isotopes of At²¹¹ , I¹³¹ , I¹²⁵ , Y⁹⁰ , Re¹⁸⁶ , Re¹⁸⁸ , Sm¹⁵³ , Bi²¹² , P³² , Pb²¹² and Lu. When a radioactive conjugate is used for detection, it may contain radioactive atoms such as tc99m or I¹²³ for scintillation imaging studies, or spin labels such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

抗原結合分子與細胞毒性劑之複合體可使用多種雙功能蛋白偶聯劑進行製備，該雙功能蛋白偶聯劑為諸如 N-琥珀醯亞胺基-3-(2-吡啶基二硫代)丙酸酯 (SPDP)、琥珀醯亞胺基-4-(N-馬來醯亞胺基甲基)環己烷-1-甲酸酯 (SMCC)、亞胺基硫烷 (IT)、亞胺基酸酯的雙功能衍生物 (諸如己二酸二甲酯鹽酸鹽 (HCl))、活性酯 (諸如雙琥珀醯亞胺辛二酸)、醛 (諸如戊二醛)、雙疊氮化合物 (諸如雙(對疊氮基苯甲醯基)己二胺)、雙重氮衍生物 (諸如雙-(對重氮苯甲醯基)-乙二胺)、二異氰酸酯 (諸如 2,6-二異氰酸甲苯酯) 及雙活性氟化合物 (諸如 1,5-二氟-2,4-二硝基苯)。舉例而言，蓖麻毒蛋白免疫毒素可如 Vitetta 等人，Science238:1098 (1987) 中所闡述進行製備。用於將放射性核苷酸結合至抗體的一種例示性螯合劑為碳-14 標記的 1-異硫氰酸芐基-3-甲基二亞乙基三胺五乙酸 (MX-DTPA)。參見 WO94/11026。連接子可以為促進細胞中細胞毒性藥物釋放的「可切割連接子」。例如，可使用酸不穩定之連接子、對肽酶敏感之連接子、光不穩定之連接子、二甲基連接子或含雙硫鍵之連接子 (Chari 等人，Cancer Res.52:127-131 (1992)；美國專利第 5,208,020 號)。The complex of the antigen binding molecule and the cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), iminothiolane (IT), Bifunctional derivatives of esters of amino acids (such as dimethyl adipate hydrochloride (HCl)), active esters (such as bissuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazonium compounds (such as bis(p-azoniumbenzyl)hexanediamine), bisdiazonium derivatives (such as bis-(p-diazoniumbenzyl)-ethylenediamine), diisocyanates (such as 2,6-diisocyanatotoluene) and bisactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al.,Science 238:1098 (1987). An exemplary chelator for conjugating radionucleotides to antibodies is carbon-14 labeled 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA). See WO94/11026. The linker can be a "cleavable linker" that promotes release of the cytotoxic drug in cells. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide bond-containing linker can be used (Chari et al.,Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

本文之免疫結合物或 ADC 明確考慮但不限於此等用交聯劑製得之複合體，該交聯劑包括但不限於可商購獲得 (例如自 Pierce Biotechnology, Inc. (Rockford, IL., U.S.A) 商購獲得) 之 BMPS、EMCS、GMBS、HBVS、LC-SMCC、MBS、MPBH、SBAP、SIA、SIAB、SMCC、SMPB、SMPH、磺基-EMCS、磺基-GMBS、磺基-KMUS、磺基-MBS、磺基-SIAB、磺基-SMCC、磺基-SMPB 及 SVSB (琥珀醯亞胺基-(4-乙烯碸)苯甲酸酯)。III.重組方法及組成物The immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such complexes made with crosslinking agents, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfonate)benzoate) commercially available (e.g., commercially available from Pierce Biotechnology, Inc. (Rockford, IL., USA).III.Recombination Methods and Compositions

本發明之抗原結合分子可使用重組方法及組成物來產生，例如，如美國專利第 4,816,567 號及美國專利申請公開號 2013/0078249 中所述，其全文以引用方式併入本文中。在一個實施例中，提供編碼本文所述之抗原結合分子的經分離核酸 (例如，多核苷酸)。此類核酸可編碼包含雙特異性抗原結合分子的 VL 之胺基酸序列及/或包含雙特異性抗原結合分子的 VH 之胺基酸序列 (例如，雙特異性抗原結合分子之任一臂的輕鏈及/或重鏈)。在另一實施例中，提供一個或多個包含此類核酸之載體 (例如，表現載體)。The antigen binding molecules of the present invention can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567 and U.S. Patent Application Publication No. 2013/0078249, which are incorporated herein by reference in their entirety. In one embodiment, an isolated nucleic acid (e.g., a polynucleotide) encoding an antigen binding molecule described herein is provided. Such nucleic acids can encode an amino acid sequence comprising the VL of a bispecific antigen binding molecule and/or an amino acid sequence comprising the VH of a bispecific antigen binding molecule (e.g., a light chain and/or a heavy chain of either arm of a bispecific antigen binding molecule). In another embodiment, one or more vectors (e.g., an expression vector) comprising such nucleic acids are provided.

編碼本發明之抗原結合分子的多核苷酸可表現為單個多核苷酸分子或表現為共表現的多個 (例如，兩個或更多個) 多核苷酸。共表現的由多核苷酸編碼的多肽可透過例如二硫鍵或其他方式締合以形成功能性雙特異性抗原結合分子。舉例而言，抗原結合分子之輕鏈部分可由與抗原結合分子之部分分開的多核苷酸編碼，該抗原結合分子包含抗原結合部分的重鏈部分、Fc 域次單元及視情況選用的另一抗原結合域。當共表現時，重鏈多肽將與輕鏈多肽締合以形成抗原結合分子。在另一實例中，包含兩個 Fc 域次單元中之一者及視情況選用的一個或多個抗原結合域的抗原結合分子的部分可由與包含兩個 Fc 域次單元中之另一者及視情況選用的抗原結合域的 T 細胞活化抗原結合分子的部分不同的多核苷酸進行編碼。當共表現時，Fc 域次單元將締合以形成 Fc 域。The polynucleotides encoding the antigen binding molecules of the present invention may be expressed as a single polynucleotide molecule or as a plurality (e.g., two or more) of co-expressed polynucleotides. The co-expressed polypeptides encoded by the polynucleotides may be associated, for example, by disulfide bonds or other means to form a functional bispecific antigen binding molecule. For example, the light chain portion of the antigen binding molecule may be encoded by a polynucleotide separate from a portion of the antigen binding molecule comprising the heavy chain portion of the antigen binding portion, an Fc domain subunit, and optionally another antigen binding domain. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form the antigen binding molecule. In another example, the portion of the antigen binding molecule comprising one of the two Fc domain subunits and optionally one or more antigen binding domains may be encoded by a different polynucleotide than the portion of the T cell activating antigen binding molecule comprising the other of the two Fc domain subunits and optionally an antigen binding domain. When co-expressed, the Fc domain subunits will associate to form an Fc domain.

如果是天然抗體或天然抗體片段，則需要兩個核酸，一個用於輕鏈或其片段，且另一個用於重鏈或其片段。此等核酸編碼包含 VL 之胺基酸序列及/或包含抗體的 VH 的胺基酸序列 (例如，抗體之輕鏈及/或重鏈)。這些核酸可以在同一表現載體上，也可以在不同表現載體上。In the case of natural antibodies or natural antibody fragments, two nucleic acids are required, one for the light chain or its fragment and the other for the heavy chain or its fragment. These nucleic acids encode the amino acid sequence comprising the VL and/or the amino acid sequence comprising the VH of the antibody (e.g., the light chain and/or the heavy chain of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

如果是具有異二聚體重鏈的雙特異性抗體，需要四個核酸，一個用於第一輕鏈，一個用於第一重鏈 (其包含第一異源單體 Fc 區多肽)，一個用於第二輕鏈，且一個用於第二重鏈 (其包含第二異源單體 Fc 區多肽)。這四個核酸可包含在一個或多個核酸分子或表現載體中。此等核酸編碼包含第一 VL 之胺基酸序列、及/或包含第一 VH (包括第一異源 Fc 區域) 之胺基酸序列、及/或包含第二 VL 之胺基酸序列、及/或包含第二 VH (包括抗體之第二異源 Fc 區域) 之胺基酸序列 (例如，抗體之第一及/或第二輕鏈、及/或第一及/或第二重鏈)。這些核酸可以在同一表現載體上，也可以在不同表現載體上，通常這些核酸位於兩個或三個表現載體上，亦即一個載體可包含一個以上的這些核酸。這些雙特異性抗體的實例為 CrossMabs (參見例如，Schaefer 等人，Proc. Natl. Acad. Sci. USA108:11187-11191 (2011))。舉例而言，異源單體重鏈中之一者包含所謂「杵突變」(T366W，且視情況為 S354C 或 Y349C 中之一者)，且另一個包含所謂「臼突變」(T366S、L368A 及 Y407V，且視情況為 Y349C 或 S354C) (參見例如，Carter, P. 等人，Immunotechnol.2:73，1996) (根據 EU 索引編號)。In the case of a bispecific antibody with heterodimeric heavy chains, four nucleic acids are required, one for the first light chain, one for the first heavy chain (which comprises the first heterologous monomeric Fc region polypeptide), one for the second light chain, and one for the second heavy chain (which comprises the second heterologous monomeric Fc region polypeptide). These four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors. These nucleic acids encode an amino acid sequence comprising a first VL, and/or an amino acid sequence comprising a first VH (comprising a first heterologous Fc region), and/or an amino acid sequence comprising a second VL, and/or an amino acid sequence comprising a second VH (comprising a second heterologous Fc region of the antibody) (e.g., the first and/or second light chains, and/or the first and/or second heavy chains of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors. Usually, these nucleic acids are located on two or three expression vectors, that is, one vector can contain more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMabs (see, for example, Schaefer et al.,Proc. Natl. Acad. Sci. USA 108: 11187-11191 (2011)). For example, one of the heterologous monomer recombinants comprises a so-called "knob mutation" (T366W, and optionally one of S354C or Y349C), and the other comprises a so-called "hole mutation" (T366S, L368A and Y407V, and optionally Y349C or S354C) (see, e.g., Carter, P. et al.,Immunotechnol. 2:73, 1996) (according to EU index numbering).

在另一實施例中，提供包含此類核酸之宿主細胞。在此實施例中，宿主細胞包含 (例如，已轉化)：(1) 載體，該載體包含編碼包含抗原結合分子之至少一個 VL 的胺基酸序列及包含抗原結合分子之至少一個 VH 的胺基酸序列的核酸，或 (2) 第一載體，該第一載體包含編碼包含抗原結合分子之 VL 的胺基酸序列的核酸，以及第二載體，該第二載體包含編碼包含抗原結合分子之 VH 的胺基酸序列的核酸。在一個實施例中，宿主細胞為真核細胞，例如中國倉鼠卵巢 (CHO) 細胞或淋巴樣細胞。In another embodiment, a host cell comprising such a nucleic acid is provided. In this embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising at least one VL of the antigen-binding molecule and an amino acid sequence comprising at least one VH of the antigen-binding molecule, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of the antigen-binding molecule and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of the antigen-binding molecule. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell.

在一個實施例中，提供製備抗 STEAP1 抗原結合分子之方法，其中該方法包括在適於表現該抗 STEAP1 抗原結合分子之條件下培養如上文所提供之包含編碼該抗 STEAP1 抗原結合分子的核酸的宿主細胞，且視情況下自該宿主細胞 (或宿主細胞培養基) 中回收該抗 STEAP1 抗原結合分子。在另一實施例中，該方法進一步包括培養包含編碼抗 CD3 抗原結合分子的第二核酸的第二宿主細胞，該抗 CD3 抗原結合分子包含結合 CD3 的結合域。在一些實施例中，宿主細胞係經共培養。進一步的實施例包含從宿主細胞或培養基中回收雙特異性抗體。在一些實施例中，抗 STEAP1 及抗 CD3 抗原結合分子係於相同的宿主細胞中產生 (例如，單細胞方法)。在一些實施例中，抗 STEAP1 及抗 CD3 抗原結合分子係於單獨的宿主細胞中產生 (例如，雙細胞方法)。In one embodiment, a method of preparing an anti-STEAP1 antigen binding molecule is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the anti-STEAP1 antigen binding molecule as provided above under conditions suitable for expression of the anti-STEAP1 antigen binding molecule, and optionally recovering the anti-STEAP1 antigen binding molecule from the host cell (or host cell culture medium). In another embodiment, the method further comprises culturing a second host cell comprising a second nucleic acid encoding an anti-CD3 antigen binding molecule, the anti-CD3 antigen binding molecule comprising a binding domain that binds CD3. In some embodiments, the host cells are co-cultured. Further embodiments comprise recovering the bispecific antibody from the host cell or culture medium. In some embodiments, anti-STEAP1 and anti-CD3 antigen binding molecules are produced in the same host cell (e.g., a single cell approach). In some embodiments, anti-STEAP1 and anti-CD3 antigen binding molecules are produced in separate host cells (e.g., a two-cell approach).

在單細胞及雙細胞方法中，將編碼抗 STEAP1/CD3 TDB (例如，抗 STEAP1 半抗體及抗 CD3 半抗體) 的一種或多種質體引入一種或多種宿主細胞中用於培養及表現 TDB。在一種情況下，單個質體可編碼抗 STEAP1 半抗體及抗 CD3 半抗體兩者。替代性地，半抗體可由單獨的質體編碼。在另一情況下，每個半抗體之重鏈係於第一質體上編碼，而每個半抗體之輕鏈係於第二質體上編碼。在單細胞方法中，抗 STEAP1/CD3 TDB 係於單個宿主中產生。在雙細胞方法中，抗 STEAP1/CD3 TDB 係藉由在單獨的宿主 (例如，相同宿主細胞的單獨的培養物，或不同宿主細胞的單獨的培養物) 中表現半抗體來產生。在雙細胞方法中，兩種宿主可於同一容器或不同容器中培養。可在裂解並純化抗 STEAP1/CD3 TDB 之前合併兩種宿主培養物，或者可單獨純化兩種半抗體。In the single cell and two cell methods, one or more plasmids encoding anti-STEAP1/CD3 TDBs (e.g., anti-STEAP1 hapantibodies and anti-CD3 hapantibodies) are introduced into one or more host cells for culture and expression of the TDBs. In one instance, a single plasmid can encode both anti-STEAP1 hapantibodies and anti-CD3 hapantibodies. Alternatively, the hapantibodies can be encoded by separate plasmids. In another instance, the heavy chain of each hapantibody is encoded on a first plasmid, and the light chain of each hapantibody is encoded on a second plasmid. In the single cell method, the anti-STEAP1/CD3 TDB is produced in a single host. In the two-cell approach, anti-STEAP1/CD3 TDB is produced by expressing half antibodies in separate hosts (e.g., separate cultures of the same host cells, or separate cultures of different host cells). In the two-cell approach, the two hosts can be cultured in the same vessel or in different vessels. The two host cultures can be combined prior to lysing and purifying the anti-STEAP1/CD3 TDB, or the two half antibodies can be purified separately.

在一些實施例中，使用單細胞方法產生業經修飾以包括不對稱修飾 (例如，上述 VH、VL、CH1、CL 及/或 Fc 域之突變) 的本發明之抗 STEAP1/CD3 TDB，其與未經修飾以包括不對稱修飾的抗 STEAP1/CD3 TDB 相比，導致抗 STEAP1/CD3 TDB 的經改善之正確重鏈/輕鏈配對及/或經改善之產率。In some embodiments, single cell methods are used to generate anti-STEAP1/CD3 TDBs of the invention that have been modified to include asymmetric modifications (e.g., mutations in the VH, VL, CH1, CL and/or Fc domains as described above), which result in improved correct heavy chain/light chain pairing and/or improved yield of the anti-STEAP1/CD3 TDB compared to anti-STEAP1/CD3 TDBs that have not been modified to include asymmetric modifications.

在重組生產抗原結合分子時，將例如上述之編碼抗原結合分子之多核苷酸分離並插入一種或多種載體中，以在宿主細胞中進一步選殖及/或表現。此類核酸可藉由常規方法 (例如，使用能夠與編碼抗原結合分子之重鏈及輕鏈的基因特異性結合的寡核苷酸探針) 輕易地分離並定序。In the recombinant production of antigen-binding molecules, polynucleotides encoding antigen-binding molecules such as those described above are isolated and inserted into one or more vectors for further propagation and/or expression in host cells. Such nucleic acids can be easily isolated and sequenced by conventional methods (e.g., using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of antigen-binding molecules).

適用於選殖或表現編碼抗原結合分子之載體的宿主細胞包括本文所述之原核或真核細胞。舉例而言，抗原結合分子可以在細菌中產生，特定而言在無需醣基化及 Fc 效應功能的情況下。有關抗體片段和多肽在細菌中之表現，參見例如美國第 5,648,237、5,789,199 及 5,840,523 號專利。(另見 Charlton，Methods in Molecular Biology，第248卷(B.K.C. Lo 主編，Humana Press，Totowa，NJ，2003)，第 245-254 頁，其中描述了抗體片段在大腸桿菌中之表現。) 在表現後，抗原結合分子可與細菌細胞糊中的可溶性部分分離，並可經過進一步純化。Suitable host cells for cloning or expressing vectors encoding antigen binding molecules include prokaryotic or eukaryotic cells described herein. For example, antigen binding molecules can be produced in bacteria, particularly without glycosylation and Fc effector functions. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton,Methods in Molecular Biology,Vol .248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes expression of antibody fragments inE. coli .) Following expression, the antigen-binding molecules can be separated from the soluble portion of the bacterial cell pasteand can be further purified.

除原核生物以外，真核微生物 (如絲狀真菌或酵母菌) 也為合適的抗原結合分子編碼載體的選殖或表現宿主，包括其醣基化途徑已被「人源化」的真菌及酵母菌株，從而導致具有部分或完全人醣基化模式的抗體的產生。參見 Gerngross，Nat. Biotech.22:1409-1414 (2004)；及 Li 等人，Nat. Biotech. 24:210-215 (2004)。In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable hosts for the selection or expression of antigen-binding molecule encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross,Nat. Biotech. 22: 1409-1414 (2004); and Li et al.,Nat. Biotech . 24: 210-215 (2004).

用於表現醣基化抗原結合分子的合適的宿主細胞也來源於多細胞生物 (無脊椎動物及脊椎動物)。無脊椎動物細胞之實例包括植物及昆蟲細胞。已鑑別出許多桿狀病毒毒株，其可與昆蟲細胞聯合使用，尤其用於轉染草地貪夜蛾 (Spodoptera frugiperda) 細胞。Suitable host cells for the expression of glycosylated antigen binding molecules also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of bacilliform virus strains have been identified that can be used in conjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

植物細胞培養物亦可以用作宿主。參見例如，US 5,959,177、US 6,040,498、US 6,420,548、US 7,125,978 及 US 6,417,429 (描述了在轉基因植物中生產抗體的 PLANTIBODIES™ 技術)。Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

脊椎動物細胞也可用為宿主。例如，可使用適於在懸浮液中生長的哺乳動物細胞株。有用哺乳動物宿主細胞株之其他實例為由 SV40 (COS-7) 轉變之猴腎 CV1 細胞株；人類胚胎腎細胞株 (如 Graham 等人，J. Gen Virol.36:59-74 (1977) 中所述之 293 或 293T 細胞)；幼地鼠腎細胞 (BHK)；小鼠睾丸支持細胞 (如 Mather，Biol. Reprod.23:243-252 (1980) 中所述之 TM4 細胞)；猴腎細胞 (CV1)；非洲綠猴腎細胞 (VERO-76)；人類子宮頸癌細胞 (HELA)；犬腎細胞 (MDCK)；Buffalo 大鼠肝細胞 (BRL 3A)；人類肺細胞 (W138)；人類肝細胞 (Hep G2)；小鼠乳腺腫瘤 (MMT 060562)；TRI 細胞，如 Mather 等人，Annals N.Y.Acad. Sci.383:44-68 (1982)) 中所闡述；MRC 5 細胞；及 FS4 細胞。其他可用的哺乳動物宿主細胞株包括中國倉鼠卵巢 (CHO) 細胞，包括 DHFR- CHO 細胞 (Urlaub 等人，Proc. Natl. Acad. Sci. USA77:4216-4220 (1980))；及骨髓瘤細胞株，諸如 Y0、NS0 及 Sp2/0。關於某些適合於抗體生產的哺乳動物宿主細胞株的綜述，參見例如，Yazaki 及 Wu，Methods in Molecular Biology，第 248 卷，Lo, B.K.C. (主編)，Humana Press，Totowa，NJ (2004)，第 255-268 頁。Vertebrate cells can also be used as hosts. For example, mammalian cell strains adapted to growth in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7); human embryonic kidney cell line (e.g., 293 or 293T cells as described in Graham et al.,J. Gen Virol. 36:59-74 (1977)); baby hamster kidney cells (BHK); mouse testicular Sertoli cells (e.g., TM4 cells as described in Mather,Biol. Reprod. 23:243-252 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described in Mather et al.,Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other mammalian host cell lines that can be used include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980)); and myeloma cell lines, such as Y0, NS0, and Sp2/0. For a review of certain mammalian host cell strains suitable for antibody production, see, e.g., Yazaki and Wu,Methods in Molecular Biology , Vol. 248, Lo, BKC (Ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

在一個態樣中，宿主細胞為真核細胞，例如中國倉鼠卵巢 (CHO) 細胞或淋巴樣細胞 (例如，Y0、NS0、Sp20 細胞)。用於抗STEAP1/CD3 TDB生產的單細胞方法In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). Single cell methodfor anti-STEAP1/CD3 TDBproduction

在一種方法中，可藉由培養業已用兩種質體共轉染之宿主細胞來生產抗 STEAP1/CD3 TDB，這兩種質體各自編碼抗 STEAP1/CD3 TDB 之兩條臂中之一者 (例如，編碼抗 STEAP1 半抗體的第一質體及編碼抗 CD3 半抗體的第二質體)。宿主細胞 (例如，細菌、哺乳動物或昆蟲細胞) 之轉染可以在 96 孔盤形式中進行。為篩選 STEAP1 TDB 生產，可以挑選大約 2,000 至 3,000 個殖株，並藉由 ELISA 及完整 IgG 均相時間解析螢光 (HTRF) 來評定其結合標靶抗原 STEAP1 的能力。可以選擇生產能夠結合 STEAP1 或其片段的抗 STEAP1/CD3 TDB 的殖株用於擴增及進一步篩選 (例如，與 CD3 結合)。然後可根據所生產的雙特異性抗體 (bsAb) 百分比、效價及性能認證 (PQ) 選擇頂級殖株進行進一步分析。In one approach, anti-STEAP1/CD3 TDBs can be produced by culturing host cells that have been co-transfected with two plasmids, each encoding one of the two arms of the anti-STEAP1/CD3 TDB (e.g., a first plasmid encoding an anti-STEAP1 hapter and a second plasmid encoding an anti-CD3 hapter). Transfection of host cells (e.g., bacterial, mammalian, or insect cells) can be performed in a 96-well plate format. To screen for STEAP1 TDB production, approximately 2,000 to 3,000 clones can be picked and assessed for their ability to bind the target antigen STEAP1 by ELISA and whole IgG homogeneous time-resolved fluorescence (HTRF). Clones producing anti-STEAP1/CD3 TDBs capable of binding STEAP1 or fragments thereof can be selected for expansion and further screening (e.g., binding to CD3). Top clones can then be selected for further analysis based on percentage of bispecific antibodies (bsAbs) produced, titer, and performance qualification (PQ).

可用於生產本發明之抗 STEAP1/CD3 TBD 的示例性單細胞方法描述於國際專利申請號 PCT/US16/28850 或美國專利申請公開號 2018/0177873 中，其全文以引用方式併入本文中。Exemplary single cell methods that can be used to produce the anti-STEAP1/CD3 TBD of the present invention are described in International Patent Application No. PCT/US16/28850 or U.S. Patent Application Publication No. 2018/0177873, which are incorporated herein by reference in their entirety.

舉例而言，本文提供一種生產本文所揭示之多特異性抗原結合分子之方法，該方法包含：(a) 將編碼多特異性抗原結合分子的一種或多種多核苷酸分子引入宿主細胞中，其中該多特異性抗原結合分子包含 a) 與第一抗原結合的第一重鏈/輕鏈對，其包含第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1)，及 b) 與第二抗原結合的第二重鏈/輕鏈對，其包含第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2)，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域 (CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，且 L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基；H2 的 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，且 L2 的 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，H1 之 VH 域中的 Q39 (Kabat編號) 處之胺基酸被替換為帶正電荷的殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，且 L1 之 VL 域中的 Q38 (Kabat編號) 處之胺基酸被替換為帶負電荷的殘基；並且 H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，且 L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基；以及 (b) 將宿主細胞在特定條件下培養並持續足以生產抗原結合蛋白質的時間。在一些實施例中，帶正電荷的殘基選自 R 及 K，並且帶負電荷的殘基選自 D 及 E。在一些實施例中，帶正電荷的殘基為 R。在其他實施例中，帶正電荷的殘基為 K。在一些實施例中，帶負電荷的殘基為 D。在其他實施例中，帶負電荷的殘基為 E。在一些實施例中，第一抗原為 STEAP1 且第二抗原為 CD3。在其他實施例中，第一抗原為 CD3 並且第二抗原為 STEAP1。For example, provided herein is a method for producing a multispecific antigen-binding molecule disclosed herein, the method comprising: (a) introducing one or more polynucleotide molecules encoding the multispecific antigen-binding molecule into a host cell, wherein the multispecific antigen-binding molecule comprises a) a first heavy chain/light chain pair that binds to a first antigen, comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and b) a second heavy chain/light chain pair that binds to a second antigen, comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by a positively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a negatively charged residue, the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced by a negatively charged residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a positively charged residue; the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by a positively charged residue, and Q38 (Kabat numbering) in the VL domain of L2 is replaced by a positively charged residue; or (ii) an amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced with a negatively charged residue, an amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced with a positively charged residue, an amino acid at V133 (EU numbering) in the CL domain of L1 is replaced with a positively charged residue, and an amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced with a negatively charged residue; and an amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced with a negatively charged residue, and Q38 (Kabat numbering) in the VL domain of L2 is replaced with a negatively charged residue. (b) culturing the host cell under specific conditions and for a time sufficient to produce the antigen-binding protein. In some embodiments, the positively charged residue is selected from R and K, and the negatively charged residue is selected from D and E. In some embodiments, the positively charged residue is R. In other embodiments, the positively charged residue is K. In some embodiments, the negatively charged residue is D. In other embodiments, the negatively charged residue is E. In some embodiments, the first antigen is STEAP1 and the second antigen is CD3. In other embodiments, the first antigen is CD3 and the second antigen is STEAP1.

舉例而言，本文提供一種生產本文所揭示之多特異性抗原結合分子之方法，該方法包含：(a) 提供包含編碼多特異性抗原結合分子的一種或多種多核苷酸分子的宿主細胞，其中該多特異性抗原結合分子包含 a) 與第一抗原結合的第一重鏈/輕鏈對，其包含第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1)，及 b) 與第二抗原結合的第二重鏈/輕鏈對，其包含第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2)，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域 (CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，且 L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基；H2 的 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，且 L2 的 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，H1 之 VH 域中的 Q39 (Kabat編號) 處之胺基酸被替換為帶正電荷的殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，且 L1 之 VL 域中的 Q38 (Kabat編號) 處之胺基酸被替換為帶負電荷的殘基；並且 H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，且 L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基；以及 (b) 將宿主細胞在特定條件下培養並持續足以生產抗原結合蛋白質的時間。在一些實施例中，帶正電荷的殘基選自 R 及 K，並且帶負電荷的殘基選自 D 及 E。在一些實施例中，帶正電荷的殘基為 R。在其他實施例中，帶正電荷的殘基為 K。在一些實施例中，帶負電荷的殘基為 D。在其他實施例中，帶負電荷的殘基為 E。在一些實施例中，第一抗原為 STEAP1 且第二抗原為 CD3。在其他實施例中，第一抗原為 CD3 並且第二抗原為 STEAP1。For example, provided herein is a method for producing a multispecific antigen-binding molecule disclosed herein, the method comprising: (a) providing a host cell comprising one or more polynucleotide molecules encoding a multispecific antigen-binding molecule, wherein the multispecific antigen-binding molecule comprises a) a first heavy chain/light chain pair that binds to a first antigen, comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and b) a second heavy chain/light chain pair that binds to a second antigen, comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by a positively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a negatively charged residue, the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced by a negatively charged residue, and the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a positively charged residue; the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by a positively charged residue, and Q38 (Kabat numbering) in the VL domain of L2 is replaced by a positively charged residue; or (ii) an amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced with a negatively charged residue, an amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced with a positively charged residue, an amino acid at V133 (EU numbering) in the CL domain of L1 is replaced with a positively charged residue, and an amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced with a negatively charged residue; and an amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced with a negatively charged residue, and Q38 (Kabat numbering) in the VL domain of L2 is replaced with a negatively charged residue. (b) culturing the host cell under specific conditions and for a time sufficient to produce the antigen-binding protein. In some embodiments, the positively charged residue is selected from R and K, and the negatively charged residue is selected from D and E. In some embodiments, the positively charged residue is R. In other embodiments, the positively charged residue is K. In some embodiments, the negatively charged residue is D. In other embodiments, the negatively charged residue is E. In some embodiments, the first antigen is STEAP1 and the second antigen is CD3. In other embodiments, the first antigen is CD3 and the second antigen is STEAP1.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39K (Kabat 編號) 及 S183E (EU 編號) 取代且輕鏈含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 STEAP1 臂；以及重鏈中含有 Q39E (Kabat 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 取代的抗 CD3 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising Q39K (Kabat numbering) and S183E (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain; and an anti-CD3 arm comprising Q39E (Kabat numbering) substitution in the heavy chain and Q38K (Kabat numbering) substitution in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39K (Kabat 編號) 及 S183E (EU 編號) 取代且輕鏈含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 CD3 臂；以及重鏈中含有 Q39E (Kabat 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 取代的抗 STEAP1 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-CD3 arm comprising Q39K (Kabat numbering) and S183E (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain; and an anti-STEAP1 arm comprising Q39E (Kabat numbering) substitution in the heavy chain and Q38K (Kabat numbering) substitution in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39E (Kabat 編號) 及 S183K (EU 編號) 取代且輕鏈含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 STEAP1 臂；以及重鏈中含有 Q39K (Kabat 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 取代的抗 CD3 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising Q39E (Kabat numbering) and S183K (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain; and an anti-CD3 arm comprising Q39K (Kabat numbering) substitution in the heavy chain and Q38E (Kabat numbering) substitution in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39E (Kabat 編號) 及 S183K (EU 編號) 取代且輕鏈含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 CD3 臂；以及重鏈中含有 Q39K (Kabat 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 取代的抗 STEAP1 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-CD3 arm comprising Q39E (Kabat numbering) and S183K (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain; and an anti-STEAP1 arm comprising Q39K (Kabat numbering) substitution in the heavy chain and Q38E (Kabat numbering) substitution in the light chain.

在另一實例中，本文提供一種生產本文所揭示之多特異性抗原結合分子之方法，該方法包含：(a) 將編碼多特異性抗原結合分子的一種或多種多核苷酸分子引入宿主細胞中，其中該多特異性抗原結合分子包含 a) 與第一抗原結合的第一重鏈/輕鏈對，其包含第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1)，及 b) 與第二抗原結合的第二重鏈/輕鏈對，其包含第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2)，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域 (CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，H2 之 CH1域中的 S183 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，H2 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基；以及 (b) 將宿主細胞在特定條件下培養並持續足以生產抗原結合蛋白質的時間。在一些實施例中，帶正電荷的殘基選自 R 及 K，並且帶負電荷的殘基選自 D 及 E。在一些實施例中，帶正電荷的殘基為 R。在其他實施例中，帶正電荷的殘基為 K。在一些實施例中，帶負電荷的殘基為 D。在其他實施例中，帶負電荷的殘基為 E。在一些實施例中，第一抗原為 STEAP1 且第二抗原為 CD3。在其他實施例中，第一抗原為 CD3 並且第二抗原為 STEAP1。In another example, provided herein is a method for producing a multispecific antigen-binding molecule disclosed herein, the method comprising: (a) introducing one or more polynucleotide molecules encoding the multispecific antigen-binding molecule into a host cell, wherein the multispecific antigen-binding molecule comprises a) a first heavy chain/light chain pair that binds to a first antigen, comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and b) a second heavy chain/light chain pair that binds to a second antigen, comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by a positively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a negatively charged residue, the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced by a negatively charged residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a positively charged residue, the amino acid at S183 (EU numbering) in the CH1 domain of H2 is replaced by a negatively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by a positively charged residue, and the amino acid at L2 (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by a negatively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a positively charged residue, the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced by a positively charged residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a negatively charged residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced by a positively charged residue; or (ii) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by a negatively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a positively charged residue, the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced by a positively charged residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a negatively charged residue, and the amino acid at S183 (EU numbering) in the CH1 domain of H2 is replaced by a negatively charged residue. The invention relates to a method for preparing an antigen-binding protein comprising: (a) culturing a host cell under specific conditions and for a period of time sufficient to produce an antigen-binding protein, wherein the amino acid at S183 (EU numbering) in the VH domain of H2 is replaced with a positively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced with a negatively charged residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced with a positively charged residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced with a negatively charged residue; and (b) culturing the host cell under specific conditions and for a period of time sufficient to produce the antigen-binding protein. In some embodiments, the positively charged residue is selected from R and K, and the negatively charged residue is selected from D and E. In some embodiments, the positively charged residue is R. In other embodiments, the positively charged residue is K. In some embodiments, the negatively charged residue is D. In other embodiments, the negatively charged residue is E. In some embodiments, the first antigen is STEAP1 and the second antigen is CD3. In other embodiments, the first antigen is CD3 and the second antigen is STEAP1.

舉例而言，本文提供一種生產本文所揭示之多特異性抗原結合分子之方法，該方法包含：(a) 提供包含編碼多特異性抗原結合分子的一種或多種多核苷酸分子的宿主細胞，其中該多特異性抗原結合分子包含 a) 與第一抗原結合的第一重鏈/輕鏈對，其包含第一重鏈多肽 (H1) 及第一輕鏈多肽 (L1)，及 b) 與第二抗原結合的第二重鏈/輕鏈對，其包含第二重鏈多肽 (H2) 及第二輕鏈多肽 (L2)，其中每個 H1 及 H2 包含重鏈可變域 (VH) 及重鏈恆定域 (CH1)，並且每個 L1 及 L2 包含輕鏈可變域 (VL) 及輕鏈恆定域 (VL)，其中：(i) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，H2 之 CH1域中的 S183 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基；或 (ii) H1 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基，H1 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，L1 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，L1 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，H2 之 CH1 域中的 S183 (EU 編號) 處之胺基酸被替換為帶正電荷的殘基，H2 之 VH 域中的 Q39 (Kabat 編號) 處之胺基酸被替換為帶負電荷的殘基，L2 之 VL 域中的 Q38 (Kabat 編號) 處之胺基酸被替換為帶正電荷的殘基，並且 L2 之 CL 域中的 V133 (EU 編號) 處之胺基酸被替換為帶負電荷的殘基；以及 (b) 將宿主細胞在特定條件下培養並持續足以生產抗原結合蛋白質的時間。在一些實施例中，帶正電荷的殘基選自 R 及 K，並且帶負電荷的殘基選自 D 及 E。在一些實施例中，帶正電荷的殘基為 R。在其他實施例中，帶正電荷的殘基為 K。在一些實施例中，帶負電荷的殘基為 D。在其他實施例中，帶負電荷的殘基為 E。在一些實施例中，第一抗原為 STEAP1 且第二抗原為 CD3。在其他實施例中，第一抗原為 CD3 並且第二抗原為 STEAP1。For example, provided herein is a method for producing a multispecific antigen-binding molecule disclosed herein, the method comprising: (a) providing a host cell comprising one or more polynucleotide molecules encoding a multispecific antigen-binding molecule, wherein the multispecific antigen-binding molecule comprises a) a first heavy chain/light chain pair that binds to a first antigen, comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and b) a second heavy chain/light chain pair that binds to a second antigen, comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), wherein each H1 and H2 comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and each L1 and L2 comprises a light chain variable domain (VL) and a light chain constant domain (VL), wherein: (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by a positively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a negatively charged residue, the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced by a negatively charged residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a positively charged residue, the amino acid at S183 (EU numbering) in the CH1 domain of H2 is replaced by a negatively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced by a positively charged residue, and the amino acid at L2 (i) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by a negatively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a positively charged residue, the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced by a positively charged residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a negatively charged residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced by a positively charged residue; or (ii) the amino acid at S183 (EU numbering) in the CH1 domain of H1 is replaced by a negatively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H1 is replaced by a positively charged residue, the amino acid at V133 (EU numbering) in the CL domain of L1 is replaced by a positively charged residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L1 is replaced by a negatively charged residue, and the amino acid at S183 (EU numbering) in the CH1 domain of H2 is replaced by a negatively charged residue. The invention relates to a method for preparing an antigen-binding protein comprising: (a) culturing a host cell under specific conditions and for a period of time sufficient to produce an antigen-binding protein, wherein the amino acid at S183 (EU numbering) in the VH domain of H2 is replaced with a positively charged residue, the amino acid at Q39 (Kabat numbering) in the VH domain of H2 is replaced with a negatively charged residue, the amino acid at Q38 (Kabat numbering) in the VL domain of L2 is replaced with a positively charged residue, and the amino acid at V133 (EU numbering) in the CL domain of L2 is replaced with a negatively charged residue; and (b) culturing the host cell under specific conditions and for a period of time sufficient to produce the antigen-binding protein. In some embodiments, the positively charged residue is selected from R and K, and the negatively charged residue is selected from D and E. In some embodiments, the positively charged residue is R. In other embodiments, the positively charged residue is K. In some embodiments, the negatively charged residue is D. In other embodiments, the negatively charged residue is E. In some embodiments, the first antigen is STEAP1 and the second antigen is CD3. In other embodiments, the first antigen is CD3 and the second antigen is STEAP1.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39K (Kabat 編號) 及 S183E (EU 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 STEAP1 臂；以及重鏈中含有 Q39E (Kabat 編號) 及 S183K (EU 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 CD3 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising Q39K (Kabat numbering) and S183E (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain; and an anti-CD3 arm comprising Q39E (Kabat numbering) and S183K (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39K (Kabat 編號) 及 S183E (EU 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 CD3 臂；以及重鏈中含有 Q39E (Kabat 編號) 及 S183K (EU 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 STEAP1 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-CD3 arm comprising Q39K (Kabat numbering) and S183E (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain; and an anti-STEAP1 arm comprising Q39E (Kabat numbering) and S183K (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39E (Kabat 編號) 及 S183K (EU 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 STEAP1 臂；以及重鏈中含有 Q39K (Kabat 編號) 及 S183E (EU 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 CD3 臂。In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-STEAP1 arm comprising Q39E (Kabat numbering) and S183K (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain; and an anti-CD3 arm comprising Q39K (Kabat numbering) and S183E (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain.

在一些實施例中，本文所述之多特異性抗原結合分子包含：重鏈中含有 Q39E (Kabat 編號) 及 S183K (EU 編號) 取代且輕鏈中含有 Q38K (Kabat 編號) 及 V133E (EU 編號) 取代的抗 CD3 臂；以及重鏈中含有 Q39K (Kabat 編號) 及 S183E (EU 編號) 取代且輕鏈中含有 Q38E (Kabat 編號) 及 V133K (EU 編號) 取代的抗 STEAP1 臂。在一個實例中，本發明之抗 STEAP1/CD3 TDB 可以如 Dillon 等人於MAbs9:213-230, 2017 中所述進行生產。用於抗STEAP1/CD3 TDB生產的雙細胞方法In some embodiments, the multispecific antigen-binding molecules described herein comprise: an anti-CD3 arm comprising Q39E (Kabat numbering) and S183K (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain; and an anti-STEAP1 arm comprising Q39K (Kabat numbering) and S183E (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain. In one example, the anti-STEAP1/CD3 TDB of the present invention can be produced as described in Dillon et al.,MAbs 9:213-230, 2017.Dual Cell Methodfor Anti-STEAP1/CD3 TDB Production

替代性地，可藉由使用高細胞密度發酵單獨 (亦即，在兩種不同的細胞株中) 培養抗體半聚體 (例如，半抗體)，然後通過蛋白質 A 層析法獨立地分離各半抗體，來生產抗 STEAP1/CD3 TDB。然後可例如以 1:1 莫耳比合併經純化之半抗體，並在 50 mM Tris (pH 8.5) 中在 2 mM DTT 存在下孵育 4 小時，以允許退火並還原鉸鏈區中之二硫鍵。在不含 DTT 的相同緩衝液中透析 24 小時至 48 小時，導致鏈間二硫鍵之形成。Alternatively, anti-STEAP1/CD3 TDB can be produced by culturing antibody hemimers (e.g., half antibodies) separately (i.e., in two different cell lines) using high cell density fermentation and then isolating each half antibody independently by protein A chromatography. The purified half antibodies can then be combined, for example, at a 1:1 molar ratio and incubated in 50 mM Tris (pH 8.5) in the presence of 2 mM DTT for 4 hours to allow annealing and reduction of disulfide bonds in the hinge region. Dialysis in the same buffer without DTT for 24 to 48 hours results in the formation of interchain disulfide bonds.

TDB 可替代性地藉由將兩種質體 (各自編碼 TDB 的不同臂) 轉染到單獨的宿主細胞中來生產。宿主細胞可以經共培養或單獨培養。宿主細胞之轉染可以在 96 孔盤形式中進行。為篩選 TDB 生產，可以挑選 2,000 至 3,000 個殖株，並藉由 ELISA 及完整 IgG 均相時間解析螢光 (HTRF) 來評定其結合所選抗原 (例如，STEAP1) 的能力。可以選擇生產能夠結合 STEAP1 或其片段的 TDB 的殖株用於擴增及進一步篩選。根據所生產的雙特異性抗體 (bsAb) 百分比、效價及 PQ 選擇頂級殖株進行進一步分析。示例性生產方法TDB can alternatively be produced by transfecting two plasmids, each encoding a different arm of the TDB, into separate host cells. The host cells can be co-cultured or cultured alone. Transfection of host cells can be performed in a 96-well plate format. To screen for TDB production, 2,000 to 3,000 clones can be picked and assessed for their ability to bind to a selected antigen (e.g., STEAP1) by ELISA and whole IgG homogeneous time-resolved fluorescence (HTRF). Clones that produce TDB capable of binding STEAP1 or fragments thereof can be selected for expansion and further screening. Top clones are selected for further analysis based on the percentage of bispecific antibodies (bsAbs) produced, titer, and PQ.Exemplary Production Methods

在一個實例中，TDB 係藉由共培養策略生產，其中使用表現一種半抗體 (臼) 的大腸桿菌 (E. coli) 細胞及表現第二半抗體 (杵) 的大腸桿菌 (E. coli) 細胞以預定比例在搖瓶中一起生長，使得其產生相似量的各種半抗體 (參見，Spiess 等人，Nat. Biotechnol.31(8):753-8 (2013)；PCT 公開號 WO 2011/069104，其全文以引用方式併入本文中)。然後收穫共培養之細菌培養液，在微流化器中破碎細胞，並藉由蛋白質 A 親和層析法純化抗體。據觀察，在微流化期間，蛋白質 A 捕獲兩條經退火之臂並形成鉸鏈鏈間二硫鍵。In one example, TDB is produced by a co-culture strategy, in whichE. coli cells expressing one hapten (hole) andE. coli cells expressing a second hapten (knob) are grown together in a shake flask at a predetermined ratio so that they produce similar amounts of each hapten (see Spiess et al.,Nat. Biotechnol. 31(8):753-8 (2013); PCT Publication No. WO 2011/069104, which is incorporated herein by reference in its entirety). The co-cultured bacterial culture is then harvested, the cells are disrupted in a microfluidizer, and the antibodies are purified by protein A affinity chromatography. It was observed that during microfluidization, protein A captured both annealed arms and formed inter-hinge disulfide bonds.

在另一實例中，如前所述生產全長雙特異性抗體 (Junttila 等人Cancer Res.74:5561-5571，2014；Sun 等人Science Trans.Med.7:287ra270，2015)。簡言之，藉由瞬時轉染 CHO 細胞來表現在其 CH3 域中含有「杵」或「臼」突變的兩種半抗體 (例如，抗 STEAP1，諸如 huAb44.v6.05 及抗 CD3 諸如 40G5c 或 38E4v1.MD1)，然後用蛋白質 A 進行親和純化。將等量的兩種半抗體與 200 莫耳過量的還原型麩胱甘肽一起在 pH 8.5 及 32℃ 孵育過夜，以驅動杵-臼二硫鍵之形成。透過疏水交互作用層析法純化經組裝之雙特異性抗體 (例如，抗 STEAP1/CD3 TDB)，以去除污染物。藉由質譜、粒徑排阻層析 (SEC) 及凝膠電泳對經純化之抗 STEAP1/CD3 TDB 進行純度表徵。IV.測定In another example, full-length bispecific antibodies were produced as described previously (Junttila et al.Cancer Res. 74:5561-5571, 2014; Sun et al.Science Trans. Med. 7:287ra270, 2015). Briefly, two half antibodies containing "knob" or "hole" mutations in their CH3 domains (e.g., anti-STEAP1, such as huAb44.v6.05 and anti-CD3, such as 40G5c or 38E4v1.MD1) were expressed by transient transfection of CHO cells and then affinity purified with protein A. Equal amounts of the two half antibodies were incubated with 200 molar excess of reduced glutathione at pH 8.5 and 32°C overnight to drive the formation of knob-hole disulfide bonds. The assembled bispecific antibody (eg, anti-STEAP1/CD3 TDB) was purified by hydrophobic interaction chromatography to remove contaminants. The purified anti-STEAP1/CD3 TDB was characterized for purity by mass spectrometry, size exclusion chromatography (SEC), and gel electrophoresis.IV.Assay

可藉由此領域中已知的各種測定法對本文所提供之抗原結合分子的物理/化學性質及/或生物活性進行鑑別、篩選或表徵。The physical/chemical properties and/or biological activities of the antigen-binding molecules provided herein can be identified, screened or characterized by various assays known in the art.

在一個態樣中，利用已知方法諸如 ELISA、西方墨點法等，檢測本發明之抗原結合分子的抗原結合活性。In one embodiment, the antigen-binding activity of the antigen-binding molecules of the present invention is detected using known methods such as ELISA, Western blot, etc.

在另一態樣中，可使用競爭測定法鑑別與參考抗體競爭結合於 STEAP1 之抗原結合分子。在某些態樣中，此類競爭抗原結合分子與由參考抗體所結合的相同表位結合 (例如，線性或構形表位)。用於圖譜建立抗體結合的表位的詳細示例性方法提供於以下文獻中：Morris，「Epitope Mapping Protocols」，載於Methods in Molecular Biology第 66 卷 (Humana Press, Totowa, NJ) (1996) 中。In another aspect, a competition assay can be used to identify antigen binding molecules that compete with a reference antibody for binding to STEAP1. In certain aspects, such competing antigen binding molecules bind to the same epitope bound by the reference antibody (e.g., a linear or conformational epitope). Detailed exemplary methods for mapping epitopes to which antibodies bind are provided in Morris, "Epitope Mapping Protocols,"Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ) (1996).

在一種例示性競爭測定中，將固定化 STEAP1 置於包含與 STEAP1 結合之第一標記抗體及第二標記抗體 (正在試驗其與第一抗體競爭與 STEAP1 結合的能力) 的溶液中進行孵育。第二抗體可存在於融合瘤上清液中。作為對照，將固定化 STEAP1 置於包含第一標記抗體但不包含第二未標記抗體的溶液中進行孵育。在允許第一抗體與 STEAP1 結合的條件下孵化後，去除多餘的未結合抗體，並測量與固定化 STEAP1 相關聯之標記物的量。如果試驗樣品中與固定化 STEAP1 相關的標記物的數量相對於對照樣品而言明顯減少，則表明第二抗體正在與第一抗體競爭與 STEAP1 的結合。參見 Harlow 及 Lane (1988)Antibodies: A Laboratory Manualch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)。V.醫藥製劑In an exemplary competition assay, immobilized STEAP1 is incubated in a solution containing a first labeled antibody that binds to STEAP1 and a second labeled antibody that is being tested for its ability to compete with the first antibody for binding to STEAP1. The second antibody may be present in the fusion tumor supernatant. As a control, immobilized STEAP1 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to STEAP1, excess unbound antibody is removed and the amount of label associated with immobilized STEAP1 is measured. If the amount of label associated with immobilized STEAP1 in the test sample is significantly reduced relative to the control sample, it indicates that the second antibody is competing with the first antibody for binding to STEAP1. See Harlow and Lane (1988)Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).V.Pharmaceutical Preparations

本發明之抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 的醫藥調配物係藉由將具有所需純度之抗原結合分子與視情況選用的醫藥上可接受之載劑、賦形劑或穩定劑混合而製備為凍乾調配物或水溶液的形式以供儲存。關於製劑的一般資訊，參見例如：Gilman 等人主編，The Pharmacological Bases of Therapeutics，第 8 版，Pergamon Press，1990；A. Gennaro 主編，Remington’s Pharmaceutical Sciences，第 18 版，Mack Publishing Co.，Pennsylvania，1990；Avis 等人主編，Pharmaceutical Dosage Forms: Parenteral MedicationsDekker，New York，1993；Lieberman 等人主編，Pharmaceutical Dosage Forms: TabletsDekker，New York，1990；Lieberman 等人主編，Pharmaceutical Dosage Forms: Disperse SystemsDekker，New York，1990；及 Walters 主編Dermatological and Transdermal Formulations(Drugs and the Pharmaceutical Sciences)，第 119 卷，Marcel Dekker，2002。Pharmaceutical formulations of the antigen-binding molecules of the present invention (e.g., monospecific and/or multispecific antigen-binding molecules such as bispecific or trispecific antigen-binding molecules) are prepared by mixing antigen-binding molecules having the desired purity with a pharmaceutically acceptable carrier, excipient or stabilizer as appropriate, in the form of a lyophilized formulation or aqueous solution for storage. For general information on formulations, see, for example: Gilman et al., eds.,The Pharmacological Bases of Therapeutics , 8th ed., Pergamon Press, 1990; A. Gennaro, ed.,Remington's Pharmaceutical Sciences , 18th ed., Mack Publishing Co., Pennsylvania, 1990; Avis et al., eds.,Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York, 1993; Lieberman et al., eds.,Pharmaceutical Dosage Forms: Tablets Dekker, New York, 1990; Lieberman et al., eds.,Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York, 1990; and Walters, ed., Dermatological and Transdermal Formulations (Drugs and the Pharmaceutical Sciences), Vol. 119, Marcel Dekker, 2002.

如本文所述之抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 的醫藥調配物係藉由將具有所需純度之此類一種或多種抗體與一種或多種視情況選用的醫藥上可接受之載劑混合而製備 (Remington's Pharmaceutical Sciences第 16 版，Osol, A. 主編 (1980)) 為凍乾組成物或水溶液的形式。醫藥上可接受之載劑在採用的劑量及濃度下通常對受體無毒，其包括但不限於：緩衝劑，例如組胺酸、磷酸鹽、檸檬酸鹽、醋酸鹽及其他有機酸；抗氧化劑，包括抗壞血酸及蛋氨酸；防腐劑 (例如十八烷基二甲基芐基氯化銨；六甲基氯化銨；苯扎氯銨；芐索銨氯化物；苯酚、丁醇或芐醇；對羥基苯甲酸烷基酯，如對羥基苯甲酸甲酯或對羥基苯甲酸丙酯；鄰苯二酚；間苯二酚；環己醇；3-戊醇及間甲酚)；低分子量 (小於約 10 個殘基) 多肽；蛋白質，例如血清白蛋白、明膠或免疫球蛋白；親水性聚合物，例如聚乙烯吡咯烷酮；胺基酸，例如甘胺酸、麩醯胺酸、天冬醯胺酸、組胺酸、精胺酸或離胺酸；單醣、二糖及其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合劑 (例如 EDTA)；糖，例如蔗糖、甘露醇、海藻糖或山梨糖醇；成鹽抗衡離子，例如鈉；金屬錯合物 (例如鋅蛋白錯合物)；及/或非離子界面活性劑，例如聚乙二醇 (PEG)。本文中之示例性醫藥上可接受之載劑進一步包括間質藥物分散劑，諸如可溶性中性活性透明質酸酶醣蛋白 (sHASEGP)，例如人類可溶性 PH-20 透明質酸酶醣蛋白，諸如 rHuPH20 (HYLENEX®, Halozyme, Inc.)。某些例示性 sHASEGP 及使用方法 (包括 rHuPH20) 描述於美國專利公開號 2005/0260186 及 2006/0104968 中。在一個態樣中，sHASEGP 與一種或多種另外的糖胺聚醣酶諸如軟骨素酶結合在一起。Pharmaceutical formulations of antigen-binding molecules as described herein (e.g., monospecific and/or multispecific antigen-binding molecules such as bispecific or trispecific antigen-binding molecules) are prepared by mixing such one or more antibodies having the desired purity with one or more optionally selected pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of a lyophilized composition or an aqueous solution. Pharmaceutically acceptable carriers are generally nontoxic to the recipient at the dosages and concentrations employed and include, but are not limited to: buffers such as histidine, phosphate, citrate, acetate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzylammonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; o-catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes such as zinc protein complexes; and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersions, such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Halozyme, Inc.). Certain exemplary sHASEGPs and methods of use (including rHuPH20) are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is conjugated to one or more additional glycosaminoglycans, such as chondroitinase.

例示性凍乾抗體組成物敘述於美國專利號 6,267,958。水溶性抗體組成物包括美國專利第 6,171,586 號及 WO 2006/044908 中所述的那些，後者所述之組成物包括組胺酸-乙酸鹽緩衝劑。Exemplary lyophilized antibody compositions are described in U.S. Patent No. 6,267,958. Water-soluble antibody compositions include those described in U.S. Patent No. 6,171,586 and WO 2006/044908, the latter of which comprises a histidine-acetate buffer.

本文所述之製劑還可包含一種以上的活性化合物，優選具有互補活性且彼此無不利影響的活性化合物。舉例而言，此類藥物的類型及有效量取決於調配物中存在的拮抗劑的量及類型以及個體的臨床參數。The formulations described herein may also contain more than one active compound, preferably active compounds with complementary activities and without adverse effects on each other. For example, the type and effective amount of such drugs depends on the amount and type of antagonist present in the formulation and the clinical parameters of the individual.

活性成分也可包埋在例如透過凝聚技術或透過介面聚合製備的微囊 (例如，分別為羥甲基纖維素微囊或明膠微囊和聚(甲基丙烯酸甲酯)微囊) 中、膠體藥物遞送系統 (例如脂質體、白蛋白微球、微乳、奈米顆粒及奈米微囊 (nanocapsule)) 中或粗滴乳狀液中。此等技術揭示於Remington's Pharmaceutical Sciences(第 16 版，Osol, A. 主編，1980)。The active ingredient can also be embedded in microcapsules (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences (16th edition, Osol, A. ed., 1980).

可以製備緩釋製劑。緩釋製劑的適宜實例包括含有拮抗劑的固體疏水聚合物的半透性基質，該基質是成形物品的形式，例如膜或微囊。緩釋基質的實例包括聚酯、水凝膠 (例如，聚(2-羥乙基甲基丙烯酸酯) 或聚(乙烯醇))、聚丙交酯 (美國第 3,773,919 號專利)、L-谷胺酸及 γ-L-谷胺酸乙酯的共聚物、不可降解的乙烯-醋酸乙烯酯、可降解的乳酸-乙醇酸共聚物諸如 LUPRON DEPOT™ (由乳酸-乙醇酸共聚物和醋酸亮丙瑞林組成的可注射微球) 及聚-D-(-)-3-羥基丁酸。Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl gamma-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

用於活體內給藥的製劑必須是無菌的。這可以透過無菌濾膜過濾輕鬆實現。VI.治療方法Preparations forintravenous administration must be sterile. This is readily accomplished by filtration through sterile membranes.VI.Methods of Treatment

本發明之任何抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 或其組成物可用於本文所述之任何治療方法中。在一些實施例中，抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子，例如雙特異性或三特異性抗原結合分子) 可用於治療癌症 (例如，表現 STEAP1 之癌症) 或延緩其進展。在額外實施例中，抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 可用於抑制或減少癌細胞 (例如，表現 STEAP1 之癌症) 之增生。Any antigen binding molecule (e.g., monospecific and/or multispecific antigen binding molecule such as bispecific or trispecific antigen binding molecule) or composition thereof of the present invention can be used in any of the treatment methods described herein. In some embodiments, the antigen binding molecule (e.g., monospecific and/or multispecific antigen binding molecule such as bispecific or trispecific antigen binding molecule) can be used to treat cancer (e.g., cancer expressing STEAP1) or delay its progression. In additional embodiments, antigen binding molecules (e.g., monospecific and/or multispecific antigen binding molecules such as bispecific or trispecific antigen binding molecules) can be used to inhibit or reduce the proliferation of cancer cells (e.g., cancers expressing STEAP1).

在一些實施例中，抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 可用於治療有需要之個體的表現 STEAP1 之癌症或延緩其進展。在一些情況下，表現 STEAP1 之癌症為實性瘤。在一些情況下，表現 STEAP1 之癌症為前列腺癌、尤文氏肉瘤、肺癌、大腸直腸癌、乳癌、膀胱癌、卵巢癌或子宮頸癌。在一些情況下，表現 STEAP1 之癌症為前列腺癌。在一些情況下，表現 STEAP1 之癌症為尤文氏肉瘤。In some embodiments, antigen binding molecules (e.g., monospecific and/or multispecific antigen binding molecules such as bispecific or trispecific antigen binding molecules) can be used to treat or slow the progression of a cancer expressing STEAP1 in a subject in need thereof. In some cases, the cancer expressing STEAP1 is a solid tumor. In some cases, the cancer expressing STEAP1 is prostate cancer, Ewing's sarcoma, lung cancer, colorectal cancer, breast cancer, bladder cancer, ovarian cancer, or cervical cancer. In some cases, the cancer expressing STEAP1 is prostate cancer. In some cases, the cancer expressing STEAP1 is Ewing's sarcoma.

在一些實施例中，將抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 與額外治療劑或額外治療方案組合向個體投予。在一些情況下，額外治療劑包含化學治療劑、免疫治療劑、靶向療法、激素療法、放射療法或其組合。例示性額外治療劑包括但不限於：烷化劑，諸如六甲蜜胺、白消安、卡鉑、卡莫司汀、氯芥苯丁酸、順鉑、環磷醯胺、達卡巴嗪、洛莫司汀、美法崙、奧沙拉鉑、替莫唑胺或噻替派；抗代謝藥，諸如 5-氟尿嘧啶 (5-FU)、6-巰基嘌呤 (6-MP)、卡培他濱、阿糖胞苷、氟尿苷、氟達拉濱、吉西他濱、羥基脲、甲氨蝶呤或培美曲塞；蒽環類藥物，諸如柔紅黴素、阿黴素、表阿黴素或伊達比星；拓撲異構酶 I 抑制劑，諸如拓撲替康或伊立替康 (CPT-11)；拓撲異構酶 II 抑制劑，諸如依托泊苷 (VP-16)、替尼泊苷或米托蒽醌；有絲分裂抑制劑，諸如多西紫杉醇、雌莫司汀、伊沙匹隆、紫杉醇、長春鹼、長春新鹼或長春瑞濱；或皮質類固醇，諸如強體松、甲潑尼龍或地塞米松。In some embodiments, an antigen binding molecule (e.g., a monospecific and/or multispecific antigen binding molecule such as a bispecific or trispecific antigen binding molecule) is administered to an individual in combination with an additional therapeutic agent or additional treatment regimen. In some cases, the additional therapeutic agent comprises a chemotherapy agent, an immunotherapy agent, a targeted therapy, a hormonal therapy, a radiation therapy, or a combination thereof. Exemplary additional therapeutic agents include, but are not limited to: alkylating agents such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, olsalplatin, temozolomide, or thiotepa; anti-metabolites such as 5-fluorouracil (5-FU), 6-hydroxypurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed; anthracyclines such as daunorubicin, adriamycin, epiadriamycin, or idarubicin; topoisomerase I inhibitors, such as toponotecan or irinotecan (CPT-11); topoisomerase II inhibitors, such as etoposide (VP-16), teniposide, or mitoxantrone; mitotic inhibitors, such as docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine, or vinorelbine; or corticosteroids, such as prednisone, methylprednisolone, or dexamethasone.

在一些情況下，額外治療劑包含一線療法。在一些情況下，額外治療劑包含二線療法、三線療法、四線療法或五線療法。In some cases, the additional treatment includes first-line treatment. In some cases, the additional treatment includes second-line treatment, third-line treatment, fourth-line treatment, or fifth-line treatment.

在一些情況下，額外治療劑包含補救療法。In some cases, additional treatments include rescue therapies.

在一些情況下，額外治療劑包含姑息療法。In some cases, additional treatment includes palliative care.

在一些情況下，額外治療劑包含激素療法。示例性激素療法包括但不限於亮丙瑞林、恩雜魯胺、阿魯他胺及阿比特龍。In some cases, the additional treatment comprises hormonal therapy. Exemplary hormonal therapies include, but are not limited to, leuprolide, enzalutamide, alutamide, and abiraterone.

在一些情況下，額外治療劑包含免疫檢查點抑制劑，例如 PD-L1、PD-L2、PD-1、CTLA-4、LAG3、B7-H3、KIR、CD137、PS、TFM3、CD52、CD30、CD20、CD33、CD27、OX40、GITR、ICOS、BTLA (CD272)、CD160、2B4、LAIR1、TIGHT、LIGHT、DR3、CD226、CD2 或 SLAM 的抑制劑。在一些情況下，免疫檢查點抑制劑為帕博利珠單抗 (pembrolizumab)、納武利尤單抗 (nivolumab)、曲美木單抗 (tremelimumab) 或伊匹單抗 (ipilimumab)。In some cases, the additional treatment comprises an immune checkpoint inhibitor, such as an inhibitor of PD-L1, PD-L2, PD-1, CTLA-4, LAG3, B7-H3, KIR, CD137, PS, TFM3, CD52, CD30, CD20, CD33, CD27, OX40, GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM. In some cases, the immune checkpoint inhibitor is pembrolizumab, nivolumab, tremelimumab, or ipilimumab.

在一些情況下，額外治療劑包含抗體，諸如阿崙單抗、曲妥珠單抗、替依莫單抗 (ibritumomab tiuxetan)、本妥昔單抗維多丁 (brentuximab vedotin)、曲妥珠單抗-美坦新結合物 (ado-trastuzumab emtansine) 或博納吐單抗 (blinatumomab)。In some cases, the additional treatment includes an antibody such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumab emtansine, or blinatumomab.

在一些情況下，額外治療劑包含聚 ADP 核糖聚合酶 (PARP) 的抑制劑。示例性 PARP 抑制劑包括但不限於奧拉帕利 (olaparib) (AZD-2281，LYNPARZA®，來自 Astra Zeneca)、盧卡帕尼 (rucaparib) (PF-01367338，RUBRACA®，來自 Clovis Oncology)、尼拉帕尼 (niraparib) (MK-4827，ZEJULA®，來自 Tesaro)、他拉唑帕尼 (talazoparib) (BMN-673，來自 BioMarin Pharmaceutical Inc.)、維利帕尼 (veliparib) (ABT-888，來自 Abb Vie)、CK-102 (原 CEP 9722，來自 Teva Pharmaceutical Industries Ltd.)、E7016 (來自 Eisai)、依尼帕尼 (iniparib) (BSI 201，來自 Sanofi) 及帕米帕利 (pamiparib) (BGB-290，來自 BeiGene)。In some cases, additional treatments include inhibitors of poly ADP ribose polymerase (PARP). Exemplary PARP inhibitors include, but are not limited to, olaparib (AZD-2281, LYNPARZA®, from Astra Zeneca), rucaparib (PF-01367338, RUBRACA®, from Clovis Oncology), niraparib (MK-4827, ZEJULA®, from Tesaro), talazoparib (BMN-673, from BioMarin Pharmaceutical Inc.), veliparib (ABT-888, from Abb Vie), CK-102 (formerly CEP 9722, from Teva Pharmaceutical Industries Ltd.), E7016 (from Eisai), iniparib (BSI 201, from Sanofi), and pamiparib (BGB-290, from BeiGene).

在一些情況下，額外治療劑包含細胞激素。示例性細胞激素包括但不限於 IL-Ιβ、IL-6、IL-7、IL-10、IL-12、IL-15、IL-21 或 TNFα。In some cases, the additional therapeutic agent comprises a cytokine. Exemplary cytokines include, but are not limited to, IL-1β, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, or TNFα.

在一些實施例中，額外治療劑包含受體促效劑。在一些情況下，受體促效劑包含類鐸受體 (TLR) 配體。在一些情況下，TLR 配體包含 TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8 或 TLR9。在一些情況下，TLR 配體包含合成配體，諸如 Pam3Cys、CFA、MALP2、Pam2Cys、FSL-1、Hib-OMPC、Poly I:C、poly A:U、AGP、MPLA、RC-529、MDF2p、CFA 或鞭毛蛋白。In some embodiments, the additional therapeutic agent comprises a receptor agonist. In some cases, the receptor agonist comprises a toll-like receptor (TLR) ligand. In some cases, the TLR ligand comprises TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In some cases, the TLR ligand comprises a synthetic ligand such as Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I:C, poly A:U, AGP, MPLA, RC-529, MDF2p, CFA, or flagellin.

在一些情況下，額外治療方案為放射療法。In some cases, radiation therapy is an additional treatment option.

在一些情況下，額外治療方案為手術。In some cases, surgery is an additional treatment option.

上文述及之此等聯合療法涵蓋聯合投予 (其中兩種或多種治療劑包含同一或單獨的醫藥組成物中)，以及單獨投予，在這種情況下，本發明之抗原結合分子的投予可在投予額外一種或多種治療劑之前、同時及/或之後發生。在一個實施例中，投予抗原結合分子及投予額外治療劑彼此發生在約一個月內，或發生在約一週、兩週或三週內，或發生在約一天、兩天、三天、四天、五天、或六天內。Such combination therapies mentioned above encompass co-administration (wherein two or more therapeutic agents are included in the same or separate pharmaceutical compositions), as well as separate administration, in which case the administration of the antigen binding molecules of the present invention may occur before, simultaneously with, and/or after the administration of the additional one or more therapeutic agents. In one embodiment, administration of the antigen binding molecules and administration of the additional therapeutic agents occur within about one month, or within about one week, two weeks, or three weeks, or within about one day, two days, three days, four days, five days, or six days of each other.

本發明之抗原結合分子 (及/或任何額外治療劑) 可藉由任何合適的方式投予，包括皮下、靜脈內、肌內、局部、口服、經皮、腹膜內、眶內、藉由植入、藉由吸入、鞘內、心室內或鼻內。舉例而言，在一些實施例中，抗原結合分子係經皮下投予。在其他實施例中，抗原結合分子係經靜脈內投予。在一些實施例中，在患者中藉由皮下注射投予的抗原結合分子比藉由靜脈內注射投予相同抗原結合分子呈現出較小的毒性反應。給藥可透過任何合適的途徑進行，例如透過注射，例如靜脈內或皮下注射，部分取決於短暫投予還是長期投予。本文中考慮各種給藥方案，其包括但不限於在多種時間點單次或多次投予、快速注射投予及脈衝輸注。The antigen binding molecules of the present invention (and/or any additional therapeutic agent) can be administered by any suitable means, including subcutaneously, intravenously, intramuscularly, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. For example, in some embodiments, the antigen binding molecule is administered subcutaneously. In other embodiments, the antigen binding molecule is administered intravenously. In some embodiments, the antigen binding molecule administered by subcutaneous injection in a patient exhibits less toxicity than the same antigen binding molecule administered by intravenous injection. Administration can be performed by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether it is administered for a short period of time or for a long period of time. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations over various time points, bolus administration, and pulse infusion.

本發明之抗原結合分子可按照與良好醫學實踐一致的方式進行配製、給藥及投予。在此情況下，考慮的因素包括待治療的具體失調、待治療的具體哺乳動物、個別個體的臨床病症、失調的原因、遞送藥物的部位、投予方法、投予時程及醫療從業者已知的其他因素。抗原結合分子並非必須、但可以視情況與一種或多種目前用於預防或治療所述癌症之藥劑一起調配。此類其他藥劑的有效量取決於存在於調配物中的抗原結合分子的量、癌症或治療的類型以及上文討論的其他因素。這些藥物通常以與本文中所述相同的劑量和投予途徑，或本文中所述劑量的約 1% 至 99%，或以經驗上/臨床上確定為適當的任意劑量及藉由任意途徑使用。The antigen binding molecules of the present invention can be formulated, dosed and administered in a manner consistent with good medical practice. In this case, factors to be considered include the specific disorder to be treated, the specific mammal to be treated, the clinical condition of the individual, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to medical practitioners. The antigen binding molecules are not required, but can be formulated with one or more agents currently used to prevent or treat the cancer, as appropriate. The effective amount of such other agents depends on the amount of antigen binding molecules present in the formulation, the type of cancer or treatment, and other factors discussed above. These drugs are generally used in the same dosages and by any route as described herein, or about 1% to 99% of the dosages described herein, or in any dosage and by any route determined empirically/clinically to be appropriate.

對於癌症之預防或治療，本發明之抗原結合分子之適當劑量 (當單獨使用或與一種或多種其他額外治療劑組合使用) 將取決於待治療之癌症的類型、癌症之嚴重程度及病程、抗原結合分子係出於預防或是治療之目的而投予、先前的治療、患者的臨床病史及對該抗原結合分子的反應、以及主治醫師的判斷。抗原結合分子適於向患者投予一次或在一系列治療投予。For the prevention or treatment of cancer, the appropriate dose of the antigen binding molecules of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of cancer being treated, the severity and course of the cancer, whether the antigen binding molecule is being administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antigen binding molecule, and the judgment of the attending physician. The antigen binding molecules are suitably administered to the patient once or over a series of treatments.

作為一般性建議，向人類投予之抗原結合分子的治療有效量將在約 0.01 mg/kg 患者體重至約 100 mg/kg 患者體重之範圍內，無論藉由單次投予亦或多次投予。在一些實施例中，舉例而言，使用的抗原結合分子為約 0.01 mg/kg 至約 45 mg/kg、約 0.01 mg/kg 至約 40 mg/kg、約 0.01 mg/kg 至約 35 mg/kg、約 0.01 mg/kg 至約 30 mg/kg、約 0.01 mg/kg 至約 25 mg/kg、約 0.01 mg/kg 至約 20 mg/kg、約 0.01 mg/kg 至約 15 mg/kg、約 0.01 mg/kg 至約 10 mg/kg、約 0.01 mg/kg 至約 5 mg/kg 或約 0.01 mg/kg 至約 1 mg/kg。在一個實施例中，在 21 天週期的第 1 天，以約 100 mg、約 200 mg、約 300 mg、約 400 mg、約 500 mg、約 600 mg、約 700 mg、約 800 mg、約 900 mg、約 1000 mg、約 1100 mg、約 1200 mg、約 1300 mg 或約 1400 mg 的劑量向人類投予本文所述之抗原結合分子。該劑量可以單劑量或以多劑量 (例如 2 或 3 劑量) 投予，例如輸注。對於在幾天或更長時間內重複給藥，視病症而定，治療通常將持續直至出現所需的疾病症狀抑制。抗原結合分子的一種示例性劑量將在從 0.05 mg/kg 至約 10 mg/kg 的範圍內。因此，可以向個體投予約 0.5 mg/kg、2.0 mg/kg、4.0 mg/kg 或 10 mg/kg 中的一種或多種劑量 (或其任意組合)。此等劑量可以間歇施用，例如每週或每三周施用 (例如，使得患者接受約 2 種至約 20 種或例如約六種劑量的抗 CD3 抗體)。可投予初始較高的負載劑量，隨後投予一個或多個較低劑量。藉由習用技術及測定很容易監測此治療的進展。在本發明之前述態樣中任一者的一些實施例中，個體、患者或受試者為人類。As a general rule, a therapeutically effective amount of the antigen binding molecule administered to a human will be in the range of about 0.01 mg/kg patient body weight to about 100 mg/kg patient body weight, whether by a single administration or multiple administrations. In some embodiments, for example, the antigen binding molecule is used at about 0.01 mg/kg to about 45 mg/kg, about 0.01 mg/kg to about 40 mg/kg, about 0.01 mg/kg to about 35 mg/kg, about 0.01 mg/kg to about 30 mg/kg, about 0.01 mg/kg to about 25 mg/kg, about 0.01 mg/kg to about 20 mg/kg, about 0.01 mg/kg to about 15 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 5 mg/kg, or about 0.01 mg/kg to about 1 mg/kg. In one embodiment, an antigen binding molecule described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg on day 1 of a 21-day cycle. The dose may be administered as a single dose or in multiple doses (e.g., 2 or 3 doses), such as by infusion. For repeated dosing over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dosage of the antigen binding molecule will range from 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to an individual. Such doses may be administered intermittently, such as weekly or every three weeks (e.g., so that the patient receives about 2 to about 20 or, for example, about six doses of anti-CD3 antibodies). An initial higher loading dose may be administered, followed by one or more lower doses. The progress of this treatment is easily monitored by customary techniques and assays. In some embodiments of any of the foregoing aspects of the invention, the individual, patient or subject is a human.

在一些實施例中，抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 可用於抑制或減少表現 STEAP1 之細胞的增生。在一些情況下，細胞為前列腺癌、尤文氏肉瘤、肺癌、大腸直腸癌、乳癌、膀胱癌、卵巢癌或子宮頸癌之細胞。在一些情況下，細胞為前列腺癌細胞或尤文氏肉瘤細胞。在一些情況下，抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 係用於活體內方法中。在額外情況下，抗原結合分子 (例如，單特異性及/或多特異性抗原結合分子諸如雙特異性或三特異性抗原結合分子) 係用於活體外或離體方法中。VII.套組/製品In some embodiments, antigen binding molecules (e.g., monospecific and/or multispecific antigen binding molecules such as bispecific or trispecific antigen binding molecules) can be used to inhibit or reduce proliferation of cells expressing STEAP1. In some cases, the cells are prostate cancer, Ewing's sarcoma, lung cancer, colorectal cancer, breast cancer, bladder cancer, ovarian cancer, or cervical cancer cells. In some cases, the cells are prostate cancer cells or Ewing's sarcoma cells. In some cases, antigen binding molecules (e.g., monospecific and/or multispecific antigen binding molecules such as bispecific or trispecific antigen binding molecules) are used in an in vivo method. In additional cases, antigen binding molecules (eg, monospecific and/or multispecific antigen binding molecules such as bispecific or trispecific antigen binding molecules) are used in in vitro or ex vivo methods.VII.Kits/Articles

本文提供了一種套組或製品，該套組或製品包括本文所述之一種或多種組成物 (例如，包含本文所述之抗原結合分子中之任意一種或多種及醫藥上可接受之載劑的組成物) 及用於向個體投予該組成物以治療癌症 (例如，前列腺癌或尤文氏肉瘤) 或延緩其進展的藥品仿單。Provided herein is a kit or article of manufacture comprising one or more compositions described herein (e.g., a composition comprising any one or more of the antigen-binding molecules described herein and a pharmaceutically acceptable carrier) and a package insert for administering the composition to an individual to treat cancer (e.g., prostate cancer or Ewing's sarcoma) or delay its progression.

該套組或製品可包含容器及容器上或與容器相關之標示或藥品仿單。合適的容器包括例如瓶、小瓶、注射器、IV 溶液袋等。容器可以由多種材料例如玻璃或塑膠形成。該容器可容納組成物，該組成物本身或與有效治療、預防及/或診斷症狀的另一組成物結合使用，並可能具有無菌入口 (例如，容器可為具有可透過皮下注射針頭穿臼的塞子的靜脈內溶液袋或小管)。組成物中的至少一種活性劑為本發明之抗原結合分子。標示或藥品仿單可指示該組成物用於治療本文所揭示之任何疾患 (例如，癌症 (例如，前列腺癌或尤文氏肉瘤))。The kit or article may include a container and a label or drug leaflet on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed of a variety of materials such as glass or plastic. The container can hold a composition that is used by itself or in combination with another composition that is effective in treating, preventing and/or diagnosing symptoms, and may have a sterile access port (for example, the container may be an intravenous solution bag or tubule with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an antigen binding molecule of the present invention. The label or drug leaflet may indicate that the composition is used to treat any disease disclosed herein (for example, cancer (for example, prostate cancer or Ewing's sarcoma)).

再者，套組或製品可包含 (a) 其中包含有組成物的第一容器，其中該組成物包含本發明之抗原結合分子；及 (b) 其中包含有組成物的第二容器，其中該組成物包含其他另外的毒性劑或其他治療劑。本發明之此態樣中的套組或製品可以進一步包含指示組成物可以用於治療特定病況的藥品仿單。替代地或另外地，套組或製品可進一步包含第二 (或第三) 容器，該容器包含醫藥上可接受之緩衝劑，例如抑菌注射用水 (BWFI)、磷酸鹽緩衝鹽水、林格氏溶液及葡萄糖溶液。從商業和使用者的角度來看，它可以進一步包含其他材料，其中包括其他緩衝劑、稀釋劑、過濾器、針頭及注射器。VIII.用於治療細胞激素釋放症候群(CRS)的托珠單抗先前技術Furthermore, the kit or article may comprise (a) a first container containing a composition, wherein the composition comprises an antigen-binding molecule of the present invention; and (b) a second container containing a composition, wherein the composition comprises other additional toxic agents or other therapeutic agents. The kit or article in this aspect of the invention may further comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the kit or article may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. From a commercial and user perspective, it may further comprise other materials, including other buffers, diluents, filters, needles, and syringes.VIII.Prior Artof Tocilizumabfor the Treatment of Cytokine Release Syndrome(CRS)

涉及 T 細胞活化之雙特異性抗體治療劑伴有細胞激素釋放症候群 (CRS)。CRS 為一種潛在地危及生命之複合症狀，其係因為在擴大及持續免疫反應期間細胞激素由免疫效應子或標靶細胞過度釋放所引起。CRS 可由各種因素 (包括有毒病原體感染) 或由活化或增強免疫反應以產生明顯及持續之免疫反應的藥劑所觸發。Bispecific antibody therapy involving T cell activation is associated with cytokine release syndrome (CRS). CRS is a potentially life-threatening symptom complex caused by excessive release of cytokines from immune effector or target cells during an amplified and sustained immune response. CRS can be triggered by a variety of factors, including infection with virulent pathogens, or by agents that activate or enhance the immune response to produce a pronounced and sustained immune response.

不管何種誘發因素，嚴重或危及生命之 CRS 皆為醫學緊急情況。若不能成功管控，則其可產生顯著失能或致命結果。目前的臨床管理側重於治療個體體徵及症狀，提供支持性照護，且嘗試使用高劑量皮質類固醇來抑制炎症反應。然而，此方式並不總是成功，尤其係在後期干預之情形下。此外，類固醇可負面地影響 T 細胞功能，此可減弱免疫調節療法在癌症治療中之臨床益處。Regardless of the precipitating factors, severe or life-threatening CRS is a medical emergency. If not successfully managed, it can result in significant disability or fatal outcomes. Current clinical management focuses on treating individual signs and symptoms, providing supportive care, and attempting to suppress the inflammatory response with high-dose corticosteroids. However, this approach is not always successful, especially in late-stage intervention settings. In addition, steroids can negatively affect T-cell function, which can diminish the clinical benefit of immunomodulatory therapies in cancer treatment.

CRS 與高 IL-6 含量相關聯 (Panelli 等人，J Transl.Med.，2:17，2004；Lee 等人，Blood,124:188-195，2014；Doessegger 及 Banholzer，Clin. Transl.Immunology，4:e39，2015)，且 IL-6 與 CRS 之嚴重程度相關，與未發生 CRS 或發生較輕 CRS 反應 (NCI CTCAE 0 級至 3 級) 的患者相比，發生重度或危及生命之 CRS (NCI CTCAE 4 級或 5 級) 的患者具有高得多的 IL-6 含量多 (Chen 等人，J. Immunol. Methods，434:1-8，2016)。CRS is associated with high IL-6 levels (Panelli et al.,J Transl. Med. , 2:17, 2004; Lee et al.,Blood, 124:188-195, 2014; Doessegger and Banholzer,Clin. Transl. Immunology , 4:e39, 2015), and IL-6 is associated with the severity of CRS. Patients with severe or life-threatening CRS (NCI CTCAE grade 4 or 5) have much higher IL-6 levels than patients without CRS or with milder CRS reactions (NCI CTCAE grade 0 to 3) (Chen et al.,J. Immunol. Methods , 434:1-8, 2016).

托珠單抗 (ACTEMRA®/ROACTEMRA®) 為針對可溶性及膜結合 IL-6R 之重組、人源化、抗人單株抗體，其抑制 IL-6 介導之傳訊 (例如參見 WO 1992/019579，其全部內容以引用方式併入本文中)。托珠單抗已由美國食品藥物監督管理局 (U.S. Food and Drug Administration) 核準用於治療成人以及 2 歲及更年長兒科患者的嚴重或危及生命之 CAR-T 細胞誘導性 CRS。最初之臨床資料 (Locke 等人，Blood, 130: 1547, 2017) 表明，托珠單抗預防可藉由在細胞激素釋放之前阻斷 IL-6 受體之傳訊來減小 CAR-T 細胞誘導性 CRS 的嚴重程度。因此，托珠單抗前置用藥亦可減小與雙特異性抗體療法有關之 CRS 之頻率或降低其嚴重程度。可與托珠單抗組合使用之其他抗 IL-6R 抗體包括撒裡路單抗(sarilumab)、維巴麗珠單抗 (vobarilizumab) (ALX-0061)、SA-237 及其變異體。CRS症狀及分級Tocilizumab (ACTEMRA®/ROACTEMRA®) is a recombinant, humanized, anti-human monoclonal antibody directed against soluble and membrane-bound IL-6R that inhibits IL-6-mediated signaling (see, e.g., WO 1992/019579, the entire contents of which are incorporated herein by reference). Tocilizumab has been approved by the US Food and Drug Administration for the treatment of severe or life-threatening CAR-T cell-induced CRS in adults and pediatric patients 2 years of age and older. Initial clinical data (Locke et al.,Blood , 130: 1547, 2017) suggest that tocilizumab prophylaxis may reduce the severity of CAR-T cell-induced CRS by blocking IL-6 receptor signaling prior to cytokine release. Therefore, tocilizumab premedication may also reduce the frequency or severity of CRS associated with bispecific antibody therapy. Other anti-IL-6R antibodies that can be used in combination with tocilizumab include sarilumab, vobarilizumab (ALX-0061), SA-237 and its variants.CRSsymptoms and grades

CRS 可根據 Lee 等人Blood124:188-195，2014 或 Lee 等人，Biol Blood Marrow Transplant25(4):625-638，2019 所確立之改良細胞激素釋放症候群分級系統進行分級，如表 A 中所述。除診斷標準以外，亦提供基於其嚴重程度之推薦 CRS 管控 (包括使用皮質類固醇及/或抗細胞激素療法之早期干預) 並提及於表 A 及 B 中。表A.細胞激素釋放症候群分級系統等級修正細胞激素釋放症候群分級系統ASTCT共識分級系統第 1 級症狀不會危及生命，且僅需要對症治療 (例如發燒、噁心、疲勞、頭痛、肌痛、不適)體溫 ≥ 38℃ 無低血壓 無低氧第 2 級症狀需要適度干預且對其具有反應 氧需求 ＜ 40%；或 對流體或低劑量^a之一種血管升壓劑具有反應的低血壓；或 2 級器官毒性體溫 ≥ 38℃* 伴以不需要升壓藥之低血壓及/或^†需要低流量鼻插管^‡或漏氣之缺氧   第 3 級症狀需要侵襲性干預且對其具有反應 需氧量 ≥ 40%；或 需要高劑量^b或多種血管升壓劑之低血壓；或 3 級器官毒性或 4 級轉胺酶炎體溫 ≥ 38℃* 伴以需要具有或不具有升壓素之升壓藥的低血壓及/或^†需要高流量鼻插管^‡、面罩、非再呼吸器面罩或文丘裏面罩之缺氧第 4 級危及生命之症狀 需要通風支持或 4 級器官毒性 (排除轉胺酶炎)體溫 ≥ 38℃* 伴以需要多種升壓藥 (不包括升壓素) 之低血壓及/或^†需要正壓 (例如 CPAP、BiPAP、插管及機械換氣法) 之缺氧第 5 級死亡死亡Lee 2014 準則：Lee 等人，Blood,124: 188-195, 2014。 ASTCT 共識分級：Lee 等人，Biol Blood Marrow Transplant, 25(4): 625-638, 2019。^a低劑量升壓藥：低於表 B 所示劑量之單一升壓藥。^b高劑量升壓藥：如表 B 中所定義。 *發熱定義為體溫 ≥38℃ 且不歸因於任何其他病因。在然後接受退熱或抗細胞激素療法 (例如托珠單抗 (tocilizumab) 或類固醇) 之 CRS 患者中，不再需要發熱對後續 CRS 嚴重程度進行分級。在此情形下，CRS 分級係由低血壓及/或低氧來驅動。 †CRS 等級取決於以下較嚴重事件：不歸因於任何其他病因之低血壓或低氧。舉例而言，將體溫為 39.5℃、具有需要 1 種血管升壓劑之低血壓且具有需要低流量鼻套管之低氧的患者分類為 3 級 CRS。 ‡低流量鼻套管定義為以 ≤ 6L/分鐘進行氧遞送。低流量亦包括偶爾用於兒科中之吹氣式氣氧遞送。高流量定義為以 ＞6L/分鐘進行遞送。表B.高劑量升壓藥高劑量血管升壓劑(持續時間≥3小時)升壓劑劑量去甲腎上腺素單一療法≥ 20 µg/min多巴胺 (dopamine) 單一療法≥ 10 µg /kg/min苯福林 (phenylephrine) 單一療法≥ 200 µg/min腎上腺素單一療法≥ 10 µg/min若使用血管加壓素則血管加壓素 + 去甲腎上腺素等效物 ≥ 10 µg/min^a若使用組合或血管升壓劑 (非血管加壓素)則去甲腎上腺素等效物 ≥ 20 µg/min^amin = 分鐘；VASST = 血管加壓素及敗血性休克試驗。^aVASST 血管升壓劑當量方程式：去甲腎上腺素等效劑量 = [去甲腎上腺素 (µg /min)] + [多巴胺 (µg /kg/min) ÷ 2] + [腎上腺素 (µg /min)] + [苯福林 (µg /min) ÷ 10]。CRS can be graded according to the modified cytokine release syndrome grading system established by Lee et al.,Blood 124:188-195, 2014 or Lee et al.,Biol Blood Marrow Transplant 25(4):625-638, 2019, as described in Table A. In addition to the diagnostic criteria, recommended management of CRS based on its severity (including early intervention with corticosteroids and/or anticytokine therapy) is also provided and mentioned in Tables A and B.TableA.Cytokine Release Syndrome Grading SystemLevelRevised CRP grading systemASTCTconsensus grading system Level 1 Symptoms are not life-threatening and require only symptomatic treatment (e.g., fever, nausea, fatigue, headache, myalgia, malaise) Body temperature ≥ 38℃ No hypotension No hypoxia Level 2 Symptoms requiring moderate intervention for which the oxygen requirement is <40%; or hypotension responsive to fluids or low-dose^a vasopressor; or grade 2 organ toxicity Temperature ≥ 38°C* with hypotension not requiring vasopressors and/or^† hypoxia requiring low-flow nasal cannula^‡ or air leak Level 3 Symptoms requiring invasive intervention with a responsive oxygen requirement ≥ 40%; or hypotension requiring high-^doseb or multiple vasopressors; or grade 3 organ toxicity or grade 4 transaminitis Temperature ≥ 38°C* with hypotension requiring vasopressors with or without vasopressors and/or^† hypoxia requiring high-flow nasal cannula^‡ , face mask, non-rebreather mask, or Venturi mask Level 4 Life-threatening symptoms requiring ventilatory support or grade 4 organ toxicity (excluding transaminitis) Temperature ≥ 38°C* with hypotension requiring multiple vasopressors (excluding vasopressin) and/or^† hypoxia requiring positive pressure (e.g. CPAP, BiPAP, intubation, and mechanical ventilation) Level 5 die die Lee 2014 Guidelines: Lee et al.,Blood, 124: 188-195, 2014. ASTCT consensus grade: Lee et al.,Biol Blood Marrow Transplant , 25(4): 625-638, 2019.^aLow -dose vasopressor: A single vasopressor at a dose lower than that shown in Table B.^bHigh -dose vasopressor: As defined in Table B. * Fever is defined as a temperature ≥ 38°C not attributable to any other etiology. In patients with CRS who subsequently receive antipyretic or anticytokine therapy (e.g., tocilizumab or steroids), fever is no longer required to grade the subsequent severity of CRS. In this setting, CRS grade is driven by hypotension and/or hypoxia. †CRS grade is determined by the more severe event of: hypotension or hypoxia not attributable to any other etiology. For example, a patient with a temperature of 39.5°C, hypotension requiring 1 vasopressor, and hypoxia requiring a low-flow nasal cannula is classified as Grade 3 CRS. ‡Low-flow nasal cannula is defined as oxygen delivery at ≤ 6 L/min. Low flow also includes insufflated oxygen delivery, which is occasionally used in pediatrics. High flow is defined as delivery at > 6 L/min.TableB.High-dose vasopressorsHigh-dose vasopressors(duration≥3hours)BoostersDosage Norepinephrine monotherapy ≥ 20 µg/min Dopamine monotherapy ≥ 10 µg/kg/min Phenylephrine monotherapy ≥ 200 µg/min Epinephrine monotherapy ≥ 10 µg/min If vasopressin is used Then vasopressin + norepinephrine equivalents ≥ 10 µg/min^a If using combination or vasopressor (non-vasopressin) Norepinephrine equivalents ≥ 20 µg/min^a min = minutes; VASST = vasopressin and septic shock test.^a VASST vasopressor equivalent equation: Norepinephrine equivalent dose = [norepinephrine (µg /min)] + [dopamine (µg /kg/min) ÷ 2] + [epinephrine (µg /min)] + [phenylephrine (µg /min) ÷ 10].

CRS 及/或輸注相關反應 (IRR) 之輕度至中度表現可包括諸如發熱、頭痛及肌痛等症狀，且可使用所指示之止痛劑、退熱劑及抗組織胺劑進行症狀性治療。CRS 及/或 IRR 之嚴重或危及生命之表現 (例如低血壓、心跳過速、呼吸困難或胸部不適) 應使用所指示之支持性及復甦性措施進行侵襲性治療，包括使用高劑量皮質類固醇、IV 輸液、入住加護病房及其他支持性措施。嚴重 CRS 可與其他臨床後遺症 (例如瀰慢性血管內凝血、毛細管洩漏症候群或巨噬球活化症候群 (MAS)) 有關。基於免疫之療法所致之重度或危及生命的 CRS 的照護標準尚未確立；已經公布了使用抗細胞激素療法 (諸如托珠單抗) 之病例報告及建議 (Teachey 等人，Blood121:5154-5157 (2013)；Lee 等人，Blood，124:188-195 (2014)；Maude 等人，New Engl. J. Med.，371:1507-1517 (2014))。Mild to moderate manifestations of CRS and/or infusion-related reactions (IRRs) may include symptoms such as fever, headache, and myalgia, and may be treated symptomatically with analgesics, antipyretics, and antihistamines as indicated. Severe or life-threatening manifestations of CRS and/or IRRs (e.g., hypotension, tachycardia, dyspnea, or chest discomfort) should be treated aggressively with supportive and resuscitative measures as indicated, including high-dose corticosteroids, IV fluids, ICU admission, and other supportive measures. Severe CRS may be associated with other clinical sequelae such as chronic intravascular coagulation, capillary leak syndrome, or macrophage activation syndrome (MAS). Standards of care for severe or life-threatening CRS caused by immune-based therapies have not been established; case reports and recommendations for the use of anticytokine therapy (such as tocilizumab) have been published (Teachey et al.,Blood 121:5154-5157 (2013); Lee et al.,Blood , 124:188-195 (2014); Maude et al.,New Engl. J. Med. , 371:1507-1517 (2014)).

如表 A 所示，即使患有廣泛共病之受試者出現中度 CRS 表現亦應密切監測，且考慮入住加護病室及使用托珠單抗。托珠單抗作為預先用藥As shown in Table A, even subjects with extensive comorbidities who present with moderate manifestations of CRS should be closely monitored and consideration should be given to ICU admission and the use oftocilizumab .

在一些態樣中，將有效量之托珠單抗作為預先用藥投予，例如在投予本文所述之多特異性抗原結合分子 (例如，雙特異性或三特異性抗原結合分子，例如雙特異性抗體) 之前向個體投予。以前置用藥形式投予托珠單抗可減小 CRS 之頻率或嚴重程度。在一些態樣中，托珠單抗作為預先用藥在第 1 週期中投予，例如在多特異性抗原結合分子 (例如，雙特異性抗體) 之第一劑量 (C1D1)、第二劑量 (C1D2) 及/或第三劑量 (C1D3) 之前投予。在一些態樣中，托珠單抗以約 1 mg/kg 至約 15 mg/kg，例如約 4 mg/kg 至約 10 mg/kg，例如約 6 mg/kg 至約 10 mg/kg，例如約 8 mg/kg 之單一劑量靜脈內投予個體。在一些態樣中，托珠單抗以約 8 mg/kg 之單一劑量經靜脈內投予個體。可與托珠單抗組合使用之其他抗 IL-6R 抗體包括撒裡路單抗(sarilumab)、維巴麗珠單抗 (vobarilizumab) (ALX-0061)、SA-237 及其變異體。In some aspects, an effective amount of tocilizumab is administered as a premedication, e.g., prior to administration of a multispecific antigen-binding molecule (e.g., a bispecific or trispecific antigen-binding molecule, e.g., a bispecific antibody) described herein to an individual. Administration of tocilizumab as a premedication can reduce the frequency or severity of CRS. In some aspects, tocilizumab is administered as a premedication in cycle 1, e.g., prior to the first dose (C1D1), the second dose (C1D2), and/or the third dose (C1D3) of a multispecific antigen-binding molecule (e.g., a bispecific antibody). In some aspects, tocilizumab is administered intravenously to a subject in a single dose of about 1 mg/kg to about 15 mg/kg, such as about 4 mg/kg to about 10 mg/kg, such as about 6 mg/kg to about 10 mg/kg, such as about 8 mg/kg. In some aspects, tocilizumab is administered intravenously to a subject in a single dose of about 8 mg/kg. Other anti-IL-6R antibodies that can be used in combination with tocilizumab include sarilumab, vobarilizumab (ALX-0061), SA-237, and variants thereof.

例如，在一個態樣中，多特異性抗原結合分子 (例如，雙特異性抗體) 係與托珠單抗 (ACTEMRA® / ROACTEMRA®) 共同投予，其中首先投予個體托珠單抗 (ACTEMRA® / ROACTEMRA®)，且雙特異性抗體隨後單獨投予 (例如，個體用托珠單抗 (ACTEMRA® / ROACTEMRA®) 預治療)。For example, in one embodiment, a multispecific antigen-binding molecule (e.g., a bispecific antibody) is co-administered with tocilizumab (ACTEMRA®/ROACTEMRA®), wherein the individual is administered tocilizumab (ACTEMRA®/ROACTEMRA®) first and the bispecific antibody is subsequently administered alone (e.g., the individual is pretreated with tocilizumab (ACTEMRA®/ROACTEMRA®)).

在一些態樣中，本揭露之特徵在於本發明之多特異性抗原結合分子 (例如，雙特異性抗體) 在製造藥物中之用途，該藥物用於與一種或多種額外治療劑 (例如，托珠單抗) 組合治療患有前列腺癌或尤文氏肉瘤的個體。In some aspects, the disclosure features use of a multispecific antigen-binding molecule (e.g., a bispecific antibody) of the invention in the manufacture of a medicament for treating a subject with prostate cancer or Ewing's sarcoma in combination with one or more additional therapeutic agents (e.g., tocilizumab).

在一些態樣中，多特異性抗原結合分子 (例如，雙特異性抗體) 及一種或多種額外治療劑係經分開調配。在一些態樣中，多特異性抗原結合分子 (例如，雙特異性抗體) 係待在投予一種或多種額外治療劑之前向個體投予。在其他態樣中，多特異性抗原結合分子 (例如，雙特異性抗體) 係待在一種或多種額外治療劑之後向個體投予，例如，在投予有效量的托珠單抗之後向個體投予。In some aspects, the multispecific antigen-binding molecule (e.g., bispecific antibody) and one or more additional therapeutic agents are formulated separately. In some aspects, the multispecific antigen-binding molecule (e.g., bispecific antibody) is to be administered to the subject prior to administration of the one or more additional therapeutic agents. In other aspects, the multispecific antigen-binding molecule (e.g., bispecific antibody) is to be administered to the subject after the one or more additional therapeutic agents, for example, after administration of an effective amount of tocilizumab.

在一些態樣中，多特異性抗原結合分子 (例如，雙特異性抗體) 及一種或多種額外治療劑係經一起調配。In some aspects, a multispecific antigen-binding molecule (e.g., a bispecific antibody) and one or more additional therapeutic agents are formulated together.

在一些態樣中，本揭露之特徵在於本發明之多特異性抗原結合分子 (例如，雙特異性抗體) 與一種或多種額外治療劑組合用於治療患有前列腺癌或尤文氏肉瘤的個體。In some aspects, the disclosure features a multispecific antigen-binding molecule (e.g., a bispecific antibody) of the invention in combination with one or more additional therapeutic agents for treating a subject suffering from prostate cancer or Ewing's sarcoma.

在一些態樣中，多特異性抗原結合分子 (例如，雙特異性抗體) 及一種或多種額外治療劑係經分開調配。在一些態樣中，多特異性抗原結合分子 (例如，雙特異性抗體) 係待在投予一種或多種額外治療劑之前向個體投予。在其他態樣中，多特異性抗原結合分子 (例如，雙特異性抗體) 係待在一種或多種額外治療劑之後向個體投予，例如，在投予有效量的托珠單抗之後向個體投予。In some aspects, the multispecific antigen-binding molecule (e.g., bispecific antibody) and one or more additional therapeutic agents are formulated separately. In some aspects, the multispecific antigen-binding molecule (e.g., bispecific antibody) is to be administered to the subject prior to administration of the one or more additional therapeutic agents. In other aspects, the multispecific antigen-binding molecule (e.g., bispecific antibody) is to be administered to the subject after the one or more additional therapeutic agents, for example, after administration of an effective amount of tocilizumab.

在一些態樣中，多特異性抗原結合分子 (例如，雙特異性抗體) 及一種或多種額外治療劑係經一起調配。托珠單抗治療CRSIn some aspects, a multispecific antigen-binding molecule (e.g., a bispecific antibody) and one or more additional therapeutic agents are formulated together.Tocilizumab for the treatmentof CRS

在一些態樣中，個體在用治療性多特異性抗原結合分子 (例如，雙特異性抗體) 治療期間發生 CRS 事件且投予有效量之托珠單抗以管理 CRS 事件。In some aspects, the subject develops a CRS event during treatment with a therapeutic multispecific antigen binding molecule (e.g., a bispecific antibody) and an effective amount of tocilizumab is administered to manage the CRS event.

在一些態樣中，個體具有 CRS 事件 (例如，在用多特異性抗原結合分子 (例如，雙特異性抗體) 治療後具有 CRS 事件，例如在多特異性抗原結合分子之第一劑量或後續劑量後具有 CRS 事件)，且該方法進一步包括在中止用多特異性抗原結合分子治療時治療 CRS 事件之症狀。In some aspects, the individual has a CRS event (e.g., has a CRS event after treatment with a multispecific antigen-binding molecule (e.g., a bispecific antibody), such as after a first dose or a subsequent dose of the multispecific antigen-binding molecule), and the method further comprises treating symptoms of the CRS event upon discontinuation of treatment with the multispecific antigen-binding molecule.

在一些態樣中，個體發生 CRS 事件，且該方法進一步包括在中止多特異性抗原結合分子 (例如，雙特異性抗體) 治療時向個體投予有效量之介白素 6 受體 (IL-6R) 拮抗劑 (例如抗 IL-6R 抗體，例如托珠單抗 (ACTEMRA® / ROACTEMRA®)) 以管理 CRS 事件。在一些態樣中，IL-6R拮抗劑 (例如托珠單抗) 以約 1 mg/kg 至約 15 mg/kg，例如約 4 mg/kg 至約 10 mg/kg，例如約 6 mg/kg 至約 10 mg/kg，例如約 8 mg/kg 之單一劑量靜脈內投予個體。在一些態樣中，托珠單抗以約 8 mg/kg 之單一劑量經靜脈內投予個體。可與托珠單抗組合使用之其他抗 IL-6R 抗體包括撒裡路單抗(sarilumab)、維巴麗珠單抗 (vobarilizumab) (ALX-0061)、SA-237 及其變異體。In some aspects, the subject develops a CRS event, and the method further comprises administering to the subject an effective amount of an interleukin 6 receptor (IL-6R) antagonist (e.g., an anti-IL-6R antibody, such as tocilizumab (ACTEMRA®/ROACTEMRA®)) to manage the CRS event while discontinuing the multispecific antigen binding molecule (e.g., bispecific antibody) treatment. In some aspects, the IL-6R antagonist (e.g., tocilizumab) is administered intravenously to the subject in a single dose of about 1 mg/kg to about 15 mg/kg, such as about 4 mg/kg to about 10 mg/kg, such as about 6 mg/kg to about 10 mg/kg, such as about 8 mg/kg. In some aspects, tocilizumab is administered intravenously to a subject in a single dose of about 8 mg/kg. Other anti-IL-6R antibodies that can be used in combination with tocilizumab include sarilumab, vobarilizumab (ALX-0061), SA-237, and variants thereof.

在一些態樣中，CRS 事件在治療 CRS 事件之症狀的 24 小時內未消退或惡化，且該方法進一步包括向個體投予一個或多個額外劑量之 IL-6R 拮抗劑 (例如抗 IL-6R 抗體，例如托珠單抗) 來控制 CRS 事件，例如以約 1 mg/kg 至約 15 mg/kg，例如約 4 mg/kg 至約 10 mg/kg，例如約 6 mg/kg 至約 10 mg/kg，例如約 8 mg/kg 之劑量將一個或多個額外劑量之托珠單抗靜脈內投予個體。在一些態樣中，該一個或多個額外劑量之托珠單抗以約 8 mg/kg 之單一劑量經靜脈內投予該個體。In some aspects, the CRS event does not resolve or worsens within 24 hours of treating the symptoms of the CRS event, and the method further comprises administering to the subject one or more additional doses of an IL-6R antagonist (e.g., an anti-IL-6R antibody, such as tocilizumab) to control the CRS event, such as administering one or more additional doses of tocilizumab intravenously to the subject at a dose of about 1 mg/kg to about 15 mg/kg, such as about 4 mg/kg to about 10 mg/kg, such as about 6 mg/kg to about 10 mg/kg, such as about 8 mg/kg. In some aspects, the one or more additional doses of tocilizumab are administered intravenously to the subject in a single dose of about 8 mg/kg.

在一些態樣中，該方法進一步包括向個體投予有效量之皮質類固醇。皮質類固醇可靜脈內投予個體。在一些態樣中，皮質類固醇為甲潑尼松 (甲潑尼龍)。在一些情況下，甲潑尼松以每天約 1 mg/kg 至每天約 5 mg/kg，例如每天約 2 mg/kg 之劑量投予。在一些情況下，皮質類固醇為地塞米松。在一些情況下，地塞米松以約 10 mg 之劑量 (例如，靜脈內約 10 mg 之單一劑量) 或以約 0.5 mg/kg/天之劑量投予。In some aspects, the method further comprises administering to the individual an effective amount of a corticosteroid. The corticosteroid can be administered intravenously to the individual. In some aspects, the corticosteroid is methylprednisolone (methylprednisolone). In some cases, methylprednisolone is administered in an amount of about 1 mg/kg per day to about 5 mg/kg per day, such as about 2 mg/kg per day. In some cases, the corticosteroid is dexamethasone. In some cases, dexamethasone is administered in an amount of about 10 mg (e.g., a single dose of about 10 mg intravenously) or in an amount of about 0.5 mg/kg/day.

若單獨投予 IL-6R 拮抗劑 (例如托珠單抗) 不能管理 CRS 事件，則可向個體投予皮質類固醇，例如甲潑尼龍或地塞米松。在一些態樣中，治療 CRS 事件之症狀進一步包括用高劑量升壓藥 (例如，去甲腎上腺素、多巴胺、去羥腎上腺素、腎上腺素或升壓素及去甲腎上腺素) 治療，例如，如表 A、B 及 C 中所述。表 C 及 D 提供有關重度或危及生命的 CRS 之托珠單抗治療的詳細資訊。If administration of an IL-6R antagonist (e.g., tocilizumab) alone fails to manage the CRS event, a corticosteroid, such as methylprednisolone or dexamethasone, may be administered to the individual. In some aspects, treating the symptoms of the CRS event further comprises treatment with a high-dose vasopressor (e.g., norepinephrine, dopamine, norepinephrine, epinephrine, or vasopressin and norepinephrine), e.g., as described in Tables A, B, and C. Tables C and D provide detailed information about tocilizumab treatment of severe or life-threatening CRS.

在一些態樣中，本揭露的特徵在於托珠單抗在製備用於治療患有CRS事件的個體的藥物中的用途，其中 CRS 事件在用本發明的雙特異性抗體治療個體期間出現。In some aspects, the disclosure features use of tocilizumab in the preparation of a medicament for treating an individual suffering from a CRS event, wherein the CRS event occurs during treatment of the individual with a bispecific antibody of the invention.

在一些態樣中，在中止用本發明之多特異性抗原結合分子 (例如，雙特異性抗體) 治療的同時將藥物投予個體。In some aspects, a drug is administered to a subject simultaneously with cessation of treatment with a multispecific antigen-binding molecule (e.g., bispecific antibody) of the invention.

在一些態樣中，該藥物被配製用於以約 1 mg/kg 至約 15 mg/kg，例如約 4 mg/kg 至約 10 mg/kg，例如約 6 mg/kg 至約 10 mg/kg，例如約 8 mg/kg 之單一劑量的托珠單抗之靜脈內投予。In some aspects, the drug is formulated for intravenous administration of tocilizumab in a single dose of about 1 mg/kg to about 15 mg/kg, e.g., about 4 mg/kg to about 10 mg/kg, e.g., about 6 mg/kg to about 10 mg/kg, e.g., about 8 mg/kg.

在一些態樣中，CRS 事件在治療該 CRS 事件之症狀的 24 小時內未消退或惡化， 且向個體投予一個或多個額外劑量之托珠單抗。一個或多個額外劑量之托珠單抗以約 1 mg/kg 至約 15 mg/kg，例如約 4 mg/kg 至約 10 mg/kg，例如約 6 mg/kg 至約 10 mg/kg，例如約 8 mg/kg 之劑量靜脈內投予個體。In some aspects, the CRS event does not resolve or worsens within 24 hours of treating the symptoms of the CRS event, and one or more additional doses of tocilizumab are administered to the subject. The one or more additional doses of tocilizumab are administered intravenously to the subject at a dose of about 1 mg/kg to about 15 mg/kg, such as about 4 mg/kg to about 10 mg/kg, such as about 6 mg/kg to about 10 mg/kg, such as about 8 mg/kg.

在一些態樣中，藥物與有效量的皮質類固醇組合使用以治療 CRS 事件。托珠單抗及皮質類固醇可分開調配。In some aspects, the drug is used in combination with an effective amount of a corticosteroid to treat a CRS episode. Tocilizumab and the corticosteroid can be administered separately.

在一些態樣中，皮質類固醇經靜脈內投予個體。在一些態樣中，皮質類固醇為甲潑尼龍，例如甲潑尼龍以每天約 1 mg/kg 至每天約 5 mg/kg，例如每天約 2 mg/kg 之劑量向個體投予。在一些態樣中，皮質類固醇為地塞米松，例如，地塞米松將以每天約 10 mg 或約 0.5 mg/kg 的劑量向個體投予。In some embodiments, the corticosteroid is administered intravenously to the subject. In some embodiments, the corticosteroid is methylprednisolone, for example, methylprednisolone is administered to the subject in an amount of about 1 mg/kg per day to about 5 mg/kg per day, for example, about 2 mg/kg per day. In some embodiments, the corticosteroid is dexamethasone, for example, dexamethasone is administered to the subject in an amount of about 10 mg or about 0.5 mg/kg per day.

在一些態樣中，本揭露之特徵在於托珠單抗用於治療患有 CRS 事件的個體，其中 CRS 事件在用本發明之多特異性抗原結合分子 (例如，雙特異性抗體) 治療個體期間出現。In some aspects, the disclosure features tocilizumab for use in treating an individual who has a CRS event, wherein the CRS event occurs during treatment of the individual with a multispecific antigen-binding molecule (e.g., a bispecific antibody) of the invention.

在一些態樣中，托珠單抗將向個體投予，同時暫停用本發明的雙特異性抗體進行的治療。In some aspects, tocilizumab will be administered to an individual while treatment with the bispecific antibody of the invention is suspended.

在一些態樣中，托珠單抗係以約 1 mg/kg 至約 15 mg/kg，例如約 4 mg/kg 至約 10 mg/kg，例如約 6 mg/kg 至約 10 mg/kg，例如約 8 mg/kg 之單一劑量調配用於靜脈內投予。In some aspects, tocilizumab is formulated for intravenous administration in a single dose of about 1 mg/kg to about 15 mg/kg, e.g., about 4 mg/kg to about 10 mg/kg, e.g., about 6 mg/kg to about 10 mg/kg, e.g., about 8 mg/kg.

在一些態樣中，CRS 事件在治療該 CRS 事件之症狀的 24 小時內未消退或惡化，且向個體投予一個或多個額外劑量之托珠單抗。一個或多個額外劑量之托珠單抗以約 1 mg/kg 至約 15 mg/kg，例如約 4 mg/kg 至約 10 mg/kg，例如約 6 mg/kg 至約 10 mg/kg，例如約 8 mg/kg 之劑量靜脈內投予個體。In some aspects, the CRS event does not resolve or worsens within 24 hours of treating the symptoms of the CRS event, and one or more additional doses of tocilizumab are administered to the subject. The one or more additional doses of tocilizumab are administered intravenously to the subject at a dose of about 1 mg/kg to about 15 mg/kg, such as about 4 mg/kg to about 10 mg/kg, such as about 6 mg/kg to about 10 mg/kg, such as about 8 mg/kg.

在一些態樣中，托珠單抗與有效量的皮質類固醇聯合使用以治療 CRS 事件。托珠單抗及皮質類固醇可分開調配。在一些態樣中，皮質類固醇經靜脈內投予個體。在一些態樣中，皮質類固醇為甲潑尼龍，例如甲潑尼龍以每天約 1 mg/kg 至每天約 5 mg/kg，例如每天約 2 mg/kg 之劑量向個體投予。在一些態樣中，皮質類固醇為地塞米松，例如，地塞米松將以每天約 10 mg 或約 0.5 mg/kg 的劑量向個體投予。表 C.托珠單抗治療重度或危及生命的細胞介素釋放症候群 (CRS)TCZ 後治療^a8 天  至少每 6 小時測量一次直至恢復至基線，然後每 12 小時測量一次直至第 8 天^c或直至自 ICU出院至少每 6 小時記錄一次，直至停用升壓藥^c至少每 6 小時記錄一次，直至患者呼吸室內空氣^c至少每 6 小時測量一次直至恢復至基線，然後每 12 小時測量一次直至第 8 天^c或直至自 ICU出院當地實驗室評估xxxxx  中心實驗室評估xxx3 天xxxxx  xxx2 天xxxxx  xxx1 天xxxxx  xxx6 小時xxxxx  xxxTCZ 投予x                      x^hx^h預 TCZ 治療 (在 24 小時內)  x^cx^cx^cx^cxxxxxxxxx評估/程序TCZ 投予 (8 mg/kg)生命徵象^b加壓藥文件記錄^dFiO2脈搏血氧飽和度，靜息血液學肝功能測試 (AST、ALT、總膽紅素)血清化學及肌酸^eCRP、LDH 及血清鐵蛋白凝血 (aPTT、PT/INR、纖維蛋白原)感染檢查^f血漿細胞介素血漿 IL-6 PD 標記^g血清 TCZ 藥物動力學CRP：C-反應蛋白；eCRF：電子病例報告表；IL：介白素；PD：藥效學；PK：藥物動力學；TCZ：托珠單抗。 註：在不良事件 eCRF 上記錄異常或惡化的臨床顯著異常。^a若重複服用 TCZ，則遵循第二次服用 TCZ 後的活動時間表。^b包括患者坐位或仰臥位時之呼吸頻率、心率以及收縮壓及舒張壓，以及 體溫。^c臨床資料庫中應記錄任何 24 小時內的最大值及最小值。^d記錄伴隨藥物 eCRF 中的升壓藥類型及劑量。^e包括鈉、鉀、氯化物、碳酸氫鹽、葡萄糖及血尿素氮^f包括對細菌、真菌及病毒感染之評估。^g包括 IL-6、可溶性 IL-6R 及 sgp130。^h將在 TCZ 輸注結束時進行血清 TCZ PK 及血漿 IL-6 PD 標記的抽血，且將自未用於投予 TCZ 之手臂抽血。按級別管理CRS事件In some aspects, tocilizumab is used in combination with an effective amount of a corticosteroid to treat a CRS event. Tocilizumab and a corticosteroid can be formulated separately. In some aspects, the corticosteroid is administered intravenously to a subject. In some aspects, the corticosteroid is methylprednisolone, for example, methylprednisolone is administered to a subject in an amount of about 1 mg/kg per day to about 5 mg/kg per day, for example, about 2 mg/kg per day. In some aspects, the corticosteroid is dexamethasone, for example, dexamethasone is administered to a subject in an amount of about 10 mg or about 0.5 mg/kg per day.Table C.Tocilizumab for the treatment of severe or life-threatening CRS Post-TCZ^treatmenta 8 days Measure at least every 6 hours until recovery to baseline, then every 12 hours until day^8cor untildischarge from the ICU Record at least every 6 hours until^vasopressors are discontinuedc Record at least every 6 hours until the patient is breathing room air^. Measure at least every 6 hours until recovery to baseline, then every 12 hours until day^8cor untildischarge from the ICULocal Laboratory Assessment x x x x xCentral Laboratory Evaluation x x x 3 days x x x x x x x x 2 days x x x x x x x x 1 day x x x x x x x x 6 hours x x x x x x x x TCZ administration x x^h x^h Pre-TCZ treatment (within 24 hours) x^c x^c x^c x^c x x x x x x x x xAssessment/Procedure TCZ administration (8 mg/kg) Vital^signsb Pressurized drug^{documentation} FiO2 Pulse blood oxygen saturation, resting Hematology Liver function tests (AST, ALT, total bilirubin) Serum chemistry and^creatine CRP, LDH and serum ferritin Coagulation (aPTT, PT/INR, fibrinogen) Infection^test Plasma interleukin Plasma IL-6 PD marker^g Serum TCZ pharmacokinetics CRP: C-reactive protein; eCRF: electronic case report form; IL: interleukin; PD: pharmacodynamics; PK: pharmacokinetics; TCZ: tocilizumab. Note: Record abnormal or worsening clinically significant abnormalities on the adverse event eCRF.^aIf TCZ is repeated, follow the activity schedule after the second dose of TCZ.^bInclude respiratory rate, heart rate, and systolic and diastolic blood pressures, and temperature with the patient in the sitting or supine position. cMaximum and minimum values within any 24-hour period should be recorded in^the clinical database.^dRecord the type and dose of vasopressors in the concomitant medications eCRF.^eInclude sodium, potassium, chloride, bicarbonate, glucose, and blood urea nitrogen^fInclude assessment for bacterial, fungal, and viral infections.^gIncludes IL-6, soluble IL-6R, and sgp130.^hBlood draws for serum TCZ PK and plasma IL-6 PD markers will be performed at the end of TCZ infusion and will be drawn from the arm not used for TCZ administration. Management ofCRSeventsby grade

CRS 事件之管理可根據 CRS 之級別 (表 A 及 D) 及共病之存在進行定制。表 D 提供按級別管理 CRS 症候群之建議。表D.管理細胞介素釋放症候群之建議事件^a、b採取的行動^b第1級發燒、體質性症狀立即行動：• 若輸注仍在進行，將輸注速率減慢至 50% 或中斷輸注。 • 根據需要按指示對症治療，包括抗組胺藥、解熱藥及/或鎮痛藥。 • 若存在發燒及嗜中性球減少症，則進行治療。 • 流體平衡監測器；根據臨床指示投予 IV 輸液。重新開始輸注：• 若治療性雙特異性抗體輸注中斷，則等到事件消退後 30 分鐘，然後以原始輸注速率之 50% 重新開始輸注。第2級低血壓：對液體或單一低劑量加壓藥有反應^c缺氧：需要 ＜ 40% FiO2 來維持足夠的血紅素氧飽和度 器官毒性：第 2 級立即行動：• 遵循所有 1 級建議。 • 保持進一步的雙特異性抗體治療，直至症狀完全消退。 • 考慮用 IV 皮質類固醇 (諸如甲潑尼龍 2 mg/kg/天，或者，若存在神經系統症狀，地塞米松 0.5 mg/kg/天) 治療。^b• 考慮投予單一劑量之托珠單抗 8 mg/kg IV。 • 密切監測心臟及其他器官功能。 • 按指示提供血液動力學支持。 • 為缺氧提供氧氣。 • 酌情入住 ICU。 • 若在 24 小時內未改善，則作為 3 級事件進行管理：–開始檢查並評定 MAS/HLH 之體徵及症狀。 • 若症狀 連續 3 天消退至 ≤ 1 級，則可接受下一劑雙特異性抗體。重新開始輸注：• 等到事件消退後 30 分鐘，然後以原始輸注速率之最多 25% 時重新開始輸注。 • 若低血壓或缺氧復發，則立即停止輸注。在此週期內不應再次投予 (重新開始) 雙特異性抗體。 • 若低血壓或缺氧復發，則作為 3 級事件進行管理。下一週期：• 若症狀連續 3 天消退至 ≤ 1 級，則可接受下一劑雙特異性抗體，如下所示： 若事件發生在輸注期間或輸注後 24 小時內，則以前一週期之初始輸注速率的 50% 投予雙特異性抗體。^d後續週期：• 若在任何後續週期中發生 ≥ 3 級 IRR 或 CRS，則無論恢復情況如何，永久停用雙特異性抗體 (參見 3 級管理指南)。 若在後續週期中發生 ≤ 2 級 CRS，則按照嚴重程度指示進行管理 (參見 1 級或 2 級管理指南)。第3級低血壓：需要多種加壓藥或高劑量加壓藥^c缺氧：需要 ≥ 40% FiO2 來維持足夠的血紅素氧飽和度 器官毒性：3 級 (例如，4 級轉胺酶升高)立即行動：• 停止進一步輸注雙特異性抗體。 • 根據需要按指示對症治療，包括抗組胺藥、解熱藥及/或鎮痛藥。 • 按臨床指示 (例如發燒及嗜中性球減少症、感染) 提供其他支持性照護。 • 流體平衡監測器；根據臨床指示投予 IV 輸液。 • 使患者住院至少 24 小時。 • 用 IV 皮質類固醇 (諸如甲潑尼龍 2 mg/kg/天，或者，若存在神經系統症狀，地塞米松 0.5 mg/kg/天) 治療。 • 投予托珠單抗 8 mg/kg IV。–若 24 小時後未改善，則重複投予托珠單抗。 開始檢查且評估 MAS/HLH 之體徵及症狀。 • 在 ICU 中監測心肺及器官功能。 • 為缺氧提供氧氣。 • 建議入住 ICU。重新開始輸注：• 在此週期內不應再次投予雙特異性抗體。下一週期：• 若患者在之前的任何週期中具有 ≥ 2 級 IRR 或 CRS，則永久停用雙特異性抗體。 • 若患者在皮質類固醇及托珠單抗治療後 8 小時內未恢復 (發熱或仍在使用血管加壓藥)，則永久停用雙特異性抗體。 • 若患者在皮質類固醇及托珠單抗治療後 8 小時內恢復 (無發熱且停用升壓藥)，可在下一週期投予雙特異性抗體，如下所示：–使患者住院至少 24 小時。–若事件發生在輸注期間或輸注後 24 小時內，則以前一週期之初始輸注速率的 50% 投予雙特異性抗體。^d後續週期：• 若 ≥ 3 級 CRS 復發，則永久停用雙特異性抗體。 若在後續週期中發生 ≤ 2 級 CRS，則按照嚴重程度指示進行管理 (亦即 1 級或 2 級管理指南)。第4級需要機械換氣； 器官毒性：第 4 級 (不包括轉胺酶升高)• 遵循所有 3 級管理指南。 • 永久停止雙特異性抗體治療。CRS = 細胞介素釋放症候群；HLH = 噬血細胞性淋巴組織細胞增生症；ICU = 加護病室；IV = 靜脈內；MAS = 巨噬細胞活化症候群。 註：CRS 為一種以如下各者為特徵之病症：噁心、頭痛、心搏過速、低血壓、皮疹、呼吸急促，以及腎髒、凝血、肝髒及神經系統病症；其由自細胞釋放細胞介素引起 (Lee 等人,Blood,124: 188-195, 2014)。^a關於症狀分級之描述，參見表 A。^b基於 Lee 等人，Blood，124: 188-195，2014 之 CRS 管理指南。^c關於高劑量升壓藥之描述及計算，參見表 B。^d若患者在下一次以 50% 降低的速率輸注期間未經歷 CRS，則可在後續週期中將輸注速率增加至初始速率。然而，若此患者經歷另一 CRS 事件，則應根據事件之嚴重程度將輸注速率降低 25% 至 50%。2級CRS事件之管理Management of CRS events can be tailored based on the grade of CRS (Tables A and D) and the presence of comorbidities. Table D provides recommendations for the management of CRS syndrome by grade.TableD.Recommendations for the management of interleukin-1 release syndromeEvent^a,bActionstakenbLevel1fever , constitutional symptomsImmediate Actions: • If infusion is ongoing, slow infusion rate to 50% or interrupt infusion. • Administer symptomatic care as indicated, including antihistamines, antipyretics, and/or analgesics, as needed. • Treat fever and neutropenia, if present. • Fluid balance monitor; administer IV fluids as clinically indicated.Restarting Infusion: • If therapeutic bispecific antibody infusion is interrupted, wait 30 minutes after event resolves, then restart infusion at 50% of original infusion rate.Grade2Hypotension : Responds to fluids or low-dose pressors alone^Hypoxia : Requires < 40% FiO2 to maintain adequate hemoglobin saturation Organ toxicity: Grade 2Immediate Actions: • Follow all Level 1 recommendations. • Maintain further bispecific antibody therapy until complete resolution of symptoms. • Consider treatment with IV corticosteroids (e.g., methylprednisolone 2 mg/kg/day or, if neurologic symptoms are present, dexamethasone 0.5 mg/kg/day).^b • Consider administering a single dose of tocilizumab 8 mg/kg IV. • Closely monitor cardiac and other organ function. • Provide hemodynamic support as indicated. • Provide oxygen for hypoxia. • Admit to ICU as appropriate. • If no improvement within 24 hours, manage as a Level 3 event:–Initiate evaluation and assessment for signs and symptoms of MAS/HLH. • If symptoms resolve to ≤ Grade 1 for 3 consecutive days, the next dose of bispecific antibody may be administered.Restart infusion: • Wait 30 minutes after the event resolves, then restart the infusion at up to 25% of the original infusion rate. • If hypotension or hypoxia recurs, stop the infusion immediately. The bispecific antibody should not be re-administered (restarted) during this cycle. • If hypotension or hypoxia recurs, manage as a Grade 3 event.Next cycle: • If symptoms resolve to ≤ Grade 1 for 3 consecutive days, the next dose of bispecific antibody may be administered as follows: If the event occurred during or within 24 hours of an infusion, administer the bispecific antibody at 50% of the initial infusion rate of the previous cycle.^dSubsequentcycles: • If ≥ Grade 3 IRR or CRS occurs in any subsequent cycle, permanently discontinue bispecific antibody regardless of recovery (see Grade 3 management guidelines). If ≤ Grade 2 CRS occurs in subsequent cycles, manage as indicated by severity (see Grade 1 or 2 management guidelines).Grade3 Hypotension: Multiple or high-dose pressors required^Hypoxia : ≥ 40% FiO2 required to maintain adequate hemoglobin saturation Organ toxicity: Grade 3 (e.g., Grade 4 transaminase elevations)Immediate Actions: • Stop further infusions of bispecific antibody. • Treat symptomatically as indicated, including antihistamines, antipyretics, and/or analgesics, as needed. • Provide other supportive care as clinically indicated (e.g., fever and neutropenia, infection). • Fluid balance monitor; administer IV fluids as clinically indicated. • Hospitalize patient for at least 24 hours. • Treat with IV corticosteroids (e.g., methylprednisolone 2 mg/kg/day or, if neurologic symptoms are present, dexamethasone 0.5 mg/kg/day). • Administer tocilizumab 8 mg/kg IV.– Repeat tocilizumab if no improvement after 24 hours. Initiate testing and assess for signs and symptoms of MAS/HLH. • Monitor cardiopulmonary and organ function in the ICU. • Provide oxygen for hypoxia. • ICU admission is recommended.Restart infusion: • Bispecific antibody should not be re-administered during this cycle.Next cycle: • Permanently discontinue bispecific antibody if patient had ≥ Grade 2 IRR or CRS in any prior cycle. • Permanently discontinue bispecific antibody if patient does not recover within 8 hours after corticosteroid and tocilizumab treatment (fever or still on vasopressors). • If patient recovers within 8 hours after corticosteroid and tocilizumab treatment (no fever and off vasopressors), bispecific antibody may be administered in the next cycle as follows:– Keep patient in hospital for at least 24 hours.– If the event occurred during or within 24 hours of an infusion, administer the bispecific antibody at 50% of the initial infusion rate in the previous cycle.^dSubsequentcycles: • Permanently discontinue the bispecific antibody if ≥ Grade 3 CRS recurs. If ≤ Grade 2 CRS occurs in a subsequent cycle, manage as indicated by severity (i.e., Grade 1 or 2 management guidelines).Grade4 requiring mechanical ventilation;Organ toxicity: Grade 4 (excluding elevated transaminases) • Follow all Level 3 management guidelines. • Permanently discontinue bispecific antibody therapy. CRS = interleukin release syndrome; HLH = hemophagocytic lymphohistiocytosis; ICU = intensive care unit; IV = intravenous; MAS = macrophage activation syndrome. Note: CRS is a disorder characterized by nausea, headache, tachycardia, hypotension, rash, tachypnea, and renal, coagulation, hepatic, and nervous system symptoms; it is caused by the release of interleukins from cells (Lee et al.,Blood, 124: 188-195, 2014).^aFor a description of symptom classification, see Table A.^bBased on the CRS management guidelines of Lee et al.,Blood , 124: 188-195, 2014.^cSee Table B for a description and calculation of high-dose vasopressors.^dIf the patient does not experience CRS during the next 50% reduced rate infusion, the infusion rate can be increased to the initial rate in subsequent cycles. However, if this patient experiences another CRS event, the infusion rate should be reduced by 25% to 50% depending on the severity of the event. Management ofGrade2CRSEvents

若個體在投予治療性多特異性抗原結合分子 (例如，雙特異性抗體) 後具有 2 級 CRS 事件 (例如，不存在共病或存在最小共病之 2 級 CRS 事件)，則該方法可進一步包括治療 2 級 CRS 事件之症狀同時中止用多特異性抗原結合分子 (例如，雙特異性抗體) 治療。若隨後至少連續三天 2 級 CRS 事件消退為 ≤ 1 級 CRS 事件，則該方法可進一步包括在不改變劑量之情況下恢復用多特異性抗原結合分子 (例如，雙特異性抗體) 治療。另一方面，若 2 級 CRS 事件在治療 2 級 CRS 事件之症狀的 24 小時內未消退或惡化為 ≥ 3 級 CRS 事件，則該方法可進一步包括向受試者投予有效量之介白素 6 受體 (IL-6R) 拮抗劑 (例如抗 IL-6R 抗體，例如托珠單抗 (ACTEMRA® / ROACTEMRA®)) 來管理 2 級或 ≥ 3 級 CRS 事件。在一些情況下，托珠單抗以約 8 mg/kg 之單一劑量經靜脈內投予該受試者。可與托珠單抗組合使用之其他抗 IL-6R 抗體包括撒裡路單抗(sarilumab)、維巴麗珠單抗 (vobarilizumab) (ALX-0061)、SA-237 及其變異體。If the individual has a Grade 2 CRS event (e.g., a Grade 2 CRS event with no or minimal comorbidity) following administration of a therapeutic multispecific antigen-binding molecule (e.g., bispecific antibody), the method may further include treating the symptoms of the Grade 2 CRS event while discontinuing treatment with the multispecific antigen-binding molecule (e.g., bispecific antibody). If the Grade 2 CRS event subsequently resolves to ≤ Grade 1 CRS events for at least three consecutive days, the method may further include resuming treatment with the multispecific antigen-binding molecule (e.g., bispecific antibody) without changing the dose. On the other hand, if the Grade 2 CRS event does not resolve within 24 hours of treating the symptoms of the Grade 2 CRS event or worsens to a Grade ≥ 3 CRS event, the method can further comprise administering to the subject an effective amount of an interleukin 6 receptor (IL-6R) antagonist (e.g., an anti-IL-6R antibody, such as tocilizumab (ACTEMRA® / ROACTEMRA®)) to manage the Grade 2 or Grade ≥ 3 CRS event. In some cases, tocilizumab is administered intravenously to the subject in a single dose of about 8 mg/kg. Other anti-IL-6R antibodies that can be used in combination with tocilizumab include sarilumab, vobarilizumab (ALX-0061), SA-237 and its variants.

若在投予多特異性抗原結合分子 (例如，雙特異性抗體) 後受體具有存在廣泛共病之 2 級 CRS 事件，則該方法可進一步包括向個體投予第一劑量之 IL-6R 拮抗劑 (例如，抗 IL-6R 抗體，例如托珠單抗 (ACTEMRA® / ROACTEMRA®)) 來管理 2 級 CRS 事件，同時中止用多特異性抗原結合分子治療。在一些情況下，以約 8 mg/kg 之劑量向個體經靜脈內投予托珠單抗之第一劑量。可與托珠單抗組合使用之其他抗 IL-6R 抗體包括撒裡路單抗(sarilumab)、維巴麗珠單抗 (vobarilizumab) (ALX-0061)、SA-237 及其變異體。在一些情況下，若 2 級 CRS 事件在兩週內消退為 ≤ 1 級 CRS 事件，則該方法進一步包括重新開始用降低劑量之多特異性抗原結合分子 (例如，雙特異性抗體) 進行治療。在一些情況下，若事件發生於輸注期間或其 24 小時內，則較小劑量為前一週期之初始輸注速率的 50%。另一方面，若 2 級 CRS 事件在治療 2 級 CRS 事件之症狀的 24 小時內未消退或惡化為 ≥ 3 級 CRS 事件，則該方法可進一步包括向受試者投予一個或多個 (例如，一、二、三、四、五個或更多個) 額外劑量之 IL-6R 拮抗劑 (例如抗 IL-6R 抗體，例如托珠單抗) 來管理 2 級或 ≥ 3 級 CRS 事件。在一些特定實例中，2 級 CRS 事件在治療 2 級 CRS 事件之症狀的 24 小時內未消退或惡化為≥ 3 級 CRS 事件，且該方法可進一步包括向受試者投予一個或多個額外劑量之托珠單抗來管理 2 級或 ≥ 3 級 CRS 事件。在一些情況下，以約 1 mg/kg 至約 15 mg/kg (例如約 4 mg/kg 至約 10 mg/kg，例如約 6 mg/kg 至約 10 mg/kg，例如約 8 mg/kg) 之劑量向個體經靜脈內投予托珠單抗之一個或多個額外劑量。在一些情況下，該方法進一步包括向受試者投予有效量之皮質類固醇。可在一個或多個額外劑量之托珠單抗或其他抗 IL-6R 抗體之前、之後或與其同時投予皮質類固醇。在一些情況下，皮質類固醇經靜脈內投予受試者。在一些情況下，皮質類固醇為甲潑尼龍。在一些情況下，甲潑尼龍以每天約 1 mg/kg 至每天約 5 mg/kg，例如每天約 2 mg/kg 之劑量投予。在一些情況下，皮質類固醇為地塞米松。在一些情況下，地塞米松以約 10 mg 之劑量 (例如，靜脈內約 10 mg 之單一劑量) 或以約 0.5 mg/kg/天之劑量投予。3級CRS事件之管理If the subject has a Grade 2 CRS event in the presence of extensive comorbidities following administration of a multispecific antigen binding molecule (e.g., a bispecific antibody), the method can further comprise administering to the individual a first dose of an IL-6R antagonist (e.g., an anti-IL-6R antibody, such as tocilizumab (ACTEMRA® / ROACTEMRA®)) to manage the Grade 2 CRS event while discontinuing treatment with the multispecific antigen binding molecule. In some cases, the first dose of tocilizumab is administered intravenously to the individual at a dose of about 8 mg/kg. Other anti-IL-6R antibodies that can be used in combination with tocilizumab include sarilumab, vobarilizumab (ALX-0061), SA-237, and variants thereof. In some instances, if a Grade 2 CRS event resolves to a ≤ Grade 1 CRS event within two weeks, the approach further includes restarting treatment with a reduced dose of the multispecific antigen binding molecule (e.g., bispecific antibody). In some instances, if the event occurred during an infusion or within 24 hours of it, the smaller dose is 50% of the initial infusion rate of the previous cycle. On the other hand, if the Grade 2 CRS event does not resolve within 24 hours of treating the symptoms of the Grade 2 CRS event or worsens to a Grade 3 CRS event, the method may further include administering to the subject one or more (e.g., one, two, three, four, five, or more) additional doses of an IL-6R antagonist (e.g., an anti-IL-6R antibody, such as tocilizumab) to manage the Grade 2 or Grade 3 CRS event. In some specific instances, the Grade 2 CRS event does not resolve within 24 hours of treating the symptoms of the Grade 2 CRS event or worsens to a Grade 3 CRS event, and the method may further include administering to the subject one or more additional doses of tocilizumab to manage the Grade 2 or Grade 3 CRS event. In some cases, one or more additional doses of tocilizumab are administered intravenously to the subject in an amount of about 1 mg/kg to about 15 mg/kg (e.g., about 4 mg/kg to about 10 mg/kg, e.g., about 6 mg/kg to about 10 mg/kg, e.g., about 8 mg/kg). In some cases, the method further comprises administering to the subject an effective amount of a corticosteroid. The corticosteroid may be administered before, after, or simultaneously with the one or more additional doses of tocilizumab or other anti-IL-6R antibody. In some cases, the corticosteroid is administered intravenously to the subject. In some cases, the corticosteroid is methylprednisolone. In some cases, methylprednisolone is administered in an amount of about 1 mg/kg per day to about 5 mg/kg per day, such as about 2 mg/kg per day. In some cases, the corticosteroid is dexamethasone. In some cases, dexamethasone is administered in an amount of about 10 mg (e.g., a single dose of about 10 mg intravenously) or in an amount of about 0.5 mg/kg/day. Management ofGrade3CRSEvents

若在投予治療性多特異性抗原結合分子 (例如，雙特異性抗體) 後個體具有 3 級 CRS 事件，則該方法可進一步包括向個體投予第一劑量之 IL-6R 拮抗劑 (例如，抗 IL-6R 抗體，例如托珠單抗 (ACTEMRA® / ROACTEMRA®)) 來管理 3 級 CRS 事件，同時中止用多特異性抗原結合分子治療。在一些情況下，以約 8 mg/kg 之劑量向個體經靜脈內投予托珠單抗之第一劑量。可與托珠單抗組合使用之其他抗 IL-6R 抗體包括撒裡路單抗(sarilumab)、維巴麗珠單抗 (vobarilizumab) (ALX-0061)、SA-237 及其變異體。在一些情況下，個體在用多特異性抗原結合分子 (例如，雙特異性抗體) 治療後的 8 小時內恢復 (例如不發熱且停止使用升壓藥)，且該方法進一步包括恢復用降低劑量之多特異性抗原結合分子 (例如，雙特異性抗體) 治療。在一些情況下，若事件發生於輸注期間或其 24 小時內，則較小劑量為前一週期之初始輸注速率的 50%。在其他實例中，若 3 級 CRS 事件在治療 3 級 CRS 事件之症狀的 24 小時內未消退或惡化為 4 級 CRS 事件，則該方法可進一步包括向受試者投予一個或多個 (例如，一、二、三、四、五個或更多個) 額外劑量之 IL-6R 拮抗劑 (例如抗 IL-6R 抗體，例如托珠單抗) 來管理 3 級或 4 級 CRS 事件。在一些特定實例中，3 級 CRS 事件在治療 3 級 CRS 事件之症狀的 24 小時內未消退或惡化為 4 級 CRS 事件，且該方法進一步包括向受試者投予一個或多個額外劑量之托珠單抗來管理 3 級或 4 級 CRS 事件。在一些情況下，以約 1 mg/kg 至約 15 mg/kg (例如約 4 mg/kg 至約 10 mg/kg，例如約 6 mg/kg 至約 10 mg/kg，例如約 8 mg/kg) 之劑量向個體經靜脈內投予托珠單抗之一個或多個額外劑量。在一些情況下，該方法進一步包括向受試者投予有效量之皮質類固醇。可在一個或多個額外劑量之托珠單抗或其他抗 IL-6R 抗體之前、之後或與其同時投予皮質類固醇。在一些情況下，皮質類固醇經靜脈內投予受試者。在一些情況下，皮質類固醇為甲潑尼龍。在一些情況下，甲潑尼龍以每天約 1 mg/kg 至每天約 5 mg/kg，例如每天約 2 mg/kg 之劑量投予。在一些情況下，皮質類固醇為地塞米松。在一些情況下，地塞米松以約 10 mg 之劑量 (例如，靜脈內約 10 mg 之單一劑量) 或以約 0.5 mg/kg/天之劑量投予。4級CRS事件之管理If the individual has a Grade 3 CRS event following administration of a therapeutic multispecific antigen binding molecule (e.g., a bispecific antibody), the method can further comprise administering to the individual a first dose of an IL-6R antagonist (e.g., an anti-IL-6R antibody, such as tocilizumab (ACTEMRA® / ROACTEMRA®)) to manage the Grade 3 CRS event while discontinuing treatment with the multispecific antigen binding molecule. In some cases, the first dose of tocilizumab is administered intravenously to the individual at a dose of about 8 mg/kg. Other anti-IL-6R antibodies that can be used in combination with tocilizumab include sarilumab, vobarilizumab (ALX-0061), SA-237, and variants thereof. In some cases, the subject recovers (e.g., is afebrile and off vasopressors) within 8 hours of treatment with the multispecific antigen-binding molecule (e.g., bispecific antibody), and the method further comprises resuming treatment with a reduced dose of the multispecific antigen-binding molecule (e.g., bispecific antibody). In some cases, if the event occurred during an infusion or within 24 hours thereof, the smaller dose is 50% of the initial infusion rate of the previous cycle. In other examples, if a Grade 3 CRS event does not resolve within 24 hours of treating the symptoms of the Grade 3 CRS event or worsens to a Grade 4 CRS event, the method may further include administering to the subject one or more (e.g., one, two, three, four, five, or more) additional doses of an IL-6R antagonist (e.g., an anti-IL-6R antibody, such as tocilizumab) to manage the Grade 3 or 4 CRS event. In some specific examples, a Grade 3 CRS event does not resolve within 24 hours of treating the symptoms of the Grade 3 CRS event or worsens to a Grade 4 CRS event, and the method further includes administering to the subject one or more additional doses of tocilizumab to manage the Grade 3 or 4 CRS event. In some cases, one or more additional doses of tocilizumab are administered intravenously to the subject in an amount of about 1 mg/kg to about 15 mg/kg (e.g., about 4 mg/kg to about 10 mg/kg, e.g., about 6 mg/kg to about 10 mg/kg, e.g., about 8 mg/kg). In some cases, the method further comprises administering to the subject an effective amount of a corticosteroid. The corticosteroid may be administered before, after, or simultaneously with the one or more additional doses of tocilizumab or other anti-IL-6R antibody. In some cases, the corticosteroid is administered intravenously to the subject. In some cases, the corticosteroid is methylprednisolone. In some cases, methylprednisolone is administered in an amount of about 1 mg/kg per day to about 5 mg/kg per day, such as about 2 mg/kg per day. In some cases, the corticosteroid is dexamethasone. In some cases, dexamethasone is administered in an amount of about 10 mg (e.g., a single dose of about 10 mg intravenously) or in an amount of about 0.5 mg/kg/day. Management ofGrade4CRSEvents

若在投予治療性多特異性抗原結合分子 (例如，雙特異性抗體) 後受體具有 4 級 CRS 事件，則該方法可進一步包括向受體投予第一劑量之 IL-6R 拮抗劑 (例如，抗 IL-6R 抗體，例如托珠單抗 (ACTEMRA® / ROACTEMRA®)) 來管理 4 級 CRS 事件，並永久停止用多特異性抗原結合分子治療。在一些情況下，以約 8 mg/kg 之劑量向個體經靜脈內投予托珠單抗之第一劑量。可與托珠單抗組合使用之其他抗 IL-6R 抗體包括撒裡路單抗(sarilumab)、維巴麗珠單抗 (vobarilizumab) (ALX-0061)、SA-237 及其變異體。在一些情況下，4 級 CRS 事件可在治療 4 級 CRS 事件之症狀的 24 內解決。若 4 級 CRS 事件在治療 4 級 CRS 事件之症狀的 24 小時內未消退，則該方法可進一步包括向受試者投予一個或多個額外劑量之 IL-6R 拮抗劑 (例如抗 IL -6R 抗體，例如托珠單抗 (ACTEMRA® / ROACTEMRA®)) 來管理 4 級 CRS 事件。在一些特定實例中，4 級 CRS 事件在治療 4 級 CRS 事件之症狀的 24 小時內未消退，且該方法進一步包括向受試者投予一個或多個 (例如，一、二、三、四、或五個或更個) 額外劑量之托珠單抗來管理 4 級 CRS 事件。在一些情況下，以約 1 mg/kg 至約 15 mg/kg (例如約 4 mg/kg 至約 10 mg/kg，例如約 6 mg/kg 至約 10 mg/kg，例如約 8 mg/kg) 之劑量向個體經靜脈內投予托珠單抗之一個或多個額外劑量。在一些情況下，該方法進一步包括向受試者投予有效量之皮質類固醇。可在一個或多個額外劑量之托珠單抗或其他抗 IL-6R 抗體之前、之後或與其同時投予皮質類固醇。在一些情況下，皮質類固醇經靜脈內投予受試者。在一些情況下，皮質類固醇為甲潑尼龍。在一些情況下，甲潑尼龍以每天約 1 mg/kg 至每天約 5 mg/kg，例如每天約 2 mg/kg 之劑量投予。在一些情況下，皮質類固醇為地塞米松。在一些情況下，地塞米松以約 10 mg 之劑量 (例如，靜脈內約 10 mg 之單一劑量) 或以約 0.5 mg/kg/天之劑量投予。實例If the subject has a Grade 4 CRS event following administration of a therapeutic multispecific antigen binding molecule (e.g., a bispecific antibody), the method can further include administering to the subject a first dose of an IL-6R antagonist (e.g., an anti-IL-6R antibody, such as tocilizumab (ACTEMRA® / ROACTEMRA®)) to manage the Grade 4 CRS event and permanently discontinue treatment with the multispecific antigen binding molecule. In some cases, the first dose of tocilizumab is administered intravenously to the subject at a dose of about 8 mg/kg. Other anti-IL-6R antibodies that can be used in combination with tocilizumab include sarilumab, vobarilizumab (ALX-0061), SA-237, and variants thereof. In some cases, a Grade 4 CRS event may resolve within 24 hours of treating the symptoms of the Grade 4 CRS event. If the Grade 4 CRS event does not resolve within 24 hours of treating the symptoms of the Grade 4 CRS event, the method may further include administering to the subject one or more additional doses of an IL-6R antagonist (e.g., an anti-IL-6R antibody, such as tocilizumab (ACTEMRA®/ROACTEMRA®)) to manage the Grade 4 CRS event. In some specific instances, the Grade 4 CRS event does not resolve within 24 hours of treating the symptoms of the Grade 4 CRS event, and the method further includes administering to the subject one or more (e.g., one, two, three, four, or five or more) additional doses of tocilizumab to manage the Grade 4 CRS event. In some cases, one or more additional doses of tocilizumab are administered intravenously to the subject in an amount of about 1 mg/kg to about 15 mg/kg (e.g., about 4 mg/kg to about 10 mg/kg, e.g., about 6 mg/kg to about 10 mg/kg, e.g., about 8 mg/kg). In some cases, the method further comprises administering to the subject an effective amount of a corticosteroid. The corticosteroid may be administered before, after, or simultaneously with the one or more additional doses of tocilizumab or other anti-IL-6R antibody. In some cases, the corticosteroid is administered intravenously to the subject. In some cases, the corticosteroid is methylprednisolone. In some cases, methylprednisolone is administered in an amount of about 1 mg/kg per day to about 5 mg/kg per day, such as about 2 mg/kg per day. In some cases, the corticosteroid is dexamethasone. In some cases, dexamethasone is administered in an amount of about 10 mg (e.g., a single dose of about 10 mg intravenously) or in an amount of about 0.5 mg/kg/day.Examples

以下為本發明之方法及組成物的實例。應當理解，鑒於上文給出的一般描述，可以實施各種其他實施例。實例1.重組STEAP1表現及純化重組STEAP1表現The following are examples of methods and compositions of the present invention. It should be understood that various other embodiments may be implemented in light of the general description given above.Example1.RecombinantSTEAP1Expression and PurifiedRecombinantSTEAP1Expression

合成全長人 STEAP1 DNA 序列 (M1-L339) (Genescript)，並選殖到多面體啟動子下游的經修飾之 pAcGP67A 載體中。重組桿狀病毒係使用 BACULOGOLD™ 系統 (BD Biosciences) 按照標準方案生成。重組 STEAP1 係用帶有 C 端 FLAG 親和標籤及之後的 Avi 標籤表現；構建體之序列如下所示 (帶下劃線的序列為 FLAG 親和標籤，而 Avi 標籤以粗體示出)： MGSMESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQLGNSGLNDIFEAQKIEWHEGENLYFQSDYKDDDDKThe full-length human STEAP1 DNA sequence (M1-L339) was synthesized (Genescript) and cloned into the modified pAcGP67A vector downstream of the polyhedron promoter. Recombinant bacilli were generated using the BACULOGOLD™ system (BD Biosciences) according to standard protocols. Recombinant STEAP1 was expressed with a C-terminal FLAG affinity tag followed by an Avi tag; the sequence of the construct is shown below (the underlined sequence is the FLAG affinity tag, and the Avi tag is in bold): MGSMESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQLGNSGLNDIFEAQKIEWHE GENLYFQSDYKDDDDK

將 2 LSf9細胞分成 2x10⁶/ml，並添加至 5 L Thompson Optimum Growth 燒瓶中。將燒瓶用 2.5 mL/L P3 病毒以 0.5 的近似感染複數 (MOI) 感染。6 小時後，將以下添加劑添加至培養物中 (注：添加劑係於使用當天新鮮製備)：1 mM δ-胺乙醯丙酸 (Sigma Chemicals；對於 1 M 儲備液，將 167.5 gm/L 重懸於 H₂O 中並過濾滅菌) 及 5 μM 氯化鐵 (Sigma Chemicals；對於 1mM 儲備液，將 162 mg/L 重懸於 H₂O 中並過濾滅菌)。將培養物於 37℃ 在 120 rpm 攪拌下生長，並於 72 小時後藉由離心進行收穫。2 L ofSf9 cells were split at 2x10⁶ /ml and added to 5 L Thompson Optimum Growth flasks. Flasks were infected with 2.5 mL/L P3 virus at an approximate multiplicity of infection (MOI) of 0.5. Six hours later, the following additives were added to the culture (Note: additives were freshly prepared on the day of use): 1 mM δ-aminoacetyl propionic acid (Sigma Chemicals; for 1 M stock, 167.5 gm/L was resuspended in H₂ O and filter-sterilized) and 5 μM ferric chloride (Sigma Chemicals; for 1 mM stock, 162 mg/L was resuspended in H₂ O and filter-sterilized). Cultures were grown at 37°C with agitation at 120 rpm and harvested after 72 hours by centrifugation.

表 5 示出用於擴增來自經免疫之大鼠的 IGHV5 序列的引子。表5RT 步驟引子混合物：G1R.1        TCCAGGGGCCAGTGGATAGACG2R.1        CACACMGGGGCCAGTGGATAGACG3R.1        GCAGCCAGGGACCAAGGGATAGACIgAR1        GGAAGTAATCRTGAATCAGGCAGCC  PCR 引子混合物：G1R.2        AGATGGGGGTGTCGTTTTGGCG2R.2        CGATGGGGSTGTTGTTTTGGCG3R.2        GACAGATGGGGCTGTTGTTGTAGCIgAR2        TGTCAGTGGGTAGATGGTGGGVH5-For    TGTCCTTTTCATAAAAGGTGTCCAGTGT重組可溶性STEAP1之純化Table5 shows the primers used to amplify the IGHV5 sequences from immunized rats. RT step primer mix: G1R.1 TCCAGGGGCCAGTGGATAGAC G2R.1 CACACMGGGGCCAGTGGATAGAC G3R.1 GCAGCCAGGGACCAAGGGATAGAC IgAR1 GGAAGTAATCRTGAATCAGGCAGCC PCR Primer Mix: G1R.2 AGATGGGGGTGTCGTTTTGGC G2R.2 CGATGGGGSTGTTGTTTTGGC G3R.2 GACAGATGGGGCTGTTGTTGTAGC IgAR2 TGTCAGTGGGTAGATGGTGGG VH5-For TGTCCTTTTCATAAAAGGTGTCCAGTGTPurification ofrecombinant solubleSTEAP1

將沉澱的細胞重懸於補充有 COMPLETE™ 不含 EDTA 的蛋白酶抑制劑混合片 (羅氏) 及 Benzonase (內部製備) 的 25 mM Tris pH 7.5、150 mM NaCl、5mM MgCl₂(緩衝液 A) 中。將細胞懸液在 15,000 psi 的壓力設置下穿過微流化器一次。細胞裂解後，將懸浮液於 4℃ 以 40,000 rpm 旋轉沉降 1 小時。將經分離之膜重懸於補充有來自 Anatrace (#NG310-CH210) 的 1% (w/v) LMNG (月桂基麥芽糖新戊二醇) 及 0.1% (w/v) 膽固醇半琥珀酸酯 (CHS) 的緩衝液 A 中，並於 4℃ 輕輕攪拌 2 小時進行溶解。於 4℃ 以 40,000 rpm 超速離心 30 分鐘後，將澄清的上清液與用緩衝液 B (25 mM Tris pH 8, 150 mM NaCl, 0.02% LMNG, 0.002% CHS) 預平衡的抗 M2 FLAG 樹脂 (Sigma) 於 4℃ 混合 1 小時。藉由重力流收集抗 FLAG 樹脂，並用 5 倍柱體積之緩衝液 B 洗滌。用緩衝液 C (25 mM Tris pH 8、150 mM NaCl、0.02% LMNG、0.002% CHS，補充有 150 μg/mL 1x FLAG 肽) 洗脫 FLAG 標記的 STEAP1。使濃縮洗脫液通過在緩衝液 B 中平衡的 SUPERDEX® S200 16/60 GL 柱 (GE Healthcare)。收集峰級分並使用 AMICON® Ultra-15 離心過濾裝置 (100K MWCO, Millipore Sigma) 濃縮至 1 mg/mL。將 800 μL 溶液與來自 BirA500 套組 (Avidity) 的 100 μL BiomixA 及 100 μL BiomixB 混合。添加 15 μg BirA 生物素-蛋白質連接酶，並將混合物於 4℃ 孵育 24 小時。最後將混合物在緩衝液 B 中平衡的 SUPERDEX® S200 16/60 GL 柱 (GE Healthcare) 上精處理。藉由 SDS-PAGE 評估蛋白質純度。實例2.大鼠抗人STEAP1抗體的開發及表徵The pelleted cells were resuspended in 25 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl₂ (Buffer A) supplemented with COMPLETE™ EDTA-free Protease Inhibitor Cocktail (Roche) and Benzonase (prepared in-house). The cell suspension was passed once through the microfluidizer at a pressure setting of 15,000 psi. After cell lysis, the suspension was spun down at 40,000 rpm for 1 hour at 4°C. The separated membranes were resuspended in buffer A supplemented with 1% (w/v) LMNG (lauryl maltose neopentyl glycol) and 0.1% (w/v) cholesterol hemisuccinate (CHS) from Anatrace (#NG310-CH210) and gently stirred for 2 hours at 4°C for solubilization. After ultracentrifugation at 40,000 rpm for 30 minutes at 4°C, the clarified supernatant was mixed with anti-M2 FLAG resin (Sigma) pre-equilibrated with buffer B (25 mM Tris pH 8, 150 mM NaCl, 0.02% LMNG, 0.002% CHS) for 1 hour at 4°C. The anti-FLAG resin was collected by gravity flow and washed with 5 column volumes of buffer B. FLAG-tagged STEAP1 was eluted with buffer C (25 mM Tris pH 8, 150 mM NaCl, 0.02% LMNG, 0.002% CHS, supplemented with 150 μg/mL 1x FLAG peptide). The concentrated eluate was passed through a SUPERDEX® S200 16/60 GL column (GE Healthcare) equilibrated in buffer B. Peak fractions were collected and concentrated to 1 mg/mL using an AMICON® Ultra-15 centrifugal filter unit (100K MWCO, Millipore Sigma). 800 μL of the solution was mixed with 100 μL BiomixA and 100 μL BiomixB from the BirA500 kit (Avidity). 15 μg of BirA biotin-protein ligase was added and the mixture was incubated at 4°C for 24 h. Finally, the mixture was polished on a SUPERDEX® S200 16/60 GL column (GE Healthcare) equilibrated in buffer B. Protein purity was assessed by SDS-PAGE.Example2. Development and characterization ofrat anti-humanSTEAP1antibodies

將 Sprague Dawley 大鼠 (Charles River, Hollister, CA) 分開於以下部位用 100 µg 編碼人 STEAP1 的質體 DNA 以及編碼大鼠 Flt3L 及大鼠 GM-CSF (建南德克，South San Francisco，CA) 的質體 DNA 進行免疫：經腹腔、皮下 (在尾巴根部)、皮下 (在頸背) 及皮下 (在兩個跗部)。在接受四個劑量之 DNA 後，大鼠分開於多個部位接受兩個劑量之 100 µg 在表面上表現人 STEAP1 的複數個細胞外囊泡。相反，將一些大鼠分開於多個部位用初始劑量之 30 µg 人 STEAP1 蛋白進行免疫，該蛋白質溶解於洗滌劑中，該洗滌劑與 MPL+TDM 佐劑 (Sigma-Aldrich, St. Louis, MO) 混合或與 TLR 促效劑組合混合：50 μg MPL (Sigma-Aldrich)、20 μg R848 (Invivogen, San Diego, CA)、10 μg PolyI:C (Invivogen) 及 10 μg CpG (Invivogen)。為了用額外蛋白質追加接種，大鼠接受用 PBS 稀釋的 15 µg 蛋白質。大鼠係經每兩週給藥一次。將來自這些大鼠的多株抗血清純化並藉由 FACS 測試與 293 或 RBA 細胞表面上的人 STEAP1 的結合。在最後一次免疫三天後，從顯示出可檢測之針對人 STEAP1 的 FACS 反應性的大鼠中收穫多個淋巴結。經蛋白質免疫之大鼠顯示出比經 DNA/EV 免疫之大鼠觀察到的更弱的 FACS 響應，因此選擇經 DNA/EV 免疫之大鼠收穫。使用磁力分離 (Miltenyi Biotec, San Diego, CA) 自完整淋巴球純化來自該等大鼠之 IgM 陰性 B 細胞，且經由電融合 (Harvard Apparatus, Holliston, MA) 與 Sp2ab 小鼠骨髓瘤細胞 (Abeome, Athens, GA) 融合。將融合細胞在 CLONACELL™-HY 培養基 C (StemCell Technologies, Vancouver, BC, Canada) 中於 37℃、7% CO₂孵育過夜，然後離心並重懸於補充有次黃嘌呤、胺蝶呤及胸苷 (HAT) (Sigma-Aldrich) 的 CLONACELL™-HY 培養基 E (StemCell Technologies) 中，分配於 12 孔盤中並使其於 37℃、7% CO₂的條件下生長。鋪板後四天，將大鼠雜交瘤細胞用抗大鼠 IgM 抗體 (Jackson ImmunoResearch, West Grove, PA) 及表現螢光標記之病毒蛋白的細胞外囊泡 (表現或不表現人 STEAP1) 染色。Sprague Dawley rats (Charles River, Hollister, CA) were immunized with 100 µg of plasmid DNA encoding human STEAP1 and plasmid DNA encoding rat Flt3L and rat GM-CSF (Genentech, South San Francisco, CA) at the following sites: intraperitoneally, subcutaneously (at the base of the tail), subcutaneously (at the back of the neck), and subcutaneously (at both tarsi). After receiving four doses of DNA, rats received two doses of 100 µg of multiple extracellular vesicles expressing human STEAP1 on their surface at multiple sites. Instead, rats were immunized at multiple sites with a priming dose of 30 µg human STEAP1 protein dissolved in detergent mixed with either MPL+TDM adjuvant (Sigma-Aldrich, St. Louis, MO) or a combination of TLR agonists: 50 μg MPL (Sigma-Aldrich), 20 μg R848 (Invivogen, San Diego, CA), 10 μg PolyI:C (Invivogen), and 10 μg CpG (Invivogen). For booster vaccination with additional protein, rats received 15 µg protein diluted in PBS. Rats were dosed every two weeks. Polyclonal antisera from these rats were purified and tested by FACS for binding to human STEAP1 on the surface of 293 or RBA cells. Three days after the last immunization, lymph nodes were harvested from rats that showed detectable FACS reactivity against human STEAP1. Rats immunized with protein showed weaker FACS responses than those observed with DNA/EV immunized rats, so DNA/EV immunized rats were selected for harvest. IgM-negative B cells from these rats were purified from intact lymphocytes using magnetic separation (Miltenyi Biotec, San Diego, CA) and fused with Sp2ab mouse myeloma cells (Abeome, Athens, GA) by electrofusion (Harvard Apparatus, Holliston, MA). Confluent cells were incubated overnight in CLONACELL™-HY Medium C (StemCell Technologies, Vancouver, BC, Canada) at 37°C, 7% CO₂ , then centrifuged and resuspended in CLONACELL™-HY Medium E (StemCell Technologies) supplemented with hypoxanthine, aminopterin, and thymidine (HAT) (Sigma-Aldrich), plated in 12-well plates, and grown at 37°C, 7% CO_2. Four days after plating, rat hybridoma cells were stained with anti-rat IgM antibody (Jackson ImmunoResearch, West Grove, PA) and extracellular vesicles expressing fluorescently labeled viral proteins with or without human STEAP1.

替代性地，使用磁力分離 (Miltenyi Biotec, San Diego, CA) 自完整淋巴球中純化來自這些大鼠的 IgM 陰性 B 細胞，並用抗大鼠 IgM 抗體 (Jackson ImmunoResearch, West Grove, PA) 及併入奈米圓盤 (建南德克) 中的經螢光標記之 STEAP1 蛋白進行染色。使用 FACSAria™ III 分選器 (BD, Franklin Lakes, NJ) 對表現出最低大鼠 IgM 表現、同時與表現 STEAP1 之細胞外囊泡結合的細胞進行分選，並將其沉積到含有培養基 E (StemCell Technologies) 的 96 孔盤中。使用 FACSAria™ III 分選器 (BD, Franklin Lakes, NJ) 對表現出最低大鼠 IgM 表現、同時與 STEAP1 蛋白結合的大鼠 B 細胞進行分選，並將其沉積到含有滋養細胞並補充有細胞激素的培養基的 96 孔盤中。於分選之後七天，藉由 ELISA 對上清液進行抗大鼠 IgG 篩選。對展示出大鼠 IgG 表現的細胞株進行規模放大，然後收穫上清液，並藉由蛋白質 G (GAMMABIND™ Plus, GE Healthcare, Pittsburgh, PA) 純化。藉由流式細胞術 (FACS) 測試經純化之大鼠 IgG 樣品與 293 細胞以及內源性表現人 STEAP1 的多個細胞株表面上表現的人 STEAP1 的結合。於分選之後七天，藉由 ELISA 對 B 細胞殖株上清液進行抗大鼠 IgG 篩選。藉由 FACS 測試展示出大鼠 IgG 結合的上清液與 293 細胞表面上表現的人 STEAP1 的結合。自顯示出 STEAP1 FACS 結合的 B 細胞中提取 RNA，用於分子選殖及重組表現。藉由 FACS 測試重組抗體與 293 細胞以及內源性表現人 STEAP1 的多個細胞株表面上表現的人 STEAP1 的結合。Alternatively, IgM-negative B cells from these rats were purified from intact lymphocytes using magnetic separation (Miltenyi Biotec, San Diego, CA) and stained with anti-rat IgM antibody (Jackson ImmunoResearch, West Grove, PA) and fluorescently labeled STEAP1 protein incorporated into nanodiscs (Genentech). Cells that expressed minimal rat IgM and that were associated with extracellular vesicles expressing STEAP1 were sorted using a FACSAria™ III sorter (BD, Franklin Lakes, NJ) and deposited into 96-well plates containing medium E (StemCell Technologies). Rat B cells that expressed minimal rat IgM and bound to STEAP1 were sorted using a FACSAria™ III sorter (BD, Franklin Lakes, NJ) and deposited into 96-well plates containing trophoblasts and cytokine-supplemented medium. Seven days after sorting, supernatants were screened for anti-rat IgG by ELISA. Cell lines that displayed rat IgG expression were scaled up, and supernatants were harvested and purified by protein G (GAMMABIND™ Plus, GE Healthcare, Pittsburgh, PA). Purified rat IgG samples were tested by flow cytometry (FACS) for binding to human STEAP1 expressed on the surface of 293 cells and multiple cell lines that endogenously express human STEAP1. Seven days after sorting, B cell line supernatants were screened by ELISA for anti-rat IgG. Supernatants that exhibited rat IgG binding were tested by FACS for binding to human STEAP1 expressed on the surface of 293 cells. RNA was extracted from B cells that exhibited STEAP1 FACS binding and used for molecular selection and recombinant expression. Recombinant antibodies were tested by FACS for binding to human STEAP1 expressed on the surface of 293 cells and multiple cell lines that endogenously express human STEAP1.

在與表現人 STEAP1 的 293 細胞結合的 21 種大鼠抗體中，2 個殖株顯示出特異性結合，而對親本 293 細胞、殖株 STEAP1-44 及 STEAP1-69 無任何反應性(圖 1A 及圖 1B)。在這兩種抗體中，抗體 STEAP1-44 (本文中亦稱為 Ab44) 在 7.5 μg/ml 的濃度下表現出與表現 STEAP-1 的細胞的強結合，與 2 μg/ml 的 Ab120 對照相當或更好。在以不同位準 (293-hSTEAP1 ＞ LNCaP X1.2 ＞ PC2-hSTEAP1 ＞ 22Rv1) 表現 STEAP1 的細胞及對照 293 細胞中對抗體 STEAP1-44 進行滴定測試，同時在 2 µg/ml 下測試對照 Ab120。觀察到 STEAP1-44 與 293-hSTEAP1、LNCaP X1.2、PC2-hSTEAP1、22Rv1 及 PC3-hSTEAP1 細胞結合的最小濃度分別為 0.39 nM、3.125 nM、6.25 nM 及 12.5 nM (圖 1C)。STEAP1-44 以 1.875 µg/ml 與不同細胞株的結合類似於對照 Ab120 以 2 µg/ml 與相同細胞的結合。對抗體 STEAP1-44 之可變區進行定序 (圖 2)。實例3.兔抗人STEAP1抗體的開發及表徵Among the 21 rat antibodies that bound to 293 cells expressing human STEAP1, 2 clones showed specific binding, while no reactivity was observed against the parental 293 cells, clones STEAP1-44 and STEAP1-69 (Fig. 1A and Fig. 1B). Of these two antibodies, antibody STEAP1-44 (also referred to herein as Ab44) showed strong binding to cells expressing STEAP-1 at a concentration of 7.5 μg/ml, comparable to or better than Ab120 at 2 μg/ml. Antibody STEAP1-44 was titrated in cells expressing STEAP1 at different levels (293-hSTEAP1 > LNCaP X1.2 > PC2-hSTEAP1 > 22Rv1) and control 293 cells, while control Ab120 was tested at 2 µg/ml. The minimum concentrations at which STEAP1-44 bound to 293-hSTEAP1, LNCaP X1.2, PC2-hSTEAP1, 22Rv1, and PC3-hSTEAP1 cells were observed to be 0.39 nM, 3.125 nM, 6.25 nM, and 12.5 nM, respectively (Figure 1C). The binding of STEAP1-44 at 1.875 µg/ml to different cell lines was similar to the binding of control Ab120 at 2 µg/ml to the same cells. The variable regions of antibody STEAP1-44 were sequenced (Figure 2).Example3. Development and characterization ofrabbit anti-humanSTEAP1antibodies

將紐西蘭白兔 (Charles River, Hollister, CA) 分開於沿背部的多個部位用 600 µg 編碼人 STEAP1 的質體 DNA 以及編碼兔 Flt3L 及 兔 GM-CSF (建南德克，South San Francisco，CA) 的質體 DNA 進行免疫。在接受四個劑量之 DNA 後，兔分開於多個部位接受三個劑量之 250 µg 在表面上表現人 STEAP1 的複數個細胞外囊泡。在三個劑量之囊泡後，兔分開於多個部位接受三個劑量之 50 µg 用洗滌劑溶解的 STEAP1 蛋白。兔係經每兩週給藥一次。將來自這些兔的多株抗血清純化並藉由 FACS 測試與 293 或 RK13 細胞表面上的人 STEAP1 的結合。在最後一次免疫三天後，從顯示出可檢測之針對人 STEAP1 的 FACS 反應性的兔身上抽取 20 mL 血液。使用磁力分離 (Miltenyi Biotec, San Diego, CA) 從 PBMC 中純化來自這些兔的 IgM 陰性 B 細胞。將兔細胞用抗兔 IgG 抗體 (Jackson ImmunoResearch, West Grove, PA) 及併入奈米圓盤 (建南德克) 中的經螢光標記之 STEAP1 蛋白進行染色。使用 FACSAria™ III 分選器 (BD, Franklin Lakes, NJ) 對表現出兔 IgG 表現、同時與 STEAP1 蛋白結合的 B 細胞進行分選，並將其沉積到包含滋養細胞並補充有細胞激素的培養基的 96 孔盤中。於分選之後七天，藉由 ELISA 對上清液進行抗兔 IgG 篩選。藉由 FACS 測試展示出兔 IgG 結合的上清液與 293 細胞表面上表現的人 STEAP1 的結合。自顯示出 STEAP1 FACS 結合的 B 細胞中提取 RNA，用於分子選殖及重組表現。藉由 FACS 測試重組抗體與 293 細胞以及內源性表現人 STEAP1 的多個細胞株表面上表現的人 STEAP1 的結合。New Zealand white rabbits (Charles River, Hollister, CA) were immunized at multiple sites along the back with 600 µg of plasmid DNA encoding human STEAP1 and plasmid DNA encoding rabbit Flt3L and rabbit GM-CSF (Genentech, South San Francisco, CA). After receiving four doses of DNA, rabbits received three doses of 250 µg of multiple extracellular vesicles expressing human STEAP1 on the surface at multiple sites. After three doses of vesicles, rabbits received three doses of 50 µg of STEAP1 protein solubilized with detergent at multiple sites. Rabbits were dosed every two weeks. Polyclonal antisera from these rabbits were purified and tested by FACS for binding to human STEAP1 on the surface of 293 or RK13 cells. Three days after the last immunization, 20 mL of blood was drawn from rabbits that showed detectable FACS reactivity against human STEAP1. IgM-negative B cells from these rabbits were purified from PBMCs using magnetic separation (Miltenyi Biotec, San Diego, CA). Rabbit cells were stained with anti-rabbit IgG antibodies (Jackson ImmunoResearch, West Grove, PA) and fluorescently labeled STEAP1 protein incorporated into nanodiscs (Invitrogen). B cells expressing rabbit IgG that also bind to the STEAP1 protein were sorted using a FACSAria™ III sorter (BD, Franklin Lakes, NJ) and deposited into 96-well plates containing trophoblasts and cytokine-supplemented medium. Seven days after sorting, supernatants were screened by ELISA against rabbit IgG. Supernatants that demonstrated rabbit IgG binding were tested by FACS for binding to human STEAP1 expressed on the surface of 293 cells. RNA was extracted from B cells that showed STEAP1 FACS binding and used for molecular selection and recombinant expression. The recombinant antibodies were tested by FACS for binding to human STEAP1 expressed on the surface of 293 cells and multiple cell lines that endogenously express human STEAP1.

圖 21 示出 rbAb3404 及 rbAb3349 之可變區序列。實例4.藉由庫新一代定序(NGS)的STEAP1-44的親和力成熟Figure 21 shows the variable region sequences of rbAb3404 and rbAb3349.Example4.Affinity maturation ofSTEAP1-44bynext generation sequencing(NGS)

藉由大鼠 NGS 庫資料挖掘，使抗體 STEAP1-44 親和力成熟 (Hsiao 等人，2019)。簡言之，使用淋巴結組織藉由雜交瘤衍生出 STEAP1-44 的同一隻大鼠的骨髓及脾臟組織提取總 RNA，並將其用於 RT-PCR 步驟，以用大鼠 IGHV5-22 種系片段 (與 STEAP1-44 使用相同的種系片段) 擴增 V_H片段。將擴增子提交以在 ILLUMINA® MiSeq® 儀器中進行雙末端定序。將序列讀段組裝成全長 VH 序列，並使用 IgBlast 解析種系片段及 CDR 邊界 (Goldstein 等人，2019)。從資料集選擇總共 17,902 個序列讀段，其中包含 IGHV5-22 及 IGHJ4 種系片段序列以及屬於與 STEAP1-44 抗體相同的株性型的長度為 13 個胺基酸殘基的 Kabat CDR H3。將它們與 IGHV5-22 種系片段序列進行比對，並對突變進行評分 (圖 3)。選擇總共 5 種變異體，其中包括資料集中 CDR 區中最常見的體細胞突變 (圖 2 及圖3)，用於藉由與 STEAP1-44 輕鏈組合來表現全長 IgG。藉由瞬時轉染 Expi293 細胞表現 IgG，並藉由蛋白質 A 層析進行純化 (Bos 等人，2015；Luan 等人，2018)。Affinity maturation of the antibody STEAP1-44 was performed by mining rat NGS libraries (Hsiao et al., 2019). Briefly, total RNA was extracted from bone marrow and spleen tissues of the same rat from which STEAP1-44 was derived by hybridoma using lymph node tissue and used in an RT-PCR step to amplify the V_H segments with rat IGHV5-22 germline segments (the same germline segments used for STEAP1-44). The amplicon was submitted for double-end sequencing in an ILLUMINA® MiSeq® instrument. Sequence reads were assembled into full-length VH sequences, and the germline segments and CDR boundaries were resolved using IgBlast (Goldstein et al., 2019). A total of 17,902 sequence reads were selected from the dataset, which contained IGHV5-22 and IGHJ4 germline segment sequences and a 13-amino acid residue-long Kabat CDR H3 belonging to the same strain type as the STEAP1-44 antibody. They were aligned with the IGHV5-22 germline segment sequence and the mutations were scored (Figure 3). A total of 5 variants, including the most common somatic mutations in the CDR region in the dataset (Figures 2 and 3), were selected for the expression of full-length IgG by combination with the STEAP1-44 light chain. IgG was expressed by transient transfection of Expi293 cells and purified by protein A chromatography (Bos et al., 2015; Luan et al., 2018).

藉由在表現 STEAP1 之細胞上的 FACS 並藉由使用可溶性生物素化重組 STEAP1 的 ELISA，對株性變異體進行測試。連續稀釋液顯示所有變異體的結合比 STEAP1-44 更強 (圖4A 及 4B)。然而，變異體之排序在測定之間變化，其中 STEAP1-44.NGS.HC3 藉由 FACS 表現出最強的結合，STEAP1-44.NGS.HC5 藉由 ELISA 表現出與重組 STEAP1 的最強結合，而 STEAP1-44.NGS.HC4 在兩種測定中皆表現出強結合。因此，在人源化後對這 3 種變異體進行更詳細的進一步檢查。實例5.  STEAP1-44的人源化The strain variants were tested by FACS on cells expressing STEAP1 and by ELISA using soluble biotinylated recombinant STEAP1. Serial dilutions showed that all variants bound more strongly than STEAP1-44 (Figures 4A and 4B). However, the ranking of the variants varied between the assays, with STEAP1-44.NGS.HC3 showing the strongest binding by FACS, STEAP1-44.NGS.HC5 showing the strongest binding to recombinant STEAP1 by ELISA, and STEAP1-44.NGS.HC4 showing strong binding in both assays. Therefore, these 3 variants were further examined in more detail after humanization.Example5.Humanization of STEAP1-44

藉由 CDR 移植將抗體 STEAP1-44 在 VK2 及 VH3 框架中人源化。簡言之，將大鼠 STEAP1-44 抗體之 CDR 區移植到輕鏈 IGKV2-29 及重鏈 IGKV3-74 骨架中，該等骨架為 STEAP1-44 的最接近之人種系片段。對於輕鏈，CDR 區包括 Kabat 位置 24 至 34 (CDR L1)、50 至 56 (CDR L2) 及 89 至 97 (CDR L3)，且對於重鏈，包括 Kabat 位置 26 至 35 (CDR H1 )、50 至 65 (CDR H2) 及 93 至 102 (CDR H3)。將稱為「游標 (Vernier)」位置以及在大鼠 STEAP1-44 與人類種系之間不同的域界面中的大鼠骨架殘基添加到 CDR 移植物中以挽救與抗原的結合，如其在 CDR 移植物人源化中正常地進行者 (參見例如，美國專利第 8,426,147 號)。在這種情況下，此等殘基包括輕鏈殘基 2 及 4 以及重鏈殘基 49 及 76。包括對大鼠殘基的骨架改變的最終 CDR 移植物稱為 huAb44.v1 (圖 5A 及 5B)。將具有不同游標位置組合的變異體 (總共 16 種變異體) 表現為人 IgG1，藉由蛋白質 A 及粒徑排阻層析純化，並在 FACS 中測試與表現 STEAP1 之細胞的結合。在所有變異體中，huAb44.v6 與 LNCaP-X1.2 細胞的結合最高，並被選為最終的人源化變異體 (圖 6A)。Antibody STEAP1-44 was humanized in VK2 and VH3 frameworks by CDR grafting. Briefly, the CDR regions of the rat STEAP1-44 antibody were grafted into the light chain IGKV2-29 and heavy chain IGKV3-74 frameworks, which are the closest human germline fragments of STEAP1-44. For the light chain, the CDR regions included Kabat positions 24 to 34 (CDR L1), 50 to 56 (CDR L2), and 89 to 97 (CDR L3), and for the heavy chain, Kabat positions 26 to 35 (CDR H1), 50 to 65 (CDR H2), and 93 to 102 (CDR H3). Rat framework residues at so-called "Vernier" positions and in the domain interfaces that differ between rat STEAP1-44 and human germline were added to the CDR graft to rescue binding to the antigen, as is normally done in CDR graft humanization (see, e.g., U.S. Patent No. 8,426,147). In this case, these residues included light chain residues 2 and 4 and heavy chain residues 49 and 76. The final CDR graft including the framework changes to the rat residues was called huAb44.v1 ( FIGS. 5A and 5B ). Variants with different combinations of cursor positions (16 variants in total) were expressed as human IgG1, purified by protein A and size exclusion chromatography, and tested for binding to cells expressing STEAP1 in FACS. Of all the variants, huAb44.v6 had the highest binding to LNCaP-X1.2 cells and was selected as the final humanized variant (Figure 6A).

將來自 NGS 庫深度定序的突變併入 huAb44.v6 中。將來自 STEAP1.NGS.HC1 至 5 的體細胞突變添加至 huAb44.v6 中，分別創建變異體 huAb44.v6.01 至 05。藉由在表現 STEAP1 的細胞上的 FACS 測試這些變異體的結合。(圖 6B)。與 huAb44.v6 相比，併入 huAb44.v6 中的所有 NGS 突變異體皆表現出改善的與 LNCaP-X1.2 細胞的結合。Mutations from deep sequencing of NGS libraries were incorporated into huAb44.v6. Somatic mutations from STEAP1.NGS.HC1 to 5 were added to huAb44.v6 to create variants huAb44.v6.01 to 05, respectively. Binding of these variants was tested by FACS on cells expressing STEAP1. (Fig. 6B). All NGS mutants incorporated into huAb44.v6 showed improved binding to LNCaP-X1.2 cells compared to huAb44.v6.

圖 6C 示出人源化抗體變異體 huAb44.v6.05 與人源化抗體 huAb44.v6 之比對。huAb44.v6.05 相對於 huAb44.v6 的差異以黑色背景示出。根據比對上方所示之 Kabat 及 Chothia 系統的 CDR 邊界，其中 Kabat CDR 邊界帶下劃線。根據 Kabat 系統進行殘基編號。實例6.  STEAP1-TDB分子媒介的T細胞毒殺Figure 6C shows an alignment of humanized antibody variant huAb44.v6.05 with humanized antibody huAb44.v6. Differences of huAb44.v6.05 relative to huAb44.v6 are shown in black background. CDR boundaries according to the Kabat and Chothia systems are shown above the alignment, with the Kabat CDR boundaries underlined. Residue numbering is according to the Kabat system.Example6.Tcell cytotoxicitymediated by STEAP1-TDB molecules

將人源化抗體 huAb44.v6 的變異體與抗 CD3 抗體 MD1 (對 CD3 的親和力較高) 及 40G5c (對 CD3 的親和力較低) 組裝為雙特異性分子，並在 T 細胞媒介的靶細胞毒殺測定中進行評估。在 STEAP1 表現高的 LNCaP-X1.2 細胞株上評定 T 細胞媒介的細胞毒殺。簡言之，人類血液樣品係透過過建南德克員工捐贈計劃獲自健康志願者。使用 FICOLL®-Paque 及 LEUCOSEP™ 管從健康志願者的血液中分離出 PBMC。使用 Miltenyi CD8⁺T 細胞分離套組 (目錄號 130-096-495) 及 Miltenyi XS 柱 (目錄號 130-041-202) 從 PBMC 中分離 CD8⁺T 細胞。將 LNCaP-X1.2 細胞 (4000 個細胞/孔) 接種到透明底 96 孔盤 (Corning，目錄號 3904) 中。次日，將標靶細胞與新鮮分離之 CD8⁺細胞以 1:4 之比率共培養。將與抗 CD3 抗體 MD1 組裝的 STEAP1 TDB 以指定濃度 (範圍 0 nM 至 5 nM) 以三重複方式添加，並於 37℃ 孵育 72 小時後評定細胞毒殺。使用 CELLTITER-GLO® 發光細胞活力試劑 (Promega，目錄號 G7570) 測量細胞活力。對於所有變異體，STEAP1-TDB 媒介之細胞毒殺的 EC₅₀值均在皮莫耳範圍內。抗體 huAb44.v6.05 展現出最高效力，EC₅₀為 90 pM，相對於親本 huAb44.v6 明顯改善約 6 倍 (圖 7A)。Variants of the humanized antibody huAb44.v6 were assembled as bispecific molecules with the anti-CD3 antibodies MD1 (higher affinity for CD3) and 40G5c (lower affinity for CD3) and evaluated in a T cell-mediated target cytotoxicity assay. T cell-mediated cytotoxicity was assessed on the LNCaP-X1.2 cell line with high STEAP1 expression. Briefly, human blood samples were obtained from healthy volunteers through the Jiannan Deke employee donation program. PBMCs were isolated from the blood of healthy volunteers using FICOLL®-Paque and LEUCOSEP™ tubes. CD8⁺ T cells were isolated from PBMC using Miltenyi CD8⁺ T Cell Isolation Kit (Cat. No. 130-096-495) and Miltenyi XS columns (Cat. No. 130-041-202). LNCaP-X1.2 cells (4000 cells/well) were plated in clear-bottom 96-well plates (Corning, Cat. No. 3904). The next day, target cells were co-cultured with freshly isolated CD8⁺ cells at a ratio of 1:4. STEAP1 TDB assembled with anti-CD3 antibody MD1 was added in triplicate at the indicated concentrations (range 0 nM to 5 nM) and cytotoxicity was assessed after incubation at 37°C for 72 hours. Cell viability was measured using CELLTITER-GLO® Luminescent Cell Viability Assay (Promega, Catalog No. G7570). EC₅₀ values for STEAP1-TDB-mediated cytotoxicity were in the picomolar range for all variants. Antibody huAb44.v6.05 showed the highest potency with an EC₅₀ of 90 pM, a significant improvement of approximately 6-fold over the parental huAb44.v6 (Figure 7A).

亦將三種最有效的抗體 (huAb44v6.03、huAb44v6.04 及 huAb44v6.05) 與抗 CD3 抗體 40G5c 組合，並使用不同 PBMC 供體在 LNCaP-X1.2 細胞中測量所有抗 STEAP1 TDB 的細胞毒殺作用。實驗設置與上述相同。再次，當用任一抗 CD3 臂測試 huAb44.v6.03、huAb44.v6.04 及 huAb44.v6.05 抗體時觀察到相同的排序。使用 huAb44.v6.05-MD1 及 huAb44.v6.05-40G5c 觀察到最有效的細胞毒殺，EC₅₀值分別為 150pM 及 160pM (圖 7B)。The three most potent antibodies (huAb44v6.03, huAb44v6.04, and huAb44v6.05) were also combined with the anti-CD3 antibody 40G5c and the cytotoxicity of all anti-STEAP1 TDBs was measured in LNCaP-X1.2 cells using different PBMC donors. The experimental setup was the same as above. Again, the same ranking was observed when huAb44.v6.03, huAb44.v6.04, and huAb44.v6.05 antibodies were tested with either anti-CD3 arm. The most potent cytotoxicity was observed with huAb44.v6.05-MD1 and huAb44.v6.05-40G5c with EC₅₀ values of 150pM and 160pM, respectively (Figure 7B).

為確定在 STEAP1 表現較低的細胞中的細胞毒殺效力，使用基因編輯技術生成 LNCaP-X1.2 衍生物 (LNCaP-X1.2 KO-3-13 及 LNCaP-X1.2 KO-2-11)。這些線代表在前列腺癌中觀察到的各種 STEAP1 位準。另外，創建缺乏 STEAP1 表現的細胞株 (LNCaP-X1.2KO-2-8) 作為對照株 (圖 8A)。在這些細胞株中測試 HuAb44.v6.05-MD1 及 huAb44.v6.05-40G5c。在所有表現 STEAP1 之細胞株中皆觀察到有效的細胞毒殺作用，但在 STEAP1 陰性細胞株 LNCaP-X1.2KO-2-8 中則未觀察到。huAb44.v6.05-MD1 之效力在 STEAP1 表現較低的細胞株 (LNCaP-X1.2、LNCaP-X1.2 KO-3-13、LNCaP-X1.2 KO-2-11) 中略有下降，EC₅₀值分別在 80 pM 至 200 pM 及 450 pM 的範圍內。一般而言，與 huAb44.v6.05-MD1 相比，huAb44.v6.05-40G5c 顯示出降低的功效；然而，在這些條件下，在 LNCaP-X1.2 及 LNCaP-X1.2-KO-3-13 中仍然觀察到 100% 細胞毒殺 (圖 8B)。確認 STEAP1-TDB 活性具有標靶依賴性的額外實驗顯示於圖 8C (於 24 小時的標靶標依賴性 T 細胞活化) 及圖 8D (於 24 小時的細胞激素分泌) 中。實例7.  STEAP1-TDB的活體內活性To determine cytotoxicity in cells with low STEAP1 expression, LNCaP-X1.2 derivatives (LNCaP-X1.2 KO-3-13 and LNCaP-X1.2 KO-2-11) were generated using gene editing technology. These lines represent the various STEAP1 levels observed in prostate cancer. In addition, a cell line lacking STEAP1 expression (LNCaP-X1.2KO-2-8) was created as a control line (Figure 8A). HuAb44.v6.05-MD1 and huAb44.v6.05-40G5c were tested in these cell lines. Effective cytotoxicity was observed in all cell lines expressing STEAP1, but not in the STEAP1-negative cell line LNCaP-X1.2KO-2-8. The potency of huAb44.v6.05-MD1 was slightly reduced in cell lines with lower STEAP1 expression (LNCaP-X1.2, LNCaP-X1.2 KO-3-13, LNCaP-X1.2 KO-2-11), with_EC50 values ranging from 80 pM to 200 pM and 450 pM, respectively. In general, huAb44.v6.05-40G5c showed reduced efficacy compared to huAb44.v6.05-MD1; however, under these conditions, 100% cytotoxicity was still observed in LNCaP-X1.2 and LNCaP-X1.2-KO-3-13 (Figure 8B). Additional experiments confirming that STEAP1-TDB activity is target-dependent are shown in Figure 8C (target-dependent T cell activation at 24 hours) and Figure 8D (cytokine secretion at 24 hours).Example 7.In vivo activity ofSTEAP1-TDB

使用接種以 LNCaP-X1.2、LNCaP-X1.2KO-3-13 或 LNCaP-X1.2KO-2-11 人前列腺癌細胞 (具有或不具有 PBMC) 的 NSG 小鼠評定 STEAP1-TDB 的抗腫瘤活性。簡言之，8 至 9 週齡的雌性 NOD SCID γ (NSG) 小鼠獲自 Jackson Laboratory (Sacramento, CA)。這些細胞表現出一系列代表前列腺癌患者的 STEAP1 表現：低 STEAP1 (LNCaPX1.2KO-2-11)、中 STEAP1 (LNCaPX1.2 KO-3-13) 及高 STEAP1 (LNCaPX1.2)。將小鼠飼養在建南德克公司，且所有實驗程序皆符合美國生理學會的指導原則並得到建南德克機構動物照護及使用委員會的批准。在每隻小鼠的右側皮下接種懸浮於 0.1 mL Hank 平衡鹽溶液 (HBSS) 中的 1000 萬個 LNCAP-X1.2 細胞或次選殖株。於細胞接種後一天，向小鼠腹膜內注射在非活化條件下培養過夜的 1000 萬個人類 PBMC。於細胞接種後 7 至 10 天，基於相似大小的腫瘤 (平均體積為 200 mm³) 將荷瘤小鼠隨機分為對照組及治療組，並於第 0 天開始給藥。所有治療皆以單次靜脈內 (IV) 注射濃度範圍為 0.1 mg/kg 至 0.5 mg/kg 的媒劑或 STEAP1-TDB 進行給予，體積為每隻小鼠 100 μL。在研究過程中每週測量兩次腫瘤體積及體重。當腫瘤體積超過約 2000 mm³或者若腫瘤變得潰爛，則將小鼠處以安樂死。對 STEAP1-TDB 的個體腫瘤體積反應如圖 9 所示。在 LNCaP-X1.2、LNCaPX1.2-KO3-13 及 LNCaP-X1.2-2-11 異種移植模型中測試的所有濃度下，使用 huAb44.v6.05-MD1 或 huAb44.v6.05-40G5c 觀察到腫瘤完全消退。媒劑對照組中的腫瘤皆未顯示出消退徵象，證明功效取決於 STEAP1-TDB 的存在。實例8.  huAb44.v6.05的親和力The antitumor activity of STEAP1-TDB was assessed using NSG mice inoculated with LNCaP-X1.2, LNCaP-X1.2KO-3-13, or LNCaP-X1.2KO-2-11 human prostate cancer cells (with or without PBMCs). Briefly, female NOD SCID γ (NSG) mice aged 8 to 9 weeks were obtained from the Jackson Laboratory (Sacramento, CA). These cells expressed a spectrum of STEAP1 expression representative of prostate cancer patients: low STEAP1 (LNCaPX1.2KO-2-11), medium STEAP1 (LNCaPX1.2KO-3-13), and high STEAP1 (LNCaPX1.2). Mice were maintained at Jianan Deke, and all experimental procedures were in accordance with the guidelines of the American Physiological Society and approved by the Jianan Deke Institutional Animal Care and Use Committee. Each mouse was inoculated subcutaneously on the right flank with 10 million LNCAP-X1.2 cells or subselected strains suspended in 0.1 mL Hank's balanced salt solution (HBSS). One day after cell inoculation, mice were injected intraperitoneally with 10 million human PBMCs cultured overnight under non-activated conditions. Seven to ten days after cell inoculation, tumor-bearing mice were randomly divided into control and treatment groups based on similar tumor size (average volume of 200 mm³ ), and drug administration began on day 0. All treatments were given as a single intravenous (IV) injection of vehicle or STEAP1-TDB at concentrations ranging from 0.1 mg/kg to 0.5 mg/kg in a volume of 100 μL per mouse. Tumor volume and body weight were measured twice weekly during the study. Mice were euthanized when tumor volume exceeded approximately 2000^mm3 or if tumors became necrotic. Individual tumor volume responses to STEAP1-TDB are shown in Figure 9. Complete tumor regression was observed with huAb44.v6.05-MD1 or huAb44.v6.05-40G5c at all concentrations tested in the LNCaP-X1.2, LNCaPX1.2-KO3-13, and LNCaP-X1.2-2-11 xenograft models. No tumors in the vehicle control group showed signs of regression, demonstrating that efficacy is dependent on the presence of STEAP1-TDB.Example8.Affinity of huAb44.v6.05

抗體 huAb44.v6.05-40G5c 對 STEAP1 的單價親和力係於無標記系統中用穩定表現人類或石蟹獼猴 STEAP1 的活 293 細胞來確定。藉由 Sapidyne Instruments (Boise, ID) 執行的動力學篩析測定 (KinExA®) 測量溶液中之平衡結合親和力及動力學。平衡解離常數 K_D及締合常數k_a係藉由實驗確定，而解離速率k_d係基於方程式k_d= K_D×k_a來計算。藉由 KinExA® 測定所確定的細胞上 huAb44.v6.05-40G5c 對人類及石蟹獼猴 STEAP1 的單價結合動力學為：實例9.在雌性SCID小鼠中靜脈給藥後抗STEAP1/CD3 T細胞依賴性雙特異性抗體的藥物動力學The monovalent affinity of antibody huAb44.v6.05-40G5c for STEAP1 was determined in a label-free system using live 293 cells stably expressing human or red macaque STEAP1. Equilibrium binding affinity and kinetics in solution were measured by a kinetic screening assay (KinExA®) performed by Sapidyne Instruments (Boise, ID). The equilibrium dissociation constant K_D and association constantka_were determined experimentally, and the dissociation ratek_d was calculated based on the equationk_d = K_D ×ka . The monovalent binding kinetics of huAb44.v6.05-40G5c for human and red macaque STEAP1 on cells determined by the KinExA® assay_were :Example9. Pharmacokinetics of an anti-STEAP1/CD3 Tcell-dependent bispecific antibodyafter intravenous administration infemaleSCID mice

在第一項研究中，在嚴重合併性免疫缺失 (SCID) 小鼠中評定投予單次靜脈內 (IV) 推注劑量 1 mg/kg 後抗 STEAP1 抗體之藥物動力學 (PK)。此研究包括嵌合 Ab44 (chAb44) 以及人源化形式的 Ab44 (huAb44.v6)。該研究的活體階段係於 Charles River Laboratories (CRL) (South San Francisco, CA) 根據當地機構動物照護及使用委員會的指導原則進行。將給藥時體重為 17 g 至 22 g、約 7 週至 9 週齡天然雌性 SCID 小鼠分配至劑量組 (每組 n = 4 隻動物)。測試品係經由尾靜脈以單次 IV 推注劑量投予 (劑量體積設定為 5 mL/kg)。使用動物限制器進行給藥。在給藥後 0.167、1、6、24、72、168、240、336 及 504 小時，經由尾部切口將連續血液樣品 (30 μL 至 40 μL) 收集到血清分離管中。收集後，將血液樣品於室溫儲存 30 分鐘至 60 分鐘，並藉由離心處理以將血清慢慢倒入 2D 條形碼管中。所有樣品皆儲存於 ‑80℃，直至使用 GRIP (通用) ELISA 測定法分析藥物濃度 (Yang 2008)。將測試品的定量極限設定為 0.0156 µg/mL。In the first study, the pharmacokinetics (PK) of anti-STEAP1 antibodies following a single intravenous (IV) bolus dose of 1 mg/kg were evaluated in severely confluent immunodeficient (SCID) mice. This study included chimeric Ab44 (chAb44) as well as a humanized form of Ab44 (huAb44.v6). The in vivo phase of the study was conducted at Charles River Laboratories (CRL) (South San Francisco, CA) in accordance with the guidelines of the local Institutional Animal Care and Use Committee. Naïve female SCID mice weighing 17 g to 22 g at the time of dosing, approximately 7 to 9 weeks of age, were assigned to dose groups (n = 4 animals per group). The test article was administered as a single IV bolus via the caudal vein (dose volume set at 5 mL/kg). Dosing was performed using an animal restrainer. Serial blood samples (30 μL to 40 μL) were collected into serum separation tubes via tail nick at 0.167, 1, 6, 24, 72, 168, 240, 336, and 504 h after dosing. After collection, blood samples were stored at room temperature for 30 min to 60 min and centrifuged to decant serum into 2D barcoded tubes. All samples were stored at ‑80°C until analyzed for drug concentration using the GRIP (Universal) ELISA assay (Yang 2008). The limit of quantitation for the test article was set at 0.0156 µg/mL.

chAb44 及 huAb44.v6 的 PK 型態如圖 10A 所示，且 PK 參數列於表 6 中。一般而言，兩種抗體皆表現出雙指數 PK 行為，全身清除率 (CL) 較慢，範圍為約 2.6 mL/天/kg 至 2.7 mL/天/kg，且終末消除半衰期 (t_1/2) 相對較長，範圍為 16 天至 19 天。兩種抗體的最大血清濃度 (C_max) 相當 (大約 24 μg/mL)。chAb44 及 huAb44.v6 的 PK 特徵被認為適合進一步開發。The PK profiles of chAb44 and huAb44.v6 are shown in Figure 10A, and the PK parameters are listed in Table 6. In general, both antibodies exhibited biexponential PK behavior, with slow systemic clearance (CL) ranging from approximately 2.6 mL/day/kg to 2.7 mL/day/kg, and relatively long terminal elimination half-lives (t_1/2 ) ranging from 16 days to 19 days. The maximum serum concentrations (C_max ) of both antibodies were comparable (approximately 24 μg/mL). The PK characteristics of chAb44 and huAb44.v6 were considered suitable for further development.

在第二項研究中，在嚴重合併性免疫缺失 (SCID) 小鼠中評定投予單次靜脈內 (IV) 推注劑量 1 mg/kg 後抗 STEAP1/CD3 T 細胞依賴性雙特異性 (TDB) 抗體的 PK。該研究包括 huAb44.v6.05-MD1 及 huAb44.v6.05-40G5c TDB，以及作為對照的抗 gD IgG (以 1 mg/kg 給藥)。該研究的活體階段係於 Charles River Laboratories (CRL) (South San Francisco, CA) 根據當地機構動物照護及使用委員會的指導原則進行。將給藥時體重為 17 g 至 22 g、約 7 週至 9 週齡天然雌性 SCID 小鼠分配至治療組 (每組 n = 4 隻動物)。測試品及對照品係經由尾靜脈以單次 IV 推注劑量投予 (劑量體積設定為 5 mL/kg)。使用動物限制器進行 IV 給藥。在給藥後 0.167、1、6、24、72、168、240、336 及 504 小時，經由尾部切口將連續血液樣品 (30 μL 至 40 μL) 收集到血清分離管中。收集後，將血液樣品於室溫保存 30 分鐘至 60 分鐘，並藉由離心處理以將血清慢慢倒入 2D 條形碼管中。所有樣品皆儲存於 -80℃，直至使用 GRIP ELISA 測定法分析藥物濃度 (Yang 2008)。將測試品及對照品的定量極限皆設定為 0.0156 µg/mL。In the second study, the PK of anti-STEAP1/CD3 T cell-dependent bispecific (TDB) antibodies was evaluated in severely confluent immunodeficient (SCID) mice after a single intravenous (IV) bolus dose of 1 mg/kg. The study included huAb44.v6.05-MD1 and huAb44.v6.05-40G5c TDB, as well as anti-gD IgG (administered at 1 mg/kg) as a control. The in vivo phase of the study was conducted at Charles River Laboratories (CRL) (South San Francisco, CA) in accordance with the guidelines of the local Institutional Animal Care and Use Committee. Naive female SCID mice weighing 17 g to 22 g at the time of dosing, approximately 7 to 9 weeks of age, were assigned to treatment groups (n = 4 animals per group). Test and control articles were administered as a single IV bolus via the tail vein (dose volume set at 5 mL/kg). IV dosing was performed using an animal restrainer. Serial blood samples (30 μL to 40 μL) were collected via tail nick into serum separation tubes at 0.167, 1, 6, 24, 72, 168, 240, 336, and 504 hours after dosing. After collection, blood samples were kept at room temperature for 30 to 60 minutes and centrifuged to decant serum into 2D barcoded tubes. All samples were stored at -80°C until analyzed for drug concentration using the GRIP ELISA assay (Yang 2008). The limit of quantification was set at 0.0156 µg/mL for both the test and control articles.

抗 gD、huAb44.v6.05-MD1 TDB 及 huAb44.v6.05-40G5c TDB 的 PK 型態如圖 10B 所示，且 PK 參數列於表 7 中。huAb44.v6.05-MD1 及 huAb44.v6.05-40G5c TDB 的 PK 特徵與抗 gD 對照相當。所有 3 種測試品皆表現出雙指數 PK 行為，緩慢的 CL 的範圍為大約 3.30 mL/天/kg 至 3.74 mL/天/kg，且 t_1/2的範圍為 13.5 天至 17.1 天。對於測試品，最大血清濃度 (C_max) 在 20.3 μg/mL 至 26.2 μg/mL 的範圍內，且劑量標準化之 C_max(C_max/實際劑量) 相當 (28.6 μg/mL 至 29.2 μg/mL)。總之，huAb44.v6.05-MD1 TDB 及 huAb44.v6.05-40G5c TDB 的 PK 特徵符合對典型 IgG 抗體的預期 (Ovacik 等人，MAbs11(2):422-433 (2019))，並在石蟹獼猴研究中進行了進一步評估。The PK profiles of anti-gD, huAb44.v6.05-MD1 TDB, and huAb44.v6.05-40G5c TDB are shown in Figure 10B, and the PK parameters are listed in Table 7. The PK characteristics of huAb44.v6.05-MD1 and huAb44.v6.05-40G5c TDB were comparable to those of anti-gD. All 3 test products showed biexponential PK behavior, with slow CL ranging from approximately 3.30 mL/day/kg to 3.74 mL/day/kg and t1_/2 ranging from 13.5 days to 17.1 days. For the test articles, maximum serum concentrations (_Cmax ) ranged from 20.3 μg/mL to 26.2 μg/mL, and dose-normalized_Cmax (_Cmax /actual dose) were comparable (28.6 μg/mL to 29.2 μg/mL). In summary, the PK characteristics of huAb44.v6.05-MD1 TDB and huAb44.v6.05-40G5c TDB were consistent with expectations for typical IgG antibodies (Ovacik et al.,MAbs 11(2):422-433 (2019)) and were further evaluated in a study in stone macaques.

使用非區室分析 (NCA) 方法及 Phoenix™ WinNonlin® 6.4 版軟體 (Pharsight Corp, Mountain View, CA) 進行血清濃度-時間資料的 PK 分析。對於這兩項研究，皆使用 IV 推注輸入及實際劑量進行分析。表6.抗 STEAP1 抗體在雌性 SCID 小鼠中單一靜脈內劑量 1 mg/kg 後的藥物動力學參數組C_max(μg/mL)AUC_INF(天*μg/mL)CL(mL/天/kg)Vss(mL/kg)t_1/2(天)huAb44.v623.7 ± 2.09273 ± 48.92.59 ± 0.5159.2 ± 8.4916.0 ± 2.07chAb4423.9 ± 0.25250 ± 29.32.71 ± 0.3470.7 ± 2.5618.6 ± 2.58C_max= 最大觀察濃度；AUC_INF：從時間零至無窮大的曲線下面積；CL = 清除率；Vss = 穩態分佈體積；t_1/2= 消除半衰期表7.抗 STEAP1/CD3 TDB 及抗 gD 在雌性 SCID 小鼠中單一靜脈內劑量 1 mg/kg 後的藥物動力學參數組C_max(μg/mL)AUC_INF(天*ug/mL)CL(mL/天/kg)Vss(mL/kg)t_1/2(天)抗 gD26.2 ± 2.54293 ± 89.93.30 ± 0.8476.2 ± 9.0317.1 ± 6.23huAb44.v6.05-MD123.1 ± 3.15214 ± 29.53.74 ± 0.5178.1 ± 14.514.4 ± 2.84huAb44.v6.05-40G5c20.3 ± 1.72211 ± 40.23.47 ± 0.7165.2 ± 11.613.5 ± 3.29C_max= 最大觀察濃度；AUC_INF：從時間零至無窮大的曲線下面積；CL = 清除率；Vss = 穩態分佈體積；t_1/2= 消除半衰期實例10.抗STEAP1 T細胞依賴性雙特異性(TDB)抗體在雄性石蟹獼猴中的靜脈內藥物動力學PK analysis of serum concentration-time data was performed using noncompartmental analysis (NCA) methods and Phoenix™ WinNonlin® version 6.4 software (Pharsight Corp, Mountain View, CA). For both studies, IV bolus infusion and actual dosing were used for analysis.Table6. Pharmacokinetic parameters of anti-STEAP1 antibodies following a single intravenous dose of 1 mg/kg in female SCID miceGroupC_max(μg/mL)AUC_INF(day*μg/mL)CL(mL/day/kg)Vss(mL/kg)t_1/2(day) huAb44.v6 23.7 ± 2.09 273 ± 48.9 2.59 ± 0.51 59.2 ± 8.49 16.0 ± 2.07 chAb44 23.9 ± 0.25 250 ± 29.3 2.71 ± 0.34 70.7 ± 2.56 18.6 ± 2.58_Cmax = maximum observed concentration;_AUCINF : area under the curve from time zero to infinity; CL = clearance; Vss = steady-state volume of distribution; t1_/2 = elimination half-lifeTable7. Pharmacokinetic parameters of anti-STEAP1/CD3 TDB and anti-gD after a single intravenous dose of 1 mg/kg in female SCID miceGroupC_max(μg/mL)AUC_INF(day*ug/mL)CL(mL/day/kg)Vss(mL/kg)t_1/2(day) Anti-gD 26.2 ± 2.54 293 ± 89.9 3.30 ± 0.84 76.2 ± 9.03 17.1 ± 6.23 huAb44.v6.05-MD1 23.1 ± 3.15 214 ± 29.5 3.74 ± 0.51 78.1 ± 14.5 14.4 ± 2.84 huAb44.v6.05-40G5c 20.3 ± 1.72 211 ± 40.2 3.47 ± 0.71 65.2 ± 11.6 13.5 ± 3.29_Cmax = maximum observed concentration;_AUCINF : area under the curve from time zero to infinity; CL = clearance; Vss = steady-state volume of distribution; t1_/2 = elimination half-lifeExample10. Intravenous pharmacokinetics of ananti-STEAP1 Tcell-dependent bispecific(TDB)antibody in male stone crab macaques

在雄性石蟹獼猴中進行單次 IV 輸注 (持續時間 1 小時) 1 mg/kg 或 0.5 mg/kg 單一劑量以及 0.5 mg/kg (第 1 天) 及 7 天後之 1 mg/kg 或 2 mg/kg 的重複給藥方案後評定 huAb44.v6.05/40G5c 及 huAb44.v6.05/MD1 (抗 STEAP1 T 細胞依賴性雙特異性 (TDB) 抗體) 的藥物動力學。該研究在 Charles River Laboratories (CRL) (Reno, NV) 進行，且動物照護係根據「動物福祉法案：實驗室動物照護及使用指南」以及 CRL 實驗室動物福祉辦公室規定者進行。將給藥時體重為 2.3 kg 至 3 kg 且大約 2.5 嵗至 6 歲的天然雄性石蟹獼猴分配至治療組。在單一劑量組中觀察動物長達 7 天，並在重複劑量組觀察動物 14 天。在給藥後 0.25 小時、2 小時、6 小時、24 小時、2 天、4 天及 7 天，經由靜脈穿刺將血液樣品 (最多 1 mL 全血) 收集到血清分離管中，用於 PK 估計。在重複劑量組中，在第二次給藥後 0.25 小時、2 小時、6 小時、24 小時、2 天、4 天及 7 天收集額外血液樣品，用於 PK 估計。收集後，將收集的血液樣品於室溫靜置 60 分鐘，並藉由離心處理以將血清慢慢倒入 2D 條形碼管中。所有血清樣品皆儲存於 -70℃，直至使用 GRIP ELISA 測定法分析抗體濃度 (Yang 2008)。將測試品的定量極限設定為 0.0156 µg/mL。亦在單一劑量組的 -14 天、給藥前及 7天以及重複劑量組的給藥前及 15天時收集血液樣品用於抗藥物抗體 (ADA) 評定。The pharmacokinetics of huAb44.v6.05/40G5c and huAb44.v6.05/MD1 (anti-STEAP1 T cell-dependent bispecific (TDB) antibodies) were evaluated in male stone crab macaques after a single IV infusion (duration 1 hour) of 1 mg/kg or 0.5 mg/kg and a repeated dosing regimen of 0.5 mg/kg (day 1) and 1 mg/kg or 2 mg/kg 7 days later. The study was conducted at Charles River Laboratories (CRL) (Reno, NV), and animal care was conducted in accordance with the Animal Welfare Act: Guide for the Care and Use of Laboratory Animals and CRL Office of Laboratory Animal Welfare regulations. Natural male crab macaques weighing 2.3 kg to 3 kg at the time of dosing and approximately 2.5 to 6 years of age were assigned to treatment groups. Animals were observed for up to 7 days in the single-dose group and 14 days in the repeated-dose group. Blood samples (up to 1 mL of whole blood) were collected by venous puncture into serum separation tubes for PK estimates at 0.25 hours, 2 hours, 6 hours, 24 hours, 2 days, 4 days, and 7 days after dosing. In the repeated-dose group, additional blood samples were collected at 0.25 hours, 2 hours, 6 hours, 24 hours, 2 days, 4 days, and 7 days after the second dose for PK estimates. After collection, the collected blood samples were kept at room temperature for 60 minutes and centrifuged to decant the serum into 2D barcoded tubes. All serum samples were stored at -70°C until analysis of antibody concentration using the GRIP ELISA assay (Yang 2008). The limit of quantification of the assay was set at 0.0156 µg/mL. Blood samples were also collected for anti-drug antibody (ADA) assessment at -14 days, pre-dose and 7 days for the single-dose group and pre-dose and 15 days for the repeated-dose group.

使用非區室分析 (NCA) (Phoenix™ WinNonlin® 6.4 版，Pharsight Corp，Mountain View，CA) 及 IV 輸注輸入對濃度-時間資料進行 PK 分析。濃度-時間型態如圖 10C 所示，且對應的 PK 參數估計值列於表 8 中。HuAb44.v6.05-40G5c TDB 展現出預期的雙指數分佈 (Ferl 等人，2018；Sun 等人，2015；Staflin 等人，2020)，0.5 mg/kg (動物 3001) 及 1 mg/kg (動物 7101、8101、9201) 劑量組中的全身清除率 (CL) 在 14 mL/天/kg 至 19 mL/天/kg 的範圍內。0.5 mg/kg 及 1 mg/kg 劑量下的 C_max值與預期一致，並且在整個 0.5 mg/kg 組具有可比性。對於 0.5 mg/kg 及 1 mg/kg 劑量組，huAb44.v6.05-40G5c 的劑量標準化暴露量 (AUC_0-7/劑量) 相當。分析血清樣品中的抗藥物抗體 (ADA)。在兩隻動物 (7101 及 9201) 中觀察到 ADA，並且在一隻動物 (9201) 中，ADA 的發生與全身暴露量減少相關。PK analysis of concentration-time data was performed using noncompartmental analysis (NCA) (Phoenix™ WinNonlin® version 6.4, Pharsight Corp, Mountain View, CA) and IV infusion input. The concentration-time profile is shown in Figure 10C, and the corresponding PK parameter estimates are listed in Table 8. The HuAb44.v6.05-40G5c TDB exhibited the expected biexponential distribution (Ferl et al., 2018; Sun et al., 2015; Staflin et al., 2020), with systemic clearance (CL) ranging from 14 mL/day/kg to 19 mL/day/kg in the 0.5 mg/kg (animal 3001) and 1 mg/kg (animals 7101, 8101, 9201) dose groups._Cmax values at the 0.5 mg/kg and 1 mg/kg doses were as expected and comparable across the 0.5 mg/kg group. Dose-normalized exposure (AUC_0-7 /dose) of huAb44.v6.05-40G5c was equivalent for the 0.5 mg/kg and 1 mg/kg dose groups. Serum samples were analyzed for anti-drug antibodies (ADA). ADA were observed in two animals (7101 and 9201), and in one animal (9201), the occurrence of ADA was associated with a decrease in systemic exposure.

總之，huAb44.v6.05-MD1 TDB 及 huAb44.v6.05-40G5c TDB 的 PK 特徵與對典型 IgG 抗體的預期一致。預期 huAb44.v6.05 TDB 之投予對於表現 STEAP1 之癌症 (包括但不限於前列腺癌或尤文氏肉瘤) 的治療為安全且可耐受的。表8.huAb44.v6.05/40G5c 及 huAb44.v6.05/MD1 TDB 在石蟹獼猴靜脈內投予後的藥物動力學參數動物治療劑量(mg/kg)第一劑量後第二劑量後第1天第8天C_max(µg/mL)AUC_0-7(天*µg/mL)AUC_0-7/劑量(天*µg*kg/mL/mg)CL(mL/天/kg)Cmax(µg/mL)AUC_7-14(天*µg/mL)3001huAb44.v6.05-40G5c1NA27.949.549.515.3NANA5001huAb44.v6.05-40G5c1NA29.1NDNDNDNANA6001huAb44.v6.05-MD11NA23.4NDNDNDNANA7101^{^}huAb44.v6.05-40G5c0.5NA11.523.346.614.0NANA8101huAb44.v6.05-40G5c0.5115.325.050.017.522.4ND9201^{^}huAb44.v6.05-40G5c0.5112.621.242.419.121.933.510101huAb44.v6.05-40G5c0.5113.5NDNDNDNDND11101huAb44.v6.05-40G5c0.5212.1NDNDNDNDND11102huAb44.v6.05-40G5c0.5213.2NDNDNDNDNDC_max= 最大觀察濃度；CL = 清除率；AUC_0-7/劑量：按劑量標準化的給藥後第 0 天至第 7 天的血清濃度-時間曲線下面積；AUC_7-14/劑量：按第二劑量標準化的給藥後 7 至 14 天的血清濃度-時間曲線下面積；NA = 不適用；ND = 由於資料不足而未確定；^{^}ADA 陽性實例11.與STEAP1結合之Ab-44的結構In summary, the PK characteristics of huAb44.v6.05-MD1 TDB and huAb44.v6.05-40G5c TDB are consistent with expectations for typical IgG antibodies. Administration of huAb44.v6.05 TDB is expected to be safe and tolerable for the treatment of cancers expressing STEAP1, including but not limited to prostate cancer or Ewing's sarcoma.Table8. Pharmacokinetic parameters of huAb44.v6.05/40G5c and huAb44.v6.05/MD1 TDB after intravenous administration in stone macaquesanimaltreatmentDosage(mg/kg)After the first doseAfter the second doseDay1​Day8​C_max(µg/mL)AUC_0-7(day*µg/mL)AUC_0-7 /dose(day*µg*kg/mL/mg)CL(mL/day/kg)Cmax(µg/mL)AUC_7-14(day*µg/mL) 3001 huAb44.v6.05-40G5c 1 NA 27.9 49.5 49.5 15.3 NA NA 5001 huAb44.v6.05-40G5c 1 NA 29.1 ND ND ND NA NA 6001 huAb44.v6.05-MD1 1 NA 23.4 ND ND ND NA NA 7101^{^} huAb44.v6.05-40G5c 0.5 NA 11.5 23.3 46.6 14.0 NA NA 8101 huAb44.v6.05-40G5c 0.5 1 15.3 25.0 50.0 17.5 22.4 ND 9201^{^} huAb44.v6.05-40G5c 0.5 1 12.6 21.2 42.4 19.1 21.9 33.5 10101 huAb44.v6.05-40G5c 0.5 1 13.5 ND ND ND ND ND 11101 huAb44.v6.05-40G5c 0.5 2 12.1 ND ND ND ND ND 11102 huAb44.v6.05-40G5c 0.5 2 13.2 ND ND ND ND ND_Cmax = maximum observed concentration; CL = clearance;_AUC0-7 /dose: area under the serum concentration-time curve from day 0 to day 7 after dosing normalized by dose;_AUC7-14 /dose: area under the serum concentration-time curve from day 7 to day 14 after dosing normalized by the second dose; NA = not applicable; ND = not determined due to insufficient data;^{^} ADA-positiveexample11.Structure ofAb-44bindingtoSTEAP1

將重組 STEAP1 與具有 STEAP1-44 輕鏈的抗體 STEAP1-44.NGS.HC2 之 Fab 片段以 1:1.2 莫耳比一起孵育，並於冰上孵育 30 分鐘。將混合物注射到在 25 mM Tris pH 8、150 mM NaCl、0.02% LMNG、0.002% CHS 中平衡的 SUPEROSE® 6 Improve 3.2/300 柱 (GE Healthcare) 上。將3.5μL 濃度為 1 mg/mL 的複合體的峰級分施加至塗有薄層金的輝光放電多孔碳網格 (25 nm ultrafoil 網格 2/2 孔，200 目，來自 Quantifoil) 上。在 VITROBOT™ Mark IV (Thermo Fisher) 中使用 5 秒的印跡時間在 100% 的濕度下印跡網格，然後在液氮冷卻的液態乙烷中進行活塞冷凍。Recombinant STEAP1 was incubated with the Fab fragment of the antibody STEAP1-44.NGS.HC2 with the light chain of STEAP1-44 at a molar ratio of 1:1.2 and incubated on ice for 30 minutes. The mixture was injected onto a SUPEROSE® 6 Improve 3.2/300 column (GE Healthcare) equilibrated in 25 mM Tris pH 8, 150 mM NaCl, 0.02% LMNG, 0.002% CHS. 3.5 μL of the peak fraction of the complex at a concentration of 1 mg/mL was applied to a GD holey carbon grid (25 nm ultrafoil grid 2/2 hole, 200 mesh from Quantifoil) coated with a thin layer of gold. Grids were blotted in a VITROBOT™ Mark IV (Thermo Fisher) using a 5-second blot time at 100% humidity and then piston-frozen in liquid ethane cooled with liquid nitrogen.

在 Titan KRIOS™ 電子顯微鏡 (Thermo Fisher) 上使用 SerialEM (Mastronarde, 2005) 收集總共 6225 個動態影像堆疊，該顯微鏡在 300 kV 下運行並配備 BioQuantum 能量過濾器，該能量過濾器用 20eV 能量狹縫及 K2 Summit 直接電子探測器照相機 (Gatan) 運行。影像以 165,000 x 的標稱放大倍率記錄，對應於每個畫素的畫素尺寸為 0.824 Å。每個影像堆疊包含每 0.2 秒記錄的 50 個訊框，累積劑量為 46 e-/A²且總曝光時間為 10 秒。以 0.5 μm 至 1.5 μm 的設定散焦範圍記錄影像。為了克服複合體的優先取向並減少方向分辨率各向異性，以 40 度的傾斜角度收集完整資料集中的 1288 個動態影像 (Tan, 2017)。A total of 6225 dynamic image stacks were collected using SerialEM (Mastronarde, 2005) on a Titan KRIOS™ electron microscope (Thermo Fisher) operated at 300 kV and equipped with a BioQuantum energy filter with a 20 eV energy slit and a K2 Summit direct electron detector camera (Gatan). Images were recorded at a nominal magnification of 165,000 x, corresponding to a pixel size of 0.824 Å per pixel. Each image stack consisted of 50 frames recorded every 0.2 s, with a cumulative dose of 46 e-/A² and a total exposure time of 10 s. Images were recorded with a set defocus range of 0.5 μm to 1.5 μm. To overcome the preferential orientation of the complex and reduce directional resolution anisotropy, 1288 dynamic images in the full dataset were collected at a tilt angle of 40 degrees (Tan, 2017).

使用 cisTEM 進行從訊框堆疊到精細 3D 體積圖的影像處理 (Grant, 2018)。對於由 Fab44 結合的 STEAP1 之 3D 結構，使用 163759 個顆粒；在重建期間應用 C3 對稱性並生成了 3 Å 模型。對於原子模型構建，針對 STEAP1 及 Fab44 兩者的模型皆使用 Swiss Model (swissmodel.expasy.org/) 構建，然後使用交互式分子動力學 (Croll, 2018) 重建完整模型，並在真實空間中細化 (Afonine, 2018)。Image processing from frame stacking to detailed 3D volume maps was performed using cisTEM (Grant, 2018). For the 3D structure of STEAP1 bound by Fab44, 163,759 particles were used; C3 symmetry was applied during reconstruction and a 3 Å model was generated. For atomic model building, models for both STEAP1 and Fab44 were built using Swiss Model (swissmodel.expasy.org/), and the full model was then reconstructed using interactive molecular dynamics (Croll, 2018) and refined in real space (Afonine, 2018).

在與 STEAP1 複合的 Ab44 的冷凍電鏡結構中，發現人 STEAP1 蛋白次單元形成六個穿膜螺旋，正如基於疏水性分析所預測的那樣。基於冷凍電鏡結構及靜電表面分析，STEAP1 的穿膜螺旋 1 (TM1) 橫跨 Trp71 至 Ile95，STEAP1 的 TM2 橫跨 Ile109 至 Tyr130，STEAP1 的 TM3 橫跨 Lys162 至 Met184，STEAP1 的 TM4 橫跨 Trp214 至 Trp236，STEAP1 的 TM5 橫跨 Trp247 至 Phe272，且 STEAP1 的 TM6 橫跨 Phe291 至 Phe309。因此，每個 STEAP1 蛋白次單元內亦存在三個不同的細胞外環 (ECL)，並可定義為：STEAP1 的細胞外環 1 (ECL1)，其橫跨 His96 至 Lys108；STEAP1 的 ECL2，其橫跨 Arg185 至 Val213；STEAP1 的 ECL3，其橫跨 Ala273 至 Thr290。總體而言，STEAP1 形成六穿膜螺旋束，其間形成溶劑可及的裂隙並暴露於細胞外側。發現血紅素輔因子結合於該裂隙深處，主要由 TM2、TM3、TM4 及 TM5 接觸並協調，其中 TM6 發揮重要作用。ECL2 在細胞外隙上延伸，與結合的血紅素呈近似 V 狀構象，其中 ECL2 的頂點位於血紅素配體最近基團上方約 25 Å 處。In the cryo-EM structure of Ab44 in complex with STEAP1, the human STEAP1 protein subunit was found to form six transmembrane helices, as predicted based on hydrophobicity analysis. Based on the cryo-EM structure and electrostatic surface analysis, the transmembrane helix 1 (TM1) of STEAP1 spans Trp71 to Ile95, TM2 of STEAP1 spans Ile109 to Tyr130, TM3 of STEAP1 spans Lys162 to Met184, TM4 of STEAP1 spans Trp214 to Trp236, TM5 of STEAP1 spans Trp247 to Phe272, and TM6 of STEAP1 spans Phe291 to Phe309. Therefore, three different extracellular loops (ECLs) are also present within each STEAP1 protein subunit and can be defined as: extracellular loop 1 (ECL1) of STEAP1, which spans His96 to Lys108; ECL2 of STEAP1, which spans Arg185 to Val213; and ECL3 of STEAP1, which spans Ala273 to Thr290. Overall, STEAP1 forms a six-transmembrane helical bundle with a solvent-accessible cleft in between and exposed to the extracellular side. The heme cofactor is found to bind deep within this cleft, primarily contacted and coordinated by TM2, TM3, TM4, and TM5, with TM6 playing an important role. ECL2 extends over the extracellular space and assumes a nearly V-shaped conformation with bound heme, with the apex of ECL2 positioned approximately 25 Å above the nearest group of the heme ligand.

在冷凍電鏡結構中，人 STEAP1 蛋白次單元與兩個等效的 STEAP1 蛋白次單元形成同源三聚體。近似三重對稱軸沿 STEAP1 三聚體垂直於膜雙層的平面延伸。在 STEAP1 次單元之間形成廣泛而複合的三聚體界面，該界面主要由細胞外側的 ECL2 媒介。ECL1 及 TM2 對 STEAP1 的總體三聚體次單元界面具有較小但重要的影響，而 ECL3、TM5 及 TM6 為該界面的最外圍。當從膜的細胞外側觀察時，來自三個次單元中之各者的 ECL2 在三聚體的中心形成中央帽狀結構，當考慮到各蛋白鏈的 N 端到 C 端時，其中次單元係以逆時針方向排列。三聚體 STEAP1 組裝體及各 ECL2 的弧形 V 狀構象的一個結果是，一個次單元與各相鄰次單元相互交叉。總體而言，實際上，任何一種單元體中的細胞外隙皆以複合方式形成，兩個相鄰次單元皆有重要作用。具體而言，來自次單元 B 的 TM4 區將其自身 (及一些側鏈) 插入次單元 A 的 TM2 及 TM3 之間，而來自次單元 C 的 TM4 將其自身 (及一些側鏈) 插入次單元 A 的 TM4 及 TM5 之間。Ab44-STEAP1複合體的總體結構描述In the cryo-EM structure, the human STEAP1 protein subunit forms a homotrimer with two equivalent STEAP1 protein subunits. Approximate three-fold symmetry axes extend along the plane of the STEAP1 trimer perpendicular to the membrane bilayer. An extensive and complex trimer interface is formed between the STEAP1 subunits, which is mainly mediated by ECL2 on the cell extracellular side. ECL1 and TM2 have a smaller but significant impact on the overall trimer subunit interface of STEAP1, while ECL3, TM5 and TM6 are the outermost of this interface. When viewed from the cell extracellular side of the membrane, ECL2 from each of the three subunits forms a central cap-like structure in the center of the trimer, in which the subunits are arranged in a counterclockwise direction when considering the N-terminus to the C-terminus of each protein chain. A consequence of the curved V-shaped conformation of the trimeric STEAP1 assembly and each ECL2 is that one subunit interdigitates with each neighboring subunit. Overall, the extracellular space in virtually any single unit is formed in a complex manner, with both neighboring subunits having important roles. Specifically, the TM4 region from subunit B inserts itself (and some side chains) between TM2 and TM3 of subunit A, while TM4 from subunit C inserts itself (and some side chains) between TM4 and TM5 of subunit A. Overall structural description of theAb44-STEAP1complex

在冷凍電鏡結構中，發現 Ab44 之 Fab 對接於 STEAP1 三聚體的細胞外表面上，並且主要 (但非排他地) 與一個 STEAP1 啟動子結合。從結合單個 STEAP1 啟動子的角度來看，Ab44 之重鏈 (HC) 與 ECL1 (最廣泛)、ECL2 及 ECL3 接觸。從結合單個 STEAP1 啟動子的角度來看，Ab44 之輕鏈 (LC) 僅與 ECL2 接觸。除預期的 Ab44-STEAP1 次單元交互作用以外，各 Ab44 亦透過其重鏈與 STEAP1 三聚體的相鄰次單元進行特定但輕微的接觸。In the cryo-EM structure, the Fab of Ab44 was found to be docked on the extracellular surface of the STEAP1 trimer and to bind primarily (but not exclusively) to one STEAP1 promoter. From the perspective of binding to a single STEAP1 promoter, the heavy chain (HC) of Ab44 contacts ECL1 (most extensively), ECL2, and ECL3. From the perspective of binding to a single STEAP1 promoter, the light chain (LC) of Ab44 contacts only ECL2. In addition to the expected Ab44-STEAP1 subunit interactions, each Ab44 also makes specific but subtle contacts with neighboring subunits of the STEAP1 trimer through its heavy chain.

除 Ab44-STEAP1 交互作用以外，Ab44 之 Fab 亦形成廣泛的同型 Ab44-Ab44 界面。沿該結構的三重軸的 LC-LC-LC 配對之間產生多個極性同型 Fab 交互作用。除這些 LC-LC 同型 Ab44-Ab44 交互作用以外，在冷凍電鏡結構中亦觀察到 HC-LC 同型交互作用。參見圖 11A 及 11B。Ab44與STEAP1之間的殘基及表面區域接觸：In addition to Ab44-STEAP1 interactions, the Fab of Ab44 also forms extensive homotypic Ab44-Ab44 interfaces. Multiple polar homotypic Fab interactions occur between LC-LC-LC pairs along the three-fold axis of the structure. In addition to these LC-LC homotypic Ab44-Ab44 interactions, HC-LC homotypic interactions are also observed in the cryo-EM structure. See Figures 11A and 11B.Residue and surface region contacts betweenAb44andSTEAP1 :

Ab44 HC CDR 所媒介之交互作用總結如下所示：Tyr103-HC 羥基與來自 Trp195 側鏈 (ECL2) 的 NH 基團之間的氫鍵；Gly101-HC 主鏈羰基與 Gln202 (ECL2) 醯胺側鏈之間的氫鍵；Tyr107-HC 羥基與 Gln198 (ECL2) 醯胺側鏈之間的氫鍵；Leu56-HC 主鏈羰基與 Lys281 (ECL3) 的胺側鏈之間的氫鍵；Ser73-HC 羥基與 His102 (ECL1) 咪唑側鏈之間的氫鍵；Asn74-HC 主鏈醯胺與 Ser101 (ECL1) 主鏈羰基之間的氫鍵；Asn74-HC 與 Gln103 側鏈 (ECL1) 之間的氫鍵。亦參見表 3。總體而言，HC:STEAP1 交互作用掩蓋 680 Ų的溶劑暴露區域。The interactions mediated by the Ab44 HC CDR are summarized as follows: hydrogen bond between Tyr103-HC hydroxyl and the NH group from the Trp195 side chain (ECL2); hydrogen bond between Gly101-HC main chain carbonyl and the amide side chain of Gln202 (ECL2); hydrogen bond between Tyr107-HC hydroxyl and the amide side chain of Gln198 (ECL2); hydrogen bond between Leu56-HC main chain carbonyl and the amine side chain of Lys281 (ECL3); hydrogen bond between Ser73-HC hydroxyl and the imidazole side chain of His102 (ECL1); hydrogen bond between Asn74-HC Hydrogen bonds between the main-chain amide and the main-chain carbonyl of Ser101 (ECL1); hydrogen bonds between Asn74-HC and the side-chain of Gln103 (ECL1). See also Table 3. Overall, the HC:STEAP1 interaction masks a solvent-exposed region of 680 Ų .

Ab44 LC CDR 所媒介之交互作用總結如下所示：Tyr35-LC 羥基與 Gln202 (ECL2) 的主鏈羰基之間的氫鍵；Tyr54-LC 羥基與 Gln201 (ECL2) 的主鏈羰基之間的氫鍵；殘基 Asn203 及 Lys204 參與與 LC CDR 殘基的凡得瓦交互作用。總體而言，LC:STEAP1 交互作用掩蓋 170 Ų的適度溶劑暴露區域。A summary of the interactions mediated by the Ab44 LC CDR is as follows: hydrogen bond between the Tyr35-LC hydroxyl and the main-chain carbonyl of Gln202 (ECL2); hydrogen bond between the Tyr54-LC hydroxyl and the main-chain carbonyl of Gln201 (ECL2); residues Asn203 and Lys204 participate in van der Waals interactions with LC CDR residues. Overall, the LC:STEAP1 interaction covers a moderate solvent-exposed region of 170 Ų .

各 Ab44 亦與 STEAP1 三聚體的相鄰次單元接觸。具體而言，Ab44 之 Thr28-HC (主要與次單元 A 結合) 與次單元 B 的 Asp206 側鏈形成直接氫鍵交互作用，而 Arg98-HC 似乎與 Asp205 的羧基發生靜電交互作用。此交互作用使溶劑暴露面積增加 88 Ų。亦參見圖 12A 及 12B。同型Ab44界面之間的殘基及表面區域接觸Each Ab44 also contacts adjacent subunits of the STEAP1 trimer. Specifically, Thr28-HC of Ab44 (primarily bound to subunit A) forms a direct hydrogen-bond interaction with the Asp206 side chain of subunit B, while Arg98-HC appears to interact electrostatically with the carboxyl group of Asp205. This interaction increases the solvent-exposed area by 88 Ų . See also Figures 12A and 12B.Residue and surface area contacts betweenhomotypicAb44 interfaces

來自 Ab44 Fab A 的 Arg82-LC 與來自鄰近 Ab44 (Fab C) 的 Glu84-LC 及 Glu86-LC 形成鹽橋交互作用，並且該三分離子交互作用在冷凍電鏡結構中的所有 LC 啟動子中皆可見。來自 Ab44 Fab A 的 Asp65-LC 亦與來自分別結合在 STEAP1 的次單元 B 及次單元 C 上的 Fab 的 Asp65-LC 直接在三重對稱通路上形成氫鍵合網路。來自 Ab44 Fab A 及 Ab44 Fab C 的 LC 之間的交互作用掩蓋約 180 Ų的溶劑暴露區域 (來自 Ab44 Fab A 的 HC 不與 Ab44 Fab C 交互作用)。Arg82-LC from Ab44 Fab A forms salt-bridge interactions with Glu84-LC and Glu86-LC from neighboring Ab44 (Fab C), and this triionic interaction is visible in all LC promoters in the cryo-EM structure. Asp65-LC from Ab44 Fab A also forms a hydrogen-bonding network directly in a three-fold symmetric pathway with Asp65-LC from Fabs bound to subunit B and subunit C of STEAP1, respectively. The interaction between LC from Ab44 Fab A and Ab44 Fab C covers a solvent-exposed region of approximately 180 Ų (HC from Ab44 Fab A does not interact with Ab44 Fab C).

Glu1-HC 與鄰近 Ab44 Fab B 之 Ser72-LC 形成凡得瓦交互作用及表面互補性，並且這兩個側鏈之間亦可能存在水媒介的氫鍵。Fab A 與 LC-Fab B 之間的交互作用掩蓋總共約 220 Ų的溶劑暴露區域。參見圖 13A 及 13B。Glu1-HC forms van der Waals interactions and surface complementarity with Ser72-LC of the neighboring Ab44 Fab B, and water-mediated hydrogen bonds may also exist between these two side chains. The interaction between Fab A and LC-Fab B covers a total of approximately 220^Å2 of solvent-exposed area. See Figures 13A and 13B.

預期其他 Ab44 衍生的變異體 (包括 huAb44.v6.05) 以與具有如本實例中所述之 STEAP1-44 輕鏈的抗體 STEAP1-44.NGS.HC2 類似的方式結合至 STEAP1。正如 Hsiao 等人如上所討論的，在抗原結合後，體細胞突變及殖株擴增導致株性相關但不同的 B 細胞譜系表現以在殖株之間可能不同的親和力結合抗原的抗體。此類體細胞突變 (例如，如實例 4 中所評估的) 預計不會改變抗體的總體結合模式，而是影響特定殘基交互作用之詳情，例如，藉由添加新的有益交互作用或去除不利的交互作用或藉由減少結合中的熵罰點 (Schmidt 等人Proc. Natl. Acad. Sci. USA110:264-269，2013；Vajda 等人，Curr. Opin. Struct. Biol.67;226-231，2021)。Other Ab44-derived variants, including huAb44.v6.05, are expected to bind to STEAP1 in a similar manner as the antibody STEAP1-44.NGS.HC2 having the STEAP1-44 light chain as described in this example. As discussed by Hsiao et al., above, after antigen binding, somatic cell mutation and strain expansion result in strain-related but distinct B cell repertoires expressing antibodies that bind antigen with affinities that may differ between strains. Such somatic mutations (e.g., as assessed in Example 4) are not expected to change the overall binding pattern of the antibody, but rather to affect the details of specific residue interactions, for example, by adding new beneficial interactions or removing unfavorable interactions or by reducing the entropy penalty in binding (Schmidt et al. Proc. Natl. Acad. Sci. USA 110:264-269, 2013; Vajda et al.,Curr. Opin. Struct. Biol. 67;226-231, 2021).

Ab44 與 mAb120.545 (亦稱為萬多妥珠單抗) 與 STEAP1 結合的比較 (Oosterheert 等人，J. Biol. Chem.295:9502-9512 (2020)) 揭示了不同的結合模式。萬多妥珠單抗的主要表位係以 ECL2 為中心，其中重鏈及輕鏈對結合的貢獻幾乎相同 (分別為 410 Ų及 340 Ų)。萬多妥珠單抗 CDR H3 潛入由位於血紅素正上方的鹼性殘基所形成的口袋中；基於用 STEAP4 獲得的結構資料 (Oosterheert, 2018)，預計該基本環將協調螯合離子 Fe³⁺受質。雖然萬多妥珠單抗預計會競爭受質結合，但 Ab44 似乎並不具備此特徵。參見圖 14。Comparison of Ab44 and mAb120.545 (also known as vendotumab) binding to STEAP1 (Oosterheert et al.,J. Biol. Chem. 295:9502-9512 (2020)) revealed different binding modes. The major epitope of vendotumab is centered on ECL2, with almost identical contributions of the heavy and light chains to binding (410 Ų and 340 Ų , respectively). The vendotumab CDR H3 nestles in a pocket formed by the basic residues located just above the heme; based on structural data obtained with STEAP4 (Oosterheert, 2018), this basic ring is expected to coordinate the chelation of ionic Fe³⁺ substrates. While vendotumab would be expected to compete for substrate binding, Ab44 does not appear to have this characteristic. See Figure 14.

如 Oosterheert 等人，J. Biol.Chem.295:9502-9512 (2020) 所報導的 mAb120.545 HC CDR 媒介的關鍵交互作用總結如下：Tyr104-HC 之主鏈羰基與 Trp195 (ECL2) 吲哚側鏈之 NH 之間的氫鍵；Tyr108-HC 羥基側鏈與 Gln202 側鏈 (ECL2) 之間的氫鍵；Thr58-HC 之主鏈羰基與 Asn203 (ECL2) 之側鏈之間的氫鍵；Tyr104-HC 羥基側鏈與 Lys204 側鏈之 NH 之間的氫鍵；Tyr108-HC 羥基側鏈與 Gln198 (ECL2) 主鏈羰基之間的氫鍵；Tyr102-HC 羥基側鏈與 Gln198 側鏈 (ECL2) 之間的氫鍵；Ser59-HC 之羥基側鏈與 Gln201(ECL2) 之主鏈羰基之間的氫鍵；Tyr51-HC 羥基側鏈與 Gln202 (ECL2) 主鏈羰基之間的氫鍵；Ser57-HC 羥基側鏈與 Glu205 (ECL2) 之羧基側鏈之間的氫鍵；Tyr104-HC 羥基側鏈與 Ala207 之主鏈羰基之間的氫鍵。總體而言，HC:STEAP1 交互作用掩蓋 444 Ų的溶劑暴露區域。As reported by Oosterheert et al.,J. Biol.Chem. 295:9502-9512 (2020), the key interactions mediated by the mAb120.545 HC CDR are summarized as follows: a hydrogen bond between the main chain carbonyl of Tyr104-HC and the NH of the indole side chain of Trp195 (ECL2); a hydrogen bond between the hydroxyl side chain of Tyr108-HC and the side chain of Gln202 (ECL2); a hydrogen bond between the main chain carbonyl of Thr58-HC and the side chain of Asn203 (ECL2); a hydrogen bond between the hydroxyl side chain of Tyr104-HC and the NH of the side chain of Lys204 ; hydrogen bond between Tyr108-HC hydroxyl side chain and Gln198 (ECL2) main chain carbonyl group; hydrogen bond between Tyr102-HC hydroxyl side chain and Gln198 (ECL2) side chain; hydrogen bond between Ser59-HC hydroxyl side chain and Gln201 (ECL2) main chain carbonyl group; hydrogen bond between Tyr51-HC hydroxyl side chain and Gln202 (ECL2) main chain carbonyl group; hydrogen bond between Ser57-HC hydroxyl side chain and Glu205 (ECL2) and a hydrogen bond between the hydroxyl side chain of Tyr104-HC and the main chain carbonyl group of Ala207. Overall, the HC:STEAP1 interaction masks a solvent-exposed region of 444 Ų .

如 Oosterheert 等人，J. Biol.Chem.295:9502-9512 (2020) 所報導的 mAb120.545 LC CDR 媒介的交互作用總結如下：Ser33-LC 之主鏈羰基與 Tyr107 側鏈 (ECL1) 之羥基之間的氫鍵；Ser33-LC 羥基側鏈與 Asn194 側鏈 (ECL2) 之 NH 基團之間的氫鍵；Gln35-LC 側鏈與 Gln104 (ECL2) 主鏈醯胺之間的氫鍵；Tyr100-LC 之主鏈羰基與 Gln201 (ECL2) 側鏈之間的氫鍵；Tyr100-LC 羥基側鏈與 Gln202 (ECL2) 之側鏈之間的氫鍵；Gln35-LC 及 Gln104 (ECL2) 側鏈之間的氫鍵。總體而言，LC:STEAP1 交互作用掩蓋 367 Ų的溶劑暴露區域。As reported by Oosterheert et al.,J. Biol.Chem. 295:9502-9512 (2020), the interactions mediated by the CDRs of mAb120.545 LC are summarized as follows: a hydrogen bond between the main chain carbonyl of Ser33-LC and the hydroxyl group of the Tyr107 side chain (ECL1); a hydrogen bond between the hydroxyl side chain of Ser33-LC and the NH group of the Asn194 side chain (ECL2); a hydrogen bond between the Gln35-LC side chain and the main chain amide of Gln104 (ECL2); a hydrogen bond between the main chain carbonyl of Tyr100-LC and the NH group of Asn194 side chain (ECL2); a hydrogen bond between the main chain carbonyl of Gln35-LC and the main chain amide of Gln104 (ECL2); a hydrogen bond between the main chain carbonyl of Gln100-LC and the NH group of Asn194 side chain ... hydrogen bonds between the side chains of LC:STEAP1; hydrogen bonds between the hydroxyl side chain of Tyr100-LC and the side chain of Gln202 (ECL2); hydrogen bonds between the side chains of Gln35-LC and Gln104 (ECL2). Overall, the LC:STEAP1 interaction masks a solvent-exposed region of 367 Ų .

來自對稱性相關 Fab' 之一的 mAb120.545 輕鏈係透過以下交互作用貢獻了 270 Ų溶劑暴露區域的額外交互作用：Arg32-LC' 側鏈與 Asp206 (ECL2) 側鏈之間的鹽橋；Tyr98-LC’ 羥基側鏈與 Glu205 (ECL2) 之羧基鏈之間的氫鍵；Gln27-LC’ 之側鏈與 Glu205 (ECL2) 之羧基鏈之間的氫鍵；Asn99-LC’ 之側鏈與 Glu205 (ECL2) 之羧基鏈之間的氫鍵；Arg32-LC' 之胍側鏈與 Asp206 (ECL2) 之主鏈羰基之間的氫鍵；Ser33-LC 之主鏈醯胺與 Glu205 (ECL2) 之羧基鏈之間的氫鍵；Ser33-LC 羥基側鏈與 Glu205 (ECL2) 之羧基鏈之間的氫鍵；Arg32-LC 之主鏈醯胺與 Glu205 (ECL2) 之羧基鏈之間的氫鍵。The mAb120.545 light chain from one of the symmetry-related Fab's contributes additional interactions to the 270 Ų solvent-exposed region through the following interactions: a salt bridge between the Arg32-LC' side chain and the Asp206 (ECL2) side chain; a hydrogen bond between the Tyr98-LC' hydroxyl side chain and the Glu205 (ECL2) carboxyl chain; a hydrogen bond between the Gln27-LC' side chain and the Glu205 (ECL2) carboxyl chain; a hydrogen bond between the Asn99-LC' side chain and the Glu205 (ECL2) carboxyl chain; and a hydrogen bond between the Arg32-LC' side chain and the Asp206 (ECL2) side chain. hydrogen bond between the guanidine side chain of Ser33-LC and the main chain carbonyl group of Asp206 (ECL2); hydrogen bond between the main chain amide of Ser33-LC and the carboxyl chain of Glu205 (ECL2); hydrogen bond between the hydroxyl side chain of Ser33-LC and the carboxyl chain of Glu205 (ECL2); hydrogen bond between the main chain amide of Arg32-LC and the carboxyl chain of Glu205 (ECL2).

總之，本實例中描述的資料證明，Ab44 及本文所揭示之其他抗 STEAP1 抗體結合至 STEAP1 之獨特表位，該表位不同於文獻中所述之現有抗 STEAP1 抗體。實例12.  Ab44與石蟹獼猴STEAP1的預期結合In summary, the data described in this example demonstrate that Ab44 and other anti-STEAP1 antibodies disclosed herein bind to a unique epitope of STEAP1 that is distinct from existing anti-STEAP1 antibodies described in the literature.Example12.Expected Binding of Ab44to Macaca mulattaSTEAP1

人類與靈長類 STEAP1 之間的序列同源性程度很高。所有三個細胞外環皆嚴格保留，表明 Ab44 對這些蛋白質具有交叉反應性。圖 15 示出人類、石蟹獼猴及紅毛猩猩的序列比對。另外，發現 STEAP1 在石蟹獼猴及人類正常組織中表現出相似的表現模式。實例13.與現有的抗STEAP1抗體相比，huAb44.v6.05表現出出乎意料的優異特性The degree of sequence homology between human and primate STEAP1 is high. All three extracellular loops are strictly conserved, indicating that Ab44 has cross-reactivity to these proteins. Figure 15 shows the sequence alignment of human, macaque and orangutan. In addition, STEAP1 was found to exhibit similar expression patterns in macaque and human normal tissues.Example13.huAb44.v6.05exhibits unexpectedly superior properties comparedto existing anti-STEAP1antibodies

將本文所述之 huAb44.v6.05 抗 STEAP1 抗體與現有抗 STEAP1 抗體萬多妥珠單抗在 TDB 背景下的結合親和力及細胞毒殺效力進行比較。The binding affinity and cytotoxic potency of the huAb44.v6.05 anti-STEAP1 antibody described herein were compared with those of the existing anti-STEAP1 antibody vendotumumab in the context of TDB.

首先，在直接頭對頭比較中將 huAb44.v6.05 的結合親和力與萬多妥珠單抗進行比較。圖 22 示出藉由 FACS 所評定之 huAb44.v6.05 及萬多妥珠單抗 IgG 與表現 STEAP1 之 LNCaP-X1.2細胞的結合的比較。與萬多妥珠單抗相比，huAb44.v6.05 表現出更強的結合。huAb44.v6.05 的 EC50 為 1.9 nM，與萬多妥珠單抗的 EC50 3.5 nM 相比，其改善至大約 1.8 倍。更令人驚訝的是，與萬多妥珠單抗相比，huAb44.v6.05 表現出與細胞更高的最大結合，其亦為親和力的函數。這些資料證明，與現有的抗 STEAP1 抗體 (諸如萬多妥珠單抗) 相比，huAb44.v6.05 以更高的結合親和力與表現 STEAP1 之細胞結合。First, the binding affinity of huAb44.v6.05 was compared to vendotumab in a direct head-to-head comparison. Figure 22 shows a comparison of the binding of huAb44.v6.05 and vendotumab IgG to LNCaP-X1.2 cells expressing STEAP1 as assessed by FACS. huAb44.v6.05 exhibited stronger binding compared to vendotumab. The EC50 of huAb44.v6.05 was 1.9 nM, which is an improvement of approximately 1.8-fold compared to the EC50 of 3.5 nM for vendotumab. Even more surprising, huAb44.v6.05 exhibited higher maximal binding to cells than vendotumab, which was also a function of affinity. These data demonstrate that huAb44.v6.05 binds to cells expressing STEAP1 with higher binding affinity than existing anti-STEAP1 antibodies such as vendotumab.

接著，將 huAb44.v6.05 TDB 的細胞毒殺效力與萬多妥珠單抗 TDB 進行比較。圖 23A 及 23B 示出在細胞毒殺測定中測試的雙特異性抗 STEAP1/抗 CD3 抗體分別與人類 CD8+T 細胞及表現 STEAP1 之 LNCaP-X1.2 細胞及 LNCaPX1.2KO3-13 細胞的細胞毒殺效力的直接頭對頭比較。LNCaP-X1.2 細胞株為來自可公開獲得的 LNCaP 殖株 FGC 細胞的亞系。LNCaPX1.2 細胞株係由 LNCaP 殖株 FGC 細胞的異種移植腫瘤片確立，該等細胞在免疫受損的小鼠中作為異種移植物生長。LNCaPX1.2KO3-13 為藉由基因編輯技術降低 STEAP1 表現而衍生自 LNCaP-X1.2 細胞的細胞株。在這些實驗中，對 MD1 及 40G5c 抗 CD3 臂兩者皆進行了測試。Next, the cytotoxic potency of huAb44.v6.05 TDB was compared to that of vendotumab TDB. Figures 23A and 23B show a direct head-to-head comparison of the cytotoxic potency of the bispecific anti-STEAP1/anti-CD3 antibodies tested in the cytotoxicity assay against human CD8+ T cells and LNCaP-X1.2 cells and LNCaPX1.2KO3-13 cells expressing STEAP1, respectively. The LNCaP-X1.2 cell line is a subline of the publicly available LNCaP strain FGC cells. The LNCaPX1.2 cell line was established from xenograft tumor pieces of LNCaP clone FGC cells grown as xenografts in immunocompromised mice. LNCaPX1.2KO3-13 is a cell line derived from LNCaP-X1.2 cells by gene editing to reduce STEAP1 expression. In these experiments, both the MD1 and 40G5c anti-CD3 arms were tested.

圖 23A 及 23B 顯示，如 EC50 值所示之萬多妥珠單抗-TDB 的細胞毒殺效力與 huAb44.v6.05-TDB 相比顯著較低。在 MD1 及 40G5c抗 CD3 臂皆觀察到 huAb44.v6.05-TDB 之改善。再者，與 huAb44.v6.05-TDB 相比，萬多妥珠單抗-TDB 未達到 100% 細胞毒殺效力。因此，這些結果證明，與利用現有抗 STEAP1 抗體諸如萬多妥珠單抗的 TDB 相比，huAb44.v6.05 TDB 具有令人驚訝地改善之細胞毒殺效力。Figures 23A and 23B show that the cytotoxic potency of vendotozumab-TDB, as indicated by the EC50 value, was significantly lower than that of huAb44.v6.05-TDB. Improvements of huAb44.v6.05-TDB were observed in both the MD1 and 40G5c anti-CD3 arms. Furthermore, vendotozumab-TDB did not achieve 100% cytotoxic potency compared to huAb44.v6.05-TDB. Therefore, these results demonstrate that huAb44.v6.05 TDB has surprisingly improved cytotoxic potency compared to TDBs utilizing existing anti-STEAP1 antibodies such as vendotozumab.

另外，在 T 細胞活化測定中，以直接頭對頭比較的方式對 huAb44.v6.05-TDB 的活性與萬多妥珠單抗 TDB 進行了比較。如圖 24A 至 24D 所示，與萬多妥珠單抗-TDB 相比，huAb44.v6.05-TDB 導致 CD4 及 CD8 T 細胞兩者之更高的活化。In addition, the activity of huAb44.v6.05-TDB was compared to vendotumumab TDB in a direct head-to-head comparison in a T cell activation assay. As shown in Figures 24A to 24D, huAb44.v6.05-TDB resulted in higher activation of both CD4 and CD8 T cells compared to vendotumumab-TDB.

再者，在標靶依賴性細胞激素分泌 (包括 IFNγ、TNFα、IL-2、IL-6 及顆粒酶 B 之分泌) 方面對 huAb44.v6.05-TDB 之活性與萬多妥珠單抗 TDB 進行比較。如圖 25A 至 25J 所示，huAb44.v.05-TDB 導致與萬多妥珠單抗-TDB 相比，與 LNCaP-X1.2 (圖25A 至 25E) 或 LNCaP-X1.2-KO-3-13 細胞 (圖25F 至 25J) 一起孵育 24 小時的 T 細胞的細胞激素分泌含量明顯更高。Furthermore, the activity of huAb44.v6.05-TDB was compared with vendotumumab TDB in target-dependent cytokine secretion, including secretion of IFNγ, TNFα, IL-2, IL-6, and granzyme B. As shown in Figures 25A to 25J, huAb44.v.05-TDB resulted in significantly higher levels of cytokine secretion from T cells incubated with LNCaP-X1.2 (Figures 25A to 25E) or LNCaP-X1.2-KO-3-13 cells (Figures 25F to 25J) for 24 hours compared to vendotumumab-TDB.

huAb44.v6.05 的這些出人意料的優異特性預計將在 huAb44.v6.05 TDB 用於治療表現 STEAP1 之癌症 (包括但不限於前列腺癌或尤文氏肉瘤) 的臨床功效方面具有優勢。再者，與文獻中所述之與 STEAP1 二價結合的其他抗 STEAP1 雙特異性抗體構建體不同，本實例中所用的 huAb44.v6.05 TDB 以單價方式與 STEAP1 結合。因此，本文所揭示之抗 STEAP1 TDB 即使在以單價方式結合至 STEAP1 的構建體中也能夠達到出人意料地有利的結合親和力以及對表現 STEAP1 之細胞 (包括腫瘤細胞) 的有效細胞毒殺。參考文獻1.      Bos AB, Luan P, Duque JN, Reilly D, Harms PD, Wong AW. Optimization and automation of an end-to-end high throughput microscale transient protein production process. Biotechnol Bioeng. 2015;112:1832–42. doi:10.1002/bit.25601。 2.      Luan P, Lee S, Arena TA, Paluch M, Kansopon J, Viajar S, Begum Z, Chiang N, Nakamura G, Hass PE, 等人. Automated high throughput microscale antibody purification workflows for accelerating antibody discovery.MAbs. 2018;10:624–35. doi:10.1080/19420862.2018.1445450。 3.      Hsiao YC, Shang Y, DiCara D., Yee A, Lai J, Kim SH, Ellerman D, Corpuz R, Chen Y, Rajan S, Cai H, Wu Y, Seshasayee D, Hötzel I. 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Biol.74, 531-544 (2018). doi:10.1107/S2059798318006551。序列表SEQ ID NO: 1 - NYYMA SEQ ID NO: 2 - YIDYDGGSTSYGDSVKG SEQ ID NO: 3 - RSGYYHVGYAMDA SEQ ID NO: 4 - RSSQSLEYSDGYTYLE SEQ ID NO: 5 - GVSNRFS SEQ ID NO: 6 - FQATHDPLT SEQ ID NO: 7 – STEAP1-44 重鏈可變區 EVQLVESGGGLVRPGRSLKLSCAASGFTFSNYYMAWVRQAPTRGLEWVAYIDYDGGSTSYGDSVKGRFTISRNNAKSTLYLQMNSLRSEDMATYYCARRSGYYHVGYAMDAWGQGTSVTVSS SEQ ID NO: 8 – STEAP1-44 輕鏈可變區 DDVLTQTPVSLSVTLGDQASISCRSSQSLEYSDGYTYLEWYLQKPGQSPQLLIYGVSNRFSGVPDRFIGSGSGTDFTLKISRVEPEDLGVYYCFQATHDPLTFGSGTKLEIK SEQ ID NO: 9 - NFYMA SEQ ID NO: 10 - DHYMA SEQ ID NO: 11 - YISYDGDSTYYGDSVKG SEQ ID NO: 12 - YISYDGLDTYYGDSVKG SEQ ID NO: 13 - YISYDGDNTYYGDSVKG SEQ ID NO: 14 - RSGYYHVGYAMDG SEQ ID NO: 15 - RSGYYHVGYAMNA SEQ ID NO: 16 - RSGFYHVGYAMNA SEQ ID NO: 17 – STEAP1-44.NGS.HC1 重鏈可變區 EVQLVESGGGLVQPGRSLKLSCAASGFTFSNFYMAWVRQAPTKGLEWVAYISYDGDSTYYGDSVKGRFTISRNNAKRTLYLQMNSLRSEDMATYYCARRSGYYHVGYAMDAWGQGTSVTVSS SEQ ID NO: 18 – STEAP1-44.NGS.HC2 重鏈可變區 EVQLVESGGGLVQPGRSLKLSCAASGFTFSNFYMAWVRQAPTKGLEWVAYISYDGLDTYYGDSVKGRFTISRSNAKSTLYLQMNSLRSEDMATYYCARRSGYYHVGYAMDAWGQGTSVTVSS SEQ ID NO: 19 – STEAP1-44.NGS.HC3 重鏈可變區 EVQLVESGGGLVQPGRSLKLSCAASGFTFSNFYMAWVRQAPTKGLEWVAYISYDGLDTYYGDSVKGRFTISRSNAKSTLYLQMNSLRSEDMATYYCARRSGYYHVGYAMDGWGQGTSVTVSS SEQ ID NO: 20 – STEAP1-44.NGS.HC4 重鏈可變區 EVQLVESGGGLVQPGRSLKLSCAASGFTFSDHYMAWVRQAPTKGLEWVAYISYDGDNTYYGDSVKGRFTISRNNAKSTLYLQMNSLRSEDMATYYCARRSGYYHVGYAMNAWGPGTSVTVSS SEQ ID NO: 21 – STEAP1-44.NGS.HC5 重鏈可變區 EVRLVESGGGLVQPGRSLKLSCTASGFTFSDHYMAWVRQVPTKGLEWVAYIDYDGGSTSYGDSVKGRFTISRNNAKSTLYLQMNSLRSEDMATYYCARRSGFYHVGYAMNAWGQGTSVTVSS SEQ ID NO: 22 - huAb44.v1/v2/v3/v4 重鏈可變區 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLVWVAYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSS SEQ ID NO: 23 - huAb44.v5/v6/v7/v8 重鏈可變區 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSS SEQ ID NO: 24 - huAb44.v9/v10/v11/v12 重鏈可變區 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLVWVAYIDYDGGSTSYGDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSS SEQ ID NO: 25 - huAb44.v13/v14/v15/v16 重鏈可變區 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSS SEQ ID NO: 26 - huAb44.v1/v5/v9/v13 輕鏈可變區 DDVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLQKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIK SEQ ID NO: 27 - huAb44.v2/v6/v10/v14 輕鏈可變區 DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLQKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIK SEQ ID NO: 28 - huAb44.v3/v7/v11/v15 輕鏈可變區 DDVMTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLQKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIK SEQ ID NO: 29 - huAb44.v4/v8/v12/v16 輕鏈可變區 DIVMTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLQKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIK SEQ ID NO: 30 - huAb44.v6.01 重鏈可變區 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFYMAWVRQAPGKGLVWVSYISYDGDSTYYGDSVKGRFTISRDNAKRTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSS SEQ ID NO: 31 - huAb44.v6.02 重鏈可變區 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFYMAWVRQAPGKGLVWVSYISYDGLDTYYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSS SEQ ID NO: 32 - huAb44.v6.03 重鏈可變區 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFYMAWVRQAPGKGLVWVSYISYDGLDTYYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDGWGQGTTVTVSS SEQ ID NO: 33 - huAb44.v6.04 重鏈可變區 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVRQAPGKGLVWVSYISYDGDNTYYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMNAWGQGTTVTVSS SEQ ID NO: 34 - huAb44.v6.05 重鏈可變區 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVRQAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSS SEQ ID NO: 35 - Xaa¹Xaa²YMA (Xaa¹=D/N; Xaa²=H/Y/F) SEQ ID NO: 36 - YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (Xaa³=D/S; Xaa⁴=G/D/L; Xaa⁵=S/D/N; Xaa⁶=S/Y) SEQ ID NO: 37 - RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(Xaa⁷=F/Y; Xaa⁸=N/D; Xaa⁹=A/G) SEQ ID NO: 38 - huAb44 VH 共通序列 EVQLVESGGGLVQPGGSLRLSCAASGFTFSXaa¹Xaa²YMAWVRXaa¹⁰APGKGLVWVXaa¹¹YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKGRFTISRDNAKXaa¹²TLYLQMNSLRAEDTAVYYCARRSGXaa⁷YHVGYAMXaa⁸Xaa⁹WGQGTTVTVSS (Xaa¹=N/D; Xaa²=Y/F/H; Xaa³=D/S; Xaa⁴=G/D/L; Xaa⁵=S/D/N; Xaa⁶=S/Y; Xaa⁷=Y/F; Xaa⁸=D/N; Xaa⁹=A/G; Xaa¹⁰=Q/E/K; Xaa¹¹=A/S; Xaa¹²=S/R/N) SEQ ID NO: 39 - huAb44 VL 共通序列 DXaa¹³VXaa¹⁴TQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLXaa¹⁵KPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIK (Xaa¹³=I/D; Xaa¹⁴=L/M; Xaa¹⁵=Q/K/E) SEQ ID NO: 40 - SYYIH SEQ ID NO: 41 - WIYPENDNTKYNEKFKD SEQ ID NO: 42 - DGYSRYYFDY SEQ ID NO: 43 - KSSQSLLNSRTRKNYLA SEQ ID NO: 44 - WASTRES SEQ ID NO: 45 - TQSFILRT SEQ ID NO: 46 - huAb44.v6.05/38E4v1.MD1 1C.bsAb 重鏈可變區 (MD1) EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRKAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSS SEQ ID NO: 47 - huAb44.v6.05/38E4v1.MD1 1C.bsAb 輕鏈可變區 (MD1) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQEKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIK SEQ ID NO: 48 - NYYIH SEQ ID NO: 49 - WIYPGDGNTKYNEKFKG SEQ ID NO: 50 - DSYSNYYFDY SEQ ID NO: 51 - KSSQSLLNSRTRKNYLA SEQ ID NO: 52 - WASTRES SEQ ID NO: 53 - TQSFILRT SEQ ID NO: 54 - huAb44.v6.05/40G5c 1C.bsAb 重鏈可變區 (40G5c) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRKAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSS SEQ ID NO: 55 - huAb44.v6.05/40G5c 1C.bsAb 輕鏈可變區 (40G5c) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQEKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIK SEQ ID NO: 56 - GFTFSNY SEQ ID NO: 57 - DYDGGS SEQ ID NO: 58 - GFTFSNF SEQ ID NO: 59 - GFTFSDH SEQ ID NO: 60 - SYDGDS SEQ ID NO: 61 - SYDGLD SEQ ID NO: 62 - SYDGDN SEQ ID NO: 63 - GFTFSXaa¹Xaa²(Xaa¹=N/D; Xaa²=Y/F/H) SEQ ID NO: 64 - Xaa³YDGXaa⁴Xaa⁵(Xaa³=D/S; Xaa⁴=G/D/L; Xaa⁵=S/D/N) SEQ ID NO: 65 - 人 STEAP1 MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL (SEQ ID NO: 65)。 SEQ ID NO: 66 - 石蟹獼猴 (Macaca fascicularis) STEAP1 MESRKDITNEEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLATIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPVVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKMEISSQL (SEQ ID NO: 66)。 SEQ ID NO: 67 - 紅毛猩猩 (Pongo abelii) STEAP1 MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQQTRELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVILPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEISSQL (SEQ ID NO: 67)。 SEQ ID NO: 68 - huAb44.v6.05.hIgG1.EKKE.N297G.Knob 重鏈可變區 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVREAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSS SEQ ID NO: 69 - huAb44.v6.05.hIgG1.EKKE.N297G.杵輕鏈可變區 DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLKKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIK SEQ ID NO: 70 - huAb44.v6.hIgG1.N297G.杵輕鏈 DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLQKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 71 - huAb44.v6.hIgG1.N297G.杵重鏈 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 72 - huAb44.v6.05.hIgG1.EKKE.N297G.杵輕鏈 DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLKKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 73 - huAb44.v6.05.hIgG1.EKKE.N297G.杵重鏈 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVREAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 74 - huAb44.v6.05/38E4v1.MD1 1C.bsAb 輕鏈 (aSTEAP1 臂) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLKKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 75 - huAb44.v6.05/38E4v1.MD1 1C.bsAb 重鏈 (aSTEAP1 臂) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVREAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 76 - huAb44.v6.05/40G5c 1C.bsAb 輕鏈 (aSTEAP1 臂) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLKKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 77 - huAb44.v6.05/40G5c 1C.bsAb 重鏈 (aSTEAP1 臂) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVREAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 78 - huAb44.v6.05/38E4v1.MD1 1C.bsAb 輕鏈 (aCD3 臂) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQEKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 79 - huAb44.v6.05/38E4v1.MD1 1C.bsAb 重鏈 (aCD3 臂) EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRKAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 80 - huAb44.v6.05/40G5c 1C.bsAb 輕鏈 (aCD3 臂) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQEKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 81 - huAb44.v6.05/40G5c 1C.bsAb 重鏈 (aCD3 臂) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRKAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 82 - 杵輕鏈恆定區 RTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 83 - 杵重鏈恆定區 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 84 - 臼輕鏈恆定區 RTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 85 - 臼重鏈恆定區 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 86 - GFTFTSY SEQ ID NO: 87 - YPENDN SEQ ID NO: 88 - GYTFTNY SEQ ID NO: 89 – YPGDGN SEQ ID NO: 90 - TRYYIS SEQ ID NO: 91 - CIYAGSRGITYYASWAKG SEQ ID NO: 92 - ADLRPSDITDDYGDYVMDSFDP SEQ ID NO: 93 - QASQNVGSYLA SEQ ID NO: 94 - RASILSS SEQ ID NO: 95 - QSFYYITSTTYAYS SEQ ID NO: 96 - rbAb3404 重鏈可變區 QSLEESGGDLVKPGASLTLTCNASGFDFSTRYYISWVRQAPGKGLEWIACIYAGSRGITYYASWAKGRFTISKTSS-TTVTLQATSLTAADTATYFCARADLRPSDITDDYGDYVMDSFDPWGPGTLVTVSS SEQ ID NO: 97 - rbAb3404 輕鏈可變區 DIVMTQTPASVEAAVGGTVTIKCQASQNVGSYLAWYQQKPGQPPKRLIYRASILSSGVPSRFKGSGSGTQFTLTINDLEAADAATYYCQSFYYITSTTYAYSFGGGTEVVVK SEQ ID NO: 98 - RRYYIS SEQ ID NO: 99 - CIYAGSRGITYYATWAKG SEQ ID NO: 100 - ADLRPGDIADDYGDYVMDALHP SEQ ID NO: 101 - QASQNIGSYLA SEQ ID NO: 102 - RTSILSS SEQ ID NO: 103 - QSFYYLTSTTYGYA SEQ ID NO: 104 - rbAb3349 重鏈可變區 QEQLEESGGGLVKPGASLTLTCPASGVSLSRRYYISWVRQAPGKGLEWIACIYAGSRGITYYATWAKGRFTVSKTSSTTVTLQMTSLTAADTATYFCARADLRPGDIADDYGDYVMDALHPWGPGTLVTVSS SEQ ID NO: 105 - rbAb3349 輕鏈可變區 DIVMTQSPASVEAVVGGTVTIKCQASQNIGSYLAWYQQKSGQPPRLLIYRTSILSSGVPSRFKGSGSGTQFTLTISDLEAADAATYYCQSFYYLTSTTYGYAFGGGTEVVVR SEQ ID NO: 106 - GFDFSTRY SEQ ID NO: 107 - YAGSRGI SEQ ID NO: 108 - GVSLSRRY SEQ ID NO: 109 - YAGSRGI SEQ ID NO: 110 – huAb44.v6.05 及 huAb44.v6.05.hIgG1.EKKE FR-H1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS SEQ ID NO: 111 – huAb44.v6.05 FR-H2 WVRQAPGKGLVWVS SEQ ID NO: 112 – huAb44.v6.05 及 huAb44.v6.05.hIgG1.EKKE FR-H3 RFTISRDNAKSTLYLQMNSLRAEDTAVYYCAR SEQ ID NO: 113 – huAb44.v6.05 及 huAb44.v6.05.hIgG1.EKKE FR-H4 WGQGTTVTVSS SEQ ID NO: 114 – huAb44.v6.05 及 huAb44.v6.05.hIgG1.EKKE FR-L1 DIVLTQTPLSLSVTPGQPASISC SEQ ID NO: 115 – huAb44.v6.05 FR-L2 WYLQKPGQSPQLLIY SEQ ID NO: 116 – huAb44.v6.05 及 huAb44.v6.05.hIgG1.EKKE FR-L3 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SEQ ID NO: 117 – huAb44.v6.05 及 huAb44.v6.05.hIgG1.EKKE FR-L4 FGGGTKVEIK SEQ ID NO: 118 – huAb44.v6.05.hIgG1.EKKE FR-H2WVREAPGKGLVWVS SEQ ID NO: 119 – huAb44.v6.05.hIgG1.EKKE FR-L2 WYLKKPGQSPQLLIY SEQ ID NO: 120 – huAb44.v6.05.hIgG1.KEEK FR-H2 WVRKAPGKGLVWVS SEQ ID NO: 121 – huAb44.v6.05.hIgG1.KEEK FR-L2 WYLEKPGQSPQLLIY SEQ ID NO: 122 – huAb44.v6.05/38E4v1.MD1 1C.bsAb2 重鏈 (aSTEAP1 臂) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVREAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 123 – huAb44.v6.05/38E4v1.MD1 1C.bsAb2 輕鏈 (aSTEAP1 臂) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLKKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 124 – huAb44.v6.05/38E4v1.MD1 1C.bsAb2 重鏈 (aCD3 臂) EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRKAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 125 – huAb44.v6.05/38E4v1.MD1 1C.bsAb2 輕鏈 (aCD3 臂) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQEKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 126 – huAb44.v6.05/40G5c 1C.bsAb2 重鏈 (aSTEAP1 臂) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVREAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 127 – huAb44.v6.05/40G5c 1C.bsAb2 輕鏈 (aSTEAP1 臂) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLKKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 128 – huAb44.v6.05/40G5c 1C.bsAb2 重鏈 (aCD3 臂) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRKAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 129 – huAb44.v6.05/40G5c 1C.bsAb2 輕鏈 (aCD3 臂) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQEKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 130 – huAb44.v6.05/38E4v1.MD1 1C.bsAb3 重鏈 (aSTEAP1 臂) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVRKAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 131 – huAb44.v6.05/38E4v1.MD1 1C.bsAb3 輕鏈 (aSTEAP1 臂) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLEKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 132 – huAb44.v6.05/38E4v1.MD1 1C.bsAb3 重鏈 (aCD3 臂) EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVREAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 133 – huAb44.v6.05/38E4v1.MD1 1C.bsAb3 輕鏈 (aCD3 臂) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQKKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 134 – huAb44.v6.05/40G5c 1C.bsAb3 重鏈 (aSTEAP1 臂) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVRKAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 135 – huAb44.v6.05/40G5c 1C.bsAb3 輕鏈 (aSTEAP1 臂) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLEKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 136 – huAb44.v6.05/40G5c 1C.bsAb3 重鏈 (aCD3 臂) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVREAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 137 – huAb44.v6.05/40G5c 1C.bsAb3 輕鏈 (aCD3 臂) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQKKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 138 – huAb44.v6.05/38E4v1.MD1 1C.bsAb4 重鏈 (aSTEAP1 臂) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVRKAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 139 – huAb44.v6.05/38E4v1.MD1 1C.bsAb4 輕鏈 (aSTEAP1 臂) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLEKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 140 – huAb44.v6.05/38E4v1.MD1 1C.bsAb4 重鏈 (aCD3 臂) EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVREAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 141 – huAb44.v6.05/38E4v1.MD1 1C.bsAb4 輕鏈 (aCD3 臂) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQKKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 142 – huAb44.v6.05/40G5c 1C.bsAb4 重鏈 (aSTEAP1 臂) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVRKAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 143 – huAb44.v6.05/40G5c 1C.bsAb4 輕鏈 (aSTEAP1 臂) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLEKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 144 – huAb44.v6.05/40G5c 1C.bsAb4 重鏈 (aCD3 臂) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVREAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 145 – huAb44.v6.05/40G5c 1C.bsAb4 輕鏈 (aCD3 臂) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQKKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECThese unexpectedly superior properties of huAb44.v6.05 are expected to have advantages in the clinical efficacy of huAb44.v6.05 TDB for the treatment of cancers expressing STEAP1, including but not limited to prostate cancer or Ewing's sarcoma. 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Biol.74 , 531-544 (2018). doi:10.1107/S2059798318006551.Sequence Listing SEQ ID NO: 1 - NYYMA SEQ ID NO: 2 - YIDYDGGSTSYGDSVKG SEQ ID NO: 3 - RSGYYHVGYAMDA SEQ ID NO: 4 - RSSQSLEYSDGYTYLE SEQ ID NO: 5 - GVSNRFS SEQ ID NO: 6 - FQATHDPLT SEQ ID NO: 7 - STEAP1-44 Heavy Chain Variable Region EVQLVESGGGLVRPGRSLKLSCAASGFTFSNYYMAWVRQAPTRGLEWVAYIDYDGGSTSYGDSVKGRFTISRNNAKSTLYLQMNSLRSEDMATYYCARRSGYYHVGYAMDAWGQGTSVTVSS SEQ ID NO: 8 - STEAP1-44 : 14 - YISYDGDNTYYGDSVKG SEQ ID NO: 15 - RSGYYHVGYAMDG SEQ ID NO: 16 - RSGYYHVGYAMNA SEQ ID NO: 17 - STEAP1-44.NGS.HC1 SEQ ID NO: 18 - NFYMA SEQ ID NO: 19 - DHYMA SEQ ID NO: 20 - YISYDGDSTYYGDSVKG SEQ ID NO: 21 - YISYDGLDTYYGDSVKG SEQ ID NO: 22 - YISYDGDNTYYGDSVKG SEQ ID NO: 23 - RSGYYHVGYAMDG SEQ ID NO: 24 - RSGYYHVGYAMNA SEQ ID NO: 25 - RSGYYHVGYAMNA SEQ ID NO: 26 - RSGFYHVGYAMNA SEQ ID NO: 27 - STEAP1-44.NGS.HC1 Heavy chain variable region EVQLVESGGGLVQPGRSLKLSCAASGFTFSNFYMAWVRQAPTKGLEWVAYISYDGDSTYYGDSVKGRFTISRNNAKRTLYLQMNSLRSEDMATYYCARRSGYYHVGYAMDAWGQGTSVTVSS SEQ ID NO: 18 – STEAP1-44.NGS.HC2 Heavy chain variable region EVQLVESGGGLVQPGRSLKLSCAASGFTFSNFYMAWVRQAPTKGLEWVAYISYDGLDTYYGDSVKGRFTISRSNAKSTLYLQMNSLRSEDMATYYCARRSGYYHVGYAMDAWGQGTSVTVSS SEQ ID NO: 19 – STEAP1-44.NGS.HC3 Heavy chain variable region EVQLVESGGGLVQPGRSLKLSCAASGFTFSNFYMAWVRQAPTKGLEWVAYISYDGLDTYYGDSVKGRFTISRSNAKSTLYLQMNSLRSEDMATYYCARRSGYYHVGYAMDGWGQGTSVTVSS SEQ ID NO: 20 – STEAP1-44.NGS.HC4 Heavy chain variable region EVQLVESGGGLVQPGRSLKLSCAASGFTFSDHYMAWVRQAPTKGLEWVAYISYDGDNTYYGDSVKGRFTISRNNAKSTLYLQMNSLRSEDMATYYCARRSGYYHVGYAMNAWGPGTSVTVSS SEQ ID NO: 21 – STEAP1-44.NGS.HC5 Heavy chain variable region EVRLVESGGGLVQPGRSLKLSCTASGFTFSDHYMAWVRQVPTKGLEWVAYIDYDGGSTSYGDSVKGRFTISRNNAKSTLYLQMNSLRSEDMATYYCARRSGFYHVGYAMNAWGQGTSVTVSS SEQ ID NO: 22 - huAb44.v1/v2/v3/v4 Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLVWVAYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSS SEQ ID NO: 23 - huAb44.v5/v6/v7/v8 Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSS SEQ ID NO: 24 - huAb44.v9/v10/v11/v12 Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLVWVAYIDYDGGSTSYGDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSS SEQ ID NO: 25 - huAb44.v13/v14/v15/v16 Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSS SEQ ID NO: 26 - huAb44.v1/v5/v9/v13 Light chain variable region DDVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLQKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIK SEQ ID NO: 27 - huAb44.v2/v6/v10/v14 Light chain variable region DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLQKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIK SEQ ID NO: 28 - huAb44.v3/v7/v11/v15 Light chain variable region DDVMTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLQKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIK SEQ ID NO: 29 - huAb44.v4/v8/v12/v16 Light chain variable regionDIVMTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLQKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIK SEQ ID NO: 30 - huAb44.v6.01 Heavy chain variable regionEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFYMAWVRQAPGKGLVWVSYISYDGDSTYYGDSVKGRFTISRDNAKRTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSS SEQ ID NO: 31 - huAb44.v6.02 Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFYMAWVRQAPGKGLVWVSYISYDGLDTYYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSS SEQ ID NO: 32 - huAb44.v6.03 Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFYMAWVRQAPGKGLVWVSYISYDGLDTYYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDGWGQGTTVTVSS SEQ ID NO: 33 - huAb44.v6.04 Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVRQAPGKGLVWVSYISYDGDNTYYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMNAWGQGTTVTVSS SEQ ID NO: 34 - huAb44.v6.05 Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVRQAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSS SEQ ID NO: 35 - Xaa¹ Xaa² YMA (Xaa¹ =D/N; Xaa² =H/Y/F) SEQ ID NO: 36 - YIXaa³ Xaa 2 YMAWVRXaa^{10 APGKGLVWVXaa 11 YIXaa 3 YDGXaa 4 Xaa 5 TXaa 6 YGDSVKGRFTISRDNAKXaa 12 Xaa 2 YVDGXaa 4 Xaa 5 TXaa 6YGDSVKGRFTISRDNAKXaa13SEQID} NO: 37 - RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (Xaa⁷ =F/Y; Xaa⁸ =N/D; Xaa⁹ =A/G) SEQ ID NO: 38 - huAb44 VH consensus sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSXaa^{1Xaa 2YMAWVRXaa10APGKGLVWVXaa11} YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKGRFTISRDNAKXaa¹² TLYLQMNSLRAEDTAVYYCARRSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ WGQGTTVTVSS (Xaa¹ =N/D; Xaa² =Y/F/H; Xaa³ =D/S; Xaa⁴ =G/D/L; Xaa⁵ =S/D/N; Xaa⁶ =S/Y; Xaa⁷ =Y/F; Xaa⁸ =D/N; Xaa⁹ =A/G; Xaa¹⁰ =Q/E/K; Xaa¹¹ =A/S; Xaa¹² =S/R/N) SEQ ID NO: 39 - huAb44 VL consensus sequence DXaa¹³ VXaa¹⁴ TQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLXaa^1544- WASTRES SEQ ID NO: 45 -^TQSFILRT SEQ ID NO: 46 - huAb44.v6.05/38E4v1.MD1 1C. bsAb heavy chain variable region (MD1) EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRKAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSS SEQ ID NO: 47 - huAb44.v6.05/38E4v1.MD1 1C.bsAb light chain variable region (MD1) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQEKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIK SEQ ID NO: 48 - NYYIH SEQ ID NO: 49 - WIYPGDGNTKYNEKFKG SEQ ID NO: 50 - DSYSNYYFDY SEQ ID NO: 51 54 - huAb44.v6.05/40G5c 1C. bsAb heavy chain variable region (40G5c) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRKAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSS SEQ ID NO: 55 - huAb44.v6.05/40G5c 1C. bsAb light chain variable region (40G5c) 54 - DYDGGS SEQ ID NO: 55 - GFTFSNF SEQ ID NO: 56 - GFTFSNY SEQ ID NO: 57 - DYDGGS SEQ ID NO: 58 - GFTFSNF SEQ ID NO: 59 - GFTFSDH SEQ ID NO: 60 - SYDGDS SEQ ID NO: 61 - SYDGLD SEQ ID NO: 62 - SYDGDN SEQ ID NO: 63 - GFTFSXaa¹ Xaa² (Xaa¹ =N/D; Xaa² =Y/F/H) SEQ ID NO: 64 - Xaa³ YDGXaa⁴ Xaa⁵ (Xaa³ =D/S; Xaa⁽ SEQ ID NO: 65⁾ . SEQ ID NO: 66 -Macaca fascicularis STEAP1 MESRKDITNEEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLATIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPVVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKMEISSQL (SEQ ID NO: 66). SEQ ID NO: 67 - Orangutan (Pongo abelii ) STEAP1 MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQQTRELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVILPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEISSQL (SEQ ID NO: 67). SEQ ID NO: 68 - huAb44.v6.05.hIgG1.EKKE.N297G.Knob Heavy Chain Variable RegionEVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVREAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSS SEQ ID NO: 69 - huAb44.v6.05.hIgG1.EKKE.N297G.Knob Light Chain Variable RegionDIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLKKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIK SEQ ID NO: 70 - huAb44.v6.hIgG1.N297G.Pole light chainDIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLQKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 71 - huAb44.v6.hIgG1.N297G.Pestle heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGYYHVGYAMDAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 72 - huAb44.v6.05.hIgG1.EKKE.N297G.Pole Light ChainDIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLKKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 73 - huAb44.v6.05.hIgG1.EKKE.N297G.Pestle heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVREAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 74 - huAb44.v6.05/38E4v1.MD11C. bsAb light chain (aSTEAP1 arm) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLKKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 75 - huAb44.v6.05/38E4v1.MD11C. bsAb heavy chain (aSTEAP1 arm) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVREAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 76 - huAb44.v6.05/40G5c 1C. bsAb light chain (aSTEAP1 arm) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLKKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 77 - huAb44.v6.05/40G5c 1C. bsAb heavy chain (aSTEAP1 arm) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVREAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 78 - huAb44.v6.05/38E4v1.MD11C. bsAb light chain (aCD3 arm) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQEKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 79 - huAb44.v6.05/38E4v1.MD11C. bsAb heavy chain (aCD3 arm) EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRKAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 80 - huAb44.v6.05/40G5c 1C. bsAb light chain (aCD3 arm) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQEKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 81 - huAb44.v6.05/40G5c 1C. bsAb heavy chain (aCD3 arm) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRKAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 82 - Knob light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 83 - Knob heavy chain constant regionASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 84 - Morpholight chain constant region RTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 85 - Mortar heavy chain constant regionASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 86 - GFTFTSY SEQ ID NO: 87 - YPENDN SEQ ID NO: 88 - GYTFTNY SEQ ID NO: 89 - YPGDGN SEQ ID NO: 90 - TRYYIS SEQ ID NO: 91 - CIYAGSRGITYYASWAKG SEQ ID NO: 92 - ADLRPSDITDDYGDYVMDSFDP SEQ ID NO: 93 - QASQNVGSYLA SEQ ID NO: 94 - RASILSS SEQ ID NO: 95 - QSFYYITSTTYAYS SEQ ID NO: 96 - rbAb3404 Heavy Chain Variable Region QSLEESGGDLVKPGASLTLTCNASGFDFSTRYYISWVRQAPGKGLEWIACIYAGSRGITYYASWAKGRFTISKTSS-TTVTLQATSLTAADTATYFCARADLRPSDITDDYGDYVMDSFDPWGPGTLVTVSS SEQ ID NO: 97 - rbAb3404 SEQ ID NO: 98 - RRYYIS SEQ ID NO: 99 - CIYAGSRGITYYATWAKG SEQ ID NO: 100 - ADLRPGDIADDYGDYVMDALHP SEQ ID NO: 101 - QASQNIGSYLA SEQ ID NO: 102 - RTSILSS SEQ ID NO: 103 - QSFYYLTSTTYGYA SEQ ID NO: 104 - rbAb3349 Heavy chain variable region QEQLEESGGGLVKPGASLTLTCPASGVSLSRRYYISWVRQAPGKGLEWIACIYAGSRGITYYATWAKGRFTVSKTSSTTVTLQMTSLTAADTATYFCARADLRPGDIADDYGDYVMDALHPWGPGTLVTVSS SEQ ID NO: 105 - rbAb3349 Light chain variable region DIVMTQSPASVEAVVGGTVTIKCQASQNIGSYLAWYQQKSGQPPRLLIYRTSILSSGVPSRFKGSGSGTQFTLTISDLEAADAATYYCQSFYYLTSTTYGYAFGGGTEVVVR SEQ ID NO: 106 - GFDFSTRY SEQ ID NO: 107 - YAGSRGI SEQ ID NO: 108 - GVSLSRRY SEQ ID NO: 109 - YAGSRGI SEQ ID NO: 110 - .EKKE FR-H3 RFTISRDNAKSTLYLQMNSLRAEDTAVYYCAR SEQ ID NO: 113 – huAb44.v6.05 and huAb44.v6.05.hIgG1.EKKE FR-H4 WGQGTTVTVSS SEQ ID NO: 114 – huAb44.v6.05 and huAb44.v6.05.hIgG1.EKKE FR-H5 WGQGTTVTVSS SEQ ID NO: 115 – huAb44.v6.05 and huAb44.v6.05.hIgG1.EKKE FR-H6 WGQGTTVTVSS SEQ ID NO: 116 .EKKE FR-L3 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SEQ ID NO: 117 – huAb44.v6.05 and huAb44.v6.05.hIgG1.EKKE FR-L4 FGGGTKVEIK SEQ ID NO: 118 – huAb44.v6.05.hIgG1.EKKE FR-H2 WYLQKPGQSPQLLIY SEQ ID NO: 119 – huAb44.v6.05 and huAb44.v6.05.hIgG1.EKKE FR-L3 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SEQ ID NO: 120 – huAb44.v6.05 and huAb44.v6.05.hIgG1.EKKE FR-L4 FGGGTKVEIK SEQ ID NO: 121 119 – huAb44.v6.05.hIgG1.EKKE FR-L2 WYLKKPGQSPQLLIY SEQ ID NO: 120 – huAb44.v6.05.hIgG1.KEEK FR-H2 WVRKAPGKGLVWVS SEQ ID NO: 121 – huAb44.v6.05.hIgG1.KEEK FR-L2 WYLEKPGQSPQLLIY SEQ ID NO: 122 – huAb44.v6.05/38E4v1.MD1 1C.bsAb2 heavy chain (aSTEAP1 arm) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVREAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 123 - huAb44.v6.05/38E4v1.MD1 1C.bsAb2 light chain (aSTEAP1 arm) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLKKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 124 - huAb44.v6.05/38E4v1.MD1 1C.bsAb2 heavy chain (aCD3 arm) EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRKAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 125 - huAb44.v6.05/38E4v1.MD1 1C.bsAb2 light chain (aCD3 arm) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQEKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 126 - huAb44.v6.05/40G5c 1C.bsAb2 heavy chain (aSTEAP1 arm) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVREAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 127 - huAb44.v6.05/40G5c 1C.bsAb2 light chain (aSTEAP1 arm) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLKKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 128 - huAb44.v6.05/40G5c 1C.bsAb2 heavy chain (aCD3 arm) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRKAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 129 - huAb44.v6.05/40G5c 1C.bsAb2 light chain (aCD3 arm) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQEKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 130 - huAb44.v6.05/38E4v1.MD1 1C.bsAb3 heavy chain (aSTEAP1 arm) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVRKAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 131 - huAb44.v6.05/38E4v1.MD1 1C. bsAb3 light chain (aSTEAP1 arm) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLEKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 132 - huAb44.v6.05/38E4v1.MD1 1C. bsAb3 heavy chain (aCD3 arm) EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVREAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 133 - huAb44.v6.05/38E4v1.MD1 1C. bsAb3 light chain (aCD3 arm) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQKKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 134 - huAb44.v6.05/40G5c 1C. bsAb3 heavy chain (aSTEAP1 arm) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVRKAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 135 - huAb44.v6.05/40G5c 1C. bsAb3 light chain (aSTEAP1 arm) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLEKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 136 - huAb44.v6.05/40G5c 1C. bsAb3 heavy chain (aCD3 arm) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVREAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 137 - huAb44.v6.05/40G5c 1C. bsAb3 light chain (aCD3 arm) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQKKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 138 - huAb44.v6.05/38E4v1.MD1 1C. bsAb4 heavy chain (aSTEAP1 arm) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVRKAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 139 – huAb44.v6.05/38E4v1.MD1 1C.bsAb4 light chain (aSTEAP1 arm) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLEKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 140 – huAb44.v6.05/38E4v1.MD1 1C.bsAb4 heavy chain (aCD3 arm) EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVREAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 141 - huAb44.v6.05/38E4v1.MD1 1C. bsAb4 light chain (aCD3 arm) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQKKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 142 - huAb44.v6.05/40G5c 1C. bsAb4 heavy chain (aSTEAP1 arm) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMAWVRKAPGKGLVWVSYIDYDGGSTSYGDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRSGFYHVGYAMNAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 143 - huAb44.v6.05/40G5c 1C. bsAb4 light chain (aSTEAP1 arm) DIVLTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTYLEWYLEKPGQSPQLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 144 - huAb44.v6.05/40G5c 1C. bsAb4 heavy chain (aCD3 arm) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVREAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 145 - huAb44.v6.05/40G5c 1C.bsAb4 light chain (aCD3 arm) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQKKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

圖 1A 示出篩選與表現 STEAP1 之細胞特異性結合的選定大鼠單株抗體。大鼠單株抗體為 STEAP1-44、STEAP1-45、STEAP1-81、STEAP1-75、STEAP1-103、STEAP1-10、STEAP1-23、STEAP1-67、STEAP1-34、STEAP1-19 及 STEAP1-92。 圖 1B 示出篩選與表現 STEAP1 之細胞特異性結合的選定大鼠單株抗體。大鼠單株抗體為 STEAP1-48、STEAP1-15、STEAP1-28、STEAP1-95、STEAP1-54、STEAP1-69、STEAP1-2、STEAP1-59、STEAP1-49 及 STEAP1-62。Ab120 為對照抗體。 圖 1C 示出滴定測定中抗體 STEAP1-44 之結合。 圖 2 示出藉由深度定序獲得的抗體 STEAP1-44 及 STEAP1-44 殖株變異體的可變區序列，與最接近的大鼠種系片段序列 (頂部序列) 進行比對。對於藉由深度定序 (「NGS」) 所獲得的殖株變異體，僅提供重鏈。它們與 STEAP1-44 輕鏈合併以產生全長抗體，從而共享輕鏈。假定的體細胞突變係以黑色背景顯示。根據比對上方所示之 Kabat 及 Chothia 系統的 CDR 邊界，其中 Kabat CDR 邊界帶下劃線。根據 Kabat 系統進行殘基編號。點代表由比對軟體引入的序列差異。種系片段名稱顯示於比對中。Kabat CDR 以及 VH 及 VL 序列之 SEQ ID NO 顯示於表 1 中。Chothia CDR 的 SEQ ID NO 顯示於表 2 中。 圖 3 示出深度定序資料集中 STEAP1-44 株性型組中體細胞突變的評分。示出各位置的胺基酸突變中之各者的序列讀段計數。黑框指示種系片段野生型殘基。以灰色突出顯示的殘基示出 CDR 區中最常見的體細胞突變。 圖 4A 示出與細胞表面上之 STEAP1 結合的 STEAP1-44 殖株變異體之測試。 圖 4B 示出與可溶性重組 STEAP1 結合的 STEAP1-44 殖株變異體之測試。 圖 5A 示出與人源化骨架比對的 STEAP1-44 人源化變異體之輕鏈可變區序列。變異體與人源化變異體的差異以黑色背景顯示。根據比對上方所示之 Kabat 及 Chothia 系統的 CDR 邊界，其中 Kabat CDR 邊界帶下劃線。根據 Kabat 系統進行殘基編號。點代表由比對軟體引入的序列差異。Kabat CDR 及 VL 序列的 SEQ ID NO 顯示於表 1 中。Chothia CDR 的 SEQ ID NO 顯示於表 2 中。 圖 5B 示出與人源化骨架比對的 STEAP1-44 人源化變異體之重鏈可變區序列。變異體與人源化骨架的差異以黑色背景顯示。根據比對上方所示之 Kabat 及 Chothia 系統的 CDR 邊界，其中 Kabat CDR 邊界帶下劃線。根據 Kabat 系統進行殘基編號。點代表由比對軟體引入的序列差異。Kabat CDR 及 VH 序列的 SEQ ID NO 顯示於表 1 中。Chothia CDR 的 SEQ ID NO 顯示於表 2 中。 圖 6A 示出在兩種不同濃度下測試的抗體人源化變異體的中位螢光單位 (MFI)。變異體從左到右按最強到最弱結合排序，在兩種測試的濃度中按相同的順序排序。 圖 6B 示出測試具有 5 種基於 NGS 的變異體的體細胞突變的人源化變異體 huAb44.v6 與表現 STEAP1 之 LNCaP-X1.2 細胞的結合。huAb44.v6 為親代人源化殖株，chAb44 為具有 Ab44 的大鼠可變域的人類 IgG1，且 huAb44.v6.01 至 05 為 huAb44.v6 的具有藉由庫 NGS 所鑑別之體細胞突變的序列變異體。 圖 6C 示出與人源化抗體 huAb44.v6 比對的人源化抗體變異體 huAb44.v6.05 之序列。huAb44.v6.05 相對於 huAb44.v6 的差異以黑色背景示出。根據比對上方所示之 Kabat 及 Chothia 系統的 CDR 邊界，其中 Kabat CDR 邊界帶下劃線。根據 Kabat 系統進行殘基編號。Kabat CDR 以及 VH 及 VL 區序列之 SEQ ID NO 顯示於表1中。Chothia CDR 的 SEQ ID NO 顯示於表 2 中。 圖 7A 示出在細胞毒殺測定中用人類 CD8+T 細胞及表現 STEAP1 之 LNCaP-X1.2 細胞測試的雙特異性抗 STEAP1/抗 CD3 抗體。TDB 分子之抗 CD3 臂表示為 MD1。 圖 7B 示出在細胞毒殺測定中用人類 CD8+T 細胞及表現 STEAP1 之 LNCaP-X1.2 細胞測試的雙特異性抗 STEAP1/抗 CD3 抗體。TDB 分子之抗 CD3 臂表示為 MD1 或 40G5c。 圖 8A 示出藉由螢光激活細胞分選 (FACS) 分析或西方墨點法所測量之經基因編輯修飾的 LNCaPX1.2 亞系 (LNCaP-X1.2-KO-3-13, LNCaP-X1.KO-2-11) 中的 STEAP1 含量。LNCaP-X1.2-KO-2-8 缺乏 STEAP1 表現。 圖 8B 示出在細胞毒殺測定中用人類 CD8+T 細胞及指定細胞株測試 huAb44v6.05 TDB。 圖 8C 示出 huAb44.v6.05/40G5c 的標靶依賴性 T 細胞活化 (24 小時)。 圖 8D 示出 huAb44.v6.05/40G5c 的標靶依賴性細胞激素分泌 (24 小時)。 圖 9 示出 STEAP1-TDB 抑制補充有人類 PBMC 的 NSG 小鼠中已確立的 LNCaP-X1.2、LNCaP-X1.2-KO-3-13 及 LNCaP-X1.KO-2-11 腫瘤之生長。動物於指定的 STEAP1-TDB 第 0 天接受單一 IV 劑量。劑量從 0.1 mg/kg 到 0.5 mg/kg 變化。針對各治療組繪製個別腫瘤體積。虛線指示對照組 (載體) 的擬合腫瘤體積，實線指示個別腫瘤。 圖 10A 示出抗 STEAP1 抗體在雌性 SCID 小鼠中單一靜脈內劑量 1 mg/kg 後的濃度-時間曲線。 圖 10B 示出抗 STEAP1/CD3 TDB 及抗 gD 在雌性 SCID 小鼠中單一靜脈內劑量 1 mg/kg 後的濃度-時間曲線。 圖 10C 示出 huAb44.v6.05/40G5c 及 huAb44.v6.05/MD1 TDB 在石蟹獼猴中靜脈內投予後的濃度-時間曲線。 圖 11A 示出與 Ab44 複合的 STEAP1 的冷凍電鏡重建，分辨率為約 3 Å。示出與三個 Ab44 Fab (重鏈著色為品紅色，輕鏈著色為淺粉色，恆定區著色為紫色) 複合的 STEAP1 同型三聚體 (著色為青色、灰色及小麥色的單元體) 的等值面渲染。STEAP1 細胞外環 ECL1、ECL2 及 ECL3 分別著色為綠色、紅色及橙色。 圖 11B 示出該結構的條帶渲染。示出側視圖 (沿膜的平面，與圖 A 等同) 及俯視圖。為清楚起見，僅示出來自一個次單元的血紅素 (著色為棕色)。 圖 12A 示出 Ab44 Fab 與 STEAP1 次單元 A 之間的交互作用 (示出旋轉 90 度的兩個正交側視圖)。位於 Ab44 Fab 的 4 Å 以內的 STEAP1 殘基以球體示出。著色方案類似於圖 1。為清楚起見，已省去 STEAP1 次單元 B 及 C。 圖 12B 示出 Ab44 Fab 與 STEAP1 相鄰次單元 B 之間的交互作用。視圖與圖 A 相同。為清楚起見，已省去 STEAP1 次單元 A 及 C。 圖 13A 示出 STEAP1:Fab44 複合體之表面圖示。由複合體形成所隱藏的表面被著色為紅色 (Fab A 至 STEAP1 次單元 A) 及綠色 (Fab A 至 STEAP1 次單元 B)；代表同型交互作用的隱藏表面被著色為黃色 (Fab A 至 Fab B) 或藍色 (Fab A 至 Fab C)。STEAP1 單體被著色為青色、淺灰色或深灰色。輕鏈被著色為淺粉色，且重鏈被著色為品紅色、藍灰色或小麥色。 圖 13B 示出相同表面的詳細圖示，其中示出各隱藏表面的表面積測量值。 圖 14 示出 Ab44 及萬多妥珠單抗與 STEAP1 結合後的組織。沿 STEAP1:Ab44 結構的三重軸觀察到多個極性同型 Fab 交互作用 (以球體示出)，且主要由 LC-LC 接觸驅動 (左圖，從細胞外側觀察)。相比之下，在萬多妥珠單抗 Fab 之間觀察到有限的交互作用 (右圖)。輕鏈被著色為淺粉色，且重鏈被著色為品紅色、灰色或小麥色。為清楚起見，未示出 STEAP1 同型三聚體 (位於 Fab A 的 4 Å 以內的殘基除外，該等殘基係以球體表示)。 圖 15 示出人類、石蟹獼猴及紅毛猩猩的序列比對。預期的穿膜螺旋係以黃色突出顯示，且細胞外環 1、2 及 3 分別以綠色、紅色及橙色框突出顯示。 圖 16 示出人源化抗體變異體 huAb44.v6、huAb44.v6.01、huAb44.v6.02、huAb44.v6.03、huAb44.v6.04 及 huAb44.v6.05 之可變區的序列比對。根據 Kabat 定義的 CDR 序列以底線表示。Kabat CDR 以及 VH 及 VL 序列之 SEQ ID NO 顯示於表 1 中。Chothia CDR 的 SEQ ID NO 顯示於表 2 中。 圖 17 示出雙特異性抗體之 STEAP1 特異性可變區的序列比對。根據 Kabat 定義的 CDR 序列以底線表示。Kabat CDR 以及 VH 及 VL 序列之 SEQ ID NO 顯示於表 1 中。Chothia CDR 的 SEQ ID NO 顯示於表 2 中。 圖 18 示出雙特異性抗體之 CD3 特異性可變區的序列比對。根據 Kabat 定義的 CDR 序列以底線表示。Kabat CDR 以及 VH 及 VL 序列之 SEQ ID NO 顯示於表 1 中。Chothia CDR 的 SEQ ID NO 顯示於表 2 中。 圖 19 示出 STEAP1 特異性鏈恆定區的序列比對。 圖 20 示出 CD3 特異性鏈恆定區的序列比對。 圖 21 示出兔抗人 STEAP1 抗體的可變區的序列比對。根據比對上方所示之 Kabat 及 Chothia 系統的 CDR 邊界，其中 Kabat CDR 邊界帶下劃線。根據 Kabat 系統進行殘基編號。點代表由比對軟體引入的序列差異。Kabat CDR 以及 VH 及 VL 序列之 SEQ ID NO 顯示於表 1 中。Chothia CDR 的 SEQ ID NO 顯示於表 2 中。 圖 22 示出藉由 FACS 所評定之 huAb44v6.05 及萬多妥珠單抗與表現 STEAP1 之 LNCaP-X1.2 細胞的結合的比較。在指定濃度下測試抗體變異體的 MFI。萬多妥珠單抗的 EC50 為 3.5 nM，且 huAb44.v6.05 的 EC50 為 1.9 nM。 圖 23A 示出在細胞毒殺測定中用人類 CD8+T 細胞及表現 STEAP1 之 LNCaP-X1.2 細胞測試的雙特異性抗 STEAP1/抗 CD3 抗體。對含有萬多妥珠單抗及 huAb44v6.05 的 TDB 進行比較。如圖所示，TDB 分子之抗 CD3 臂為 MD1 或 40G5c。 圖 23B 示出在細胞毒殺測定中用人類 CD8+T 細胞及表現 STEAP1 之 LNCaPX1.2KO3-13 細胞測試的雙特異性抗 STEAP1/抗 CD3 抗體。對含有萬多妥珠單抗及 huAb44v6.05 的 TDB 進行比較。如圖所示，TDB 分子之抗 CD3 臂為 MD1 或 40G5c。 圖 24A 至 24D 為示出 T 細胞活化測定中 huAb44.v6.05/40G5c 及萬多妥珠單抗/40G5c TDB 之比較的一系列圖。圖 24A 示出使用 LNCaP-X12 細胞的 CD4 T 細胞活化的結果。圖 24B 示出使用 LNCaP-X12 細胞的 CD8 T 細胞活化的結果。圖 24C 示出使用 LNCaP-X1.2-KO-3-13 細胞的 CD4 T 細胞活化的結果。圖 24D 示出使用 LNCaP-X1.2-KO-3-13 細胞的 CD8 T 細胞活化的結果。對於這些實驗中之各者，PBMC 與靶標之比率為 10:1，且孵育時間為 24 小時。 圖 25A 至 25J 為示出細胞激素分泌測定中於 24 小時的 huAb44.v6.05/40G5c 及萬多妥珠單抗/40G5c 之比較的一系列圖。圖25A 至 25E 示出 LNCaP-X1.2 細胞的結果，且圖25F 至 25J 示出 LNCaP-X1.2-KO-3-13 細胞的結果。圖25A 及 25F 示出干擾素 (IFN) γ 分泌。圖25B 及 25G 示出腫瘤壞死因子 (TNF) α 分泌。圖 25C 及 25H 示出 IL-2 分泌。圖25D 及 25I 示出 IL-6 分泌。圖25E 及 25J 示出顆粒酶 B 分泌。FIG. 1A shows selected rat monoclonal antibodies that were screened for specific binding to cells expressing STEAP1. The rat monoclonal antibodies are STEAP1-44, STEAP1-45, STEAP1-81, STEAP1-75, STEAP1-103, STEAP1-10, STEAP1-23, STEAP1-67, STEAP1-34, STEAP1-19, and STEAP1-92. FIG. 1B shows selected rat monoclonal antibodies that were screened for specific binding to cells expressing STEAP1. The rat monoclonal antibodies are STEAP1-48, STEAP1-15, STEAP1-28, STEAP1-95, STEAP1-54, STEAP1-69, STEAP1-2, STEAP1-59, STEAP1-49 and STEAP1-62. Ab120 is a control antibody. FIG1C shows the binding of antibody STEAP1-44 in the titration assay. FIG2 shows the variable region sequences of antibody STEAP1-44 and STEAP1-44 strain variants obtained by deep sequencing, aligned with the closest rat germline fragment sequence (top sequence). For strain variants obtained by deep sequencing ("NGS"), only reconstructs are provided. They are combined with the STEAP1-44 light chain to generate full-length antibodies, thereby sharing the light chain. Putative somatic mutations are shown with a black background. CDR boundaries according to the Kabat and Chothia systems are shown above the alignment, with the Kabat CDR boundaries underlined. Residue numbering is according to the Kabat system. Dots represent sequence differences introduced by the alignment software. The germline segment names are shown in the alignment. The SEQ ID NOs of the Kabat CDRs and VH and VL sequences are shown in Table 1. The SEQ ID NOs of the Chothia CDRs are shown in Table 2. Figure 3 shows the scoring of somatic mutations in the STEAP1-44 strain type group in the deep sequencing dataset. The sequence read counts for each of the amino acid mutations at each position are shown. Black boxes indicate wild-type residues of the germline fragment. Residues highlighted in gray show the most common somatic mutations in the CDR regions. Figure 4A shows the testing of STEAP1-44 clone variants binding to STEAP1 on the cell surface. Figure 4B shows the testing of STEAP1-44 clone variants binding to soluble recombinant STEAP1. Figure 5A shows the light chain variable region sequence of the STEAP1-44 humanized variant aligned with the humanized backbone. The differences between the variants and the humanized variants are shown in black background. CDR boundaries according to the Kabat and Chothia systems shown above the alignment, with the Kabat CDR boundaries underlined. Residue numbering is performed according to the Kabat system. Dots represent sequence differences introduced by the alignment software. The SEQ ID NOs for Kabat CDR and VL sequences are shown in Table 1. The SEQ ID NOs for Chothia CDRs are shown in Table 2. Figure 5B shows the heavy chain variable region sequences of STEAP1-44 humanized variants aligned with the humanized backbone. The differences between the variants and the humanized backbone are shown in black background. CDR boundaries according to the Kabat and Chothia systems are shown above the alignment, with the Kabat CDR boundaries underlined. Residue numbering is performed according to the Kabat system. Dots represent sequence differences introduced by the alignment software. The SEQ ID NOs for Kabat CDR and VH sequences are shown in Table 1. The SEQ ID NOs for Chothia CDRs are shown in Table 2. FIG6A shows the median fluorescence units (MFI) of the humanized variants of the antibody tested at two different concentrations. The variants are ranked from left to right from strongest to weakest binding, in the same order at both concentrations tested. FIG6B shows the humanized variant huAb44.v6 with somatic mutations tested for binding to LNCaP-X1.2 cells expressing STEAP1 with 5 NGS-based variants. huAb44.v6 is the parental humanized strain, chAb44 is a human IgG1 with the rat variable domains of Ab44, and huAb44.v6.01 to 05 are sequence variants of huAb44.v6 with somatic mutations identified by library NGS. FIG6C shows the sequence of humanized antibody variant huAb44.v6.05 aligned with humanized antibody huAb44.v6. Differences of huAb44.v6.05 relative to huAb44.v6 are shown in black background. CDR boundaries according to the Kabat and Chothia systems are shown above the alignment, with the Kabat CDR boundaries underlined. Residue numbering is according to the Kabat system. The SEQ ID NOs of the Kabat CDR and VH and VL region sequences are shown in Table 1. The SEQ ID NOs of the Chothia CDRs are shown in Table 2. FIG7A shows bispecific anti-STEAP1/anti-CD3 antibodies tested in a cytotoxicity assay using human CD8+ T cells and LNCaP-X1.2 cells expressing STEAP1. The anti-CD3 arm of the TDB molecule is denoted as MD1. FIG7B shows bispecific anti-STEAP1/anti-CD3 antibodies tested in a cytotoxicity assay using human CD8+ T cells and LNCaP-X1.2 cells expressing STEAP1. The anti-CD3 arm of the TDB molecule is denoted as MD1 or 40G5c. Figure 8A shows STEAP1 levels in gene-edited LNCaPX1.2 sublines (LNCaP-X1.2-KO-3-13, LNCaP-X1.KO-2-11) measured by fluorescence activated cell sorting (FACS) analysis or Western blotting. LNCaP-X1.2-KO-2-8 lacks STEAP1 expression. Figure 8B shows huAb44v6.05 TDB tested in cytotoxicity assays with human CD8+ T cells and the indicated cell lines. Figure 8C shows target-dependent T cell activation of huAb44.v6.05/40G5c (24 hours). Figure 8D shows target-dependent cytokine secretion (24 hours) of huAb44.v6.05/40G5c. Figure 9 shows that STEAP1-TDB inhibits the growth of established LNCaP-X1.2, LNCaP-X1.2-KO-3-13, and LNCaP-X1.KO-2-11 tumors in NSG mice supplemented with human PBMCs. Animals received a single IV dose of STEAP1-TDB on day 0 as indicated. Doses varied from 0.1 mg/kg to 0.5 mg/kg. Individual tumor volumes are plotted for each treatment group. The dashed line indicates the simulated tumor volume for the control group (vehicle), and the solid line indicates individual tumors. Figure 10A shows the concentration-time curves of anti-STEAP1 antibody after a single intravenous dose of 1 mg/kg in female SCID mice. Figure 10B shows the concentration-time curves of anti-STEAP1/CD3 TDB and anti-gD after a single intravenous dose of 1 mg/kg in female SCID mice. Figure 10C shows the concentration-time curves of huAb44.v6.05/40G5c and huAb44.v6.05/MD1 TDB after intravenous administration in stone crab macaques. Figure 11A shows a cryo-EM reconstruction of STEAP1 complexed with Ab44 at a resolution of approximately 3 Å. An isosurface rendering of a STEAP1 homotrimer (monomers colored cyan, gray, and wheat) in complex with three Ab44 Fabs (heavy chains colored magenta, light chains colored light pink, homeostasis regions colored purple) is shown. The STEAP1 extracellular loops ECL1, ECL2, and ECL3 are colored green, red, and orange, respectively. FIG11B shows a ribbon rendering of the structure. A side view (along the plane of the membrane, equivalent to FIGA) and a top view are shown. For clarity, only heme from one subunit is shown (colored brown). FIG12A shows the interaction between Ab44 Fab and STEAP1 subunit A (two orthogonal side views rotated 90 degrees are shown). STEAP1 residues within 4 Å of Ab44 Fab are shown as spheres. The coloring scheme is similar to Figure 1. STEAP1 subunits B and C have been omitted for clarity. Figure 12B shows the interaction between Ab44 Fab and the neighboring subunit B of STEAP1. The view is the same as Figure A. STEAP1 subunits A and C have been omitted for clarity. Figure 13A shows a surface representation of the STEAP1:Fab44 complex. Surfaces hidden by complex formation are colored red (Fab A to STEAP1 subunit A) and green (Fab A to STEAP1 subunit B); hidden surfaces representing homotypic interactions are colored yellow (Fab A to Fab B) or blue (Fab A to Fab C). STEAP1 monomers are colored cyan, light gray, or dark gray. Light chains are colored light pink, and heavy chains are colored magenta, blue-gray, or wheat. Figure 13B shows a detailed illustration of the same surface, showing the surface area measurements of each hidden surface. Figure 14 shows the organization of Ab44 and vendotumab after binding to STEAP1. Multiple polar isotypic Fab interactions (shown as spheres) are observed along the three-fold axis of the STEAP1:Ab44 structure and are primarily driven by LC-LC contacts (left, viewed from the outside of the cell). In contrast, limited interactions were observed between vendotumab Fabs (right). The light chain is colored light pink, and the heavy chain is colored magenta, gray, or wheat. For clarity, the STEAP1 homotrimer is not shown (except for residues within 4 Å of Fab A, which are represented by spheres). Figure 15 shows the sequence alignment of human,red macaque , andorangutan . The expected transmembrane helix is highlighted in yellow, and the extracellular loops 1, 2, and 3 are highlighted in green, red, and orange boxes, respectively. Figure 16 shows the sequence alignment of the variable regions of the humanized antibody variants huAb44.v6, huAb44.v6.01, huAb44.v6.02, huAb44.v6.03, huAb44.v6.04, and huAb44.v6.05. The CDR sequences according to the Kabat definition are underlined. The SEQ ID NOs of the Kabat CDRs and the VH and VL sequences are shown in Table 1. The SEQ ID NOs of the Chothia CDRs are shown in Table 2. FIG. 17 shows the sequence alignment of the STEAP1-specific variable regions of the bispecific antibodies. The CDR sequences according to the Kabat definition are underlined. The SEQ ID NOs of the Kabat CDRs and the VH and VL sequences are shown in Table 1. The SEQ ID NOs of the Chothia CDRs are shown in Table 2. FIG. 18 shows the sequence alignment of the CD3-specific variable regions of the bispecific antibodies. The CDR sequences according to the Kabat definition are underlined. The SEQ ID NOs of the Kabat CDRs and the VH and VL sequences are shown in Table 1. The SEQ ID NOs of Chothia CDRs are shown in Table 2. Figure 19 shows the sequence alignment of the constant regions of the STEAP1 specific chain. Figure 20 shows the sequence alignment of the constant regions of the CD3 specific chain. Figure 21 shows the sequence alignment of the variable regions of the rabbit anti-human STEAP1 antibodies. The CDR boundaries according to the Kabat and Chothia systems are shown above the alignment, with the Kabat CDR boundaries underlined. Residue numbering is performed according to the Kabat system. Dots represent sequence differences introduced by the alignment software. The SEQ ID NOs of the Kabat CDRs and the VH and VL sequences are shown in Table 1. The SEQ ID NOs of the Chothia CDRs are shown in Table 2. FIG. 22 shows a comparison of huAb44v6.05 and vendotozumab binding to LNCaP-X1.2 cells expressing STEAP1 as assessed by FACS. The MFI of antibody variants was tested at the indicated concentrations. The EC50 for vendotozumab was 3.5 nM and the EC50 for huAb44.v6.05 was 1.9 nM. FIG. 23A shows bispecific anti-STEAP1/anti-CD3 antibodies tested in a cytotoxicity assay with human CD8+ T cells and LNCaP-X1.2 cells expressing STEAP1. TDBs containing vendotozumab and huAb44v6.05 were compared. As indicated, the anti-CD3 arm of the TDB molecule is either MD1 or 40G5c. FIG. 23B shows bispecific anti-STEAP1/anti-CD3 antibodies tested in a cytotoxicity assay with human CD8+ T cells and LNCaPX1.2KO3-13 cells expressing STEAP1. TDBs containing vendotumab and huAb44v6.05 are compared. As indicated, the anti-CD3 arm of the TDB molecule is either MD1 or 40G5c. FIG. 24A to 24D are a series of graphs showing a comparison of huAb44.v6.05/40G5c and vendotumab/40G5c TDBs in a T cell activation assay. Figure 24A shows the results of CD4 T cell activation using LNCaP-X12 cells. Figure 24B shows the results of CD8 T cell activation using LNCaP-X12 cells. Figure 24C shows the results of CD4 T cell activation using LNCaP-X1.2-KO-3-13 cells. Figure 24D shows the results of CD8 T cell activation using LNCaP-X1.2-KO-3-13 cells. For each of these experiments, the ratio of PBMC to target was 10:1, and the incubation time was 24 hours. Figures 25A to 25J are a series of graphs showing a comparison of huAb44.v6.05/40G5c and vendotumumab/40G5c in a cytokine secretion assay at 24 hours. Figures 25A to 25E show the results for LNCaP-X1.2 cells, and Figures 25F to 25J show the results for LNCaP-X1.2-KO-3-13 cells. Figures 25A and 25F show interferon (IFN) γ secretion. Figures 25B and 25G show tumor necrosis factor (TNF) α secretion. Figures 25C and 25H show IL-2 secretion. Figures 25D and 25I show IL-6 secretion. Figures 25E and 25J show granzyme B secretion.

TW202417504A_112127378_SEQL.xmlTW202417504A_112127378_SEQL.xml

Claims (164)

Translated fromChinese

一種多特異性抗原結合分子，其包含：(A) 第一抗原結合域，其與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含選自 SEQ ID NO: 7、17 至 25、30 至 34、38 及 68 的 VH 序列之 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含選自 SEQ ID NO: 8、26 至 29、39 及 69 的 VL 序列之 CDR-L1、CDR-L2 及 CDR-L3；以及 (B) 第二抗原結合域，其與 T 細胞受體結合。A multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3 of a VH sequence selected from SEQ ID NOs: 7, 17 to 25, 30 to 34, 38 and 68; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3 of a VL sequence selected from SEQ ID NOs: 8, 26 to 29, 39 and 69; and (B) a second antigen-binding domain that binds to a T cell receptor.一種多特異性抗原結合分子，其包含： (A) 第一抗原結合域，其與 STEAP1 結合且包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)； 其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)； 其中 Xaa³為 Asp (D) 或 Ser (S)； Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)； Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)；且 CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列；及 (B) 第二抗原結合域，其與 T 細胞受體結合。A multispecific antigen-binding molecule comprises: (A) a first antigen-binding domain that binds to STEAP1 and comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Ser (S); Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (N); and Xaa⁶ is Ser (S) or Tyr (Y); and CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6; and (B) The second antigen binding domain, which binds to the T cell receptor.如請求項 1 或 2 之多特異性抗原結合分子，其中該 CDR-H1 包含 SEQ ID NO: 10、1 或 9 之胺基酸序列。The multispecific antigen-binding molecule of claim 1 or 2, wherein the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 10, 1 or 9.如請求項 1 至 3 中任一項之多特異性抗原結合分子，其中該 CDR-H2 包含 SEQ ID NO: 2、11、12 或 13 之胺基酸序列。The multispecific antigen-binding molecule of any one of claims 1 to 3, wherein the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 2, 11, 12 or 13.如請求項 1 至 4 中任一項之多特異性抗原結合分子，其中該 CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列。The multispecific antigen-binding molecule of any one of claims 1 to 4, wherein the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 16, 3, 14 or 15.如請求項 1 至 5 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含： CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列； CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列； CDR-H3，其包含 SEQ ID NO: 16 之胺基酸序列； CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。A multispecific antigen-binding molecule as claimed in any one of claims 1 to 5, wherein the first antigen-binding domain comprises:CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 10;CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 2;CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 16;CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 4;CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 5; andCDR-L3, which comprises the amino acid sequence of SEQ ID NO: 6.如請求項 1 至 5 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含： CDR-H1，其包含 SEQ ID NO: 1 之胺基酸序列； CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列； CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列； CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。A multispecific antigen-binding molecule as claimed in any one of claims 1 to 5, wherein the first antigen-binding domain comprises:CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 1;CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 2;CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 3;CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 4;CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 5; andCDR-L3, which comprises the amino acid sequence of SEQ ID NO: 6.如請求項 1 至 5 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含： CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列； CDR-H2，其包含 SEQ ID NO: 11 之胺基酸序列； CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列； CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。A multispecific antigen-binding molecule as claimed in any one of claims 1 to 5, wherein the first antigen-binding domain comprises:CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 9;CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 11;CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 3;CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 4;CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 5; andCDR-L3, which comprises the amino acid sequence of SEQ ID NO: 6.如請求項 1 至 5 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含： CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列； CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列； CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列； CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。A multispecific antigen-binding molecule as claimed in any one of claims 1 to 5, wherein the first antigen-binding domain comprises:CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 9;CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 12;CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 3;CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 4;CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 5; andCDR-L3, which comprises the amino acid sequence of SEQ ID NO: 6.如請求項 1 至 5 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含： CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列； CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列； CDR-H3，其包含 SEQ ID NO: 14 之胺基酸序列； CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。A multispecific antigen-binding molecule as claimed in any one of claims 1 to 5, wherein the first antigen-binding domain comprises:CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 9;CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 12;CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 14;CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 4;CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 5; andCDR-L3, which comprises the amino acid sequence of SEQ ID NO: 6.如請求項 1 至 5 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含： CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列； CDR-H2，其包含 SEQ ID NO: 13 之胺基酸序列； CDR-H3，其包含 SEQ ID NO: 15 之胺基酸序列； CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。A multispecific antigen-binding molecule as claimed in any one of claims 1 to 5, wherein the first antigen-binding domain comprises:CDR-H1 comprising an amino acid sequence of SEQ ID NO: 10;CDR-H2 comprising an amino acid sequence of SEQ ID NO: 13;CDR-H3 comprising an amino acid sequence of SEQ ID NO: 15;CDR-L1 comprising an amino acid sequence of SEQ ID NO: 4;CDR-L2 comprising an amino acid sequence of SEQ ID NO: 5; andCDR-L3 comprising an amino acid sequence of SEQ ID NO: 6.如請求項 1 至 11 中任一項之多特異性抗原結合分子，其中該多特異性抗原結合分子為人源化抗體。The multispecific antigen-binding molecule of any one of claims 1 to 11, wherein the multispecific antigen-binding molecule is a humanized antibody.如請求項 1 至 12 中任一項之多特異性抗原結合分子，其中該第一抗原結合域及該第二抗原結合域各自獨立地包含 IgG 骨架區，視情況為 IgG₁或 IgG₄骨架區。The multispecific antigen-binding molecule of any one of claims 1 to 12, wherein the first antigen-binding domain and the second antigen-binding domain each independently comprise an IgG framework region, optionally an IgG₁ or IgG₄ framework region.如請求項 1 至 13 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列。The multispecific antigen-binding molecule of any one of claims 1 to 13, wherein the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 7, 17 to 25, 30 to 34, 38 or 68.如請求項 1 至 14 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含：抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 8、26 至 29、39 或 69 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The multispecific antigen-binding molecule of any one of claims 1 to 14, wherein the first antigen-binding domain comprises: an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 8, 26 to 29, 39 or 69.如請求項 1 至 15 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 68 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 69 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The multispecific antigen-binding molecule of any one of claims 1 to 15, wherein the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 68; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 69.如請求項 1 至 15 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 34 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The multispecific antigen-binding molecule of any one of claims 1 to 15, wherein the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 34; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.如請求項 1 至 15 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 30 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The multispecific antigen-binding molecule of any one of claims 1 to 15, wherein the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 30; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.如請求項 1 至 15 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 31 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The multispecific antigen-binding molecule of any one of claims 1 to 15, wherein the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 31; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.如請求項 1 至 15 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 32 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The multispecific antigen-binding molecule of any one of claims 1 to 15, wherein the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 32; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.如請求項 1 至 15 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 33 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The multispecific antigen-binding molecule of any one of claims 1 to 15, wherein the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 33; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.如請求項 1 至 21 中任一項之多特異性抗原結合分子，其中該 T 細胞受體為分化簇 3 (CD3)。The multispecific antigen-binding molecule of any one of claims 1 to 21, wherein the T cell receptor is cluster of differentiation 3 (CD3).如請求項 22 之多特異性抗原結合分子，其中與 CD3 結合的該第二抗原結合域包含含有 CDR-H1、CDR-H2 及 CDR-H3 的抗 CD3 重鏈可變區 (VH) 及含有 CDR-L1、CDR-L2 及 CDR-L3 的抗 CD3 輕鏈可變區 (VL)，其中該 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3 分別包含 SEQ ID NO: 48 至 53 之序列。The multispecific antigen-binding molecule of claim 22, wherein the second antigen-binding domain that binds to CD3 comprises an anti-CD3 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3 and an anti-CD3 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 48 to 53, respectively.如請求項 23 之多特異性抗原結合分子，其中與 CD3 結合的該第二抗原結合域包含：抗 CD3 重鏈可變區，其包含與 SEQ ID NO: 54 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及與 SEQ ID NO: 55 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The multispecific antigen-binding molecule of claim 23, wherein the second antigen-binding domain that binds to CD3 comprises: an anti-CD3 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 54; and a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 55.如請求項 22 之多特異性抗原結合分子，其中與 CD3 結合的該第二抗原結合域包含含有 CDR-H1、CDR-H2 及 CDR-H3 的抗 CD3 重鏈可變區 (VH) 及含有 CDR-L1、CDR-L2 及 CDR-L3 的抗 CD3 輕鏈可變區 (VL)，其中該 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3 分別包含 SEQ ID NO: 40 至 45 之序列。The multispecific antigen-binding molecule of claim 22, wherein the second antigen-binding domain that binds to CD3 comprises an anti-CD3 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3 and an anti-CD3 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 40 to 45, respectively.如請求項 25 之多特異性抗原結合分子，其中與 CD3 結合的該第二抗原結合域包含：抗 CD3 重鏈可變區，其包含與 SEQ ID NO: 46 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 CD3 輕鏈可變區，其包含與 SEQ ID NO: 47 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The multispecific antigen-binding molecule of claim 25, wherein the second antigen-binding domain that binds to CD3 comprises: an anti-CD3 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 46; and an anti-CD3 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 47.如請求項 1 至 26 中任一項之多特異性抗原結合分子，其中該多特異性抗原結合分子包含一個或多個重鏈恆定域，其中該一個或多個重鏈恆定域係選自第一 CH1 (CH1₁) 域、第一 CH2 (CH2₁) 域、第一 CH3 (CH3₁) 域、第二 CH1 (CH1₂) 域、第二 CH2 (CH2₂) 域及第二 CH3 (CH3₂) 域。A multispecific antigen-binding molecule as claimed in any one of claims 1 to 26, wherein the multispecific antigen-binding molecule comprises one or more heavy chain constant domains, wherein the one or more heavy chain constant domains are selected from a first CH1 (CH1₁ ) domain, a first CH2 (CH2₁ ) domain, a first CH3 (CH3₁ ) domain, a second CH1 (CH1₂ ) domain, a second CH2 (CH2₂ ) domain and a second CH3 (CH3₂ ) domain.如請求項 27 之多特異性抗原結合分子，其中該一個或多個重鏈恆定域中之至少一者與另一重鏈恆定域配對。A multispecific antigen-binding molecule as claimed in claim 27, wherein at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain.如請求項 28 之多特異性抗原結合分子，其中該 CH3₁域及該 CH3₂域各包含隆凸或腔窩，且其中該 CH3₁域中的該隆凸或腔窩分別可定位於該 CH3₂域中的該腔窩或隆凸中。The multispecific antigen-binding molecule of claim 28, wherein the_CH31 domain and the_CH32 domain each comprise a protuberance or a cavity, and wherein the protuberance or the cavity in the_CH31 domain can be positioned in the cavity or the protuberance in the_CH32 domain, respectively.如請求項 29 之多特異性抗原結合分子，其中該 CH3₁域及該 CH3₂域在該隆凸與腔窩之間的界面處相接。The multispecific antigen-binding molecule of claim 29, wherein the_CH31 domain and the_CH32 domain are connected at the interface between the protuberance and the cavity.如請求項 27 至 30 中任一項之多特異性抗原結合分子，其中該 CH2₁域及該 CH2₂域各包含隆凸或腔窩，且其中該 CH2₁域中的該隆凸或腔窩分別可定位於該 CH2₂域中的該腔窩或隆凸中。A multispecific antigen-binding molecule according to any one of claims 27 to 30, wherein the_CH21 domain and the_CH22 domain each comprise a protuberance or a cavity, and wherein the protuberance or the cavity in the_CH21 domain can be positioned in the cavity or the protuberance in the_CH22 domain, respectively.如請求項 31 之多特異性抗原結合分子，其中該 CH2₁域及該 CH2₂域在該隆凸與腔窩之間的界面處相接。The multispecific antigen-binding molecule of claim 31, wherein the_CH21 domain and the_CH22 domain are connected at the interface between the protuberance and the cavity.如請求項 22 及 27 至 32 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含與 SEQ ID NO: 83 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 重鏈恆定區序列及與 SEQ ID NO: 82 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 輕鏈恆定區序列；且其中該第二抗原結合域包含與 SEQ ID NO: 85 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 CD3 重鏈恆定區序列及與 SEQ ID NO: 84 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 CD3 輕鏈恆定區序列。The multispecific antigen-binding molecule of any one of claims 22 and 27 to 32, wherein the first antigen-binding domain comprises an anti-STEAP1 heavy chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 83 and an anti-STEAP1 light chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 82; and wherein the second antigen-binding domain comprises an anti-CD3 heavy chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 85 and an anti-CD3 light chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 84. Light chain constant region sequence.如請求項 22 及 27 至 32 中任一項之多特異性抗原結合分子，其中該第一抗原結合域包含與 SEQ ID NO: 85 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 重鏈恆定區序列及與 SEQ ID NO: 84 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 輕鏈恆定區序列；且其中該第二抗原結合域包含與 SEQ ID NO: 83 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 CD3 重鏈恆定區序列及與 SEQ ID NO: 82 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 CD3 輕鏈恆定區序列。The multispecific antigen-binding molecule of any one of claims 22 and 27 to 32, wherein the first antigen-binding domain comprises an anti-STEAP1 heavy chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 85 and an anti-STEAP1 light chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 84; and wherein the second antigen-binding domain comprises an anti-CD3 heavy chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 83 and an anti-CD3 light chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 82. Light chain constant region sequence.如請求項 22 及 27 至 32 中任一項之多特異性抗原結合分子，其中該多特異性抗原結合分子包含含有 SEQ ID NO: 73 的抗 STEAP1 重鏈、含有 SEQ ID NO: 72 的抗 STEAP1 輕鏈、含有 SEQ ID NO: 81 的抗 CD3 重鏈及含有 SEQ ID NO: 80 的抗 CD3 輕鏈。The multispecific antigen-binding molecule of any one of claims 22 and 27 to 32, wherein the multispecific antigen-binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 73, an anti-STEAP1 light chain comprising SEQ ID NO: 72, an anti-CD3 heavy chain comprising SEQ ID NO: 81, and an anti-CD3 light chain comprising SEQ ID NO: 80.如請求項 22 及 27 至 32 中任一項之多特異性抗原結合分子，其中該多特異性抗原結合分子包含含有 SEQ ID NO: 73 的抗 STEAP1 重鏈、含有 SEQ ID NO: 72 的抗 STEAP1 輕鏈、含有 SEQ ID NO: 79 的抗 CD3 重鏈及含有 SEQ ID NO: 78 的抗 CD3 輕鏈。The multispecific antigen-binding molecule of any one of claims 22 and 27 to 32, wherein the multispecific antigen-binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 73, an anti-STEAP1 light chain comprising SEQ ID NO: 72, an anti-CD3 heavy chain comprising SEQ ID NO: 79, and an anti-CD3 light chain comprising SEQ ID NO: 78.如請求項 1 至 36 中任一項之多特異性抗原結合分子，其中該抗原結合分子為單鏈 Fv (scFv)、三特異性 (Fab₃)、雙特異性 (Fab₂)、雙功能抗體 (diabody) ((V_L-V_H)₂或 (V_H-V_L)₂)、三功能抗體 (triabody) (三價)、四功能抗體 (tetrabody) (四價)、微抗體 (minibody) ((scF_V-C_H)₂)、雙特異性單鏈 Fv (雙-scFv)、IgGδCH2、scFv-Fc 或 (scFv)₂-Fc。The multispecific antigen-binding molecule of any one of claims 1 to 36, wherein the antigen-binding molecule is a single-chain Fv (scFv), a trispecific (Fab₃ ), a bispecific (Fab₂ ), a diabody ((V_L -V_H )₂ or (V_H -V_L )₂ ), a triabody (trivalent), a tetrabody (tetravalent), a minibody ((scF_V-CH )₂ ), a bispecific single-chain Fv (bi-scFv), IgGδCH2, scFv-Fc or (scFv)₂ -Fc.如請求項 1 至 37 中任一項之多特異性抗原結合分子，其中該多特異性抗原結合分子為多特異性抗體，較佳為雙特異性或三特異性抗體。A multispecific antigen-binding molecule according to any one of claims 1 to 37, wherein the multispecific antigen-binding molecule is a multispecific antibody, preferably a bispecific or trispecific antibody.如請求項 1 至 38 中任一項之多特異性抗原結合分子，其中該多特異性抗原結合分子進一步包含與腫瘤相關抗原結合的第三抗原結合域。A multispecific antigen-binding molecule as claimed in any one of claims 1 to 38, wherein the multispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to a tumor-associated antigen.如請求項 39 之多特異性抗原結合分子，其中該腫瘤相關抗原為在前列腺癌細胞或尤文氏肉瘤 (Ewing sarcoma) 上表現的受體。The multispecific antigen-binding molecule of claim 39, wherein the tumor-associated antigen is a receptor expressed on prostate cancer cells or Ewing sarcoma.如請求項 39 或 40 之多特異性抗原結合分子，其中該腫瘤相關抗原為前列腺特異性膜抗原 (PSMA)、STEAP2、前列腺幹細胞抗原 (PSCA)、上皮細胞黏著分子 (EpCAM)、前列腺特異性抗原 (PSA)、前列腺酸性磷酸酶 (PAP) 或 HBA-71。The multispecific antigen binding molecule of claim 39 or 40, wherein the tumor-associated antigen is prostate-specific membrane antigen (PSMA), STEAP2, prostate stem cell antigen (PSCA), epithelial cell adhesion molecule (EpCAM), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), or HBA-71.如請求項 1 至 41 中任一項之多特異性抗原結合分子，其中該多特異性抗原結合分子與人 STEAP1、石蟹獼猴 STEAP1 或其組合結合，較佳地其中該多特異性抗原結合分子與 SEQ ID NO: 65 之人 STEAP1 結合。The multispecific antigen-binding molecule of any one of claims 1 to 41, wherein the multispecific antigen-binding molecule binds to human STEAP1, red macaque STEAP1 or a combination thereof, preferably wherein the multispecific antigen-binding molecule binds to human STEAP1 of SEQ ID NO: 65.如請求項 1 至 42 中任一項之多特異性抗原結合分子，其中該第一抗原結合域與選自 STEAP1 之 Ser101、His102、Gln103 及 Lys281 的至少一個殘基結合，其中殘基位置 101、102、103 及 281 對應於 SEQ ID NO: 65 中所示之位置 101、102、103 及 281。The multispecific antigen-binding molecule of any one of claims 1 to 42, wherein the first antigen-binding domain binds to at least one residue selected from Ser101, His102, Gln103 and Lys281 of STEAP1, wherein residue positions 101, 102, 103 and 281 correspond to positions 101, 102, 103 and 281 shown in SEQ ID NO: 65.如請求項 1 至 43 中任一項之多特異性抗原結合分子，其中該第一抗原結合域與 STEAP1 之 Ser101、His102、Gln103 及 Lys281 結合。The multispecific antigen-binding molecule of any one of claims 1 to 43, wherein the first antigen-binding domain binds to Ser101, His102, Gln103 and Lys281 of STEAP1.如請求項 1 至 44 中任一項之多特異性抗原結合分子，其中該多特異性抗原結合分子具有約 11、11.5、12、12.6、13、13.5、15、18、20、23.4、25、27.9、29.1 或 30 µg/mL 之 C_max。The multispecific antigen-binding molecule of any one of claims 1 to 44, wherein the multispecific antigen-binding molecule has a C_max of about 11, 11.5, 12, 12.6, 13, 13.5, 15, 18, 20, 23.4, 25, 27.9, 29.1 or 30 µg/mL.如請求項 1 至 44 中任一項之多特異性抗原結合分子，其中該多特異性抗原結合分子具有約 11 µg/mL 至約 30 µg/mL 之 C_max。The multispecific antigen-binding molecule of any one of claims 1 to 44, wherein the multispecific antigen-binding molecule has a C_max of about 11 µg/mL to about 30 µg/mL.如請求項 46 之多特異性抗原結合分子，其中該多特異性抗原結合分子具有約 20 µg/mL 至約 28 µg/mL 之 C_max。The multispecific antigen-binding molecule of claim 46, wherein the multispecific antigen-binding molecule has a C_max of about 20 µg/mL to about 28 µg/mL.如請求項 47 之多特異性抗原結合分子，其中該多特異性抗原結合分子具有約 24 µg/mL 之 C_max。The multispecific antigen-binding molecule of claim 47, wherein the multispecific antigen-binding molecule has a C_max of about 24 µg/mL.如請求項 1 至 48 中任一項之多特異性抗原結合分子，其中該多特異性抗原結合分子具有約 0.6、0.56、0.5、0.45、0.4、0.35、0.3、0.25、0.2、0.15、0.1、0.09、0.05 或更低之 EC₅₀。The multispecific antigen-binding molecule of any one of claims 1 to 48, wherein the multispecific antigen-binding molecule has an EC₅₀ of about 0.6, 0.56, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.09, 0.05 or less.如請求項 1 至 48 中任一項之多特異性抗原結合分子，其中該多特異性抗原結合分子具有約 0.05 至約 0.8 之 EC₅₀。The multispecific antigen-binding molecule of any one of claims 1 to 48, wherein the multispecific antigen-binding molecule has an EC₅₀ of about 0.05 to about 0.8.如請求項 50 之多特異性抗原結合分子，其中該 EC₅₀係用人 CD8+ T 細胞及表現 STEAP1 之 LNCaP-X1.2 細胞在細胞毒殺測定中在 72 小時所判定，且該 EC₅₀為約 0.05 至約 0.4。The multispecific antigen-binding molecule of claim 50, wherein the_EC50 is determined in a cytotoxicity assay using human CD8+ T cells and LNCaP-X1.2 cells expressing STEAP1 at 72 hours, and the_EC50 is about 0.05 to about 0.4.如請求項 51 之多特異性抗原結合分子，其中該 EC₅₀為約 0.08 或約 0.3。The multispecific antigen-binding molecule of claim 51, wherein the_EC50 is about 0.08 or about 0.3.如請求項 50 之多特異性抗原結合分子，其中該 EC₅₀係用人 CD8+ T 細胞及表現 STEAP1 之 LNCaPX1.2KO3-13 細胞在細胞毒殺測定中在 72 小時所判定，且該 EC₅₀為約 0.1 至約 0.8。The multispecific antigen-binding molecule of claim 50, wherein the_EC50 is determined in a cytotoxicity assay using human CD8+ T cells and LNCaPX1.2KO3-13 cells expressing STEAP1 at 72 hours, and the_EC50 is about 0.1 to about 0.8.如請求項 53 之多特異性抗原結合分子，其中該 EC₅₀為約 0.1 或約 0.7。The multispecific antigen-binding molecule of claim 53, wherein the EC₅₀ is about 0.1 or about 0.7.一種與 STEAP1 結合的抗原結合分子，其包含：抗 STEAP1 重鏈可變區 (VH)，其包含選自 SEQ ID NO: 7、17 至 25、30 至 34、38 及 68 的 VH 序列之 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含選自 SEQ ID NO: 8、26 至 29、39 及 69 的 VL 序列之 CDR-L1、CDR-L2 及 CDR-L3。An antigen-binding molecule that binds to STEAP1 comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3 of a VH sequence selected from SEQ ID NOs: 7, 17 to 25, 30 to 34, 38 and 68; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3 of a VL sequence selected from SEQ ID NOs: 8, 26 to 29, 39 and 69.一種與 STEAP1 結合的抗原結合分子，其包含：抗 STEAP1 重鏈可變區 (VH)，其包含 CDR-H1、CDR-H2 及 CDR-H3；及抗 STEAP1 輕鏈可變區 (VL)，其包含 CDR-L1、CDR-L2 及 CDR-L3； 其中 CDR-H1 包含 Xaa¹Xaa²YMA (SEQ ID NO: 35)； 其中 Xaa¹為 Asp (D) 或 Asn (N)；且 Xaa²為 His (H)、Tyr (Y) 或 Phe (F)； CDR-H2 包含 YIXaa³YDGXaa⁴Xaa⁵TXaa⁶YGDSVKG (SEQ ID NO: 36)； 其中 Xaa³為 Asp (D) 或 Ser (S)； Xaa⁴為 Gly (G)、Asp (D) 或 Leu (L)； Xaa⁵為 Ser (S)、Asp (D) 或 Asn (N)；且 Xaa⁶為 Ser (S) 或 Tyr (Y)； CDR-H3 包含 RSGXaa⁷YHVGYAMXaa⁸Xaa⁹(SEQ ID NO: 37)； 其中 Xaa⁷為 Phe (F) 或 Tyr (Y)； Xaa⁸為 Asn (N) 或 Asp (D)；且 Xaa⁹為 Ala (A) 或 Gly (G)； CDR-L1 包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2 包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3 包含 SEQ ID NO: 6 之胺基酸序列。An antigen-binding molecule that binds to STEAP1, comprising: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3; and an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3; wherein CDR-H1 comprises Xaa¹ Xaa² YMA (SEQ ID NO: 35); wherein Xaa¹ is Asp (D) or Asn (N); and Xaa² is His (H), Tyr (Y) or Phe (F); CDR-H2 comprises YIXaa³ YDGXaa⁴ Xaa⁵ TXaa⁶ YGDSVKG (SEQ ID NO: 36); wherein Xaa³ is Asp (D) or Ser (S); and Xaa⁴ is Gly (G), Asp (D) or Leu (L); Xaa⁵ is Ser (S), Asp (D) or Asn (N); and Xaa⁶ is Ser (S) or Tyr (Y); CDR-H3 comprises RSGXaa⁷ YHVGYAMXaa⁸ Xaa⁹ (SEQ ID NO: 37); wherein Xaa⁷ is Phe (F) or Tyr (Y); Xaa⁸ is Asn (N) or Asp (D); and Xaa⁹ is Ala (A) or Gly (G); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 5; and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6.如請求項 55 或 56 之抗原結合分子，其中該 CDR-H1 包含 SEQ ID NO: 10、1 或 9 之胺基酸序列。The antigen-binding molecule of claim 55 or 56, wherein the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 10, 1 or 9.如請求項 55 至 57 中任一項之抗原結合分子，其中該 CDR-H2 包含 SEQ ID NO: 2、11、12 或 13 之胺基酸序列。The antigen-binding molecule of any one of claims 55 to 57, wherein the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 2, 11, 12 or 13.如請求項 55 至 58 中任一項之抗原結合分子，其中該 CDR-H3 包含 SEQ ID NO: 16、3、14 或 15 之胺基酸序列。The antigen-binding molecule of any one of claims 55 to 58, wherein the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 16, 3, 14 or 15.如請求項 55 至 59 中任一項之抗原結合分子，其中該抗原結合分子包含： CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列； CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列； CDR-H3，其包含 SEQ ID NO: 16 之胺基酸序列； CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。An antigen-binding molecule as claimed in any one of claims 55 to 59, wherein the antigen-binding molecule comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 10; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 2; CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 16; CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 6.如請求項 55 至 59 中任一項之抗原結合分子，其中該抗原結合分子包含： CDR-H1，其包含 SEQ ID NO: 1 之胺基酸序列； CDR-H2，其包含 SEQ ID NO: 2 之胺基酸序列； CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列； CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。An antigen-binding molecule as claimed in any one of claims 55 to 59, wherein the antigen-binding molecule comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 1; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 2; CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 6.如請求項 55 至 59 中任一項之抗原結合分子，其中該抗原結合分子包含： CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列； CDR-H2，其包含 SEQ ID NO: 11 之胺基酸序列； CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列； CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。An antigen binding molecule as claimed in any one of claims 55 to 59, wherein the antigen binding molecule comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 9; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 11; CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 6.如請求項 55 至 59 中任一項之抗原結合分子，其中該抗原結合分子包含： CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列； CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列； CDR-H3，其包含 SEQ ID NO: 3 之胺基酸序列； CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。An antigen-binding molecule as claimed in any one of claims 55 to 59, wherein the antigen-binding molecule comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 9; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 12; CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 3; CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 6.如請求項 55 至 59 中任一項之抗原結合分子，其中該抗原結合分子包含： CDR-H1，其包含 SEQ ID NO: 9 之胺基酸序列； CDR-H2，其包含 SEQ ID NO: 12 之胺基酸序列； CDR-H3，其包含 SEQ ID NO: 14 之胺基酸序列； CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。An antigen-binding molecule as claimed in any one of claims 55 to 59, wherein the antigen-binding molecule comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 9; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 12; CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 14; CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 4; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 5; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 6.如請求項 55 至 59 中任一項之抗原結合分子，其中該抗原結合分子包含： CDR-H1，其包含 SEQ ID NO: 10 之胺基酸序列； CDR-H2，其包含 SEQ ID NO: 13 之胺基酸序列； CDR-H3，其包含 SEQ ID NO: 15 之胺基酸序列； CDR-L1，其包含 SEQ ID NO: 4 之胺基酸序列； CDR-L2，其包含 SEQ ID NO: 5 之胺基酸序列；且 CDR-L3，其包含 SEQ ID NO: 6 之胺基酸序列。An antigen-binding molecule as claimed in any one of claims 55 to 59, wherein the antigen-binding molecule comprises:CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 10;CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 13;CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 15;CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 4;CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 5; andCDR-L3, which comprises the amino acid sequence of SEQ ID NO: 6.如請求項 55 至 65 中任一項之抗原結合分子，其中該抗原結合分子為人源化抗體。An antigen-binding molecule as claimed in any one of claims 55 to 65, wherein the antigen-binding molecule is a humanized antibody.如請求項 55 至 66 中任一項之抗原結合分子，其中該抗原結合分子包含 IgG 骨架區，視情況為 IgG₁骨架區。An antigen binding molecule as claimed in any one of claims 55 to 66, wherein the antigen binding molecule comprises an IgG framework region, optionally an_IgG1 framework region.如請求項 55 至 67 中任一項之抗原結合分子，其包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列。An antigen binding molecule as claimed in any one of claims 55 to 67, comprising: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 7, 17 to 25, 30 to 34, 38 or 68.如請求項 55 至 68 中任一項之抗原結合分子，其包含：抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 8、26 至 29、39 或 69 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。An antigen binding molecule as claimed in any one of claims 55 to 68, comprising: an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 8, 26 to 29, 39 or 69.如請求項 55 至 69 中任一項之抗原結合分子，其包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 68 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 69 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The antigen-binding molecule of any one of claims 55 to 69, comprising: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 68; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 69.如請求項 55 至 69 中任一項之抗原結合分子，其包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 34 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The antigen-binding molecule of any one of claims 55 to 69, comprising: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 34; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.如請求項 55 至 69 中任一項之抗原結合分子，其包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 30 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The antigen-binding molecule of any one of claims 55 to 69, comprising: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 30; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.如請求項 55 至 69 中任一項之抗原結合分子，其包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 31 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The antigen-binding molecule of any one of claims 55 to 69, comprising: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 31; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.如請求項 55 至 69 中任一項之抗原結合分子，其包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 32 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The antigen-binding molecule of any one of claims 55 to 69, comprising: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 32; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.如請求項 55 至 69 中任一項之抗原結合分子，其包含：抗 STEAP1 重鏈可變區，其包含與 SEQ ID NO: 33 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 STEAP1 輕鏈可變區，其包含與 SEQ ID NO: 27 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The antigen-binding molecule of any one of claims 55 to 69, comprising: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 33; and an anti-STEAP1 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 27.如請求項 55 至 75 中任一項之抗原結合分子，其中該抗原結合分子包含與 SEQ ID NO: 83 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 重鏈恆定區序列及與 SEQ ID NO: 82 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 輕鏈恆定區序列；或其中該抗原結合分子包含與 SEQ ID NO: 85 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 重鏈恆定區序列及與 SEQ ID NO: 84 具有至少 80%、85%、90%、95% 或 100% 序列同一性的抗 STEAP1 輕鏈恆定區序列。The antigen binding molecule of any one of claims 55 to 75, wherein the antigen binding molecule comprises an anti-STEAP1 heavy chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 83 and an anti-STEAP1 light chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 82; or wherein the antigen binding molecule comprises an anti-STEAP1 heavy chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 85 and an anti-STEAP1 light chain constant region sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 84.如請求項 76 之抗原結合分子，其中該抗原結合分子包含含有 SEQ ID NO: 73 的抗 STEAP1 重鏈及含有 SEQ ID NO: 72 的抗 STEAP1 輕鏈。The antigen-binding molecule of claim 76, wherein the antigen-binding molecule comprises an anti-STEAP1 heavy chain comprising SEQ ID NO: 73 and an anti-STEAP1 light chain comprising SEQ ID NO: 72.如請求項 55 至 77 中任一項之抗原結合分子，其中該抗原結合分子為全長抗體或其片段。An antigen-binding molecule as claimed in any one of claims 55 to 77, wherein the antigen-binding molecule is a full-length antibody or a fragment thereof.如請求項 55 至 78 中任一項之抗原結合分子，其中該抗原結合分子為 Fab、Fab'、F(ab')₂、Fv、Fd、單鏈 Fv (scFv)、三特異性 (Fab₃)、雙特異性 (Fab₂)、雙功能抗體 ((V_L-V_H)₂或 (V_H-V_L)₂)、三功能抗體 (三價)、四功能抗體 (四價)、微抗體 ((scF_V-C_H)₂)、雙特異性單鏈 Fv (雙-scFv)、IgGδCH2、scFv-Fc 或 (scFv)₂-Fc。The antigen-binding molecule of any one of claims 55 to 78, wherein the antigen-binding molecule is Fab, Fab', F(ab')₂ , Fv, Fd, single-chain Fv (scFv), trispecificity (Fab₃ ), bispecificity (Fab₂ ), bifunctional antibody ((_VL -_VH )₂ or (_VH -_VL )₂ ), trifunctional antibody (trivalent), tetrafunctional antibody (tetravalent), miniantibody ((_scFV -_CH )₂ ), bispecific single-chain Fv (bi-scFv), IgGδCH2, scFv-Fc or (scFv)₂ -Fc.如請求項 55 至 79 中任一項之抗原結合分子，其中該抗原結合分子進一步包含與 T 細胞受體結合的抗原結合域。An antigen binding molecule as claimed in any one of claims 55 to 79, wherein the antigen binding molecule further comprises an antigen binding domain that binds to a T cell receptor.如請求項 55 至 80 中任一項之抗原結合分子，其中該抗原結合分子為多特異性抗體，較佳地其中該抗原結合分子為雙特異性抗體或三特異性抗體。An antigen-binding molecule as claimed in any one of claims 55 to 80, wherein the antigen-binding molecule is a multispecific antibody, preferably wherein the antigen-binding molecule is a bispecific antibody or a trispecific antibody.如請求項 80 或 81 之抗原結合分子，其中該 T 細胞受體為分化簇 3 (CD3)。The antigen binding molecule of claim 80 or 81, wherein the T cell receptor is cluster of differentiation 3 (CD3).如請求項 82 之抗原結合分子，其中與 CD3 結合的該抗原結合域包含含有 CDR-H1、CDR-H2 及 CDR-H3 的抗 CD3 重鏈可變區 (VH) 及含有 CDR-L1、CDR-L2 及 CDR-L3 的抗 CD3 輕鏈可變區 (VL)，其中 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3 分別包含 SEQ ID NO: 48 至 53 之胺基酸序列。The antigen-binding molecule of claim 82, wherein the antigen-binding domain that binds to CD3 comprises an anti-CD3 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3, and an anti-CD3 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 respectively comprise the amino acid sequences of SEQ ID NOs: 48 to 53.如請求項 83 之抗原結合分子，其中與 CD3 結合的該抗原結合域包含：抗 CD3 重鏈可變區，其包含與 SEQ ID NO: 54 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 CD3 輕鏈可變區，其包含與 SEQ ID NO: 55 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。An antigen-binding molecule as claimed in claim 83, wherein the antigen-binding domain that binds to CD3 comprises: an anti-CD3 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO: 54; and an anti-CD3 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO: 55.如請求項 82 之抗原結合分子，其中與 CD3 結合的該抗原結合域包含含有 CDR-H1、CDR-H2 及 CDR-H3 的抗 CD3 重鏈可變區 (VH) 及含有 CDR-L1、CDR-L2 及 CDR-L3 的抗 CD3 輕鏈可變區 (VL)，其中 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3 分別包含 SEQ ID NO: 40 至 45 之胺基酸序列。The antigen-binding molecule of claim 82, wherein the antigen-binding domain that binds to CD3 comprises an anti-CD3 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3, and an anti-CD3 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 respectively comprise the amino acid sequences of SEQ ID NOs: 40 to 45.如請求項 83 之抗原結合分子，其中與 CD3 結合的該抗原結合域包含：抗 CD3 重鏈可變區，其包含與 SEQ ID NO: 46 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 CD3 輕鏈可變區，其包含與 SEQ ID NO: 47 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。An antigen-binding molecule as claimed in claim 83, wherein the antigen-binding domain that binds to CD3 comprises: an anti-CD3 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO: 46; and an anti-CD3 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO: 47.如請求項 82 之抗原結合分子，其中該抗原結合分子包含含有 SEQ ID NO: 79 的抗 CD3 重鏈及含有 SEQ ID NO: 78 的抗 CD3 輕鏈。The antigen-binding molecule of claim 82, wherein the antigen-binding molecule comprises an anti-CD3 heavy chain comprising SEQ ID NO: 79 and an anti-CD3 light chain comprising SEQ ID NO: 78.如請求項 82 之抗原結合分子，其中該抗原結合分子包含含有 SEQ ID NO: 81 的抗 CD3 重鏈及含有 SEQ ID NO: 80 的抗 CD3 輕鏈。The antigen-binding molecule of claim 82, wherein the antigen-binding molecule comprises an anti-CD3 heavy chain comprising SEQ ID NO: 81 and an anti-CD3 light chain comprising SEQ ID NO: 80.如請求項 55 至 88 中任一項之抗原結合分子，其中該抗原結合分子進一步包含與腫瘤相關抗原結合的額外抗原結合域。An antigen binding molecule as in any one of claims 55 to 88, wherein the antigen binding molecule further comprises an additional antigen binding domain that binds to a tumor-associated antigen.如請求項 89 之抗原結合分子，其中該腫瘤相關抗原為在前列腺癌細胞或尤文氏肉瘤上表現的受體。The antigen-binding molecule of claim 89, wherein the tumor-associated antigen is a receptor expressed on prostate cancer cells or Ewing's sarcoma.如請求項 89 或 90 之抗原結合分子，其中該腫瘤相關抗原為前列腺特異性膜抗原 (PSMA)、STEAP2、前列腺幹細胞抗原 (PSCA)、上皮細胞黏著分子 (EpCAM)、前列腺特異性抗原 (PSA)、前列腺酸性磷酸酶 (PAP) 或 HBA-71。The antigen-binding molecule of claim 89 or 90, wherein the tumor-associated antigen is prostate-specific membrane antigen (PSMA), STEAP2, prostate stem cell antigen (PSCA), epithelial cell adhesion molecule (EpCAM), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), or HBA-71.如請求項 55 至 91 中任一項之抗原結合分子，其中該抗原結合分子與人 STEAP1、石蟹獼猴 STEAP1 或其組合結合，較佳地其中該抗原結合分子與 SEQ ID NO: 65 之人 STEAP1 結合。The antigen binding molecule of any one of claims 55 to 91, wherein the antigen binding molecule binds to human STEAP1, macaque STEAP1 or a combination thereof, preferably wherein the antigen binding molecule binds to human STEAP1 of SEQ ID NO: 65.如請求項 55 至 92 中任一項之抗原結合分子，其中該抗原結合分子與選自 STEAP1 之 Ser101、His102、Gln103 及 Lys281 的至少一個殘基結合，其中殘基位置 101、102、103 及 281 對應於 SEQ ID NO: 65 中所示之位置 101、102、103 及 281。The antigen-binding molecule of any one of claims 55 to 92, wherein the antigen-binding molecule binds to at least one residue selected from Ser101, His102, Gln103 and Lys281 of STEAP1, wherein residue positions 101, 102, 103 and 281 correspond to positions 101, 102, 103 and 281 shown in SEQ ID NO: 65.如請求項 55 至 93 中任一項之抗原結合分子，其中該抗原結合分子與 SEQ ID NO: 65 之 Ser101、His102、Gln103 及 Lys281 結合。The antigen-binding molecule of any one of claims 55 to 93, wherein the antigen-binding molecule binds to Ser101, His102, Gln103 and Lys281 of SEQ ID NO: 65.一種抗體，其包含在選自 Ser101、His102、Gln103 及 Lys281 的一個或多個殘基處與人 STEAP1 結合的第一抗原結合域，其中殘基位置 101、102、103 及 281 對應於 SEQ ID NO: 65 中所示之位置 101、102、103 及 281。An antibody comprising a first antigen-binding domain that binds to human STEAP1 at one or more residues selected from Ser101, His102, Gln103 and Lys281, wherein residue positions 101, 102, 103 and 281 correspond to positions 101, 102, 103 and 281 shown in SEQ ID NO: 65.如請求項 95 之抗體，其中該第一抗原結合域與 SEQ ID NO: 65 之 Ser101、His102、Gln103 及 Lys281 結合。The antibody of claim 95, wherein the first antigen-binding domain binds to Ser101, His102, Gln103 and Lys281 of SEQ ID NO: 65.如請求項 95 或 96 之抗體，其中該第一抗原結合域之重鏈可變區與選自 Ser101、His102、Gln103 及 Lys281 的至少一個殘基形成氫鍵。The antibody of claim 95 or 96, wherein the heavy chain variable region of the first antigen-binding domain forms a hydrogen bond with at least one residue selected from Ser101, His102, Gln103 and Lys281.如請求項 95 至 97 中任一項之抗體，其中該第一抗原結合域之該重鏈可變區的 Leu56、Ser73 及 Asn74 與 STEAP1 之 Ser101、His102、Gln103 及 Lys281 形成氫鍵，其中該重鏈可變區之殘基位置對應於 SEQ ID NO: 18 中所示之位置 56、73 及 74。The antibody of any one of claims 95 to 97, wherein Leu56, Ser73 and Asn74 of the heavy chain variable region of the first antigen binding domain form hydrogen bonds with Ser101, His102, Gln103 and Lys281 of STEAP1, wherein the residue positions of the heavy chain variable region correspond to positions 56, 73 and 74 shown in SEQ ID NO: 18.如請求項 95 至 98 中任一項之抗體，其中該第一抗原結合域包含：抗 STEAP1 重鏈可變區 (VH)，其包含選自 SEQ ID NO: 7、17 至 25、30 至 34、38 及 68 的 VH 序列之 CDR-H1、CDR-H2 及 CDR-H3；以及抗 STEAP1 輕鏈可變區 (VL) 之三個 CDR，其包含選自 SEQ ID NO: 8、26 至 29、39 及 69 的 VL 序列之 CDR-L1、CDR-L2 及 CDR-L3。The antibody of any one of claims 95 to 98, wherein the first antigen-binding domain comprises: an anti-STEAP1 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3 of a VH sequence selected from SEQ ID NOs: 7, 17 to 25, 30 to 34, 38 and 68; and three CDRs of an anti-STEAP1 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3 of a VL sequence selected from SEQ ID NOs: 8, 26 to 29, 39 and 69.如請求項 95 至 99 中任一項之抗體，其中該第一抗原結合域包含：抗 STEAP1 重鏈可變區，其包含含有與 SEQ ID NO: 7、17 至 25、30 至 34、38 或 68 至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列。The antibody of any one of claims 95 to 99, wherein the first antigen binding domain comprises: an anti-STEAP1 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 7, 17 to 25, 30 to 34, 38 or 68.如請求項 95 至 100 中任一項之抗體，其中該第一抗原結合域包含抗 STEAP1 輕鏈可變區，其包含含有與 SEQ ID NO: 8、26 至 29、39 或 69 至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The antibody of any one of claims 95 to 100, wherein the first antigen-binding domain comprises an anti-STEAP1 light chain variable region comprising a VL sequence comprising at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 8, 26 to 29, 39 or 69.如請求項 95 至 101 中任一項之抗體，其中該抗體進一步包含與 T 細胞受體結合的第二抗原結合域。The antibody of any one of claims 95 to 101, wherein the antibody further comprises a second antigen binding domain that binds to a T cell receptor.如請求項 102 之抗體，其中該 T 細胞受體為分化簇 3 (CD3)。The antibody of claim 102, wherein the T cell receptor is cluster of differentiation 3 (CD3).如請求項 103 之抗體，其中與 CD3 結合的該第二抗原結合域包含含有 CDR-H1、CDR-H2 及 CDR-H3 的抗 CD3 重鏈可變區 (VH) 及含有 CDR-L1、CDR-L2 及 CDR-L3 的抗 CD3 輕鏈可變區 (VL)，其中該 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3 分別包含 SEQ ID NO: 48 至 53 之胺基酸序列。The antibody of claim 103, wherein the second antigen-binding domain that binds to CD3 comprises an anti-CD3 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3 and an anti-CD3 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 respectively comprise the amino acid sequences of SEQ ID NOs: 48 to 53.如請求項 104 之抗體，其中與 CD3 結合的該第二抗原結合域包含：抗 CD3 重鏈可變區，其包含與 SEQ ID NO: 54 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 CD3 輕鏈可變區，其包含與 SEQ ID NO: 55 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The antibody of claim 104, wherein the second antigen binding domain that binds to CD3 comprises: an anti-CD3 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 54; and an anti-CD3 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 55.如請求項 103 之抗體，其中與 CD3 結合的該第二抗原結合域包含含有 CDR-H1、CDR-H2 及 CDR-H3 的抗 CD3 重鏈可變區 (VH) 及含有 CDR-L1、CDR-L2 及 CDR-L3 的抗 CD3 輕鏈可變區 (VL)，其中該 CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2 及 CDR-L3 分別包含 SEQ ID NO: 40 至 45 之胺基酸序列。The antibody of claim 103, wherein the second antigen-binding domain that binds to CD3 comprises an anti-CD3 heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3 and an anti-CD3 light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 respectively comprise the amino acid sequences of SEQ ID NOs: 40 to 45.如請求項 106 之抗體，其中與 CD3 結合的該第二抗原結合域包含：抗 CD3 重鏈可變區，其包含與 SEQ ID NO: 46 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VH 序列；及抗 CD3 輕鏈可變區，其包含與 SEQ ID NO: 47 具有至少 80%、85%、90%、95% 或 100% 序列同一性的 VL 序列。The antibody of claim 106, wherein the second antigen binding domain that binds to CD3 comprises: an anti-CD3 heavy chain variable region comprising a VH sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 46; and an anti-CD3 light chain variable region comprising a VL sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 47.如請求項 95 至 107 中任一項之抗體，其中多特異性抗原結合分子進一步包含與腫瘤相關抗原結合的第三抗原結合域。The antibody of any one of claims 95 to 107, wherein the multispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to a tumor-associated antigen.如請求項 108 之抗體，其中該腫瘤相關抗原為在前列腺癌細胞上表現的受體。The antibody of claim 108, wherein the tumor-associated antigen is a receptor expressed on prostate cancer cells.如請求項 108 或 109 之抗體，其中該腫瘤相關抗原為前列腺特異性膜抗原 (PSMA)、STEAP2、前列腺幹細胞抗原 (PSCA)、上皮細胞黏著分子 (EpCAM)、前列腺特異性抗原 (PSA) 或前列腺酸性磷酸酶 (PAP)。The antibody of claim 108 or 109, wherein the tumor-associated antigen is prostate-specific membrane antigen (PSMA), STEAP2, prostate stem cell antigen (PSCA), epithelial cell adhesion molecule (EpCAM), prostate-specific antigen (PSA), or prostatic acid phosphatase (PAP).一種或多種經分離核酸，其個別或一起編碼如請求項 1 至 54 中任一項之多特異性抗原結合分子、如請求項 55 至 94 中任一項之抗原結合分子、或如請求項 95 至 110 中任一項之抗體。One or more isolated nucleic acids, which individually or together encode a multispecific antigen-binding molecule as described in any one of claims 1 to 54, an antigen-binding molecule as described in any one of claims 55 to 94, or an antibody as described in any one of claims 95 to 110.一種或多種載體，其個別或一起包含如請求項 111 之經分離核酸。One or more vectors, which individually or together contain the isolated nucleic acid of claim 111.一種或多種宿主細胞，其個別或一起包含如請求項 111 之經分離核酸或如請求項 112 之載體。One or more host cells, which individually or collectively contain the isolated nucleic acid of claim 111 or the vector of claim 112.如請求項 113 之宿主細胞，其中該宿主細胞為哺乳動物細胞。The host cell of claim 113, wherein the host cell is a mammalian cell.如請求項 114 之宿主細胞，其中該哺乳動物細胞為中國倉鼠卵巢 (CHO) 細胞。The host cell of claim 114, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.如請求項 113 之宿主細胞，其中該宿主細胞為昆蟲細胞。The host cell of claim 113, wherein the host cell is an insect cell.如請求項 113 之宿主細胞，其中該宿主細胞為原核細胞。The host cell of claim 113, wherein the host cell is a prokaryotic cell.一種生產如請求項 1 至 54 中任一項之多特異性抗原結合分子、如請求項 55 至 94 中任一項之抗原結合分子、或如請求項 95 至 110 中任一項之抗體的方法，其包含在培養基中培養如請求項 113 至 117 中任一項之宿主細胞。A method for producing a multispecific antigen-binding molecule of any one of claims 1 to 54, an antigen-binding molecule of any one of claims 55 to 94, or an antibody of any one of claims 95 to 110, comprising culturing a host cell of any one of claims 113 to 117 in a culture medium.如請求項 118 之方法，其進一步包含從該(等)宿主細胞或該培養基收穫如請求項 1 至 54 中任一項之多特異性抗原結合分子、如請求項 55 至 94 中任一項之抗原結合分子、或如請求項 95 至 110 中任一項之抗體。The method of claim 118, further comprising harvesting the multispecific antigen-binding molecule of any one of claims 1 to 54, the antigen-binding molecule of any one of claims 55 to 94, or the antibody of any one of claims 95 to 110 from the host cell(s) or the culture medium.一種醫藥組成物，其包含如請求項 1 至 54 中任一項之多特異性抗原結合分子、如請求項 55 至 94 中任一項之抗原結合分子、或如請求項 95 至 110 中任一項之抗體。A pharmaceutical composition comprising the multispecific antigen-binding molecule of any one of claims 1 to 54, the antigen-binding molecule of any one of claims 55 to 94, or the antibody of any one of claims 95 to 110.如請求項 120 之醫藥組成物，其進一步包含醫藥上可接受之載劑、賦形劑或稀釋劑。The pharmaceutical composition of claim 120 further comprises a pharmaceutically acceptable carrier, excipient or diluent.如請求項 120 或 121 之醫藥組成物，其用為藥物。If the pharmaceutical composition of claim 120 or 121 is used as a medicine.如請求項 120 或 121 之醫藥組成物，其用於治療表現 STEAP1 之癌症或延緩其進展。The pharmaceutical composition of claim 120 or 121, which is used to treat or delay the progression of a cancer expressing STEAP1.如請求項 120 或 121 之醫藥組成物，其用於治療前列腺癌或尤文氏肉瘤或延緩其進展。A pharmaceutical composition as claimed in claim 120 or 121 for treating or delaying the progression of prostate cancer or Ewing's sarcoma.如請求項 1 至 54 中任一項之多特異性抗原結合分子、如請求項 55 至 94 中任一項之抗原結合分子、或如請求項 95 至 110 中任一項之抗體，其用於治療有需要之個體的表現 STEAP1 之癌症或延緩其進展。A multispecific antigen-binding molecule of any one of claims 1 to 54, an antigen-binding molecule of any one of claims 55 to 94, or an antibody of any one of claims 95 to 110 for treating or delaying the progression of a cancer expressing STEAP1 in a subject in need thereof.如請求項 1 至 54 中任一項之多特異性抗原結合分子、如請求項 55 至 94 中任一項之抗原結合分子、或如請求項 95 至 110 中任一項之抗體，其用於抑制或減少表現 STEAP1 之癌症細胞的增生。A multispecific antigen-binding molecule according to any one of claims 1 to 54, an antigen-binding molecule according to any one of claims 55 to 94, or an antibody according to any one of claims 95 to 110, for inhibiting or reducing the proliferation of cancer cells expressing STEAP1.如請求項 125 或 126 之多特異性抗原結合分子、抗原結合分子或抗體，其中該表現 STEAP1 之癌症為實性瘤。The multispecific antigen-binding molecule, antigen-binding molecule or antibody of claim 125 or 126, wherein the cancer expressing STEAP1 is a solid tumor.如請求項 125 至 127 中任一項之多特異性抗原結合分子、抗原結合分子或抗體，其中該表現 STEAP1 之癌症為前列腺癌或尤文氏肉瘤。The multispecific antigen-binding molecule, antigen-binding molecule or antibody of any one of claims 125 to 127, wherein the cancer expressing STEAP1 is prostate cancer or Ewing's sarcoma.如請求項 125 至 128 中任一項之多特異性抗原結合分子、抗原結合分子或抗體，其係待與額外治療劑或額外治療方案組合使用。A multispecific antigen-binding molecule, antigen-binding molecule or antibody as claimed in any one of claims 125 to 128, to be used in combination with an additional therapeutic agent or additional treatment regimen.如請求項 129 之多特異性抗原結合分子、抗原結合分子或抗體，其中該額外治療劑包含化學治療劑、免疫治療劑、靶向療法、放射療法或其組合。The multispecific antigen-binding molecule, antigen-binding molecule or antibody of claim 129, wherein the additional therapeutic agent comprises chemotherapy, immunotherapy, targeted therapy, radiation therapy or a combination thereof.如請求項 129 或 130 之多特異性抗原結合分子、抗原結合分子或抗體，其中該額外治療劑包含一線、二線或三線療法。The multispecific antigen-binding molecule, antigen-binding molecule, or antibody of claim 129 or 130, wherein the additional therapy comprises a first-line, second-line, or third-line therapy.如請求項 129 之多特異性抗原結合分子、抗原結合分子或抗體，其中該額外治療方案包含手術。The multispecific antigen-binding molecule, antigen-binding molecule or antibody of claim 129, wherein the additional treatment comprises surgery.如請求項 129 至 131 中任一項之多特異性抗原結合分子、抗原結合分子或抗體，其中該多特異性抗原結合分子、該抗原結合分子或該抗體與該額外治療劑係待同時投予。The multispecific antigen-binding molecule, antigen-binding molecule or antibody of any one of claims 129 to 131, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody and the additional therapeutic agent are to be administered simultaneously.如請求項 129 至 131 中任一項之多特異性抗原結合分子、抗原結合分子或抗體，其中該多特異性抗原結合分子、該抗原結合分子或該抗體與該額外治療劑係待依序投予。A multispecific antigen-binding molecule, antigen-binding molecule or antibody as claimed in any one of claims 129 to 131, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody and the additional therapeutic agent are to be administered sequentially.如請求項 129 至 131 中任一項之多特異性抗原結合分子、抗原結合分子或抗體，其中該多特異性抗原結合分子、該抗原結合分子或該抗體係待在投予該額外治療劑之前先投予。The multispecific antigen-binding molecule, antigen-binding molecule or antibody of any one of claims 129 to 131, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody is administered before the additional therapeutic agent.如請求項 129 至 131 中任一項之多特異性抗原結合分子、抗原結合分子或抗體，其中該多特異性抗原結合分子、該抗原結合分子或該抗體係待在投予該額外治療劑之後投予。The multispecific antigen-binding molecule, antigen-binding molecule or antibody of any one of claims 129 to 131, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody is to be administered after the additional therapeutic agent is administered.如請求項 125 至 136 中任一項之多特異性抗原結合分子、抗原結合分子或抗體，其中該多特異性抗原結合分子、該抗原結合分子或該抗體係待經全身性投予。The multispecific antigen-binding molecule, antigen-binding molecule or antibody of any one of claims 125 to 136, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody is to be administered systemically.如請求項 125 至 136 中任一項之多特異性抗原結合分子、抗原結合分子或抗體，其中該多特異性抗原結合分子、該抗原結合分子或該抗體係待經局部性投予。The multispecific antigen-binding molecule, antigen-binding molecule or antibody of any one of claims 125 to 136, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody is to be administered locally.如請求項 125 至 136 中任一項之多特異性抗原結合分子、抗原結合分子或抗體，其中該多特異性抗原結合分子、該抗原結合分子或該抗體係待藉由腸胃外投予來投予。The multispecific antigen-binding molecule, antigen-binding molecule or antibody of any one of claims 125 to 136, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody is to be administered by parenteral administration.如請求項 125 至 136 中任一項之多特異性抗原結合分子、抗原結合分子或抗體，其中該多特異性抗原結合分子、該抗原結合分子或該抗體係待經靜脈內或經皮下投予。A multispecific antigen-binding molecule, an antigen-binding molecule or an antibody as claimed in any one of claims 125 to 136, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody is to be administered intravenously or subcutaneously.如請求項 125 至 136 中任一項之多特異性抗原結合分子、抗原結合分子或抗體，其中該個體為人類。A multispecific antigen-binding molecule, antigen-binding molecule or antibody as claimed in any one of claims 125 to 136, wherein the individual is a human.一種如請求項 1 至 54 中任一項之多特異性抗原結合分子、如請求項 55 至 94 中任一項之抗原結合分子、或如請求項 95 至 110 中任一項之抗體在製造藥物中之用途，該藥物用於治療有需要之個體的表現 STEAP1 之癌症或延緩其進展，視情況用於治療前列腺癌或尤文氏肉瘤或延緩其進展。A use of a multispecific antigen-binding molecule of any one of claims 1 to 54, an antigen-binding molecule of any one of claims 55 to 94, or an antibody of any one of claims 95 to 110 in the manufacture of a medicament for treating or delaying the progression of a cancer expressing STEAP1 in a subject in need thereof, optionally for treating or delaying the progression of prostate cancer or Ewing's sarcoma.一種如請求項 1 至 54 中任一項之多特異性抗原結合分子、如請求項 55 至 94 中任一項之抗原結合分子、或如請求項 95 至 110 中任一項之抗體在製造藥物中之用途 16，視情況前列腺癌細胞或尤文氏肉瘤細胞。Use of a multispecific antigen-binding molecule of any one of claims 1 to 54, an antigen-binding molecule of any one of claims 55 to 94, or an antibody of any one of claims 95 to 110 in the manufacture of a medicament 16, wherein the antibody comprises a prostate cancer cell or an Ewing's sarcoma cell.一種治療有需要之個體的表現 STEAP1 之癌症或延緩其進展之方法，其包含向該個體投予有效量之如請求項 1 至 54 中任一項之多特異性抗原結合分子、如請求項 55 至 94 中任一項之抗原結合分子、或如請求項 95 至 110 中任一項之抗體。A method for treating or delaying the progression of a cancer expressing STEAP1 in a subject in need thereof, comprising administering to the subject an effective amount of the multispecific antigen-binding molecule of any one of claims 1 to 54, the antigen-binding molecule of any one of claims 55 to 94, or the antibody of any one of claims 95 to 110.如請求項 144 之方法，其中該表現 STEAP1 之癌症為實性瘤。The method of claim 144, wherein the cancer expressing STEAP1 is a solid tumor.如請求項 144 或 145 之方法，其中該表現 STEAP1 之癌症為前列腺癌或尤文氏肉瘤。The method of claim 144 or 145, wherein the cancer expressing STEAP1 is prostate cancer or Ewing's sarcoma.如請求項 144 至 146 中任一項之方法，其進一步包含向該個體投予額外治療劑或額外治療方案。The method of any of claims 144 to 146, further comprising administering to the individual an additional therapeutic agent or an additional treatment regimen.如請求項 147 之方法，其中該額外治療劑包含化學治療劑、免疫治療劑、靶向療法、放射療法或其組合。The method of claim 147, wherein the additional therapeutic agent comprises chemotherapy, immunotherapy, targeted therapy, radiation therapy, or a combination thereof.如請求項 148 之方法，其中該額外治療劑包含一線、二線或三線療法。The method of claim 148, wherein the additional treatment comprises a first-line, second-line, or third-line therapy.如請求項 148 之方法，其中該額外治療方案包含手術。The method of claim 148, wherein the additional treatment comprises surgery.如請求項 144 至 150 中任一項之方法，其中該多特異性抗原結合分子、該抗原結合分子或該抗體與該額外治療劑係同時投予。The method of any one of claims 144 to 150, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody and the additional therapeutic agent are administered simultaneously.如請求項 144 至 150 中任一項之方法，其中該多特異性抗原結合分子、該抗原結合分子或該抗體與該額外治療劑係依序投予。The method of any one of claims 144 to 150, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody and the additional therapeutic agent are administered sequentially.如請求項 152 之方法，其中該多特異性抗原結合分子、該抗原結合分子或該抗體係在投予該額外治療劑之前先投予。The method of claim 152, wherein the multispecific antigen-binding molecule, the antigen-binding molecule, or the antibody is administered prior to administering the additional therapeutic agent.如請求項 152 之方法，其中該多特異性抗原結合分子、該抗原結合分子或該抗體係在投予該額外治療劑之後投予。The method of claim 152, wherein the multispecific antigen-binding molecule, the antigen-binding molecule, or the antibody is administered after administration of the additional therapeutic agent.如請求項 144 至 154 中任一項之方法，其中該多特異性抗原結合分子、該抗原結合分子或該抗體係經全身性投予。The method of any one of claims 144 to 154, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody is administered systemically.如請求項 144 至 154 中任一項之方法，其中該多特異性抗原結合分子、該抗原結合分子或該抗體係經局部性投予。The method of any one of claims 144 to 154, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody is administered locally.如請求項 144 至 156 中任一項之方法，其中該多特異性抗原結合分子、該抗原結合分子或該抗體係藉由腸胃外投予來投予。The method of any one of claims 144 to 156, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody is administered by parenteral administration.如請求項 144 至 157 中任一項之方法，其中該多特異性抗原結合分子、該抗原結合分子或該抗體係經靜脈內或經皮下投予。The method of any one of claims 144 to 157, wherein the multispecific antigen-binding molecule, the antigen-binding molecule or the antibody is administered intravenously or subcutaneously.如請求項 144 至 158 中任一項之方法，其中該個體為人類。The method of any one of claims 144 to 158, wherein the individual is a human.一種抑制或減少表現 STEAP1 之細胞的增生之方法，其包含使該細胞與如請求項 1 至 54 中任一項之多特異性抗原結合分子、如請求項 55 至 94 中任一項之抗原結合分子、或如請求項 95 至 110 中任一項之抗體接觸足以抑制該細胞之增生的時間。A method for inhibiting or reducing proliferation of a cell expressing STEAP1, comprising contacting the cell with a multispecific antigen-binding molecule of any one of claims 1 to 54, an antigen-binding molecule of any one of claims 55 to 94, or an antibody of any one of claims 95 to 110 for a time sufficient to inhibit proliferation of the cell.如請求項 160 之方法，其中該細胞為前列腺癌細胞或尤文氏肉瘤細胞。The method of claim 160, wherein the cell is a prostate cancer cell or an Ewing's sarcoma cell.如請求項 160 或 161 之方法，其中該方法為活體內方法。The method of claim 160 or 161, wherein the method is an in vivo method.如請求項 160 或 161 之方法，其中該方法為活體外 (in vitro) 或離體 (ex vivo) 方法。The method of claim 160 or 161, wherein the method is anin vitro orex vivo method.一種套組，其包含如請求項 1 至 54 中任一項之多特異性抗原結合分子、如請求項 55 至 94 中任一項之抗原結合分子、或如請求項 95 至 110 中任一項之抗體、如請求項 111 之經分離核酸、如請求項 112 之載體、如請求項 113 至 117 中任一項之宿主細胞、或如請求項 120 至 124 中任一項之醫藥組成物，視情況包含一組說明。A kit comprising a multispecific antigen-binding molecule as described in any one of claims 1 to 54, an antigen-binding molecule as described in any one of claims 55 to 94, or an antibody as described in any one of claims 95 to 110, an isolated nucleic acid as described in claim 111, a vector as described in claim 112, a host cell as described in any one of claims 113 to 117, or a pharmaceutical composition as described in any one of claims 120 to 124, optionally comprising a set of instructions.