TW202033218A - Multi-specific protein molecules - Google Patents

Abstract

The present disclosure relates to multi-specific protein molecules. In particular, the invention relates to a multi-specific antibody with a new structural form. The multi-specific antibody can combine with CD3 and other tumor related antigens at the same time, combine with tumor related antigen expression cells, and activate CD3 positive T cells, so as to promote the specific killing of T cells to tumor cells expressing tumor related antigens. At the same time, the present disclosure also provides the preparation and application of multi-specific antibodies.

Description

Translated fromChinese

多特異性蛋白分子Multispecific protein molecule

本發明涉及多特異性抗體，如結合CD3和腫瘤相關抗原的多特異性抗體。The present invention relates to multispecific antibodies, such as multispecific antibodies that bind CD3 and tumor-associated antigens.

這裡的陳述僅提供與本發明有關的背景信息，而不必然地構成現有技術。The statements herein only provide background information related to the present invention, and do not necessarily constitute prior art.

CD3是由四條不同的鏈組成的T細胞共受體(Wucherpfennig,K.W.等(2010)“Structural Biology Of The T cell Receptor：Insights Into Receptor Assembly,Ligand Recognition,And Initiation Of Signaling,”Cold Spring Harb.Perspect.Biol.2(4)：a005140；1-14頁；Chetty,R.等(1994)“CD3：Structure,Function,And Role Of Immunostaining In Clinical Practice,”J.Pathol.173(4)：303-307；Guy,C.S.等(2009)“Organization Of Proximal Signal Initiation At The TCR：CD3 Complex,”Immunol.Rev.232(1)：7-21)。CD3 is a T cell co-receptor composed of four different chains (Wucherpfennig, KW et al. (2010) "Structural Biology Of The T cell Receptor: Insights Into Receptor Assembly, Ligand Recognition, And Initiation Of Signaling," Cold Spring Harb.Perspect . Biol. 2(4): a005140; 1-14 pages; Chetty, R. et al. (1994) "CD3: Structure, Function, And Role Of Immunostaining In Clinical Practice," J. Pathol. 173(4): 303- 307; Guy, CS et al. (2009) "Organization Of Proximal Signal Initiation At The TCR: CD3 Complex," Immunol. Rev. 232(1): 7-21).

在哺乳動物中，CD3多亞基形成的複合物與T細胞受體(TCR)的分子締合，以便在T淋巴細胞中產生激活信號(Smith-Garvin,J.E.等(2009)“T Cell Activation,”Annu.Rev.Immunol.27：591-619)。在沒有CD3的情況下，TCR不能適當組裝並且降解(Thomas,S.等(2010)“Molecular Immunology Lessons From Therapeutic T cell Receptor Gene Transfer,”Immunology 129(2)：170-177)。研究發現CD3結合所有成熟的T細胞的膜，並且幾乎不與其他細胞類型結合(Janeway,C.A.等(2005)：Immunobiology：The Immune System In Health And Disease,”第六版，Garland Science Publishing,NY,pp.214-216；Sun,Z.J.等(2001)“Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By The Solution Structure Of An Ectodomain Fragment Of The CD3ε：γHeterodimer,”Cell 105(7)：913-923；Kuhns,M.S.等(2006)“Deconstructing The Form And Function Of The TCR/CD3 Complex,”Immunity.2006 Feb，24(2)：133-139)。In mammals, CD3 multi-subunit complexes associate with T cell receptor (TCR) molecules to generate activation signals in T lymphocytes (Smith-Garvin, JE et al. (2009) "T Cell Activation, "Annu.Rev.Immunol.27:591-619). In the absence of CD3, TCR cannot be properly assembled and degraded (Thomas, S. et al. (2010) "Molecular Immunology Lessons FromTherapeutic T cell Receptor Gene Transfer, "Immunology 129(2): 170-177). Studies have found that CD3 binds to the membranes of all mature T cells and hardly binds to other cell types (Janeway, CA, etc. (2005): Immunobiology: The Immune System In Health And Disease,” Sixth Edition, Garland Science Publishing, NY, pp.214-216; Sun, ZJ, etc. (2001) “Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By The Solution Structure Of An Ectodomain Fragment Of The CD3ε: γHeterodimer, "Cell 105(7): 913-923; Kuhns, MS et al. (2006) "Deconstructing The Form And Function Of The TCR/CD3 Complex," Immunity. 2006 Feb, 24(2): 133 -139).

T細胞上T細胞受體(TCR)複合物的恆定的CD3ε信號傳導組分已經被用作靶，以促使在T細胞和腫瘤細胞之間形成免疫學突觸。CD3和腫瘤抗原的共接合(co-engagement)激活了T細胞，引起表達腫瘤抗原的腫瘤細胞的裂解(Baeuerle等(2011)“Bispecific T Cell Engager For Cancer Therapy,”In：Bispecific Antibodies,Kontermann,R.E.(Ed.)Springer-Verlag；2011：273-287)。該方法允許雙特異性抗體以對腫瘤細胞的高特異性與T細胞小室(compartment)全面相互作用並且廣泛適用於大量的細胞表面腫瘤抗原。The constant CD3 epsilon signaling component of the T cell receptor (TCR) complex on T cells has been used as a target to promote the formation of immunological synapses between T cells and tumor cells. The co-engagement of CD3 and tumor antigen activates T cells, causing the lysis of tumor cells expressing tumor antigens (Baeuerle et al. (2011) "Bispecific T Cell Engager For Cancer Therapy," In: Bispecific Antibodies, Kontermann, RE (Ed.) Springer-Verlag; 2011:273-287). This method allows bispecific antibodies to fully interact with T cell compartments with high specificity for tumor cells and is widely applicable to a large number of cell surface tumor antigens.

B7H3是B7家族的成員之一，屬於I型跨膜蛋白，包含胺基端的一個信號肽，一個細胞外的免疫球蛋白樣可變區(IgV)和恆定區(IgC)、一個跨膜區和一個含有45個胺基酸的胞質尾區(Tissue Antigens.2007 Aug；70(2)：96-104)。目前，B7H3主要存在2種剪切體，B7H3a和B7H3b。B7H3a胞外段由IgV-IgC 2個免疫球蛋白結構域組成，又稱為2IgB7H3，而B7H3b胞外段由IgV-IgC-IgV-IgC 4個免疫球蛋白結構域組成，又稱為4IgB7H3。B7H3 is a member of the B7 family and belongs to the type I transmembrane protein. It contains a signal peptide at the amino end, an extracellular immunoglobulin-like variable region (IgV) and constant region (IgC), a transmembrane region and A cytoplasmic tail containing 45 amino acids (Tissue Antigens. 2007 Aug; 70(2): 96-104). Currently, there are two main types of spliceosome in B7H3, B7H3a and B7H3b. The extracellular segment of B7H3a is composed of two immunoglobulin domains of IgV-IgC, also known as 2IgB7H3, while the extracellular segment of B7H3b is composed of four immunoglobulin domains of IgV-IgC-IgV-IgC, also known as 4IgB7H3.

B7H3蛋白質在正常組織、細胞中不表達或極低表達，卻高表達於多種腫瘤組織，並與腫瘤的進展、患者的生存及預後密切相關。臨床上已經報道，B7H3在許多癌症類型中、特別是在非小細胞肺癌、腎癌、泌尿道上皮癌、結直腸癌、前列腺癌、多形性膠質母細胞瘤、卵巢癌和胰腺癌中過表達(Lung Cancer.2009 Nov；66(2)：245-249；Clin Cancer Res.2008 Aug 15；14(16)：5150-5157)。此外，也有文獻報道，在前列腺癌中，B7H3的表達強度與臨床病理學惡性(諸如腫瘤體積、前列腺外侵襲或Gleason評分)正相關，且也與癌症進展相關(Cancer Res.2007 Aug 15；67(16)：7893-7900)。類似地，在多形性膠質母細胞瘤中，B7H3的表達與無事件存活負相關，且在胰腺癌中，B7H3的表達與淋巴結轉移和病理學進展相關。因此，B7H3被認為是一種新的腫瘤標誌物和潛在的治療靶點。B7H3 protein is not expressed or very low expressed in normal tissues and cells, but is highly expressed in a variety of tumor tissues, and is closely related to tumor progression, patient survival and prognosis. It has been clinically reported that B7H3 has been used in many cancer types, especially non-small cell lung cancer, kidney cancer, urinary tract epithelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer and pancreatic cancer. Expression (Lung Cancer. 2009 Nov; 66(2): 245-249; Clin Cancer Res. 2008 Aug 15; 14(16): 5150-5157). In addition, there are also reports in the literature that in prostate cancer, the expression intensity of B7H3 is positively correlated with clinicopathological malignancy (such as tumor volume, extra-prostatic invasion or Gleason score), and is also correlated with cancer progression (Cancer Res. 2007 Aug 15; 67 (16): 7893-7900). Similarly, in glioblastoma multiforme, B7H3 expression is negatively correlated with event-free survival, and in pancreatic cancer, B7H3 expression is correlated with lymph node metastasis and pathological progression. Therefore, B7H3 is considered as a new tumor marker and a potential therapeutic target.

現有技術中公開了一些CD3抗體分子，如OKT3、UCHT-1、SP34(Silvana Pessano et al.The EMBO Journal.1985,4(2)：337-344)，同時，如CN103703024、WO2017055389、CN102171248等也公開了一些CD3抗體分子。但在藥物研發時，仍需開發具有更加安全和有效的CD3抗體分子。Some CD3 antibody molecules are disclosed in the prior art, such as OKT3, UCHT-1, SP34 (Silvana Pessano et al. The EMBO Journal.1985, 4(2): 337-344), and at the same time, such as CN103703024, WO2017055389, CN102171248, etc. Some CD3 antibody molecules are disclosed. However, during drug development, it is still necessary to develop safer and more effective CD3 antibody molecules.

一方面，本公開提供一種多特異性蛋白分子，其包含第一多肽鏈和第二多肽鏈，其中：In one aspect, the present disclosure provides a multispecific protein molecule comprising a first polypeptide chain and a second polypeptide chain, wherein:

該第一多肽鏈從胺基末端至羧基末端依次包含針對第一靶抗原的第一結合區、針對第二靶抗原的第二結合區和第一Fc區，The first polypeptide chain includes a first binding region for a first target antigen, a second binding region for a second target antigen, and a first Fc region in sequence from the amino terminal to the carboxy terminal,

該第二多肽鏈從胺基末端至羧基末端依次包含針對第三靶抗原的第三結合區和第二Fc區，The second polypeptide chain includes a third binding region and a second Fc region for the third target antigen in sequence from the amino terminal to the carboxy terminal,

該第二結合區和/或第三結合區不包含抗體的恆定區結構域，The second binding region and/or the third binding region do not contain the constant region domain of an antibody,

該第一多肽鏈和該第二多肽鏈鏈內各區域由肽鍵和/或接頭連接。The regions in the first polypeptide chain and the second polypeptide chain are connected by peptide bonds and/or linkers.

在一些實施方式中，其中該多特異性蛋白分子的抗原結合區，具體是該第一結合區、和/或該第二結合區、和/或該第三結合區是單鏈抗體(scFv)。In some embodiments, the antigen binding region of the multispecific protein molecule, specifically the first binding region, and/or the second binding region, and/or the third binding region is a single chain antibody (scFv) .

在一些實施方式中，其中該多特異性蛋白分子的該第二靶抗原為CD3，且該第一靶抗原和第三靶抗原為相同或不同的腫瘤相關抗原(TAA)；或該第一靶抗原為CD3，且該第二靶抗原和第三靶抗原為相同或不同的腫瘤相關抗原(TAA)。In some embodiments, wherein the second target antigen of the multispecific protein molecule is CD3, and the first target antigen and the third target antigen are the same or different tumor-associated antigens (TAA); or the first target The antigen is CD3, and the second target antigen and the third target antigen are the same or different tumor-associated antigens (TAA).

在一些實施方式中，其中該多特異性蛋白分子的該腫瘤相關抗原(TAA)選自AFP、ALK、B7H3、BAGE蛋白質、BCMA、BIRC5(存活素)、BIRC7、β-連環蛋白(β-catenin)、brc-ab1、BRCA1、BORIS、CA9、CA125、碳酸酐酶IX、半胱天冬酶-8(caspase-8)、CALR、CCR5、CD19、CD20(MS4A1)、CD22、CD30、CD33、CD38、CD40、CD123、CD133、CD138、CDK4、CEA、Claudin 18.2、週期素-B1、CYP1B1、EGFR、EGFRvIII、ErbB2/Her2、ErbB3、ErbB4、ETV6-AML、EpCAM、EphA2、Fra-1、FOLR1、GAGE蛋白(例如GAGE-1、-2)、GD2、GD3、GloboH、磷脂醯肌醇蛋白聚糖-3(glypican-3)、GM3、gp100、Her2、HLA/B-raf、HLA/k-ras、HLA/MAGE-A3、hTERT、IL13Rα2、LMP2、κ-Light、LeY、MAGE蛋白(例如MAGE-1、-2、-3、-4、-6和-12)、MART-1、間皮素(mesothelin)、ML-IAP、MOv-γ、Muc1、Muc2、Muc3、Muc4、Muc5、Muc16(CA-125)、MUM1、NA17、NKG2D、NY-BR1、NY-BR62、NY-BR85、NY-ESO1、OX40、p15、p53、PAP、PAX3、PAX5、PCTA-1、PLAC1、PRLR、PRAME、PSMA(FOLH1)、RAGE蛋白質、Ras、RGS5、Rho、ROR1、SART-1、SART-3、STEAP1、STEAP2、TAG-72、TGF-β、TMPRSS2、湯-諾氏抗原(Thompson-nouvelle antigen；Tn)、TRP-1、TRP-2、酪胺酸酶和尿溶蛋白-3和5T4(Trophoblast glycoprotein)。較佳地，該腫瘤相關抗原(TAA)選自B7H3、BCMA、CEA、CD19、CD20、CD38、CD138、Claudin18.2、PSMA和間皮素。更佳In some embodiments, the tumor-associated antigen (TAA) of the multispecific protein molecule is selected from the group consisting of AFP, ALK, B7H3, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, β-catenin (β-catenin ), brc-ab1, BRCA1, BORIS, CA9, CA125, Carbonic Anhydrase IX, Caspase-8 (caspase-8), CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD33, CD38 , CD40, CD123, CD133, CD138, CDK4, CEA, Claudin 18.2, Cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE Protein (e.g. GAGE-1, -2), GD2, GD3, GloboH, glypican-3 (glypican-3), GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, IL13Rα2, LMP2, κ-Light, LeY, MAGE protein (e.g. MAGE-1, -2, -3, -4, -6 and -12), MART-1, Mesothelin ( mesothelin), ML-IAP, MOv-γ, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ESO1 OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, ROR1, SART-1, SART-3, STEP1, STEP2 TAG-72. TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase and urinary protein-3 and 5T4 (Trophoblast glycoprotein). Preferably, the tumor-associated antigen (TAA) is selected from B7H3, BCMA, CEA, CD19, CD20, CD38, CD138, Claudin 18.2, PSMA and mesothelin. Better

在一些實施方式中，其中該多特異性蛋白分子的該第一多肽鏈具有如下式I的結構：In some embodiments, wherein the first polypeptide chain of the multispecific protein molecule has the structure of Formula I as follows:

V_a1-L1-V_b1-L2-V_c2-L2-V_d2-L4-Fc1 式I，該第二多肽鏈具有如下式II的結構：V_a 1-L1-V_b 1-L2-V_c 2-L2-V_d 2-L4-Fc1 Formula I, the second polypeptide chain has the structure of Formula II as follows:

V_e3-L5-V_f3-L6-Fc2 式II，該V_a1、V_b1、V_c2、V_d2、V_e3和V_f3為抗體的輕鏈可變區或重鏈可變區，且該V_a1和V_b1，該V_c2和V_d2，與該V_e3和V_f3分別不同時為輕鏈可變區或不同時為重鏈可變區。V_e 3-L5-V_f 3-L6-Fc2 formula II, theV_a 1,V_b 1,V_c 2,V_d 2,V_e 3 andV_f 3 are the light chain variable regions or heavy Chain variable region, and theV_a 1 andV_b 1, theV_c 2 andV_d 2, and theV_e 3 andV_f 3 are not the light chain variable region or the heavy chain variable region at the same time, respectively .

在一些實施方式中，其中該多特異性蛋白分子的第一多肽鏈具有如下所示的結構：In some embodiments, the first polypeptide chain of the multispecific protein molecule has the structure shown below:

VH_TAA-L1-VL_TAA-L2-VH_CD3-L3-VL_CD3-L4-Fc1，VH_TAA -L1-VL_TAA -L2-VH_CD3 -L3-VL_CD3 -L4-Fc1,

VH_TAA-L1-VL_TAA-L2-VL_CD3-L3-VH_CD3-L4-Fc1，VH_TAA -L1-VL_TAA -L2-VL_CD3 -L3-VH_CD3 -L4-Fc1,

VL_TAA-L1-VH_TAA-L2-VH_CD3-L3-VL_CD3-L4-Fc1，VL_TAA -L1-VH_TAA- L2-VH_CD3 -L3-VL_CD3 -L4-Fc1,

VL_TAA-L1-VH_TAA-L2-VL_CD3-L3-VH_CD3-L4-Fc1，VL_TAA -L1-VH_TAA -L2-VL_CD3 -L3-VH_CD3 -L4-Fc1,

VH_CD3-L1-VL_CD3-L2-VH_TAA-L3-VL_TAA-L4-Fc1，VH_CD3 -L1-VL_CD3 -L2-VH_TAA -L3-VL_TAA -L4-Fc1,

VH_CD3-L1-VL_CD3-L2-VL_TAA-L3-VH_TAA-L4-Fc1，VH_CD3 -L1-VL_CD3 -L2-VL_TAA -L3-VH_TAA -L4-Fc1,

VL_CD3-L1-VH_CD3-L2-VH_TAA-L3-VL_TAA-L4-Fc1或VL_CD3 -L1-VH_CD3 -L2-VH_TAA -L3-VL_TAA -L4-Fc1 or

VL_CD3-L1-VH_CD3-L2-VL_TAA-L3-VH_TAA-L4-Fc1；且該第二多肽鏈具有如下所示的結構：VL_CD3 -L1-VH_CD3 -L2-VL_TAA -L3-VH_TAA -L4-Fc1; and the second polypeptide chain has the following structure:

VL_TAA-L5-VH_TAA-L6-F_C2或VH_TAA-L5-VL_TAA-L6-F_C2。VL_TAA -L5-VH_TAA -L6-F_C 2 or VH_TAA -L5-VL_TAA -L6-F_C 2.

在一些實施方式中，其中該多特異性蛋白分子的第一多肽鏈具有VL_TAA-L1-VH_TAA-L2-VH_CD3-L3-VL_CD3-L4-Fc1的結構，且該第二條鏈具有VL_TAA-L5-VH_TAA-L6-F_C2的結構。In some embodiments, the first polypeptide chain of the multispecific protein molecule has the structure of VL_TAA -L1-VH_TAA- L2-VH_CD3 -L3-VL_CD3 -L4-Fc1, and the second chain having_{VL TAA -L5-VH TAA -L6-}F C structure 2.

在一些實施方式中，其中該多特異性蛋白分子的第一多肽鏈具有VH_CD3-L1-VL_CD3-L2-VL_TAA-L3-VH_TAA-L4-Fc1的結構，且該第二條鏈具有VH_TAA-L5-VL_TAA-L6-F_C2的結構。In some embodiments, the first polypeptide chain wherein the multispecific protein molecule having_{CD3 -L1-VL CD3 -L2-VL} TAA -L3-VH TAA -L4-Fc1 structure of the VH, and the second strand having_{VH TAA -L5-VL TAA -L6-}F C structure 2.

在一些實施方式中，其中該TAA為B7H3。In some embodiments, wherein the TAA is B7H3.

在一些實施方式中，其中該多特異性蛋白分子的該第一Fc區和該第二Fc區是相同的Fc或不同的Fc。較佳地，該第一Fc區為knob-Fc，該第二Fc區為hole-Fc；或該第一Fc區為hole-Fc，該第二Fc區為knob-Fc。In some embodiments, wherein the first Fc region and the second Fc region of the multispecific protein molecule are the same Fc or different Fc. Preferably, the first Fc region is knob-Fc and the second Fc region is hole-Fc; or the first Fc region is hole-Fc and the second Fc region is knob-Fc.

在一些實施方式中，其中該多特異性蛋白分子的該第一Fc區的羧基端連接His標簽(His tag)或第二Fc區的羧基端連接His tag。In some embodiments, the carboxyl end of the first Fc region of the multispecific protein molecule is connected to a His tag or the carboxyl end of the second Fc region is connected to a His tag.

在一些實施方式中，其中該多特異性蛋白分子中針對CD3的抗原結合區包含抗體輕鏈可變區和重鏈可變區，其中該輕鏈可變區包含分別如SEQ ID NO：48、49和50所示的LCDR1、LCDR2和LCDR3，且該重鏈可變區包含選自以下i)至v)任一項的HCDR1、HCDR2和HCDR3：In some embodiments, the antigen-binding region against CD3 in the multispecific protein molecule comprises an antibody light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises SEQ ID NO: 48, The LCDR1, LCDR2, and LCDR3 shown in 49 and 50, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 selected from any one of i) to v) below:

i)分別如SEQ ID NO：37、38和39所示的HCDR1、HCDR2和HCDR3；i) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 37, 38 and 39, respectively;

ii)分別如SEQ ID NO：37、40和41所示的HCDR1、HCDR2和HCDR3；ii) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 37, 40 and 41, respectively;

iii)分別如SEQ ID NO：37、40和42所示的HCDR1、HCDR2和HCDR3；iii) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 37, 40 and 42, respectively;

iv)分別如SEQ ID NO：37、40和43所示的HCDR1、HCDR2和HCDR3；或iv) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 37, 40 and 43, respectively; or

v)分別如SEQ ID NO：37、47和45所示的HCDR1、HCDR2和HCDR3。v) HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 37, 47 and 45, respectively.

在一些實施方式中，其中該多特異性蛋白分子中針對CD3的抗原結合區包含如SEQ ID NO：36所示的輕鏈可變區和/或選自如SEQ ID NO：29、30、31、32和35中任一項所示的重鏈可變區。In some embodiments, the antigen binding region for CD3 in the multispecific protein molecule comprises a light chain variable region as shown in SEQ ID NO: 36 and/or is selected from the group consisting of as SEQ ID NO: 29, 30, 31, The heavy chain variable region shown in any one of 32 and 35.

在一些實施方式中，其中該多特異性蛋白分子中針對CD3的抗原結合區包含如SEQ ID NO：55、56、57、58、61、62、63、64、65或68所示的scFv。In some embodiments, the antigen binding region against CD3 in the multispecific protein molecule comprises an scFv as shown in SEQ ID NO: 55, 56, 57, 58, 61, 62, 63, 64, 65 or 68.

在一些實施方式中，其中該多特異性蛋白分子中該腫瘤相關抗原為B7H3，針對B7H3的抗原結合區包含抗體輕鏈可變區和重鏈可變區，其中該輕鏈可變區包含分別如SEQ ID NO：12、13和14所示的LCDR1、LCDR2和LCDR3，且該重鏈可變區包含分別如SEQ ID NO：9、10和11所示的HCDR1、HCDR2和HCDR3。In some embodiments, wherein the tumor-associated antigen in the multispecific protein molecule is B7H3, the antigen-binding region for B7H3 comprises an antibody light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the respective LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 12, 13, and 14, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 9, 10, and 11, respectively.

在一些實施方式中，其中該多特異性蛋白分子中針對B7H3的抗原結合區包含：如SEQ ID NO：8所示的輕鏈可變區和/或如SEQ ID NO：7所示的重鏈可變區；或如SEQ ID NO：16所示的輕鏈可變區和/或如SEQ ID NO：15所示的重鏈可變區。In some embodiments, the antigen binding region for B7H3 in the multispecific protein molecule comprises: a light chain variable region as shown in SEQ ID NO: 8 and/or a heavy chain as shown in SEQ ID NO: 7 Variable region; or the light chain variable region shown in SEQ ID NO: 16 and/or the heavy chain variable region shown in SEQ ID NO: 15.

在一些實施方式中，其中該多特異性蛋白分子中針對B7H3的抗原結合區包含如SEQ ID NO：51、52、53或54所示的scFv。In some embodiments, the antigen binding region for B7H3 in the multispecific protein molecule comprises a scFv as shown in SEQ ID NO: 51, 52, 53 or 54.

在一些實施方式中，其中該多特異性蛋白分子包含第一多肽鏈和第二多肽鏈，該第一多肽鏈選自胺基酸序列如SEQ ID NO：72、73、74、75、76、77、78、79、80、83、84、85、86或87所示的多肽，和/或該第二多肽鏈選自胺基酸序列如SEQ ID NO：71、88或70所示的多肽。In some embodiments, wherein the multispecific protein molecule comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain is selected from amino acid sequences such as SEQ ID NO: 72, 73, 74, 75 ,The polypeptide represented by 76, 77, 78, 79, 80, 83, 84, 85, 86 or 87, and/or the second polypeptide chain is selected from an amino acid sequence as shown in SEQ ID NO: 71, 88 or 70 The peptides shown.

在一些實施方式中，其中多特異性蛋白分子包含第一多肽鏈和第二多肽鏈，該第二多肽鏈的胺基酸序列如SEQ ID NO：71、88或70所示，且：In some embodiments, wherein the multispecific protein molecule comprises a first polypeptide chain and a second polypeptide chain, the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 71, 88 or 70, and :

該第一多肽鏈的胺基酸序列如SEQ ID NO：72所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 72;

該第一多肽鏈的胺基酸序列如SEQ ID NO：73所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 73;

該第一多肽鏈的胺基酸序列如SEQ ID NO：74所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 74;

該第一多肽鏈的胺基酸序列如SEQ ID NO：75所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 75;

該第一多肽鏈的胺基酸序列如SEQ ID NO：76所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 76;

該第一多肽鏈的胺基酸序列如SEQ ID NO：77所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 77;

該第一多肽鏈的胺基酸序列如SEQ ID NO：78所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 78;

該第一多肽鏈的胺基酸序列如SEQ ID NO：79所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 79;

該第一多肽鏈的胺基酸序列如SEQ ID NO：80所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 80;

該第一多肽鏈的胺基酸序列如SEQ ID NO：83所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 83;

該第一多肽鏈的胺基酸序列如SEQ ID NO：84所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 84;

該第一多肽鏈的胺基酸序列如SEQ ID NO：85所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 85;

該第一多肽鏈的胺基酸序列如SEQ ID NO：86所示；或者The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 86; or

該第一多肽鏈的胺基酸序列如SEQ ID NO：87所示。The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO:87.

在一些實施方式中，其中多特異性蛋白分子包含第一多肽鏈和第二多肽鏈，該第一多肽鏈的胺基酸序列如SEQ ID NO：73所示，且該第二多肽鏈的胺基酸序列如SEQ ID NO：71所示。In some embodiments, wherein the multispecific protein molecule comprises a first polypeptide chain and a second polypeptide chain, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 73, and the second polypeptide chain The amino acid sequence of the peptide chain is shown in SEQ ID NO:71.

在一些實施方式中，其中多特異性蛋白分子包含第一多肽鏈和第二多肽鏈，該第一多肽鏈的胺基酸序列如SEQ ID NO：78所示，且該第二多肽鏈的胺基酸序列如SEQ ID NO：71所示。In some embodiments, wherein the multispecific protein molecule comprises a first polypeptide chain and a second polypeptide chain, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 78, and the second polypeptide chain The amino acid sequence of the peptide chain is shown in SEQ ID NO:71.

在一些實施方式中，其中多特異性蛋白分子包含第一多肽鏈和第二多肽鏈，該第一多肽鏈的胺基酸序列如SEQ ID NO：76所示，且該第二多肽鏈的胺基酸序列如SEQ ID NO：71所示。In some embodiments, wherein the multispecific protein molecule comprises a first polypeptide chain and a second polypeptide chain, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 76, and the second polypeptide chain The amino acid sequence of the peptide chain is shown in SEQ ID NO:71.

在另一方面，本公開涉及一種醫藥組成物，其含有治療有效量的根據如前所述的多特異性蛋白分子，以及一種或多種藥學上可接受的載體、稀釋劑、緩衝劑或賦形劑。較佳地，該治療有效量為單位劑量的組合物中含有0.1-3000mg(更佳為1-1000mg)的如前所述的多特異性蛋白分子。In another aspect, the present disclosure relates to a pharmaceutical composition, which contains a therapeutically effective amount of the multispecific protein molecule as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients Agent. Preferably, the therapeutically effective amount is a unit dose of the composition containing 0.1-3000 mg (more preferably 1-1000 mg) of the aforementioned multispecific protein molecule.

在另一方面，本公開涉及一種分離的核酸分子，其編碼如前所述的多特異性蛋白分子。In another aspect, the present disclosure relates to an isolated nucleic acid molecule that encodes a multispecific protein molecule as described above.

在另一方面，本公開涉及一種重組載體，其包含如前所述的分離的核酸分子。In another aspect, the present disclosure relates to a recombinant vector comprising an isolated nucleic acid molecule as described above.

在另一方面，本公開涉及一種用如前所述的重組載體轉化的宿主細胞，該宿主細胞選自原核細胞和真核細胞，較佳為真核細胞，更佳哺乳動物細胞或昆蟲細胞。In another aspect, the present disclosure relates to a host cell transformed with the aforementioned recombinant vector. The host cell is selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells or insect cells.

在另一方面，本公開涉及用於生產如前所述的多特異性蛋白分子的方法，該方法包括將如前所述的宿主細胞在培養基中進行培養以形成並積累如前所述的多特異性蛋白分子，以及從培養物回收該多特異性蛋白分子的步驟。In another aspect, the present disclosure relates to a method for producing the aforementioned multispecific protein molecule, the method comprising culturing the aforementioned host cell in a culture medium to form and accumulate the aforementioned multispecific protein molecule. Specific protein molecule, and the step of recovering the multispecific protein molecule from the culture.

在另一方面，本公開涉及作為藥物的如前所述的多特異性蛋白分子或如前所述的醫藥組成物，或如前所述的分離的核酸分子，較佳該藥物為激活T細胞的藥物，更佳該藥物為治療癌症的藥物、或治療自身免疫性或炎性疾病的藥物。In another aspect, the present disclosure relates to the aforementioned multispecific protein molecule or the aforementioned medical composition as a drug, or the aforementioned isolated nucleic acid molecule, preferably the drug is an activatorT cell drugs, more preferably, the drugs are drugs for treating cancer, or drugs for treating autoimmune or inflammatory diseases.

在另一方面，本公開涉及如前所述的多特異性蛋白分子或如前所述的醫藥組成物，或如前所述的分離的核酸分子在製備激活T細胞的藥物中的用途。In another aspect, the present disclosure relates to the use of the aforementioned multispecific protein molecule or the aforementioned pharmaceutical composition, or the aforementioned isolated nucleic acid molecule in the preparation of a medicine for activating T cells.

在另一方面，本公開涉及如前所述的多特異性蛋白分子或如前所述的醫藥組成物，或如前所述的分離的核酸分子在製備治療癌症、或治療自身免疫性或炎性疾病的藥物中的用途。In another aspect, the present disclosure relates to a multispecific protein molecule as described above or a pharmaceutical composition as described above, or an isolated nucleic acid molecule as described above in preparation for the treatment of cancer, or treatment of autoimmunity or inflammation. Use in medicine for sexual diseases.

在另一方面，本公開涉及一種激活T細胞的方法，該方法包括向受試者施用治療有效量的如前所述的多特異性蛋白分子或如前所述的醫藥組成物，或如前所述的分離的核酸分子。In another aspect, the present disclosure relates to a method for activating T cells, the method comprising administering to a subject a therapeutically effective amount of the aforementioned multispecific protein molecule or the aforementioned pharmaceutical composition, or the aforementioned The isolated nucleic acid molecule.

在另一方面，本公開涉及一種治療癌症或自身免疫性或炎性疾病的方法，該方法包括向受試者施用治療有效量的如前所述的多特異性蛋白分子或如前所述的醫藥組成物，或如前所述的分離的核酸分子。較佳地，該方法包括向受試者施用包含單位劑量的組合物中含有0.1-3000mg的如前所述的多特異性蛋白分子，或如前所述的醫藥組成物，或如前所述的分離的核酸分子。In another aspect, the present disclosure relates to a method of treating cancer or autoimmune or inflammatory diseases, the method comprising administering to a subject a therapeutically effective amount of the aforementioned multispecific protein molecule or the aforementioned A pharmaceutical composition, or an isolated nucleic acid molecule as described above. Preferably, the method comprises administering to the subject a composition containing a unit dose containing 0.1-3000 mg of the multispecific protein molecule as described above, or the pharmaceutical composition as described above, or as described above Of isolated nucleic acid molecules.

在一些實施方式中，前面任一所述癌症選自癌瘤，淋巴瘤，胚細胞瘤(blastoma)，肉瘤，和白血病或淋巴樣惡性。該癌症的更具體的例子包括鱗狀細胞癌、骨髓瘤、小細胞肺癌、非小細胞肺癌(NSCLC)、頭和頸鱗狀細胞癌(HNSCC)、神經膠質瘤、何傑金淋巴瘤、非何傑金淋巴瘤、彌漫性大B-細胞淋巴瘤(DLBCL)、濾泡性淋巴瘤、急性成淋巴細胞性白血病(ALL)、急性髓細胞樣白血病(AML)、慢性淋巴細胞性白血病(CLL)、慢性髓細胞樣白血病(CML)、原發性縱隔大B-細胞淋巴瘤、套細胞淋巴瘤(MCL)、小淋巴細胞性淋巴瘤(SLL)、富含T-細胞/組織細胞的大B-細胞淋巴瘤、多發性骨髓瘤、髓樣細胞白血病-1蛋白(Mcl-1)、骨髓異常增生綜合症(MDS)、胃腸(道)癌、腎癌、卵巢癌、肝癌、成淋巴細胞性白血病、淋巴細胞白血病、結腸直腸癌、子宮內膜癌、腎癌、前列腺癌、甲狀腺癌、黑色素瘤、軟骨肉瘤、神經母細胞瘤、胰腺癌、多形性成膠質細胞瘤、胃癌、骨癌、尤因氏肉瘤、子宮頸癌、腦癌、胃癌、膀胱癌、肝細胞瘤、乳腺癌、結腸癌、肝細胞癌(HCC)、透明細胞腎細胞癌(RCC)、頭和頸癌、咽喉癌、肝膽癌(hepatobiliary cancer)、中樞神經系統癌、食管癌、惡性胸膜間皮瘤、全身性輕鏈澱粉樣變性、淋巴漿細胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓異常增生綜合症、骨髓增生性腫瘤、神經內分泌腫瘤、梅克爾細胞癌、睾丸癌和皮膚癌。在一些實施方案中，其中該癌症為B7-H3陽性細胞相關的癌症；較佳為乳腺癌、卵巢癌、前列腺癌、胰腺癌、腎癌、肺癌、肝癌、胃癌、結腸癌、膀胱癌、食管癌、宮頸癌、膽囊癌、膠質母細胞瘤和黑色素瘤。In some embodiments, any one of the aforementioned cancers is selected from carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of this cancer include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin’s lymphoma, non Hodgkin’s lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) ), chronic myeloid leukemia (CML), the originalPrimary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), large B-cell lymphoma rich in T-cells/histiocytosis, multiple myeloma, myeloid Cell-like leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal (tract) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, uterus Endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric cancer, bone cancer, Ewing's sarcoma, cervical cancer, brain Cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, throat cancer, hepatobiliary cancer, central nervous system Systemic cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumor, neuroendocrine tumor, Merkel cell carcinoma , Testicular cancer and skin cancer. In some embodiments, wherein the cancer is a B7-H3-positive cell-related cancer; preferably breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophagus Cancer, cervical cancer, gallbladder cancer, glioblastoma and melanoma.

在一些實施方式中，前面任一所述自身免疫性疾病或炎性疾病選自：類風濕關節炎、牛皮癬、克羅恩病、強硬性脊柱炎、多發性硬化症、I型糖尿病、肝炎、心肌炎、Sjogren綜合症、移植排斥後的自體免疫性溶血性貧血、水皰性類天皰瘡、格雷夫氏病、橋本甲狀腺炎、系統性紅斑狼瘡(SLE)、重症肌無力、天皰瘡、惡性貧血。In some embodiments, any one of the aforementioned autoimmune diseases or inflammatory diseases is selected from: rheumatoid arthritis, psoriasis, Crohn's disease, ankylosing spondylitis, multiple sclerosis, type I diabetes, hepatitis, Myocarditis, Sjogren syndrome, autoimmune hemolytic anemia after transplant rejection, vesicular pemphigoid, Grave's disease, Hashimoto's thyroiditis, systemic lupus erythematosus (SLE), myasthenia gravis, pemphigus, Pernicious anemia.

[發明詳述][Detailed description of the invention]

術語the term

本公開所用胺基酸三字母代碼和單字母代碼如J.biol.chem,243,p3558(1968)中所述。The three-letter codes and one-letter codes of amino acids used in the present disclosure are as described in J. biool. chem, 243, p3558 (1968).

術語“多特異性蛋白分子”指能夠與兩個或兩個以上的目標抗原或目標抗原表位特異性結合的蛋白分子。能夠對兩個目標抗原或目標抗原表位特異性結合的蛋白分子稱為雙特異性蛋白分子，包含抗體或抗體的抗原結合片段(如單鏈抗體)的“雙特異性蛋白分子”在本文中可以與“雙特異性抗體”互換。The term "multispecific protein molecule" refers to a protein molecule that can specifically bind to two or more target antigens or target antigen epitopes. A protein molecule capable of specifically binding to two target antigens or target antigen epitopes is called a bispecific protein molecule. A "bispecific protein molecule" comprising an antibody or an antigen-binding fragment of an antibody (such as a single-chain antibody) is used herein Can be interchanged with "bispecific antibody".

術語針對抗原的“結合區”或“結合區域”是指在多特異性蛋白分子或在抗體分子中，能夠與抗原特異性結合的區域或部分(part)，抗原結合區可以是能直接與抗原結合的配體結合結構域部分、也可以是能直接與抗原結合的包含抗體可變區的結構域。The term "binding region" or "binding region" for an antigen refers to a region or part that can specifically bind to an antigen in a multispecific protein molecule or an antibody molecule. The antigen binding region can be a region or part that can directly bind to the antigen. The part of the ligand binding domain to be bound may also be a domain containing the variable region of an antibody that can directly bind to an antigen.

術語“抗體(antibody，Ab)”包含任何包括至少一個與具體抗原(例如CD3)特異性結合或相互作用的互補決定區(CDR)的抗原結合分子或分子複合物。術語“抗體”包含：包括藉由雙硫鍵相互連接的四條多肽鏈，二條重(H)鏈和二條輕(L)鏈的免疫球蛋白分子以及其多聚體(例如IgM)。各重鏈包含重鏈可變區(文中縮寫為HCVR或VH)和重鏈恆定區。這一重鏈恆定區包含三個區(結構域)，CH1、CH2和CH3。各輕鏈包含輕鏈可變區(文中縮寫為LCVR或VL)和輕鏈恆定區。輕鏈恆定區包含一個區(結構域，CL1)。VH和VL區可進一步細分為高變區，稱為互補決定區(CDR)，其間散佈著較保守性區域，稱為框架區(framework region,FR，也稱骨架區、構架區)。各VH和VL是由三個CDR和四個FR所組成，以下列順序由胺基端排列到羧基端：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。在本公開的不同實施例中，抗CD3抗體(或其抗原結合部分)、抗B7H3抗體(或其抗原結合部分)或抗其他目標抗原的FR可與人類生殖系序列相同，或可經自然或人工修飾。抗體可以是不同亞類(subclass)的抗體，例如，IgG(例如，IgG1、IgG2、IgG3、或IgG4亞類)、IgA1、IgA2、IgD、IgE或IgM抗體。The term "antibody (Ab)" includes any antigen binding molecule or molecular complex that includes at least one complementarity determining region (CDR) that specifically binds or interacts with a specific antigen (eg, CD3). The term "antibody" includes immunoglobulin molecules including four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and their multimers (for example, IgM). Each heavy chain includes a heavy chain variable region (abbreviated as HCVR or VH in the text) and a heavy chain constant region. This heavy chain constant region contains three regions (domains), CH1, CH2 and CH3. Each light chain includes a light chain variable region (abbreviated as LCVR or VL in the text) and a light chain constant region. The light chain constant region contains a region (domain, CL1). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDR), interspersed with more conservative regions, called framework regions (FR, also called framework regions, framework regions). Each VH and VL are composed of three CDRs and four FRs, arranged from the amino end to the carboxyl end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the anti-CD3 antibody (or antigen-binding portion thereof), anti-B7H3 antibody (or antigen-binding portion thereof), or FR against other target antigens may be the same as the human germline sequence, or may be natural or Artificial modification. The antibodies may be antibodies of different subclasses, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subclass), IgA1, IgA2, IgD, IgE, or IgM antibodies.

術語“抗體”還包含完全抗體分子的抗原結合片段。術語抗體的“抗原結合部分”、“抗原結合結構域”、“抗原結合片段”等，如文中所用，包含任何與抗原特異性結合形成複合物的天然生成、酶製得、合成或基因工程改造的多肽或糖蛋白。抗體的抗原結合片段可使用任何適合的標準技術，例如蛋白質水解消化作用或涉及編碼抗體可變區和(視需要)恆定區的DNA操作和表達的重組基因工程技術，來源於例如全抗體分子。這一DNA是已知的和/或可容易地從例如市售來源、DNA資料庫(包含，例如噬菌體-抗體資料庫)取得或可經合成。這一DNA可用化學或藉由使用分子生物技術來定序和操作，例如將一個或多個可變和/或恆定區排列成適合的配置，或導入密碼子，產生半胱胺酸殘基、修飾、增添或刪除胺基酸等。The term "antibody" also encompasses antigen-binding fragments of complete antibody molecules. The terms "antigen-binding portion", "antigen-binding domain", "antigen-binding fragment", etc. of an antibody, as used herein, include any naturally occurring, enzymatic, synthetic or genetically engineered complex that specifically binds to an antigen Of peptides or glycoproteins. Antigen-binding fragments of antibodies can use any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving DNA manipulation and expression of variable regions and (optionally) constant regions encoding antibodies, derived from, for example, whole antibody molecules. This DNA is known and/or can be easily obtained from, for example, commercially available sources, DNA databases (including, for example, phage-antibody databases) or can be synthesized. This DNA can be sequenced and manipulated chemically or by using molecular biotechnology, such as arranging one or more variable and/or constant regions into a suitable configuration, or introducing codons to generate cysteine residues, Modify, add or delete amino acids, etc.

抗原結合片段的非限定示例包含：(i)Fab片段；(ii)F(ab')2片段；(iii)Fd片段；(iv)Fv片段；(v)單鏈Fv(scFv)分子；(vi)dAb片段。其它工程改造分子，例如區域特異性抗體、單域抗體、區域刪除抗體、嵌合抗體、CDR-植入抗體、雙抗體、三抗體、四抗體、微抗體、奈米抗體(例如單價奈米抗體、雙價奈米抗體等)、小模塊免疫醫藥(SMIP)和鯊可變IgNAR區，也涵蓋在文中所用的“抗原結合片段”的詞語內。Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab' )2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; ( vi) dAb fragment. Other engineered molecules, such as region-specific antibodies, single-domain antibodies, region-deleted antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, tri-antibodies, tetra-antibodies, mini-antibodies, nano-antibodies (e.g. monovalent nano-antibodies , Bivalent nanoantibodies, etc.), small modular immunopharmaceuticals (SMIP) and shark variable IgNAR regions are also included in the term "antigen-binding fragment" used in the text.

抗體的抗原結合片段典型地將包含至少一個可變區。可變區可以是任何大小或胺基酸組成的區域且一般將包含與一個或多個框架序列相鄰或在其框架內的CDR。在具有VH區與VL區結合的抗原結合片段中，VH和VL區可以任何適合的排列位於彼此相對處。例如可變區可為二聚化並含有VH-VL或VL-VH二聚體。The antigen-binding fragment of an antibody will typically contain at least one variable region. The variable region can be a region of any size or amino acid composition and will generally comprise CDRs adjacent to or within the framework of one or more framework sequences. Among the antigen-binding fragments that have a combination of VH and VL regions, the VH and VL regionsThey can be located opposite each other in any suitable arrangement. For example, the variable region may be dimerized and contain VH-VL or VL-VH dimers.

在某些實施例中，抗體的抗原結合片段在任何可變區和恆定區的配置中，可變區和恆定區可直接彼此相連接或可藉由完整或部分的絞鏈或連接子區相連接。絞鏈區可由至少2個(例如5、10、15、20、40、60或更多個)胺基酸所組成，使其在單一多肽分子中於相鄰的可變和/或恆定區之間產生柔性和半柔性連結。再者，在本發明的抗體的抗原結合片段可包含以非共價彼此相互連結和/或與一個或多個單體VH或VL區相連結(例如以雙硫鍵)的任何上列的可變區和恆定區配置的同源二聚體或異源二聚體(或其它多聚體)。In some embodiments, the antigen-binding fragment of an antibody is in any variable region and constant region configuration, the variable region and constant region can be directly connected to each other or can be connected to each other by a complete or partial hinge or linker region. connection. The hinge region can be composed of at least 2 (for example, 5, 10, 15, 20, 40, 60 or more) amino acids, making it between adjacent variable and/or constant regions in a single polypeptide molecule. Produce flexible and semi-flexible connections between. Furthermore, the antigen-binding fragments of the antibodies of the present invention may include any of the above-listed materials that are non-covalently connected to each other and/or to one or more monomeric VH or VL regions (for example, disulfide bonds). The homodimer or heterodimer (or other multimer) of the variable region and constant region configuration.

“鼠源抗體”在本公開中為根據本領域知識和技能製備的來源於小鼠的單株抗體。製備時用抗原注射試驗對象，然後分離表達具有所需序列或功能特性的抗體的融合瘤，當所注射的試驗對象為小鼠時，所產生的抗體為鼠源抗體。A "murine antibody" in the present disclosure is a mouse-derived monoclonal antibody prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with antigen, and then the fusion tumor expressing the antibody with the desired sequence or functional properties is isolated. When the injected test subject is a mouse, the antibody produced is a murine antibody.

“嵌合抗體(chimeric antibody)”，是將鼠源抗體的可變區與人抗體的恆定區融合而成的抗體，可以減輕鼠源性抗體誘發的免疫應答反應。建立嵌合抗體，要先建立分泌鼠源性特異性單抗的融合瘤，然後從鼠融合瘤細胞中選殖可變區基因，再根據需要選殖人抗體的恆定區基因，將鼠可變區基因與人恆定區基因連接成嵌合基因後插入表達載體中，最後在真核系統或原核系統中表達嵌合抗體分子。在本公開一個較佳的實施方案中，該嵌合抗體的抗體輕鏈進一步包含人源κ、λ鏈或其變體的輕鏈恆定區。該嵌合抗體的抗體重鏈進一步包含人源IgG1、IgG2、IgG3、IgG4或其變體的重鏈恆定區，較佳包含人源IgG1、IgG2或IgG4重鏈恆定區，或者使用胺基酸突變(如YTE突變或回復突變，L234A和/或L235A突變，或S228P突變)的IgG1、IgG2或IgG4重鏈恆定區變體。"Chimeric antibody" is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a fusion tumor that secretes a murine-derived specific monoclonal antibody, and then select the variable region gene from the mouse fusion tumor cell, and then select the constant region gene of the human antibody according to the need, and change the mouse variable region gene. The region gene and the human constant region gene are connected to form a chimeric gene and then inserted into the expression vector, and finally the chimeric antibody molecule is expressed in the eukaryotic or prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof. The antibody heavy chain of the chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising human IgG1, IgG2 orIgG4 heavy chain constant region, or IgG1, IgG2 or IgG4 heavy chain constant region variants using amino acid mutations (such as YTE mutation or back mutation, L234A and/or L235A mutation, or S228P mutation).

術語“人源化抗體(humanized antibody)”，包括CDR移植抗體(CDR-grafted antibody)，是指將動物來源抗體，例如鼠源抗體的CDR序列移植到人的抗體可變區框架區(或構架區，framework region)中產生的抗體。可以克服嵌合抗體由於攜帶大量異源蛋白成分，從而誘導的異源性反應。此類構架序列可以從包括種系抗體基因序列的公共DNA數據庫或公開的參考文獻獲得。如人重鏈和輕鏈可變區基因的種系DNA序列可以在“VBase”人種系序列數據庫(在因特網http：//www.vbase2.org/獲得)，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。為避免免疫原性下降的同時，引起的活性下降，可對該人抗體可變區框架序列進行最少的反向突變或回復突變，以保持活性。本公開的人源化抗體也包括進一步由噬菌體展示對CDR進行親和力成熟後的人源化抗體。The term "humanized antibody", including CDR-grafted antibody, refers to transplanting the CDR sequence of an animal-derived antibody, such as a murine antibody, into a human antibody variable region framework (or framework). Region, framework region). It can overcome the heterogeneous reaction induced by the chimeric antibody carrying a large amount of heterologous protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of the human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet http://www.vbase2.org/), and in Kabat, EA, etc., Found in 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease of immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity. The humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation for CDR by phage display.

由於抗原的接觸殘基，CDR的移植可由於與抗原接觸的構架殘基而導致產生的抗體或其抗原結合片段對抗原的親和力減弱。此類相互作用可以可能是體細胞高度突變的結果。因此，可能仍然需要將此類供體構架胺基酸移植至人源化抗體的構架。來自非人抗體或其抗原結合片段的參與抗原結合的胺基酸殘基可藉由檢查動物單株抗體可變區序列和結構來鑒定。CDR供體構架中與種系不同的各殘基可被認為是相關的。如果不能確定最接近的種系，那麼可將序列與亞類共有序列或具有高相似性百分數的動物抗體序列的共有序列相比較。稀有構架殘基被認為可能是體細胞高度突變的結果，從而在結合中發揮重要作用。Due to the contact residues of the antigen, the grafting of the CDR may result in the weakened affinity of the produced antibody or its antigen-binding fragment to the antigen due to the framework residues in contact with the antigen. Such interactions may be the result of hypermutation of somatic cells. Therefore, it may still be necessary to transplant such donor framework amino acids to the framework of humanized antibodies. The amino acid residues involved in antigen binding from non-human antibodies or antigen-binding fragments thereof can be identified by examining the sequence and structure of the variable region of animal monoclonal antibodies. Residues in the CDR donor framework that are different from the germline can be considered related. If the closest germline cannot be determined, the sequence can be compared with the consensus sequence of a subclass or animal antibody sequence with a high percentage of similarity. Rare framework residues are thought to be the result of hypermutation of somatic cells and thus play an important role in binding.

在本公開一個的實施方案中，該抗體或其抗原結合片段，可進一步包含人源或鼠源κ、λ鏈或其變體的輕鏈恆定區，或進一步包含人源或鼠源IgG1、IgG2、IgG3、IgG4或其變體的重鏈恆定區。In an embodiment of the present disclosure, the antibody or antigen-binding fragment thereof may further comprise a light chain constant region of human or murine κ, λ chain or variants thereof, or further comprise human or murine IgG1, IgG2 , IgG3, IgG4 or variants of the heavy chain constant region.

“人抗體”與“人源抗體”可以互換使用，可以是源於人的抗體或者是從一種轉基因生物體中獲得的抗體，該轉基因生物體經“改造”以響應於抗原刺激而產生特異性人抗體並且可以藉由本領域已知的任何方法產生。在某些技術中，將人重鏈和輕鏈基因座的元素元件引入到源於胚胎幹細胞系的生物體的細胞株中，這些細胞系中的內源性重鏈和輕鏈基因座被靶向破壞這些細胞系中包含靶向的內源性重鏈和輕鏈基因座破壞。轉基因生物可以合成對人抗原特異的人抗體，並且該生物可以用於產生人抗體-分泌融合瘤。人抗體還可以是一種抗體，其中重鏈和輕鏈是由源於一個或多個人DNA來源的核苷酸序列編碼的。完全人抗體還可以藉由基因或染色體轉染方法以及噬菌體展示技術來構建，或者由體外活化的B細胞構建，所有的這些都是本領域已知的。"Human antibody" and "human antibody" can be used interchangeably. They can be antibodies derived from humans or antibodies obtained from a genetically modified organism that has been "engineered" to produce specificity in response to antigen stimulation Human antibodies can also be produced by any method known in the art. In some technologies, the elements of human heavy and light chain loci are introduced into cell lines of organisms derived from embryonic stem cell lines, and the endogenous heavy and light chain loci in these cell lines are targeted. Disruption of these cell lines contains the targeted endogenous heavy and light chain loci. Transgenic organisms can synthesize human antibodies specific to human antigens, and the organisms can be used to produce human antibody-secreting fusion tumors. A human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies can also be constructed by gene or chromosome transfection methods and phage display technology, or constructed from B cells activated in vitro, all of which are known in the art.

“單株抗體”是指從基本上均質抗體的群體獲得的抗體，即除可能的變體抗體(例如含有天然存在的突變或在製造單株抗體製劑的期間產生的突變，這些變體通常以少量存在)之外，構成該群體的個別抗體相同和/或結合相同表位。與通常包含針對不同決定簇(表位)的不同抗體的多株抗體製備物不同，單株抗體製備物(製劑)的每個單株抗體是針對抗原上的單一決定簇的。因此，修飾語“單株”指示如從基本上均質抗體群體獲得的抗體的特性，且不應解釋為需要藉由任何特定方法來製造抗體。例如，根據本公開使用的單株抗體可藉由各種技術製備，該技術包括但不限於融合瘤方法、重組DNA方法、噬菌體展示方法以及利用含有全部或部分人免疫球蛋白基因座的轉基因動物的方法，此類方法以及用於製備單株抗體的其他示例性方法在本文中進行描述。"Monclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, except for possible variant antibodies (for example, containing naturally occurring mutations or mutations generated during the manufacture of monoclonal antibody preparations, these variants are usually Except for a small amount), the individual antibodies constituting the population are identical and/or bind the same epitope. Unlike multi-strain antibody preparations which usually contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation (preparation) is directed against a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates the characteristics of the antibody as obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of the antibody by any specific method. For example, monoclonal antibodies used in accordance with the present disclosure can be prepared by various techniques, including but not limited to fusion tumor methods, recombinant DNA methods, phage display methods, andMethods using transgenic animals that contain all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

術語“全長抗體”、“完整抗體”、“完全抗體”和“全抗體”在本文中可互換使用，指基本上完整形式的抗體，與下文定義的抗原結合片段相區分。該術語特別指重鏈包含Fc區的抗體。The terms "full-length antibody", "whole antibody", "whole antibody" and "whole antibody" are used interchangeably herein and refer to an antibody in a substantially intact form, as distinguished from the antigen-binding fragments defined below. The term specifically refers to an antibody whose heavy chain contains an Fc region.

此外，雖然Fv片段的兩個結構域VL和VH由分開的基因編碼，但可使用重組方法，藉由合成的接頭連接它們，從而使得其能夠產生為其中VL和VH區配對形成單價分子的單個蛋白質鏈(稱為單鏈Fv(scFv)；參見，例如，Bird等人(1988)Science242：423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85：5879-5883)。此類單鏈抗體也意欲包括在術語抗體的“抗原結合片段”中。使用本領域技術人員已知的常規技術獲得此類抗體片段，並且以與對於完整抗體的方式相同的方式就功用性篩選的片段。可藉由重組DNA技術或藉由酶促或化學斷裂完整免疫球蛋白來產生抗原結合部分。In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, recombination methods can be used to connect them by a synthetic linker so that it can be produced as a single molecule in which the VL and VH regions are paired to form a monovalent molecule. Protein chains (referred to as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single chain antibodies are also intended to be included in the term "antigen-binding fragments" of antibodies. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies. The antigen binding portion can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of the intact immunoglobulin.

抗原結合片段還可併入至包含一對串聯Fv片段(VH-CH1-VH-CH1)的單鏈分子中，該對串聯Fv片段連同互補輕鏈多肽一起形成一對抗原結合區(Zapata等人，1995 Protein Eng.8(10)：1057-1062；及美國專利US5641870)。Antigen-binding fragments can also be incorporated into single-chain molecules comprising a pair of tandem Fv fragments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al. , 1995 Protein Eng. 8(10): 1057-1062; and US Patent No. 5,641,870).

Fab是藉由用蛋白酶木瓜蛋白酶(切割H鏈的224位的胺基酸殘基)處理IgG抗體分子所獲得的片段中的具有約50,000Da的分子量並具有抗原結合活性的抗體片段，其中H鏈N端側的約一半和整個L鏈藉由二硫鍵結合在一起。Fab is an antibody fragment that has a molecular weight of about 50,000 Da and has antigen-binding activity among the fragments obtained by treating IgG antibody molecules with the protease papain (cutting the amino acid residue at position 224 of the H chain), wherein the H chain About half of the N-terminal side and the entire L chain are joined by disulfide bonds.

F(ab')2是藉由用胃蛋白酶消化IgG鉸鏈區中兩個二硫鍵的下方部分而獲得的分子量為約100,000Da並具有抗原結合活性並包含在鉸鏈位置相連的兩個Fab區的抗體片段。F(ab')2 is obtained by digesting the lower part of the two disulfide bonds in the hinge region of IgG with pepsin. It has a molecular weight of about 100,000 Da and has antigen-binding activity and contains two Fab regions connected at the hinge position. Antibody fragments.

Fab'是藉由切割上述F(ab')2的鉸鏈區的二硫鍵而獲得的分子量為約50,000Da並具有抗原結合活性的抗體片段。Fab'可以藉由用還原劑例如二硫蘇糖醇處理特異性識別並結合抗原的F(ab')2來生產。Fab' is an antibody fragment with a molecular weight of about 50,000 Da and antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of F(ab')2. Fab' can be produced by treating F(ab')2 that specifically recognizes and binds antigen with a reducing agent such as dithiothreitol.

此外，可以藉由將編碼抗體的Fab'片段的DNA插入到原核生物表達載體或真核生物表達載體中並將載體導入到原核生物或真核生物中以表達Fab'來生產該Fab'。In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into a prokaryotic organism or eukaryotic organism to express the Fab'.

術語“單鏈抗體”、“單鏈Fv”或“scFv”意指包含藉由接頭連接的抗體重鏈可變結構域(或區域；VH)和抗體輕鏈可變結構域(或區域；VL)的分子。此類scFv分子可具有一般結構：NH₂-VL-接頭-VH-COOH或NH₂-VH-接頭-VL-COOH。合適的現有技術接頭由重複的GGGGS胺基酸序列或其變體組成，例如使用1-4個(包括1個、2個、3個或4個)重複的變體(Holliger等人(1993)，Proc Natl Acad Sci USA.90：6444-6448)。可用於本公開的其他接頭由Alfthan等人(1995)，Protein Eng.8：725-731，Choi等人(2001)，Eur J Immuno.31：94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J Mol Biol.293：41-56和Roovers等人(2001)，Cancer Immunol Immunother.50：51-59.描述。The term "single-chain antibody", "single-chain Fv" or "scFv" means an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. ) Numerator. Such scFv molecules may have the general structure: NH₂ -VL-linker-VH-COOH or NH₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example using 1-4 (including 1, 2, 3 or 4) repeated variants (Holliger et al. (1993) , Proc Natl Acad Sci USA. 90: 6444-6448). Other linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur J Immuno. 31: 94-106, Hu et al. (1996), Cancer Res 56: 3055-3061, Kipriyanov et al. (1999), J Mol Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol Immunother. 50: 51-59.

“多特異性抗體”是指包含兩個或更多個抗原結合結構域，能夠結合兩個或更多個不同的表位(例如，兩個、三個、四個或更多個不同的表位)，表位可以在相同或不同的抗原上的抗體。多特異性抗體的示例包括結合兩個不同表位的“雙特異性抗體”。"Multispecific antibody" refers to containing two or more antigen-binding domains capable of binding two or more different epitopes (for example, two, three, four or more different epitopes) The epitope can be on the same or different antigens. Examples of multispecific antibodies include "bispecific antibodies" that bind to two different epitopes.

術語腫瘤相關抗原的“雙價雙特異性抗體”是指雙特異性抗體中針對某個腫瘤相關抗原靶點具有兩個抗原結合區，例如B7H3雙價雙特異性抗體指在該雙特異性抗體中包含兩個針對B7H3的抗原結合區。術語“單價雙特異性抗體”是指雙特異性抗體中針對某個靶點只有一個抗原結合區，例如B7H3單價雙特異性抗體指在該雙特異性抗體中包含一個針對B7H3的抗原結合區。The term "bivalent bispecific antibody" of a tumor-associated antigen refers to a bispecific antibody that has two antigen binding regions against a tumor-associated antigen target. For example, the B7H3 bivalent bispecific antibody refers to the bispecific antibody It contains two antigen binding regions for B7H3. The term "monovalent bispecific antibody" refers to a bispecific antibody with only one antigen binding region for a certain target. For example, a B7H3 monovalent bispecific antibody refers to a bispecific antibody that contains an antigen binding region for B7H3.

“Linker”或“接頭”或“連接子”或用於連接兩個蛋白質結構域中間的“L1”指用於連接蛋白質結構域的連接性多肽序列，通常具有一定的柔性，linker的使用不會使蛋白質結構域原有的功能喪失。"Linker" or "linker" or "linker" or "L1" used to connect two protein domains refers to the connecting peptide sequence used to connect protein domains. It usually has a certain degree of flexibility. The use of linker will not The original function of the protein domain is lost.

雙抗體(diabody)是指scFv被二聚體化的抗體片段，是具有二價抗原結合活性的抗體片段。在二價抗原結合活性中，兩個抗原可以是相同或不同的。Diabodies are antibody fragments in which scFv is dimerized, and are antibody fragments with bivalent antigen binding activity. In the bivalent antigen binding activity, the two antigens may be the same or different.

dsFv是藉由將其中每個VH和VL中的一個胺基酸殘基被半胱胺酸殘基取代的多肽經由半胱胺酸殘基之間的二硫鍵相連而獲得的。可以按照已知方法(Protein Engineering.7：697(1994))基於抗體的三維結構預測來選擇被半胱胺酸殘基取代的胺基酸殘基。dsFv is obtained by connecting a polypeptide in which one amino acid residue in each of VH and VL is replaced by a cysteine residue via a disulfide bond between the cysteine residues. The amino acid residues substituted with cysteine residues can be selected according to a known method (Protein Engineering. 7: 697 (1994)) based on the three-dimensional structure prediction of the antibody.

本公開一些實施例中抗原結合片段可以藉由以下步驟來生產：獲得本公開的特異性識別並結合抗原的單株抗體的VH和/或VL及所需的其他結構域的編碼cDNA，構建編碼抗原結合片段的DNA，將該DNA插入到原核生物表達載體或真核生物表達載體中，然後將該表達載體導入到原核生物或真核生物中以表達抗原結合片段。In some embodiments of the present disclosure, the antigen-binding fragments can be produced by the following steps: obtain the cDNA encoding the VH and/or VL and other required domains of the monoclonal antibody of the present disclosure that specifically recognize and bind the antigen, and construct the encoding The DNA of the antigen-binding fragment is inserted into a prokaryotic expression vector or eukaryotic expression vector, and then the expression vector is introduced into the prokaryotic or eukaryotic organism to express the antigen-binding fragment.

"Fc區"可以是天然序列Fc區或變體Fc區。雖然免疫球蛋白重鏈的Fc區的邊界可能變化，但人IgG重鏈Fc區通常被定義成從位置Cys226上的胺基酸殘基或從Pro230延伸至其羧基端。Fc區中的殘基的編號為如Kabat中的EU索引的編號。Kabat等，Sequences of Proteins of Immunological Interest，第5版Public Health Service,National Institutes of Health,Bethesda,Md.,1991。免疫球蛋白的Fc區通常具有兩個恆定區結構域CH2和CH3。文中“第一Fc”也稱作“Fc1”，第二Fc也稱作“Fc2”。The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is generally defined as the position from Cys226.The amino acid residue may extend from Pro230 to its carboxy terminus. The numbering of residues in the Fc region is as the numbering of the EU index in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of immunoglobulin usually has two constant region domains, CH2 and CH3. In the text, the "first Fc" is also referred to as "Fc1", and the second Fc is also referred to as "Fc2".

“V_a1-L1-V_b1-L2-V_c2-L2-V_d2-L4-Fc1”與“V_e3-L5-V_f3-L6-Fc2”中V_a1、V_b1、V_c2、V_d2、V_e3和V_f3為抗體的輕鏈可變區或重鏈可變區，V_a1和V_b1結合第一抗原(第一靶抗原)表位，V_c2和V_d2結合第二抗原(第二靶抗原)表位，V_e3和V_f3結合第三抗原(第三靶抗原)表位，第一抗原表位、第二抗原表位和第三抗原表位可以相同或不同。"V_a 1-L1-V_b 1-L2-V_c 2-L2-V_d 2-L4-Fc1" and "V_e 3-L5-V_f 3-L6-Fc2" inV_a 1,V_b 1.V_c 2,V_d 2,V_e 3 andV_f 3 are light chain variable regions or heavy chain variable regions of antibodies, andV_a 1 andV_b 1 bind to the first antigen (first target antigen)table V_c 2 andV_d 2 bind to the epitope of the second antigen (second target antigen),V_e 3 andV_f 3 bind to the epitope of the third antigen (third target antigen), the first epitope, the second The epitope and the third epitope can be the same or different.

類似“VH_TAA-L1-VL_TAA-L2-VH_CD3-L3-VL_CD3-L4-Fc1”中，VH_TAA和VL_TAA表示抗體可變區結合腫瘤相關抗原的表位，VH_CD3和VL_CD3表示抗體可變區結合CD3的表位。Similar to "VH_TAA -L1-VL_TAA -L2-VH_CD3 -L3-VL_CD3 -L4-Fc1", VH_TAA and VL_TAA indicate the epitope where the variable region of the antibody binds to tumor-associated antigen, and VH_CD3 and VL_CD3 indicate The antibody variable region binds to the epitope of CD3.

本公開“knob-Fc”指在抗體Fc區包含T366W的點突變，以形成類似knob(紐)的空間結構。相對應地，“hole-Fc”指在抗體Fc區包含T366S、L368A、Y407V的點突變，以形成類似hole(孔)的空間結構。Knob-Fc和hole-Fc由於空間位阻的原因，更易形成異二聚體。為進一步地促進異二聚體的形成，還可在knob-Fc和hole-Fc分別引入S354C和Y349C的點突變，藉由二硫鍵進一步促進異二聚體的形成。同時，為消除或減弱抗體Fc引起的ADCC效應，還可向Fc引入的234A和235A的取代突變。例如，本公開較佳的knob-Fc和hole-Fc分別如SEQ ID NO：69和70所示。在雙特異性抗體中，knob-Fc或hole-Fc既可以作為第一多肽鏈的Fc區域，也可以作為第二多肽鏈的Fc區域，在同一雙特異性抗體中，第一多肽鏈和第二多肽鏈的Fc區不同時為knob-Fc或hole-Fc。The "knob-Fc" in the present disclosure refers to a point mutation containing T366W in the Fc region of an antibody to form a knob-like spatial structure. Correspondingly, "hole-Fc" refers to point mutations containing T366S, L368A, and Y407V in the Fc region of an antibody to form a hole-like spatial structure. Knob-Fc and hole-Fc are more likely to form heterodimers due to steric hindrance. To further promote the formation of heterodimers, point mutations of S354C and Y349C can be introduced into knob-Fc and hole-Fc, respectively, to further promote the formation of heterodimers through disulfide bonds. At the same time, in order to eliminate or weaken the ADCC effect caused by antibody Fc, substitution mutations of 234A and 235A can also be introduced into Fc. For example, the preferred knob-Fc and hole-Fc of the present disclosure are shown in SEQ ID NOs: 69 and 70, respectively. In bispecific antibodies, knob-Fc or hole-Fc can be used asIt is the Fc region of the first polypeptide chain, and can also be used as the Fc region of the second polypeptide chain. In the same bispecific antibody, the Fc regions of the first polypeptide chain and the second polypeptide chain are not both knob-Fc Or hole-Fc.

術語“胺基酸差異”或“胺基酸突變”是指相較於原蛋白質或多肽，變體蛋白質或多肽存在胺基酸的改變或突變，包括在原蛋白質或多肽的基礎上發生1個或數個胺基酸的插入、缺失或替換。The term "amino acid difference" or "amino acid mutation" means that compared with the original protein or polypeptide, the variant protein or polypeptide has amino acid changes or mutations, including one or more mutations on the basis of the original protein or polypeptide. Insertion, deletion or replacement of several amino acids.

抗體的“可變區”是指單獨的或組合的抗體輕鏈的可變區(VL)或抗體重鏈的可變區(VH)。如在本領域中已知的，重鏈和輕鏈的可變區各自由藉由3個互補決定區(CDR)(也稱為高變區)連接的4個框架區(FR)組成。每一條鏈中的CDR藉由FR緊密地保持在一起並且與來自另一條鏈的CDR一起促成抗體的抗原結合部位的形成。存在至少2個用於確定CDR的技術：(1)基於跨種序列變異性的方法(即，Kabat等Sequences of Proteins of Immunological Interest,(第5版，1991,National Institutes of Health,Bethesda MD))；和(2)基於抗原-抗體複合物的晶體學研究的方法(Al-Lazikani等，J.Molec.Biol.273：927-948(1997))。如本文中所用，CDR可指由任一方法或由兩種方法的組合確定的CDR。The "variable region" of an antibody refers to the variable region (VL) of the antibody light chain or the variable region (VH) of the antibody heavy chain, alone or in combination. As known in the art, the variable regions of the heavy chain and the light chain each consist of 4 framework regions (FR) connected by 3 complementarity determining regions (CDR) (also called hypervariable regions). The CDRs in each chain are held tightly by FRs and together with the CDRs from the other chain contribute to the formation of the antigen binding site of the antibody. There are at least two techniques for determining CDRs: (1) A method based on cross-species sequence variability (ie, Kabat et al. Sequences of Proteins of Immunological Interest, (5th edition, 1991, National Institutes of Health, Bethesda MD)) ; And (2) Method based on crystallographic study of antigen-antibody complex (Al-Lazikani et al., J. Molec. Biol. 273:927-948 (1997)). As used herein, CDR may refer to a CDR determined by either method or a combination of both methods.

術語“抗體框架”或“FR區”，是指可變結構域VL或VH的一部分，其用作該可變結構域的抗原結合環(CDR)的支架。從本質上講，其是不具有CDR的可變結構域。The term "antibody framework" or "FR region" refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.

術語“CDR”是指抗體的可變結構域內主要促成抗原結合的6個高變區之一。該6個CDR的最常用的定義之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中一些實施方式中使用的，CDR可以以Kabat規則(Kabat等Sequences of Proteins of Immunological Interest,(第5版，1991,National Institutes of Health,Bethesda MD))定義輕鏈可變結構域的CDR1、CDR2和CDR3(LCDR1、LCDR2、LCDR3)，以及重鏈可變結構域的CDR1、CDR2和CDR3(HCDR1、HCDR2、HCDR3)，例如在對本公開中CD3抗體CDR的定義。在另一些實施方式中，還可以採用IMGT等規則進行抗體的CDR的定義，例如在對B7H3抗體CDR即採用的是IMGT規則進行定義。The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that mainly contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used in some embodiments herein, CDRs can be based on Kabat rules (Kabat et al. Sequences of Proteins of Immunological Interest, (5th edition, 1991, National Institutes of Health, Bethesda MD))Define the CDR1, CDR2, and CDR3 of the light chain variable domain (LCDR1, LCDR2, LCDR3), and the CDR1, CDR2, and CDR3 (HCDR1, HCDR2, HCDR3) of the heavy chain variable domain, for example, in the present disclosure, the CD3 antibody CDR Definition. In other embodiments, rules such as IMGT can also be used to define the CDR of an antibody. For example, the CDR of a B7H3 antibody is defined by the IMGT rule.

“抗體恆定區結構域”指來源於抗體的輕鏈和重鏈的恆定區的結構域，包括CL和來源於不同類抗體的CH1、CH2、CH3和CH4結構域。抗體中用於連接重鏈CH1和CH2結構域的鉸鏈區(hinge region)不屬於本公開所定義的“抗體恆定區結構域”的範疇。"Antibody constant region domains" refer to the domains derived from the constant regions of the light and heavy chains of an antibody, including CL and CH1, CH2, CH3, and CH4 domains derived from different types of antibodies. The hinge region (hinge region) used to connect the CH1 and CH2 domains of the heavy chains in an antibody does not belong to the category of "antibody constant region domains" defined in the present disclosure.

術語“腫瘤抗原”是指由腫瘤細胞產生的物質，視需要是蛋白質，包括“腫瘤相關抗原”或“TAA”(其是指在腫瘤細胞中產生的且與相應的正常組織相比在癌症中差異表達的蛋白質)以及“腫瘤特異性抗原”或“TSA”(其是指在腫瘤細胞中產生的且與相應的正常組織相比在癌症中特異性表達或異常表達的腫瘤抗原)。The term "tumor antigen" refers to a substance produced by tumor cells, and optionally a protein, including "tumor-associated antigen" or "TAA" (which refers to what is produced in tumor cells and is compared with the corresponding normal tissue in cancer Differentially expressed proteins) and "tumor specific antigens" or "TSA" (which refers to tumor antigens produced in tumor cells and specifically expressed or abnormally expressed in cancer compared to the corresponding normal tissues).

“腫瘤相關抗原”的非限定示例包含，例如AFP、ALK、B7H3、BAGE蛋白質、BCMA、BIRC5(存活素)、BIRC7、β-連環蛋白(β-catenin)、brc-ab1、BRCA1、BORIS、CA9、CA125、碳酸酐酶IX、半胱天冬酶-8(caspase-8)、CALR、CCR5、CD19、CD20(MS4A1)、CD22、CD30、CD33、CD38、CD40、CD123、CD133、CD138、CDK4、CEA、Claudin 18.2、週期素-B1、CYP1B1、EGFR、EGFRvIII、ErbB2/Her2、ErbB3、ErbB4、ETV6-AML、EpCAM、EphA2、Fra-1、FOLR1、GAGE蛋白(例如GAGE-1、-2)、GD2、GD3、GloboH、磷脂醯肌醇蛋白聚糖-3(glypican-3)、GM3、gp100、Her2、HLA/B-raf、HLA/k-ras、HLA/MAGE-A3、hTERT、IL13Rα2、LMP2、κ-Light、LeY、MAGE蛋白(例如MAGE-1、-2、-3、-4、-6和-12)、MART-1、間皮素(mesothelin)、ML-IAP、MOv-γ、Muc1、Muc2、Muc3、Muc4、Muc5、Muc16(CA-125)、MUM1、NA17、NKG2D、NY-BR1、NY-BR62、NY-BR85、NY-ESO1、OX40、p15、p53、PAP、PAX3、PAX5、PCTA-1、PLAC1、PRLR、PRAME、PSMA(FOLH1)、RAGE蛋白質、Ras、RGS5、Rho、ROR1、SART-1、SART-3、STEAP1、STEAP2、TAG-72、TGF-β、TMPRSS2、湯-諾氏抗原(Thompson-nouvelle antigen；Tn)、TRP-1、TRP-2、酪胺酸酶和尿溶蛋白-3、5T4(Trophoblast glycoprotein)。Non-limiting examples of "tumor-associated antigens" include, for example, AFP, ALK, B7H3, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, β-catenin, brc-ab1, BRCA1, BORIS, CA9 , CA125, Carbonic Anhydrase IX, Caspase-8 (caspase-8), CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD33, CD38, CD40, CD123, CD133, CD138, CDK4, CEA, Claudin 18.2, Cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein (e.g. GAGE-1, -2), GD2, GD3, GloboH, glypican-3 (glypican-3), GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, IL13Rα2, LMP2, κ-Light, LeY, MAGE protein (e.g. MAGE-1, -2, -3, -4, -6 and -12), MART-1, mesothelin, ML -IAP, MOv-γ, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, ROR1, SART-1, SART-3, STEP1, STEP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase and urinary protein-3, 5T4 (Trophoblast glycoprotein).

“CD3”，指表達在T細胞上作為多分子T細胞受體(TCR)的部分的抗原，且其是由下列四條受體鏈中的二條鏈所形成的同源二聚體或異源二聚體所組成：CD3-ε、CD3-δ、CD3-ζ和CD3-γ。人類CD3-ε(hCD3ε)包含UniProtKB/Swiss-Prot：P07766.2中所述的胺基酸序列。人類CD3-δ(hCD3δ包含UniProtKB/Swiss-Prot：P04234.1中所述的胺基酸序列。因此，除非明確地指出是來自非人類物種，例如“小鼠CD3”、“猴CD3”等，否則“CD3”一詞指人類CD3。"CD3" refers to an antigen expressed on T cells as part of a multi-molecule T cell receptor (TCR), and it is a homodimer or heterodimer formed by two of the following four receptor chains The aggregate is composed of: CD3-ε, CD3-δ, CD3-ζ and CD3-γ. Human CD3-ε (hCD3ε) contains the amino acid sequence described in UniProtKB/Swiss-Prot: P07766.2. Human CD3-δ (hCD3δ contains the amino acid sequence described in UniProtKB/Swiss-Prot: P04234.1. Therefore, unless it is clearly stated that it is from a non-human species, such as "mouse CD3", "monkey CD3", etc., Otherwise the term "CD3" refers to human CD3.

“表位”或“抗原決定簇”是指抗原上免疫球蛋白或抗體特異性結合的部位。表位通常以獨特的空間構象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15個連續或非連續的胺基酸。參見，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。"Epitope" or "antigenic determinant" refers to the site on an antigen where an immunoglobulin or antibody specifically binds. Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or discontinuous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

術語“特異性結合”、“選擇性結合”、“選擇性地結合”和“特異性地結合”是指抗體對預先確定的抗原上的表位的結合。通常，抗體以大約小於10^-8M，例如大約小於10^-9M、10^-10M、10^-11M或更小的親和力(KD)結合。The terms "specifically binds", "selectively binds", "selectively binds" and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, antibodies bind with an affinity (KD) of approximately less than 10^-8 M, such as approximately less than 10^-9 M, 10^-10 M, 10^-11 M, or less.

術語“親和力”是指在單一表位處，抗體與抗原之間相互作用的強度。在各抗原位點內，抗體“臂”的可變區藉由弱非共價力與抗原在多個胺基酸位點處相互作用；相互作用愈大，親和力愈強。如本文所用，抗體或其抗原結合片段(例如Fab片段)的術語“高親和力”通常是指具有1E^-9M或更小的K_D(例如1E^-10M或更小的K_D、1E^-11M或更小的K_D、1E^-12M或更小的K_D、1E^-13M或更小的K_D、1E^-14M或更小的K_D等)的抗體或抗原結合片段。The term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single epitope. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at multiple amino acid sites through weak non-covalent forces; the greater the interaction, the stronger the affinity. As used herein, an antibody or antigen-binding fragment thereof (e.g. Fab fragments), the term "high affinity" generally refers to having a K_D 1E^-9 M in K_D or less (e.g. 1E^-10 M or less, 1E^{- 11} M or less K_D , 1E^-12 M or less K_D , 1E^-13 M or less K_D , 1E^-14 M or less K_D, etc.) antibodies or antigen-binding fragments.

術語"KD"或“K_D”是指特定抗體-抗原相互作用的解離平衡常數。通常，抗體以小於大約1E^-8M，例如小於大約1E^-9M、1E^-10M或1E^-11M或更小的解離平衡常數(KD)結合抗原，例如，如使用表面等離子體共振(SPR)技術在BIACORE儀中測定的。KD值越小，親和力越大。The term "KD" or "K_D "refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Generally, the antibody binds to the antigen with a dissociation equilibrium constant (KD) less than about 1E^-8 M, for example, less than about 1E^-9 M, 1E^-10 M, or 1E^-11 M or less, for example, as using surface plasmon resonance ( SPR) technology is measured in BIACORE instrument. The smaller the KD value, the greater the affinity.

術語“核酸分子”是指DNA分子和RNA分子。核酸分子可以是單鏈或雙鏈的，但較佳是雙鏈DNA。當將核酸與另一個核酸序列置於功能關係中時，核酸是“有效連接的”。例如，如果啟動子或增強子影響編碼序列的轉錄，那麼啟動子或增強子有效地連接至該編碼序列。The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.

術語"載體"意指能夠遞送一個或多個目標基因或序列並且較佳地在宿主細胞中表達其的構建體。載體的示例包括，但不限於，病毒載體、裸露DNA或RNA表達載體、質粒、黏粒或噬菌體載體、與陽離子凝聚劑締合的DNA或RNA表達載體、包封在脂質體中的DNA或RNA表達載體和某些真核生物細胞諸如生產細胞。The term "vector" means a construct capable of delivering one or more target genes or sequences and preferably expressing them in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic flocculants, DNA or RNA encapsulated in liposomes Expression vectors and certain eukaryotic cells such as producer cells.

現有技術中熟知生產和純化抗體和抗原結合片段的方法，如冷泉港的抗體實驗技術指南，5-8章和15章。例如，鼠可以用抗原或其片段免疫，所得到的抗體能被覆性、純化，並且可以用常規的方法進行胺基酸測序。抗原結合片段同樣可以用常規方法製備。本公開所述的抗體或抗原結合片段用基因工程方法在非人源的CDR區加上一個或多個人源FR區。人FR種系序列可以藉由比對IMGT人類抗體可變區種系基因數據庫和MOE軟體，從網站http：//www.imgt.org/得到，或者從免疫球蛋白雜誌，2001ISBN012441351上獲得。The methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as Cold Spring Harbor’s Antibody Experiment Technical Guide, Chapters 5-8 and 15. For example, mice can be immunized with antigens or fragments thereof, and the obtained antibodies can be covered and purified, and amino acid sequencing can be performed by conventional methods. Antigen bindingFragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments described in the present disclosure are genetically engineered to add one or more human FR regions to non-human CDR regions. Human FR germline sequences can be obtained from the website http://www.imgt.org/ by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351.

術語“宿主細胞”是指已向其中引入了表達載體的細胞。宿主細胞可包括細菌、微生物、植物或動物細胞。易於轉化的細菌包括腸桿菌科(enterobacteriaceae)的成員，例如大腸桿菌(Escherichia coli)或沙門氏菌(Salmonella)的菌株；芽孢桿菌科(Bacillaceae)例如枯草芽孢桿菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；鏈球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。適當的微生物包括釀酒酵母(Saccharomyces cerevisiae)和畢赤酵母(Pichia pastoris)。適當的動物宿主細胞系包括CHO(中國倉鼠卵巢細胞系)、HEK293細胞(非限制性實施例如HEK293E細胞)和NS0細胞。The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plant or animal cells. Bacteria that are easily transformed include members of the enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), HEK293 cells (non-limiting examples such as HEK293E cells), and NSO cells.

工程化的抗體或抗原結合片段可用常規方法製備和純化。比如，編碼重鏈和輕鏈的cDNA序列，可以選殖並重組至GS表達載體。重組的免疫球蛋白表達載體可以穩定地轉染CHO細胞。作為一種可選的現有技術，哺乳動物類表達系統會導致抗體的糖基化，特別是在Fc區的高度保守N端位點。藉由表達與抗原特異性結合的抗體得到穩定的純株。陽性的純株在生物反應器的無血清培養基中擴大培養以生產抗體。分泌了抗體的培養液可以用常規技術純化。比如，用含調整過的緩衝液的蛋白A或蛋白G Sepharose FF管柱進行純化。洗去非特異性結合的組分。再用pH梯度法沖提結合的抗體，用SDS-PAGE檢測抗體片段，收集。抗體可用常規方法進行過濾濃縮。可溶的混合物和多聚體，也可以用常規方法去除，比如分子篩、離子交換。得到的產物需立即冷凍，如-70℃，或者凍乾。The engineered antibody or antigen-binding fragment can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As an alternative existing technology, mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region. A stable pure strain is obtained by expressing antibodies that specifically bind to the antigen. The positive pure strains are expanded in the serum-free medium of the bioreactor to produce antibodies. The antibody-secreted culture medium can be purified by conventional techniques. For example, use a protein A or protein G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. Then, the bound antibody was extracted by pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also beUse conventional methods to remove, such as molecular sieve, ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.

“給予”和“處理”當應用於動物、人、實驗受試者、細胞、組織、器官或生物流體時，是指外源性藥物、治療劑、診斷劑或組合物與動物、人、受試者、細胞、組織、器官或生物流體的接觸。“給予”和“處理”可以指例如治療、藥物代謝動力學、診斷、研究和實驗方法。細胞的處理包括試劑與細胞的接觸，以及試劑與流體的接觸，其中該流體與細胞接觸。“給予”和“處理”還意指藉由試劑、診斷、結合組合物或藉由另一種細胞體外和離體處理例如細胞。“處理”當應用於人、獸醫學或研究受試者時，是指治療處理、預防或預防性措施，研究和診斷應用。"Administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions that interact with animals, humans, and recipients. Contact with subjects, cells, tissues, organs or biological fluids. "Administration" and "treatment" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. The treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids contact the cells. "Administration" and "treatment" also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo. "Treatment" when applied to human, veterinary or research subjects, refers to treatment, preventive or preventive measures, research and diagnostic applications.

“治療”意指給予患者內用或外用治療劑，例如包含本公開實施例的任一種化合物的組合物，該患者具有一種或多種疾病症狀，而已知該治療劑對這些症狀具有治療作用。通常，在受治療患者或群體中以有效緩解一種或多種疾病症狀的量給予治療劑，以誘導這類症狀退化或抑制這類症狀發展到任何臨床有測量的程度。有效緩解任何具體疾病症狀的治療劑的量(也稱作“治療有效量”)可根據多種因素變化，例如患者的疾病狀態、年齡和體重，以及藥物在患者產生需要療效的能力。藉由醫生或其它專業衛生保健人士通常用於評價該症狀的嚴重性或進展狀況的任何臨床檢測方法，可評價疾病症狀是否已被減輕。儘管本公開的實施方案(例如治療方法或製品)可能無法在緩解每個目標疾病症狀方面都有效，但是根據本領域已知的任何統計學檢驗方法如Student t檢驗、卡方檢驗、依據Mann和Whitney的U檢驗、Kruskal-Wallis檢驗(H檢驗)、Jonckheere-Terpstra檢驗和Wilcoxon檢驗確定，其在統計學顯著數目的患者中應當減輕目標疾病症狀。"Treatment" means administering an internal or external therapeutic agent, such as a composition containing any one of the compounds of the embodiments of the present disclosure, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce the regression of such symptoms or inhibit the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount") can vary depending on various factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. By any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms, it can be evaluated whether the symptoms of the disease have been alleviated. Although the embodiments of the present disclosure (such as treatment methods or products) may not be effective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-TerpstraThe test and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.

“胺基酸保守修飾”或“胺基酸保守取代”指蛋白質或多肽中的胺基酸被具有相似特徵(例如電荷、側鏈大小、疏水性/親水性、主鏈構象和剛性等)的其他胺基酸取代，從而使得在不改變蛋白質或多肽的生物活性或其他所需特性(例如抗原親和力和/或特異性)的情況下，可以經常進行改變。本領域技術人員認識到，通常，多肽的非必需區域中的單個胺基酸取代基本上不改變生物活性(參見，例如，Watson等人，(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.，第224頁(第4版))。此外，結構上或功能上相似的胺基酸的取代不太可能破壞生物活性。示例性保守取代於下表“示例性胺基酸保守取代”中陳述。"Conservative modification of amino acid" or "conservative substitution of amino acid" means that the amino acid in the protein or polypeptide has similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) Other amino acid substitutions allow frequent changes without changing the biological activity or other desired characteristics of the protein or polypeptide (such as antigen affinity and/or specificity). Those skilled in the art recognize that, generally, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al., (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub .Co., page 224 (4th edition)). In addition, the substitution of structurally or functionally similar amino acids is unlikely to destroy biological activity. Exemplary conservative substitutions are set out in the table "Exemplary Amino Acid Conservative Substitutions" below.

示例性胺基酸保守取代Exemplary amino acid conservative substitutions

“有效量”、“有效劑量”是指獲得任一種或多種有益的或所需的治療結果所必需的藥物、化合物或醫藥組成物的量。對於預防用途，有益的或所需的結果包括消除或降低風險、減輕嚴重性或延遲病症的發作，包括病症、其併發症和在病症的發展過程中呈現的中間病理表型的生物化學、組織學和/或行為症狀。對於治療應用，有益的或所需的結果包括臨床結果，諸如減少各種本公開靶抗原相關病症的發病率或改善該病症的一個或多個症狀，減少治療病症所需的其它藥劑的劑量，增強另一種藥劑的療效，和/或延緩患者的本公開靶抗原相關病症的進展。"Effective amount" and "effective dose" refer to the amount of the drug, compound or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results. For preventive use, beneficial or desired results include elimination or reduction of risk, reduction of severity, or delay of the onset of the disease, including the biochemistry, tissue, and organization of the disease, its complications, and intermediate pathological phenotypes that appear during the development of the disease Academic and/or behavioral symptoms. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related disorders of the present disclosure or improving one or more symptoms of the disorder, reducing the dose of other agents required to treat the disorder, and enhancing The efficacy of another agent, and/or delay the progression of the patient's target antigen-related disorder of the present disclosure.

“外源性”指根據情況在生物、細胞或人體外產生的物質。“內源性”指根據情況在細胞、生物或人體內產生的物質。"Exogenous" refers to substances produced outside organisms, cells, or humans according to circumstances. "Endogenous" refers to substances produced in cells, organisms, or human bodies according to circumstances.

“同源性”、“同一性”在本文中可以互換，是指兩個多核苷酸序列之間或兩個多肽之間的序列相似性。當兩個比較序列中的位置均被相同鹼基或胺基酸單體亞基佔據時，例如如果兩個DNA分子的每一個位置都被腺嘌呤佔據時，那麼該分子在該位置是同源的。兩個序列之間的同源性百分率是兩個序列共有的匹配或同源位置數除以比較的位置數×100的函數。例如，在序列最佳比對時，如果兩個序列中的10個位置有6個匹配或同源，那麼兩個序列為60%同源；如果兩個序列中的100個位置有95個匹配或同源，那麼兩個序列為95%同源。通常，當比對兩個序列時進行比較以給出最大百分比同源性。例如，可以藉由BLAST算法執行比較，其中選擇算法的參數以在各個參考序列的整個長度上給出各個序列之間的最大匹配。"Homology" and "identity" are interchangeable herein and refer to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecule is homologous at that position of. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous; if there are 95 matches in 100 positions in the two sequences Or homology, then the two sequences are 95% homologous. Generally, when comparing two sequences, a comparison is made to give the maximum percentage homology. For example, the comparison can be performed by the BLAST algorithm, in which the parameters of the algorithm are selected to give the maximum match between each sequence over the entire length of each reference sequence.

以下參考文獻涉及經常用於序列分析的BLAST算法：BLAST算法(BLAST ALGORITHMS)：Altschul,S.F.等人，(1990)J.Mol.Biol.215：403-410；Gish,W.等人，(1993)Nature Genet.3：266-272；Madden,T.L.等人，(1996)Meth.Enzymol.266：131-141；Altschul,S.F.等人，(1997)Nucleic Acids Res.25：3389-3402；Zhang,J.等人，(1997)Genome Res.7：649-656。其他如NCBI BLAST提供的常規BLAST算法也為本領域技術人員所熟知。The following references refer to the BLAST algorithm that is often used in sequence analysis: BLAST algorithm (BLAST ALGORITHMS): Altschul, S.F. et al. (1990) J. Mol. Biol. 215: 403-410;Gish, W. et al., (1993) Nature Genet. 3:266-272; Madden, TL et al., (1996) Meth. Enzymol. 266:131-141; Altschul, SF et al., (1997) Nucleic Acids Res .25: 3389-3402; Zhang, J. et al. (1997) Genome Res. 7:649-656. Other conventional BLAST algorithms such as NCBI BLAST are also well known to those skilled in the art.

“分離的”指純化狀態，並且在這種情況下意味著在指定的分子基本上不含其他生物分子，例如核酸、蛋白質、脂質、碳水化合物或其他材料，例如細胞碎片和生長培養基。通常，術語“分離的”並不意圖指完全不存在這些材料或不存在水、緩衝液或鹽，除非它們以顯著干擾如本文所述的化合物的實驗或治療用途的量存在。"Isolated" refers to a purified state, and in this case means that the designated molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Generally, the term "isolated" is not intended to mean the complete absence of these materials or the absence of water, buffers or salts, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compound as described herein.

“視需要”或“視需要地”意味著隨後所描述地事件或環境可以但不必發生，該說明包括該事件或環境發生或不發生的場合。例如，“視需要包含1-3個抗體重鏈可變區”意味著特定序列的抗體重鏈可變區可以但不必須存在。"As needed" or "as needed" means that the event or environment described later can but does not have to occur, and the description includes occasions where the event or environment occurs or does not occur. For example, "containing 1-3 antibody heavy chain variable regions as necessary" means that the antibody heavy chain variable regions of a specific sequence may but need not be present.

“醫藥組成物”表示含有一種或多種本公開所述化合物或其生理學上/可藥用的鹽或前體藥物與其他化學組分的混合物，該其他組分例如生理學/可藥用的載體和賦形劑。醫藥組成物的目的是促進對生物體的給藥，利於活性成分的吸收進而發揮生物活性。"Pharmaceutical composition" means a mixture containing one or more of the compounds described in the present disclosure or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable Carriers and excipients. The purpose of the medicinal composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.

術語“藥學上可接受的載體”指適合用於製劑中用於遞送抗體或抗原結合片段的任何無活性物質。載體可以是抗黏附劑、黏合劑、包衣、崩解劑、充填劑或稀釋劑、防腐劑(如抗氧化劑、抗菌劑或抗真菌劑)、增甜劑、吸收延遲劑、潤濕劑、乳化劑、緩衝劑等。合適的藥學上可接受的載體的示例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等)右旋糖、植物油(例如橄欖油)、鹽水、緩衝液、緩衝的鹽水和等滲劑例如糖、多元醇、山梨糖醇和氯化鈉。The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for the delivery of antibodies or antigen-binding fragments. The carrier can be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (such as antioxidant, antibacterial or antifungal), sweetener, absorption delaying agent, wetting agent, Emulsifier, buffer, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (e.g., olive oil), saline, buffer, buffered saline, and the like Penetrating agents such as sugars, polyols, sorbitol and sodium chloride.

術語“癌症”、“癌的”或“惡性的”指或描述哺乳動物中一般以不受調節的細胞生長為特徵的生理狀況。癌症的例子包括但不限於癌瘤，淋巴瘤，胚細胞瘤(blastoma)，肉瘤，和白血病或淋巴樣惡性。這種癌症的更具體的例子包括鱗狀細胞癌、骨髓瘤、小細胞肺癌、非小細胞肺癌(NSCLC)、頭和頸鱗狀細胞癌(HNSCC)、神經膠質瘤、何傑金淋巴瘤、非何傑金淋巴瘤、彌漫性大B-細胞淋巴瘤(DLBCL)、濾泡性淋巴瘤、急性成淋巴細胞性白血病(ALL)、急性髓細胞樣白血病(AML)、慢性淋巴細胞性白血病(CLL)、慢性髓細胞樣白血病(CML)、原發性縱隔大B-細胞淋巴瘤、套細胞淋巴瘤(MCL)、小淋巴細胞性淋巴瘤(SLL)、富含T-細胞/組織細胞的大B-細胞淋巴瘤、多發性骨髓瘤、髓樣細胞白血病-1蛋白(Mcl-1)、骨髓異常增生綜合症(MDS)、胃腸(道)癌、腎癌、卵巢癌、肝癌、成淋巴細胞性白血病、淋巴細胞白血病、結腸直腸癌、子宮內膜癌、腎癌、前列腺癌、甲狀腺癌、黑色素瘤、軟骨肉瘤、神經母細胞瘤、胰腺癌、多形性成膠質細胞瘤、胃癌、骨癌、尤因氏肉瘤、子宮頸癌、腦癌、胃癌、膀胱癌、肝細胞瘤、乳腺癌、結腸癌、肝細胞癌(HCC)、透明細胞腎細胞癌(RCC)、頭和頸癌、咽喉癌、肝膽癌(hepatobiliary cancer)、中樞神經系統癌、食管癌、惡性胸膜間皮瘤、全身性輕鏈澱粉樣變性、淋巴漿細胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓異常增生綜合症、骨髓增生性腫瘤、神經內分泌腫瘤、梅克爾細胞癌、睾丸癌和皮膚癌。The terms "cancer", "cancerous" or "malignant" refer to or describe the physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of this cancer include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin’s lymphoma, Non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia ( CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), T-cell/histiocytic-rich Large B-cell lymphoma, multiple myeloma, myeloid leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal (tract) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblast Cellular leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric cancer, Bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatocytoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer , Throat cancer, hepatobiliary cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma (lymphoplasmacytic lymphoma), myelodysplastic syndrome, Myeloproliferative tumors, neuroendocrine tumors, Merkel cell carcinoma, testicular cancer and skin cancer.

“炎性病症”是指其中過度或不受調節的炎症反應導致過度炎性症狀、宿主組織損傷或組織功能喪失的任何疾病、病症或綜合症。“炎性疾病”也是指由白細胞或嗜中性粒細胞趨化性的彙集而介導的病理狀態。"Inflammatory disorder" refers to any disease, disorder or syndrome in which an excessive or unregulated inflammatory response results in excessive inflammatory symptoms, host tissue damage, or loss of tissue function. "Inflammatory disease" also refers to a pathological state mediated by the pooling of leukocytes or neutrophil chemotaxis.

“炎症”是指由於組織損傷或破壞而引起的局部保護性反應，其用來破壞、削弱或杜絕(隔離)有害物質和受傷組織。炎症與白細胞或中性粒細胞趨化性的彙集顯著相關。炎症可以由病原生物體和病毒以及非感染性原因引起，該非感染性原因如創傷或心肌梗塞後的再灌注或中風、對外源性抗原的免疫應答和自身免疫應答。"Inflammation" refers to a local protective response caused by tissue damage or destruction, which is used to destroy, weaken or eliminate (isolate) harmful substances and injured tissues. Inflammation is significantly related to the pooling of leukocyte or neutrophil chemotaxis. Inflammation can be caused by pathogenic organisms and viruses as well as non-infectious causes such as reperfusion or stroke after trauma or myocardial infarction, immune response to foreign antigens, and autoimmune response.

“自身免疫性疾病”是指其中組織損傷與體液或細胞介導的對身體自身成分的反應相關的任何一組疾病。自身免疫疾病的非限制性示例包括類風濕關節炎、牛皮癬、克羅恩病、強硬性脊柱炎、多發性硬化症、I型糖尿病、肝炎、心肌炎、Sjogren綜合症、移植排斥後的自體免疫性溶血性貧血、水皰性類天皰瘡、格雷夫氏病、橋本甲狀腺炎、系統性紅斑狼瘡(SLE)、重症肌無力、天皰瘡、惡性貧血等。"Autoimmune disease" refers to any group of diseases in which tissue damage is associated with body fluids or cell-mediated responses to the body's own components. Non-limiting examples of autoimmune diseases include rheumatoid arthritis, psoriasis, Crohn's disease, ankylosing spondylitis, multiple sclerosis, type I diabetes, hepatitis, myocarditis, Sjogren syndrome, autoimmunity after transplant rejection Hemolytic anemia, vesicular pemphigoid, Grave’s disease, Hashimoto’s thyroiditis, systemic lupus erythematosus (SLE), myasthenia gravis, pemphigus, pernicious anemia, etc.

此外，本公開另一方面涉及用於免疫檢測或測定目標抗原的方法、用於免疫檢測或測定目標抗原的試劑、用於免疫檢測或測定表達目標抗原的細胞的方法和用於診斷與目標抗原陽性細胞相關的疾病的診斷劑，其包含本公開的特異性識別並結合目標抗原的單株抗體或抗體片段作為活性成分。In addition, another aspect of the present disclosure relates to methods for immunological detection or determination of target antigens, reagents for immunological detection or determination of target antigens, methods for immunological detection or determination of cells expressing target antigens, and methods for diagnosis and target antigen A diagnostic agent for a disease related to a positive cell, which contains the monoclonal antibody or antibody fragment of the present disclosure that specifically recognizes and binds to the target antigen as an active ingredient.

在本公開中，用於檢測或測定目標抗原的量的方法可以是任何已知方法。例如，它包括免疫檢測或測定方法。In the present disclosure, the method for detecting or measuring the amount of the target antigen may be any known method. For example, it includes immunodetection or assay methods.

免疫檢測或測定方法是使用標記的抗原或抗體檢測或測定抗體量或抗原量的方法。免疫檢測或測定方法的示例包括放射性物質標記的免疫抗體方法(RIA)、酶免疫測定法(EIA或ELISA)、螢光免疫測定法(FIA)、發光免疫測定法、蛋白質免疫印跡法、物理化學方法等。The immunoassay or measurement method is a method of detecting or measuring the amount of antibody or antigen using labeled antigen or antibody. Examples of immunodetection or measurement methods include radioactive substance-labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay, western blotting, physical chemistry Methods etc.

上述與目標抗原陽性細胞相關的疾病可以藉由用本公開的單株抗體或抗體片段檢測或測定表達目標抗原的細胞來診斷。The above-mentioned diseases related to target antigen-positive cells can be diagnosed by detecting or measuring cells expressing the target antigen using the monoclonal antibody or antibody fragment of the present disclosure.

為了檢測表達多肽的細胞，可以使用已知的免疫檢測方法，並較佳使用免疫沉澱法、螢光細胞染色法、免疫組織染色法等。此外，可以使用利用FMAT8100HTS系統(Applied Biosystem)的螢光抗體染色法等。In order to detect cells expressing polypeptides, known immunoassay methods can be used, and preferably immunoprecipitation methods, fluorescent cell staining methods, immunotissue staining methods, etc. can be used. In addition, fluorescent antibody staining using FMAT8100HTS system (Applied Biosystem), etc. can be used.

在本公開中，對用於檢測或測定目標抗原的活體樣品沒有特別限制，只要它具有包含表達目標抗原的細胞的可能性即可，例如組織細胞、血液、血漿、血清、胰液、尿液、糞便、組織液或培養液。In the present disclosure, there is no particular limitation on the living body sample used to detect or measure the target antigen, as long as it has the possibility of containing cells expressing the target antigen, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, Stool, tissue fluid or culture fluid.

根據所需的診斷方法，含有本公開的單株抗體或其抗體片段的診斷劑還可以含有用於執行抗原-抗體反應的試劑或用於檢測反應的試劑。用於執行抗原-抗體反應的試劑包括緩衝劑、鹽等。用於檢測的試劑包括通常用於免疫檢測或測定方法的試劑，例如識別該單株抗體、其抗體片段或其結合物的標記的第二抗體和與該標記對應的受質等。According to the required diagnostic method, the diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may also contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction. The reagents used to perform the antigen-antibody reaction include buffers, salts and the like. The reagents used for detection include reagents commonly used in immunological detection or determination methods, such as a labeled second antibody that recognizes the monoclonal antibody, its antibody fragment or its conjugate, and the substrate corresponding to the label.

在以上說明書中提出了本發明一種或多種實施方式的細節。雖然可使用與本文所述類似或相同的任何方法和材料來實施或測試本發明，但是以下描述較佳的方法和材料。藉由說明書和申請專利範圍，本發明的其他特點、目的和優點將是顯而易見的。在說明書和申請專利範圍中，除非上下文中有清楚的另外指明，單數形式包括複數指代物的情況。除非另有定義，本文使用的所有技術和科學術語都具有本發明所屬領域普通技術人員所理解的一般含義。說明書中引用的所有專利和出版物都藉由引用納入。提出以下實施例是為了更全面地說明本發明的較佳實施方式。這些實施例不應以任何方式理解為限制本發明的範圍，本發明的範圍由申請專利範圍限定。The details of one or more embodiments of the present invention are set forth in the above description. Although any methods and materials similar or identical to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described below. With reference to the specification and the scope of patent application, other features, purposes and advantages of the present invention will be apparent. In the specification and patent application, unless the context clearly indicates otherwise, the singular form includes the plural referents. Unless otherwise defined, all technical and scientific terms used herein have the general meanings understood by those of ordinary skill in the art to which the present invention belongs. All patents and publications cited in the specification are incorporated by reference. The following examples are presented to more fully illustrate the preferred embodiments of the present invention. These embodiments should not be construed as limiting the scope of the present invention in any way, and the scope of the present invention is defined by the scope of patent application.

第1A圖和第1B圖：第1A圖為雙價雙特異性抗體示意圖，第1B圖為單價雙特異性抗體示意圖。Figure 1A and Figure 1B: Figure 1A is a schematic diagram of a bivalent bispecific antibody, and Figure 1B is a schematic diagram of a monovalent bispecific antibody.

第2A圖至第2D圖：流式細胞術對抗體與表達或不表達相應抗原的細胞結合活性檢測。第2A圖為不同抗體與表達人B7H3的A498細胞的結合活性檢測，第2B圖為不同抗體與過表達人B7H3的CT26細胞的結合活性檢測，第2C圖為不同抗體與不表達人B7H3的CT26細胞的結合活性檢測，結果顯示各抗體均不與不表達人B7H3的CT26細胞結合，第2D圖為不同抗體與表達CD3的Jurkat重組細胞結合活性檢測。第2A圖至第2D圖中縱坐標表示螢光信號的幾何平均值。Figure 2A to Figure 2D: Flow cytometry detection of antibody binding activity to cells expressing or not expressing the corresponding antigen. Figure 2A is the detection of the binding activity of different antibodies to A498 cells expressing human B7H3, Figure 2B is the detection of the binding activity of different antibodies to CT26 cells overexpressing human B7H3, Figure 2C is the detection of different antibodies and CT26 that does not express human B7H3 The cell binding activity test showed that none of the antibodies bind to CT26 cells that do not express human B7H3. Figure 2D shows the test of the binding activity of different antibodies to Jurkat recombinant cells expressing CD3. The ordinates in Figures 2A to 2D represent the geometric average of the fluorescent signal.

第3A圖至第3B圖：含有不同CD3 scFv的雙特異性抗體對A498的殺傷活性檢測。第3A圖為B7H3單價雙特異性抗體的殺傷活性。第3B圖為B7H3雙價雙特異抗體的殺傷活性。除了155、156、185和186對A498的殺傷活性較弱之外，其餘雙特異抗體，無論B7H3單價還是雙價，均顯示出較明顯的殺傷活性。Figure 3A to Figure 3B: Detection of A498 killing activity of bispecific antibodies containing different CD3 scFvs. Figure 3A shows the killing activity of the B7H3 monovalent bispecific antibody. Figure 3B shows the killing activity of the B7H3 bivalent bispecific antibody. Except for the weaker killing activity of 155, 156, 185 and 186 against A498, the other bispecific antibodies, regardless of B7H3 monovalent or bivalent, all showed obvious killing activity.

第4A圖至第4B圖：含有相同CD3 scFv的B7H3單雙價雙特異性抗體對A498殺傷活性比較。第4A圖為含HRH1的B7H3單價(181)和雙價(131)雙特異抗體的殺傷活性比較。第4B圖為含HRH7的B7H3單價(187)和雙價(177)的殺傷活性比較。實驗結果均顯示B7H3雙價雙特異性抗體較B7H3單價雙特異性抗體都具有明顯的A498殺傷活性，同時，B7H3雙價雙特異性抗體較B7H3單價雙特異性抗體的殺傷活性顯著增強。Figure 4A to Figure 4B: Comparison of A498 killing activity of B7H3 monobivalent bispecific antibodies containing the same CD3 scFv. Figure 4A shows the comparison of the killing activity of B7H3 monovalent (181) and bivalent (131) bispecific antibodies containing HRH1. Figure 4B shows the comparison of the monovalent (187) and bivalent (177) killing activities of B7H3 containing HRH7. The experimental results show that the B7H3 bivalent bispecific antibody has obvious A498 killing activity than the B7H3 monovalent bispecific antibody. At the same time, the B7H3 bivalent bispecific antibody has a significantly stronger killing activity than the B7H3 monovalent bispecific antibody.

第5A圖至第5C圖：含有相同CD3重鏈可變區，不同結構順序的B7H3雙價雙特異性抗體對A498的殺傷活性檢測。第5A圖為含HRH2的第一多肽鏈排列順序不同的(AFF1、AFF2、AFF3、AFF4)B7H3雙價雙特異性抗體間的殺傷活性比較。第5B圖為含HRH2的第二多肽鏈排列順序不同的(AFF3、AFF3-B)B7H3雙價雙特異性抗體間的殺傷活性比較。結果顯示序列相同，VH和VL排列順序不同的B7H3雙價雙特異性抗體均具有顯著的A498細胞殺傷活性，並且不同結構順序的分子間殺傷活性相近。第5C圖為包含相同B7H3 scFv和CD3 scFv的不同結構的雙特異性抗體的殺傷活性比較，三個待測雙特異抗體127與201、202均可在體外殺傷A498腫瘤細胞活性，其中雙特異性抗體127的殺傷活性優於201和202。Figure 5A to Figure 5C: Detection of the killing activity of B7H3 bivalent bispecific antibodies with the same CD3 heavy chain variable region and different structural sequences against A498. Figure 5A shows the killing activity of B7H3 bivalent bispecific antibodies with different sequence of the first polypeptide chain containing HRH2 (AFF1, AFF2, AFF3, AFF4)Sexual comparison. Figure 5B is a comparison of the killing activity of the B7H3 bivalent bispecific antibodies with different sequence of the second polypeptide chain containing HRH2 (AFF3, AFF3-B). The results showed that the B7H3 bivalent bispecific antibodies with the same sequence and different VH and VL sequence have significant A498 cell killing activity, and the intermolecular killing activity of different structural sequences is similar. Figure 5C is a comparison of the killing activity of bispecific antibodies with different structures containing the same B7H3 scFv and CD3 scFv. The three testedbispecific antibodies 127, 201 and 202 can all kill A498 tumor cells in vitro, among which the bispecific The killing activity ofantibody 127 is better than that of 201 and 202.

第6A圖至第6B圖：不同抗體對Jurkat重組細胞的激活檢測。第6A圖是抗體在含A498細胞的情況下，抗體介導的B7H3靶點特異的Jurkat重組細胞激活；第6B圖是抗體在不含A498細胞的情況下，介導的非B7H3靶點特異的Jurkat重組細胞激活。第6A圖和第6B圖中的圖例所指示的抗體一致。Figure 6A to Figure 6B: Activation detection of Jurkat recombinant cells by different antibodies. Figure 6A shows the antibody-mediated B7H3 target-specific activation of Jurkat recombinant cells in the presence of A498 cells; Figure 6B shows the antibody-mediated non-B7H3 target-specific activation in the absence of A498 cells Jurkat recombinant cells are activated. The antibodies indicated in the legends in Figure 6A and Figure 6B are the same.

第7A圖至第7B圖：含有相同CD3scFv不同價雙特異性抗體對Jurkat重組細胞的激活檢測。第7A圖是B7H3單/雙價雙特異性抗體在含A498細胞的情況下，抗體介導的B7H3靶點特異的Jurkat重組細胞激活；第7B圖是B7H3單/雙價雙特異性抗體在不含A498細胞的情況下，介導的非B7H3靶點特異的Jurkat重組細胞激活。Figure 7A to Figure 7B: Activation detection of Jurkat recombinant cells containing bispecific antibodies of the same CD3scFv with different valences. Figure 7A shows the B7H3 mono/bivalent bispecific antibody in the presence of A498 cells, antibody-mediated B7H3 target-specific Jurkat recombinant cell activation; Figure 7B shows the B7H3 mono/bivalent bispecific antibody In the case of A498 cells, it mediates the activation of non-B7H3 target-specific Jurkat recombinant cells.

第8A圖至第8C圖：不同抗體在有A498細胞存在時，刺激PBMC產生B7H3靶點特異的細胞因子分泌測試。第8A圖為不同抗體刺激PBMC的IFNγ分泌水平比較，第8B圖為不同抗體刺激PBMC的TNFα分泌水平比較，第8C圖為不同抗體刺激PBMC的IL-2分泌水平比較。第8A圖至第8C圖顯示118、127和132抗體能顯著刺激PBMC產生B7H3靶點特異的細胞因子分泌。第8A圖至第8C圖中的圖例所指示的抗體一致。Figure 8A to Figure 8C: In the presence of A498 cells, different antibodies stimulate PBMC to produce B7H3 target-specific cytokine secretion test. Figure 8A is a comparison of IFNγ secretion levels in PBMC stimulated by different antibodies, Figure 8B is a comparison of TNFα secretion levels in PBMC stimulated by different antibodies, and Figure 8C is a comparison of IL-2 secretion levels in PBMC stimulated by different antibodies. Figures 8A to 8C show thatantibodies 118, 127 and 132 can significantly stimulate PBMC to produce B7H3 target-specific cytokine secretion. The antibodies indicated in the legends in Figures 8A to 8C are the same.

第9A圖至第9C圖：不同抗體在CHOK1細胞(不表達B7H3)存在時，刺激PBMC產生非B7H3靶點特異的細胞因子分泌測試。第9A圖為不同抗體刺激PBMC分泌的IFNγ水平比較，第9B圖為不同抗體刺激PBMC分泌的TNFα水平比較，第9C圖為不同抗體刺激PBMC分泌細胞的IL-2水平比較。第9A圖至第9C圖顯示118、127和132抗體不能刺激PBMC進行非B7H3靶點特異的細胞因子分泌，具有很強的安全性。第9A圖至第9C圖中的圖例所指示的抗體一致。Figure 9A to Figure 9C: In the presence of CHOK1 cells (not expressing B7H3), different antibodies stimulate PBMC to produce non-B7H3 target-specific cytokine secretion test. Figure 9A is a comparison of IFNγ levels secreted by PBMCs stimulated by different antibodies, Figure 9B is a comparison of TNFα levels secreted by PBMCs stimulated by different antibodies, and Figure 9C is a comparison of IL-2 levels of PBMC secreting cells stimulated by different antibodies. Figures 9A to 9C show thatantibodies 118, 127, and 132 cannot stimulate PBMC to secrete cytokines that are not specific to the B7H3 target, and have strong safety. The antibodies indicated in the legends in Figures 9A to 9C are the same.

第10A圖至第10E圖10E：雙特異抗體在人PBMC重建的小鼠A498模型中的抗腫瘤療效檢測。第10A圖為低劑量B7H3雙價雙特異性抗體的抑瘤活性檢測，低劑量的118和119抗體仍顯示出一定的抑瘤活性，並且顯示出一定的劑量依賴性。第10B圖為0.3mpk和0.6mpk劑量B7H3雙價雙特異性抗體的抑瘤活性檢測，113抗體顯示出劑量依賴的腫瘤體內抑制活性。第10C圖為0.12mpk和0.36mpk劑量B7H3雙價雙特異性抗體的抑瘤活性檢測，兩劑量下118抗體顯示出顯著的抑瘤活性。第10D圖為0.36mpk劑量下B7H3雙價雙特異性抗體的抑瘤活性檢測，126、127和128抗體均顯示出顯著的抑瘤活性。第10E圖為127抗體在不同劑量和不同給藥頻次下的抑瘤活性。第10A圖至第10E圖中，Vehicle表示PBS給藥的陰性對照組。Figure 10A to Figure 10E Figure 10E: Anti-tumor efficacy detection of bispecific antibody in mouse A498 model reconstructed with human PBMC. Figure 10A shows the anti-tumor activity test of the low-dose B7H3 bivalent bispecific antibody. The low-dose 118 and 119 antibodies still show certain anti-tumor activity and show a certain dose-dependence. Figure 10B shows the anti-tumor activity of the B7H3 bivalent bispecific antibody at doses of 0.3 mpk and 0.6 mpk. The 113 antibody showed a dose-dependent tumor inhibitory activity in vivo. Figure 10C shows the anti-tumor activity detection of the B7H3 bivalent bispecific antibody at doses of 0.12 mpk and 0.36 mpk. The 118 antibody showed significant anti-tumor activity at the two doses. Figure 10D shows the detection of anti-tumor activity of B7H3 bivalent bispecific antibody at a dose of 0.36 mpk.Antibodies 126, 127 and 128 all showed significant anti-tumor activity. Figure 10E shows the anti-tumor activity of 127 antibody at different doses and different administration frequencies. In Figure 10A to Figure 10E, Vehicle represents a negative control group administered with PBS.

第11A圖至第11B圖：雙特異抗體在hCD3 KI小鼠模型中的抗腫瘤療效。第11A圖和第11B圖分別為118和132在hCD3 KI小鼠模型中的抑瘤效果。Figure 11A to Figure 11B: Anti-tumor efficacy of bispecific antibody in hCD3 KI mouse model. Figure 11A and Figure 11B are the tumor suppressive effects of 118 and 132 in hCD3 KI mouse model, respectively.

抗體的製備和篩選Antibody preparation and screening

製備單株抗體的方法是本領域已知的。可採用的一種方法是Kohler,G.等人，(1975)“Continuous Cultures Of Fused Cells Secreting Antibody Of PredefinedSpecificity,”Nature256：495-497的方法或其修改形式。典型地，單株抗體在非人物種，諸如小鼠中形成。通常，小鼠或大鼠被用於免疫，但其他動物，如兔、羊駝也可被使用。藉由用免疫原性量的包含人CD3或其他目標抗原(如人B7H3)的細胞、細胞提取物或蛋白製劑免疫小鼠來製備抗體。免疫原可以是但不限於，原代細胞、培養的細胞系、癌性細胞、核酸或組織。Methods of preparing monoclonal antibodies are known in the art. One method that can be used is the method of Kohler, G. et al. (1975) "Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity," Nature256:495-497 or its modified form. Typically, monoclonal antibodies are formed in non-human species, such as mice. Generally, mice or rats are used for immunization, but other animals such as rabbits and alpacas can also be used. Antibodies are prepared by immunizing mice with immunogenic amounts of cells, cell extracts, or protein preparations containing human CD3 or other target antigens (such as human B7H3). The immunogen can be, but is not limited to, primary cells, cultured cell lines, cancerous cells, nucleic acids or tissues.

在一個實施方案中，藉由使用過量表達目標抗原作為免疫原的宿主細胞獲得與目標抗原結合的單株抗體。此類細胞包括，例如且不限於，人T細胞、過表達人B7H3的細胞。In one embodiment, a monoclonal antibody that binds to the target antigen is obtained by using host cells that overexpress the target antigen as an immunogen. Such cells include, for example and without limitation, human T cells, cells overexpressing human B7H3.

為了監測抗體應答，可從動物獲得小的生物樣品(例如，血液)並測試針對免疫原的抗體滴度。可移除脾和/或一些大的淋巴結並離解為單細胞。如需要，可藉由(在非特異性黏附細胞去除之後)將細胞懸液施加至抗原塗覆的平板或孔篩選脾細胞。表達膜結合的抗原特異性的免疫球蛋白的B細胞，將與平板結合，並不會被剩餘的懸液沖洗掉。隨後，可將所得B細胞或所有離解的脾細胞與骨髓瘤細胞(例如，X63-Ag8.653和來自SaIk Institute,Cell Distribution Center,San Diego,CA的細胞)融合。聚乙二醇(PEG)可被用於融合脾或淋巴細胞與骨髓瘤細胞以形成融合瘤。隨後在選擇性培養基(例如，次黃嘌呤、胺喋呤、胸苷培養基，另外稱為“HAT培養基”)中培養融合瘤。隨後，藉由有限稀釋將所得融合瘤鋪板，並使用例如FACS(螢光激活細胞分選術)或免疫組織化學(IHC)篩選分析與免疫原特異性結合的抗體的產生。隨後，體外(例如，在組織培養瓶或中空纖維反應器)或體內(例如，在小鼠中作為腹水)培養選擇分泌單株抗體-的融合瘤。To monitor the antibody response, a small biological sample (eg, blood) can be obtained from the animal and tested for antibody titers against the immunogen. The spleen and/or some large lymph nodes can be removed and dissociated into single cells. If desired, spleen cells can be selected by applying a cell suspension to antigen-coated plates or wells (after removal of non-specific adherent cells). B cells expressing membrane-bound antigen-specific immunoglobulins will bind to the plate and will not be washed away by the remaining suspension. Subsequently, the resulting B cells or all dissociated spleen cells can be fused with myeloma cells (for example, X63-Ag8.653 and cells from Salik Institute, Cell Distribution Center, San Diego, CA). Polyethylene glycol (PEG) can be used to fuse spleen or lymphocytes with myeloma cells to form fusion tumors. The fusion tumor is then cultured in a selective medium (for example, hypoxanthine, methotrexate, thymidine medium, otherwise referred to as "HAT medium"). Subsequently, by limiting dilutionThe fusion tumors are plated, and the production of antibodies that specifically bind to the immunogen is analyzed using, for example, FACS (fluorescence activated cell sorting) or immunohistochemistry (IHC) screening. Subsequently, in vitro (e.g., in a tissue culture flask or hollow fiber reactor) or in vivo (e.g., as ascites in mice), the selected monoclonal antibody-secreting fusionoma is cultured.

作為細胞融合技術的另一個替代方式，可使用Epstein-Barr病毒(EBV)永生化的B細胞以製備本發明的單株抗體。如需要，增殖並亞選殖融合瘤，並藉由傳統的分析方法(例如，FACS、IHC、放射免疫分析、酶免疫分析、螢光免疫分析等)分析上清液的抗免疫原活性。As another alternative to cell fusion technology, Epstein-Barr virus (EBV) immortalized B cells can be used to prepare the monoclonal antibody of the present invention. If necessary, the fusion tumor is proliferated and sub-selected, and the anti-immunogen activity of the supernatant is analyzed by traditional analysis methods (for example, FACS, IHC, radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, etc.).

在另一個替代方式中，可藉由本領域已知的任何方法(例如，人源化、使用轉基因小鼠製備全人抗體、噬菌體展示技術，等)測序和重組製備抗目標抗原(如CD3、B7H3)單株抗體和任何其他等同抗體。在一個實施方案中，抗目標抗原(如CD3、B7H3)單株抗體被測序且隨後多核苷酸序列被選殖進入載體用於表達或增殖。編碼目的抗體的序列可被保持在宿主細胞中的載體中且隨後該宿主細胞可被增殖並冷凍用於以後使用。In another alternative, the anti-target antigens (such as CD3, B7H3, etc.) can be sequenced and recombinantly prepared by any method known in the art (e.g., humanization, preparation of fully human antibodies using transgenic mice, phage display technology, etc.). ) Monoclonal antibodies and any other equivalent antibodies. In one embodiment, monoclonal antibodies against the target antigen (such as CD3, B7H3) are sequenced and then the polynucleotide sequence is cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in the host cell and then the host cell can be propagated and frozen for later use.

抗CD3單株抗體和任何其他等同抗體的多核苷酸序列可被用於遺傳操作以產生“人源化”抗體，以改進抗體的親和力或其他特徵。人源化抗體的一般原理包括保留抗體的抗原結合部分的基本序列，同時將該抗體的非人剩餘部分置換為人抗體序列。存在人源化單株抗體的四個一般步驟。這些步驟為：(1)確定起始抗體輕鏈和重鏈可變結構域的核苷酸和預測的胺基酸序列，(2)設計人源化抗體，即，決定在人源化過程中使用哪一個抗體構架區，(3)實際的人源化方法/技術和，(4)人源化抗體的轉染和表達。參見，例如，美國專利號US4816567、US5807715、US5866692和US6331415。The polynucleotide sequence of the anti-CD3 monoclonal antibody and any other equivalent antibody can be used for genetic manipulation to produce "humanized" antibodies to improve the affinity or other characteristics of the antibody. The general principle of humanized antibodies includes retaining the basic sequence of the antigen-binding portion of the antibody, while replacing the non-human remaining portion of the antibody with a human antibody sequence. There are four general steps for humanizing monoclonal antibodies. These steps are: (1) Determine the nucleotide and predicted amino acid sequence of the light chain and heavy chain variable domains of the starting antibody, (2) Design the humanized antibody, that is, determine the process of humanization Which antibody framework region to use, (3) the actual humanization method/technology and (4) the transfection and expression of the humanized antibody. See, for example, US Patent Nos. US4816567, US5807715, US5866692, and US6331415.

1. B7H3抗體的製備和篩選1. Preparation and screening of B7H3 antibody

利用人PBMC、脾臟、淋巴結組織分離B細胞，並提取RNA，構建天然單鏈噬菌體抗體庫。將構建的天然單鏈噬菌體抗體文庫經過包裝形成噬菌體顆粒後，採用液相法進行淘篩，噬菌體與生物素化的B7H3液相結合，再採用鏈黴親和素磁珠分離。為了獲得與人B7H3結合的陽性序列，採用生物素化的人B7H3進行淘篩，挑取若干個單株菌落包裝成噬菌體單鏈抗體，用於噬菌體ELISA測試。分別測試單株噬菌體與人B7H3和鼠B7H3的結合活性，經篩選獲得B7H3抗體。Use human PBMC, spleen and lymph node tissues to isolate B cells and extract RNA to construct a library of natural single-stranded phage antibodies. After the constructed natural single-stranded phage antibody library is packaged to form phage particles, it is panned by the liquid phase method, the phage is combined with the biotinylated B7H3 liquid phase, and then separated by streptavidin magnetic beads. In order to obtain a positive sequence that binds to human B7H3, biotinylated human B7H3 was used for panning, and several single colonies were picked to package into phage single-chain antibodies for phage ELISA test. The binding activity of single phage to human B7H3 and mouse B7H3 was tested respectively, and B7H3 antibody was obtained after screening.

檢測用B7H3相關抗原如下所示：The B7H3-related antigens used for detection are as follows:

檢測用人B7H3抗原Human B7H3 antigen for detection

商業化產品(SinoBiological cat# 11188-H08H)Commercial products (SinoBiological cat# 11188-H08H)

序列如下：

SEQ ID NO：1The sequence is as follows:

SEQ ID NO: 1

註釋：劃橫線部分為B7H3胞外區；斜體部分為His標簽(His-tag)。Note: The underlined part is the extracellular region of B7H3; the italicized part is His-tag.

檢測用猴B7H3抗原Monkey B7H3 antigen for detection

商業化產品(SinoBiological cat#90806-C08H)Commercial product (SinoBiological cat#90806-C08H)

序列如下：

SEQ ID NO：2The sequence is as follows:

SEQ ID NO: 2

註釋：劃橫線部分為B7H3胞外區；斜體部分為His標簽。Note: The underlined part is the extracellular region of B7H3; the italicized part is the His tag.

檢測用鼠B7H3抗原Mouse B7H3 antigen for detection

商業化產品(SinoBiological cat# 50973-M08H)Commercial products (SinoBiological cat# 50973-M08H)

序列如下：

SEQ ID NO：4The sequence is as follows:

SEQ ID NO: 4

註釋：劃橫線部分為B7H3胞外區；斜體部分為His標簽。Note: The underlined part is the extracellular region of B7H3; the italicized part is the His tag.

人B7H3全長胺基酸序列

SEQ ID NO：4Human B7H3 full-length amino acid sequence

SEQ ID NO: 4

註釋：Notes:

雙橫線部分為信號肽(Signal peptide：1-28)；The double horizontal line is the signal peptide (Signal peptide: 1-28);

劃橫線部分為B7H3胞外區(Extracellular domain：29-466)，其中29-139為Ig-like V-type 1 Domain，145-238為Ig-like C2-type 1 Domain；243-357為Ig-like V-type 2 Domain，363-456為Ig-like C2-type 2 Domain；The underlined part is the extracellular domain of B7H3 (Extracellular domain: 29-466), of which 29-139 are Ig-like V-type 1 Domain, 145-238 are Ig-like C2-type 1 Domain; 243-357 are Ig -like V-type 2 Domain, 363-456 is Ig-like C2-type 2 Domain;

點劃線部分為跨膜區部分(Transmembrane domain：467-487)；The dotted line is the transmembrane domain (Transmembrane domain: 467-487);

斜體部分為胞內區(Cytoplasmic domain：488-534)。The italicized part is the intracellular domain (Cytoplasmic domain: 488-534).

猴B7H3全長胺基酸序列

SEQ ID NO：5Monkey B7H3 full-length amino acid sequence

SEQ ID NO: 5

註釋：Notes:

雙橫線部分為信號肽(Signal peptide：1-28)；The double horizontal line is the signal peptide (Signal peptide: 1-28);

劃橫線部分為B7H3胞外區(Extracellular domain：29-466)，其中29-139為Ig-like V-type 1 Domain，145-238為Ig-like C2-type 1 Domain；243-357為Ig-like V-type 2 Domain，363-456為Ig-like C2-type 2 Domain；The underlined part is the extracellular domain of B7H3 (Extracellular domain: 29-466), of which 29-139 are Ig-like V-type 1 Domain, 145-238 are Ig-like C2-type 1 Domain; 243-357 are Ig -like V-type 2 Domain, 363-456 is Ig-like C2-type 2 Domain;

點劃線部分為跨膜區部分(Transmembrane domain：467-487)；The dotted line is the transmembrane domain (Transmembrane domain: 467-487);

斜體部分為胞內區(Cytoplasmic domain：488-534)。The italicized part is the intracellular domain (Cytoplasmic domain: 488-534).

>鼠B7H3全長胺基酸序列

SEQ ID NO：6>Mouse B7H3 full-length amino acid sequence

SEQ ID NO: 6

註釋：Notes:

雙橫線部分為信號肽(Signal peptide：1-28)；The double horizontal line is the signal peptide (Signal peptide: 1-28);

劃橫線部分為B7H3胞外區(Extracellular domain：29-248)；The cross-lined part is the extracellular domain of B7H3 (Extracellular domain: 29-248);

點劃線部分為跨膜區部分(Transmembrane domain：249-269)；The dotted line is the transmembrane domain (Transmembrane domain: 249-269);

斜體部分為胞內區(Cytoplasmic domain：270-316)。The italicized part is the intracellular domain (Cytoplasmic domain: 270-316).

篩選獲得的B7H3抗體h1702序列及其經IMGT編號規則定義的CDR序列如下所示：The screened B7H3 antibody h1702 sequence and its CDR sequence defined by IMGT numbering rules are as follows:

>h1702 VH

SEQ ID NO：7>h1702 VH

SEQ ID NO: 7

>h1702 VLQTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSHYPSWYQQTPGQAPRMLIYNTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCAIHVDRDIWVFGGGTKLTVL SEQ ID NO：8>h1702 VLQTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSHYPSWYQQTPGQAPRMLIYNTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCAIHVDRDIWVFGGGTKLTVL SEQ ID NO: 8

註：順序為FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜體為FR序列，下劃線為CDR序列。Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italics in the sequence are the FR sequence, and the underline is the CDR sequence.

為了進一步提高雙特異性抗體的性能，在B7H3抗體h1702的VH和VL中分別進行半胱胺酸取代突變，在輕鏈可變區中引入G103C(胺基酸自然順序編號，在SEQ ID NO：16的第103位)突變，在重鏈可變區中引入G44C(胺基酸自然順序編號，在SEQ ID NO：15的第44位)突變，以形成一對二硫鍵，突變後的抗B7H3單鏈抗體的重鏈和輕鏈可變區序列如下所示：In order to further improve the performance of the bispecific antibody, cysteine substitution mutations were carried out in the VH and VL of the B7H3 antibody h1702, respectively, and G103C (natural sequence numbering of amino acid, in SEQ ID NO: 16th position 103) mutation, the G44C (amino acid natural sequence numbering, at position 44 of SEQ ID NO: 15) mutation was introduced into the heavy chain variable region to form a pair of disulfide bonds. The heavy chain and light chain variable region sequences of the B7H3 single chain antibody are as follows:

B7H3 VH44C：

SEQ ID NO：15B7H3 VH44C:

SEQ ID NO: 15

B7H3 VL103C：

SEQ ID NO：16B7H3 VL103C:

SEQ ID NO: 16

2. CD3抗體的製備和篩選2. Preparation and screening of CD3 antibody

在鼠源CD3抗體基礎上，經突變、建庫、人源化改造及篩選等手段可獲得人源化的CD3抗體。On the basis of murine CD3 antibodies, humanized CD3 antibodies can be obtained through methods such as mutation, library construction, humanization transformation and screening.

CD3抗原相關序列信息如下CD3 antigen related sequence information is as follows

檢測用人CD3抗原Human CD3 antigen for detection

商業化產品(SinoBiological cat#CT038-H2508H)Commercialized product (SinoBiological cat#CT038-H2508H)

序列如下：The sequence is as follows:

人CD3ε(Human CD3ε)

SEQ ID NO：17Human CD3ε (Human CD3ε)

SEQ ID NO: 17

註釋：Notes:

劃橫線部分為CD3ε胞外區(Extracellular domain：23-126)；斜體部分為His標簽The cross-lined part is the extracellular domain of CD3ε (Extracellular domain: 23-126); the italicized part is the His tag

人CD3δFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVA DYKDDDDK SEQ ID NO：18Human CD3δFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVADYKDDDDK SEQ ID NO: 18

註釋：Notes:

劃橫線部分為CD3δ胞外區(Extracellular domain：22-105)；斜體部分為Flag標簽。The cross-lined part is the extracellular domain of CD3δ (Extracellular domain: 22-105); the italicized part is the Flag label.

檢測用猴CD3抗原Monkey CD3 antigen for detection

商業化產品(Acro biosystem cat# CDD-C52W4-100ug)Commercial products (Acro biosystem cat# CDD-C52W4-100ug)

序列如下：The sequence is as follows:

猴CD3εQDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMD-HHHHHHSEQ ID NO：19Monkey CD3εQDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMD -HHHHHH SEQ ID NO: 19

註釋：Notes:

劃橫線部分為CD3ε胞外區(Extracellular domain：22-117)；斜體部分為His標簽The underlined part is the extracellular domain of CD3ε (Extracellular domain: 22-117); the italicized part is the His tag

猴CD3δFKIPVEELEDRVFVKCNTSVTWVEGTVGTLLTNNTRLDLGKRILDPRGIYRCNGTDIYKDKESAVQVHYRMCQNCVELDPATLA-DYKDDDDK SEQ ID NO：20Monkey CD3δFKIPVEELEDRVFVKCNTSVTWVEGTVGTLLTNNTRLDLGKRILDPRGIYRCNGTDIYKDKESAVQVHYRMCQNCVELDPATLA -DYKDDDDK SEQ ID NO: 20

註釋：Notes:

劃橫線部分為CD3δ胞外區(Extracellular domain：22-105)；斜體部分為Flag標簽The underlined part is the extracellular domain of CD3δ (Extracellular domain: 22-105); the italicized part is the Flag label

檢測用鼠CD3抗原Mouse CD3 antigen for detection

商業化產品(SinoBiological cat# CT033-M2508H)序列如下：The sequence of the commercial product (SinoBiological cat# CT033-M2508H) is as follows:

鼠CD3εDDAENIEYKVSISGTSVELTCPLDSDENLKWEKNGQELPQKHDKHLVLQDFSEVEDSGYYVCYTPASNKNTYLYLKARVCEYCVEVD-HHHHHH SEQ ID NO：21Mouse CD3εDDAENIEYKVSISGTSVELTCPLDSDENLKWEKNGQELPQKHDKHLVLQDFSEVEDSGYYVCYTPASNKNTYLYLKARVCEYCVEVD -HHHHHH SEQ ID NO: 21

註釋：Notes:

劃橫線部分為CD3ε胞外區(Extracellular domain：22-108)；斜體部分為His標簽The underlined part is the extracellular domain of CD3ε (Extracellular domain: 22-108); the italicized part is the His tag

鼠CD3δFKIQVTEYEDKVFVTCNTSVMHLDGTVEGWFAKNKTLNLGKGVLDPRGIYLCNGTEQLAKVVSSVQVHYRMCQNCVELDSGTMA DYKDDDDK SEQ ID NO：22Mouse CD3δFKIQVTEYEDKVFVTCNTSVMHLDGTVEGWFAKNKTLNLGKGVLDPRGIYLCNGTEQLAKVVSSVQVHYRMCQNCVELDSGTMADYKDDDDK SEQ ID NO: 22

註釋：Notes:

劃橫線部分為CD3δ胞外區(Extracellular domain：22-105)；斜體部分為Flag標簽The underlined part is the extracellular domain of CD3δ (Extracellular domain: 22-105); the italicized part is the Flag label

人CD3ε全長胺基酸序列

SEQ ID NO：23Human CD3ε full-length amino acid sequence

SEQ ID NO: 23

註釋：Notes:

雙橫線部分為信號肽(Signal peptide：1-22)；The double-line part is the signal peptide (Signal peptide: 1-22);

劃橫線部分為CD3ε胞外區(Extracellular domain：23-126)，其中32-112為Ig-like Domain；The cross-lined part is CD3ε extracellular domain (Extracellular domain: 23-126), of which 32-112 are Ig-like Domain;

點劃線部分為跨膜區部分(Transmembrane domain：127-152)；The dotted line is the transmembrane domain (Transmembrane domain: 127-152);

斜體部分為胞內區(Cytoplasmic domain：153-207)。The italicized part is the intracellular domain (Cytoplasmic domain: 153-207).

人CD3δ全長胺基酸序列

SEQ ID NO：24Human CD3δ full-length amino acid sequence

SEQ ID NO: 24

註釋：Notes:

雙橫線部分為信號肽(Signal peptide：1-21)；The double horizontal line is the signal peptide (Signal peptide: 1-21);

劃橫線部分為CD3δ胞外區(Extracellular domain：22-105)；The cross-lined part is the extracellular domain of CD3δ (Extracellular domain: 22-105);

點劃線部分為跨膜區部分(Transmembrane domain：106-126)；The dotted line is the transmembrane domain (Transmembrane domain: 106-126);

斜體部分為胞內區(Cytoplasmic domain：127-171)。The italicized part is the intracellular domain (Cytoplasmic domain: 127-171).

猴CD3ε全長胺基酸序列

SEQ ID NO：25Monkey CD3ε full-length amino acid sequence

SEQ ID NO: 25

註釋：Notes:

雙橫線部分為信號肽(Signal peptide：1-21)；The double horizontal line is the signal peptide (Signal peptide: 1-21);

劃橫線部分為CD3ε胞外區(Extracellular domain：22-117)；The cross-lined part is the extracellular domain of CD3ε (Extracellular domain: 22-117);

點劃線部分為跨膜區部分(Transmembrane domain：118-138)；The dotted line is the transmembrane domain (Transmembrane domain: 118-138);

斜體部分為胞內區(Cytoplasmic domain：139-198)。The italicized part is the intracellular domain (Cytoplasmic domain: 139-198).

猴CD3δ全長胺基酸序列

SEQ ID NO：26Monkey CD3δ full-length amino acid sequence

SEQ ID NO: 26

註釋：Notes:

雙橫線部分為信號肽(Signal peptide：1-21)；The double horizontal line is the signal peptide (Signal peptide: 1-21);

劃橫線部分為CD3δ胞外區(Extracellular domain：22-105)；The cross-lined part is the extracellular domain of CD3δ (Extracellular domain: 22-105);

點劃線部分為跨膜區部分(Transmembrane domain：106-126)；The dotted line is the transmembrane domain (Transmembrane domain: 106-126);

斜體部分為胞內區(Cytoplasmic domain：127-171)。The italicized part is the intracellular domain (Cytoplasmic domain: 127-171).

鼠CD3ε全長胺基酸序列

SEQ ID NO：27Mouse CD3ε full-length amino acid sequence

SEQ ID NO: 27

註釋：Notes:

雙橫線部分為信號肽(Signal peptide：1-21)；The double horizontal line is the signal peptide (Signal peptide: 1-21);

劃橫線部分為CD3ε胞外區(Extracellular domain：22-108)；The cross-lined part is the extracellular domain of CD3ε (Extracellular domain: 22-108);

點劃線部分為跨膜區部分(Transmembrane domain：109-134)；The dotted line is the transmembrane domain (Transmembrane domain: 109-134);

斜體部分為胞內區(Cytoplasmic domain：135-189)。The italicized part is the intracellular domain (Cytoplasmic domain: 135-189).

鼠CD3δ全長胺基酸序列

SEQ ID NO：28Mouse CD3δ full-length amino acid sequence

SEQ ID NO: 28

註釋：Notes:

雙橫線部分為信號肽(Signal peptide：1-21)；The double horizontal line is the signal peptide (Signal peptide: 1-21);

劃橫線部分為CD3δ胞外區(Extracellular domain：22-105)；The cross-lined part is the extracellular domain of CD3δ (Extracellular domain: 22-105);

點劃線部分為跨膜區部分(Transmembrane domain：106-126)；The dotted line is the transmembrane domain (Transmembrane domain: 106-126);

斜體部分為胞內區(Cytoplasmic domain：127-173)。The italicized part is the intracellular domain (Cytoplasmic domain: 127-173).

經過重複分析和優化，獲得一系列抗CD3的人源化抗體序列，重鏈可變區序列如下：After repeated analysis and optimization, a series of anti-CD3 humanized antibody sequences were obtained. The heavy chain variable region sequence is as follows:

輕鏈可變區序列如下：The light chain variable region sequence is as follows:

>HRL

SEQ ID NO：36>HRL

SEQ ID NO: 36

註：順序為FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜體為FR序列，下劃線為CDR序列。此處和下表3中CD3人源化抗體的輕鏈和重鏈可變區中的CDR(LCDR1-LCDR3和HCDR1-HCDR3)胺基酸殘基在數量和位置上符合已知的Kabat編號規則。Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italics in the sequence are the FR sequence, and the underline is the CDR sequence. Here and in Table 3 below, the CDR (LCDR1-LCDR3 and HCDR1-HCDR3) amino acid residues in the light chain and heavy chain variable regions of the CD3 humanized antibody comply with the known Kabat numbering rules in number and position .

單鏈抗體的構建和製備Construction and preparation of single chain antibody

來源自上述B7H3抗體的輕重鏈可變區和來源於CD3抗體的輕重鏈可變區可分別經連接後形成針對B7H3的scFv和針對CD3的scFv，其中接頭(linker)可選自本領域內公知的接頭序列，示例性的接頭可選自：(GGGGS)n或(GGGGS)nGGG，其中n可為1、2、3或4。The light and heavy chain variable regions derived from the above-mentioned B7H3 antibody and the light and heavy chain variable regions derived from the CD3 antibody can be respectively connected to form scFv for B7H3 and scFv for CD3, wherein the linker can be selected from those known in the art An exemplary linker can be selected from: (GGGGS)n or (GGGGS)nGGG, where n can be 1, 2, 3, or 4.

示例性的抗B7H3的scFv如下：Exemplary scFvs against B7H3 are as follows:

示例性的抗CD3的scFv如下：Exemplary scFvs against CD3 are as follows:

雙特異性抗體的構建和製備B7H3雙價雙特異性抗體和B7H3單價雙特異性抗體Construction and preparation of bispecific antibodies B7H3 bivalent bispecific antibodies and B7H3 monovalent bispecific antibodies

本公開的一些實施方式中B7H3雙價雙特異抗體的結構如第1A圖所示，其C末端可以連接His標簽，也可以不連接His標簽。採用含Fc的兩條鏈非對稱結構設計，共有兩個B7H3抗原結合域和一個CD3抗原結合域，其中B7H3抗原結合域在兩條鏈上各一個，抗原結合域均為scFv的形式。Fc區能維持抗體正常半衰期和良好穩定性，兩條鏈的設計大大降低了錯配的幾率，提高了樣品的均一性和目的抗體的得率。具體的雙特異性抗體的分子結構(Format)見下表6，此外，本公開一些實施方式中使用的B7H3單價雙特異性抗體的分子結構在第二多肽鏈中僅有Fc結構域，不包含抗原結合結構域，結構示意見第1B圖。In some embodiments of the present disclosure, the structure of the B7H3 bivalent bispecific antibody is shown in Figure 1A, and the His tag may or may not be attached to the C-terminus of the antibody. The asymmetric structure of two chains containing Fc is designed, and there are two B7H3 antigen binding domains and one CD3 antigen binding domain. Among them, the B7H3 antigen binding domain is on each of the two chains, and the antigen binding domains are in the form of scFv. The Fc region can maintain the normal half-life and good stability of the antibody. The design of the two chains greatly reduces the probability of mismatches and improves the uniformity of the sample and the yield of the target antibody. The specific molecular structure (Format) of the bispecific antibody is shown in Table 6 below. In addition, the molecular structure of the B7H3 monovalent bispecific antibody used in some embodiments of the present disclosure has only the Fc domain in the second polypeptide chain. Contains the antigen binding domain, the structure is shown in Figure 1B.

其中n選自1、2、3或4；較佳的，L1中的n為2或3，更佳為3；L2中的n為1或2，更佳為1；L3或L5中的n為3。可選地，用於連接抗原結合結構域以及Fc區的接頭可選自其他任何可用於連接抗體功能結構域的接頭，而不僅限於上述序列所限制的接頭。Wherein n is selected from 1, 2, 3 or 4; preferably, n in L1 is 2 or 3, more preferably 3; n in L2 is 1 or 2, more preferably 1; n in L3 or L5 Is 3. Optionally, the linker used to connect the antigen-binding domain and the Fc region can be selected from any other linker that can be used to connect the functional domain of an antibody, and is not limited to the linker limited by the above sequence.

上述表6中的Fc1和Fc2可為相同序列的Fc，也可分別為knob-Fc和hole-Fc或分別為hole-Fc和knob-Fc，在本公開一些實施例中，knob-Fc和hole-Fc的序列較佳表8所示序列：Fc1 and Fc2 in Table 6 above can be Fc with the same sequence, or knob-Fc and hole-Fc respectively, or hole-Fc and knob-Fc respectively. In some embodiments of the present disclosure, knob-Fc and hole The sequence of -Fc is preferably the sequence shown in Table 8:

上述輕重鏈可變區、單鏈抗體、雙特異性抗體可藉由VH和/或VL及所需的其他結構域的編碼cDNA，構建編碼上述多肽或抗原結合片段的DNA，將該DNA插入到原核生物表達載體或真核生物表達載體中，然後將該表達載體導入到原核生物或真核生物中以表達該多肽或抗原結合片段。The light and heavy chain variable regions, single-chain antibodies, and bispecific antibodies can be used to construct DNA encoding the polypeptides or antigen-binding fragments by using cDNA encoding VH and/or VL and other required domains, and insert the DNA into Prokaryote expression vector or eukaryote expression vector, and then introduce the expression vector into prokaryote or eukaryote to express the polypeptide or antigen-binding fragment.

實施例1、雙特異性抗體分子及陽性對照分子、陰性對照分子的製備Example 1. Preparation of bispecific antibody molecules, positive control molecules, and negative control molecules

根據本公開雙特異性抗體分子的設計方法，設計並製備具體雙特異性抗體分子，示例性分子胺基酸序列如下表9所示：According to the method for designing bispecific antibody molecules of the present disclosure, specific bispecific antibody molecules are designed and prepared. Exemplary amino acid sequences of the molecules are shown in Table 9 below:

本公開中使用的陰性對照(NC1、NC2、NC3)及陽性對照(MGD009)的雙特異性抗體的胺基酸序列如下：The amino acid sequences of the bispecific antibodies of the negative control (NC1, NC2, NC3) and the positive control (MGD009) used in this disclosure are as follows:

NC1：B7H3結合域被替換成非相關抗體(抗螢光素抗體，anti-fluorescein)，但保留CD3結合域)，其胺基酸序列參照文獻：The anti-fluoresceinantibody used to form the control DART diabody was antibody 4-4-20(Gruber.M.et al.(1994).NC1: The B7H3 binding domain is replaced with a non-related antibody (anti-fluorescein, but the CD3 binding domain is retained). Refer to the literature for its amino acid sequence: The anti-fluorescein antibody used to form the control DART diabody was antibody 4-4-20(Gruber.M.et al. (1994).

鏈1(VH_CD3-VL_CD3-VL_ctrl-VH_ctrl-knob-Fc)

SEQ ID NO：89Chain 1 (VH_CD3 -VL_CD3 -VL_ctrl -VH_ctrl -knob-Fc)

SEQ ID NO: 89

鏈2(VL_ctrl-VH_ctrl-hole-Fc)

SEQ ID NO：90Chain 2 (VL_ctrl -VH_ctrl -hole-Fc)

SEQ ID NO: 90

NC2：保留B7H3結合域，僅是CD3結合域被替換成抗fluorescein的非相關抗體NC2: retain the B7H3 binding domain, only the CD3 binding domain is replaced with an anti-fluorescein non-related antibody

鏈1(VH_ctrl-VL_ctrl-VL_B7H3-VH_B7H3-knob-Fc)

SEQ ID NO：91Chain 1 (VH_ctrl -VL_ctrl -VL_B7H3 -VH_B7H3 -knob-Fc)

SEQ ID NO: 91

鏈2(VL_B7H3-linker-VH_B7H3-linker-Fc)

SEQ ID NO：92Chain 2 (VL_B7H3 -linker-VH_B7H3 -linker-Fc)

SEQ ID NO: 92

註：順序為VL_B7H3-linker-VH_B7H3-linker-Fc。序列中下劃線部分為B7H3抗體序列，斜體部分為hole-Fc序列。Note: The sequence is VL_B7H3 -linker-VH_B7H3 -linker-Fc. The underlined part of the sequence is the B7H3 antibody sequence, and the italicized part is the hole-Fc sequence.

NC3NC3

鏈1(VL_ctrl-VH_ctrl-VH_CD3-VL_CD3-knob-Fc-His tag)

SEQ ID NO：93Chain 1 (VL_ctrl -VH_ctrl -VH_CD3 -VL_CD3 -knob-Fc-His tag)

SEQ ID NO: 93

鏈2(VL_ctrl-VH_ctrl-hole-Fc)

SEQ ID NO：94Chain 2 (VL_ctrl -VH_ctrl -hole-Fc)

SEQ ID NO: 94

陽性對照MGD009包含三條鏈，製備及胺基酸序列參照已公開專利申請WO2017030926A1，其胺基酸序列如下：The positive control MGD009 contains three chains, and the preparation and amino acid sequence refer to the published patent application WO2017030926A1. The amino acid sequence is as follows:

鏈1(B7H3VL-CD3VH-Fc)

SEQ ID NO：95Chain 1 (B7H3VL-CD3VH-Fc)

SEQ ID NO: 95

鏈2(CD3VL-B7H3VH)

SEQ ID NO：96Chain 2 (CD3VL-B7H3VH)

SEQ ID NO: 96

鏈3(Fc)

SEQ ID NO：97Chain 3 (Fc)

SEQ ID NO: 97

201(DART-Fc三鏈結構)201鏈1(B7H3VL-CD3VH-E-Fc)

SEQ ID NO：99201 (DART-Fc three-chain structure) 201 chain 1 (B7H3VL-CD3VH-E-Fc)

SEQ ID NO: 99

201鏈2(CD3VL-B7H3VH-K)

SEQ ID NO：100201 chain 2 (CD3VL-B7H3VH-K)

SEQ ID NO: 100

201鏈3

SEQ ID NO：97201Chain 3

SEQ ID NO: 97

202(四鏈結構，其中四條鏈的物質的量比例關係是鏈1：鏈2：鏈3：鏈4=1：2：1：1)202 (Four-chain structure, in which the ratio of the amount of substances in the four chains is chain 1: chain 2: chain 3:chain 4=1: 2: 1: 1)

202鏈1(B7H3VH-CH1-Fc)

SEQ ID NO：101202 chain 1 (B7H3VH-CH1-Fc)

SEQ ID NO: 101

202鏈2(B7H3VL-CL)

SEQ ID NO：102202 Chain 2 (B7H3VL-CL)

SEQ ID NO: 102

202鏈3(B7H3VH-CH1-CD3VH-CL)

SEQ ID NO：103202 Chain 3 (B7H3VH-CH1-CD3VH-CL)

SEQ ID NO: 103

202鏈4(CD3VL-CH1)

SEQ ID NO：104202 chain 4 (CD3VL-CH1)

SEQ ID NO: 104

實施例2、CD3-B7H3雙特異性抗體的表達與純化Example 2. Expression and purification of CD3-B7H3 bispecific antibody

表達雙特異性抗體的質粒(鏈1：鏈2為1：1)轉染HEK293E細胞，6天後收集表達上清，高速離心去除雜質。澄清的上清用Ni Sepharose excel管柱(GE Healthcare)進行純化。用PBS沖洗管柱，至A280讀數降至基線，再用PBS+10mM咪唑沖洗層析管柱，除去非特異結合的雜蛋白，並收集流出液，最後用含有300mM咪唑的PBS溶液沖提目的蛋白，並收集沖提峰。沖提樣品適當濃縮後，利用550緩衝液(10mM乙酸，pH5.5,135mM NaCl)平衡好的凝膠層析Superdex200(GE)進一步純化，收集目的峰。樣品經脫鹽管柱或超濾離心管換液至559緩衝液中(10mM乙酸，pH5.5,9%蔗糖)，分裝後於-80度凍存。The plasmid expressing the bispecific antibody (chain 1:chain 2 is 1:1) was transfected into HEK293E cells, and the expression supernatant was collected 6 days later and centrifuged at high speed to remove impurities. The clarified supernatant was purified using Ni Sepharose excel column (GE Healthcare). Rinse the column with PBS until the A280 reading drops to baseline, then rinse the column with PBS+10mM imidazole to remove non-specifically bound impurity proteins, and collect the effluent, and finally use PBS solution containing 300mM imidazole to extract the target protein , And collect the red peak. After the extracted sample is properly concentrated, it is further purified by Superdex200 (GE) gel chromatography with 550 buffer (10mM acetic acid, pH5.5, 135mM NaCl) equilibrated to collect the target peak. The sample is exchanged into 559 buffer (10mM acetic acid, pH5.5, 9% sucrose) through a desalting column or ultrafiltration centrifuge tube, and then aliquoted and stored at -80°C.

測試例1、BIAcore檢測雙特異抗體對B7H3和CD3的親和力實驗Test example 1. BIAcore detects the affinity of bispecific antibodies to B7H3 and CD3

抗體對B7H3和CD3親和力的檢測，採用捕獲(capture)抗體的形式。用偶聯有Anti-Human IgG Antibody(Cat.#BR-1008-39,Lot.# 10260416,GE)的CM5生物傳感芯片(Cat.# BR-1005-30,GE)或Protein A(Cat.# 29127556,GE)生物傳感芯片親和捕獲BsAb，然後於芯片表面流經各抗原，用Biacore T200儀器實時檢測反應信號獲得結合和解離曲線。在每個實驗循環解離完成後，用再生緩衝液Glycine1.5(Cat# BR100354，GE)或3M MgCl₂(來自Human antibody capture kit，Cat.#BR100839,GE)將芯片洗淨再生。實驗結束後用GE Biacore T200 Evaluation version 3.0軟件以(1：1)Langmuir模型擬合數據，得出親和力數值。The detection of antibody affinity to B7H3 and CD3 takes the form of capture antibody. Use CM5 biosensor chip (Cat.# BR-1005-30, GE) or Protein A (Cat. #BR-1005-30, GE) coupled with Anti-Human IgG Antibody (Cat.#BR-1008-39, Lot.# 10260416, GE). # 29127556, GE) Biosensing chip affinity captures BsAb, and then flows through each antigen on the surface of the chip, and uses Biacore T200 instrument to detect the reaction signal in real time to obtain binding and dissociation curves. After the dissociation of each experimental cycle is completed, the chip is washed and regenerated with regeneration buffer Glycine 1.5 (Cat# BR100354, GE) or 3M MgCl₂ (from Human antibody capture kit, Cat. #BR100839, GE). After the experiment, GE Biacore T200 Evaluation version 3.0 software was used to fit the data with a (1:1) Langmuir model to obtain the affinity value.

固定不同V區的排列順序，採用不同CD3抗體VH序列時，雙特異抗體對CD3的親和力略有不同，當採用HRH-6和HRH-5序列時，抗體與CD3的親和力最弱，在Biacore上無法檢測到與CD3的結合。Fix the arrangement of different V regions. When using different CD3 antibody VH sequences, the affinity of bispecific antibodies to CD3 is slightly different. When HRH-6 and HRH-5 sequences are used, the affinity of the antibody to CD3 is the weakest, which is on Biacore. Unable to detect binding to CD3.

示例性的選擇包含HRH3作為CD3抗原結合結構域的重鏈可變區的抗體進行測試，其中測試抗體118，127，132與人(human)B7H3和human CD3的親和力分別為10^-9和10^-8M水平，與MGD009的親和力相當，且與猴(cyno)B7H3和human CD3均具有很強的交叉結合活性。An exemplary selection of antibodies containing HRH3 as the heavy chain variable region of the CD3 antigen-binding domain is tested, wherein the affinity of thetest antibodies 118, 127, 132 to human B7H3 and human CD3 is 10^-9 and 10^{− At 8} M level, the affinity is equivalent to MGD009, and it has strong cross-binding activity with both monkey (cyno) B7H3 and human CD3.

測試例2、細胞水平抗體結合能力測定Test Example 2: Determination of antibody binding capacity at the cellular level

採用FACS的方法對雙特異抗體與細胞表面抗原結合能力進行檢測。對細胞表面抗原B7H3和CD3的結合，分別用A498(ATCC，HTB-44)，CT26/hB7H3(在鼠細胞CT26中過表達人B7H3的重組細胞系，內部構建，CT26來源於中科院細胞庫，TCM37)和Jurkat重組細胞系(Jurkat細胞來源於ATCC，PTS-TIB-152，重組細胞系是在Jurkat細胞基礎上過表達螢光素酶(luciferase)基因，在基因上游插入NFAT的應答元件)。The FACS method was used to detect the binding ability of bispecific antibodies to cell surface antigens. For the binding of cell surface antigens B7H3 and CD3, A498 (ATCC, HTB-44), CT26/hB7H3 (a recombinant cell line overexpressing human B7H3 in mouse cell CT26, constructed in-house, CT26 is derived from the Chinese Academy of Sciences Cell Bank, TCM37 ) And Jurkat recombinant cell line (Jurkat cells are derived from ATCC, PTS-TIB-152, the recombinant cell line is based on Jurkat cells overexpressing luciferase gene, and inserting NFAT response elements upstream of the gene).

在96孔U型底板(corning,3795)中加入FACS緩衝液(buffer)(98% PBS,2% FBS)重新懸浮的細胞，並加入梯度稀釋的抗體，4℃孵育1h，FACS緩衝液清洗2次，隨後每孔加入APC anti-human IgG Fc Antibody(biolegend，Cat# 409306,1：50稀釋)，4℃孵育30min，清洗2次後用FACS緩衝液重新懸浮細胞，最後用FACS CantoII(BD)讀取螢光信號值。Add FACS buffer (98% PBS, 2% FBS) to the 96-well U-shaped bottom plate (corning, 3795) to resuspend the cells, and add serially diluted antibodies, incubate at 4°C for 1 hour, and wash withFACS buffer 2 Times, then add APC anti-human IgG Fc Antibody (biolegend, Cat#409306, 1:50 dilution), incubate at 4°C for 30min, wash twice, resuspend the cells in FACS buffer, and finally read the fluorescence signal value with FACS CantoII (BD).

結果顯示，B7H3雙價的雙特異抗體118、127和132以及陰性對照抗體NC2(保留B7H3結合域，僅是CD3結合域被替換成非相關抗體)均能與高表達B7H3的A498細胞系結合(見第2A圖)，呈梯度依賴效應，結合能力強於MGD009，且是B7H3靶點特異的，陰性對照抗體NC1(B7H3結合域被替換成非相關抗體，但保留CD3結合域)不與A498結合。同樣地，雙特異抗體118、127和132，MGD009以及NC2與CT26/hB7H3有強結合(見第2B圖)，而不與未表達B7H3的CT26細胞系(見第2C圖)結合，也充分說明測試的雙特異抗體特異地與細胞膜表面B7H3靶點結合。抗體118、127和132與MGD009結合能力的差別，在B7H3過表達細胞系CT26/hB7H3上比A498細胞系上更加明顯，預示著B7H3雙價的雙特異抗體，相比起B7H3單價的雙特異抗體MGD009，在高表達B7H3細胞上的結合優勢更為顯著，會有更好的安全窗口。The results showed that the B7H3 bivalentbispecific antibodies 118, 127, and 132 and the negative control antibody NC2 (retaining the B7H3 binding domain, only the CD3 binding domain was replaced with an unrelated antibody) can all bind to the A498 cell line with high expression of B7H3 ( (See Figure 2A), showing a gradient-dependent effect, with stronger binding capacity than MGD009, and specific for the B7H3 target. The negative control antibody NC1 (the B7H3 binding domain is replaced with an unrelated antibody, but the CD3 binding domain is retained) does not bind to A498 . Similarly, thebispecific antibodies 118, 127 and 132, MGD009 and NC2 have strong binding to CT26/hB7H3 (see Figure 2B), but do not bind to the CT26 cell line that does not express B7H3 (see Figure 2C), which also fully explains The bispecific antibody tested specifically binds to the B7H3 target on the cell membrane surface. The difference in binding ability betweenantibodies 118, 127 and 132 and MGD009 is more obvious on the B7H3 overexpressing cell line CT26/hB7H3 than on the A498 cell line, indicating that the B7H3 bivalent bispecific antibody is compared to the B7H3 monovalent bispecific antibody MGD009 has more significant binding advantages on high-expressing B7H3 cells, and will have a better safety window.

雙特異抗體118、127和132以及陰性對照抗體NC1均能與Jurkat重組細胞系結合(見第2D圖)，呈梯度依賴效應。其中118和NC1與Jurkat重組細胞結合能力與MGD009相當，而127和132的結合能力略弱，可能由於CD3結合域在B7H3結合域和FC之間，與Jurkat重組細胞結合受到一定空間位阻的影響。不含CD3結合域的陰性對照抗體NC2不與Jurkat重組細胞結合，說明雙特異抗體與Jurkat的結合是CD3靶點特異的。Thebispecific antibodies 118, 127 and 132 and the negative control antibody NC1 can all bind to the Jurkat recombinant cell line (see Figure 2D), showing a gradient-dependent effect. Among them, the binding ability of 118 and NC1 to Jurkat recombinant cells is equivalent to that of MGD009, while the binding ability of 127 and 132 is slightly weaker. This may be due to the fact that the CD3 binding domain is between the B7H3 binding domain and FC, and the binding to Jurkat recombinant cells is affected by certain steric hindrance. . The negative control antibody NC2 without the CD3 binding domain does not bind to Jurkat recombinant cells, indicating that the binding of the bispecific antibody to Jurkat is specific for the CD3 target.

測試例3、體外PBMC殺傷實驗Test Example 3. In vitro PBMC killing experiment

雙特異抗體介導的PBMC對腫瘤細胞的殺傷實驗藉由定量檢測細胞增殖情況來實現的。利用Cell Titer-glo檢測細胞中ATP的含量，而ATP是活細胞新陳代謝的一個指標，直接與培養物中的細胞數量成正比。The anti-tumor cell killing experiment of PBMC mediated by bispecific antibody is achieved by quantitatively detecting cell proliferation. Cell Titer-glo is used to detect the content of ATP in cells, and ATP is an indicator of the metabolism of living cells, directly proportional to the number of cells in culture.

共用了四種不同的靶細胞(target cell，T)，包括三種不同B7H3表達水平的腫瘤細胞系(A498，U87(中科院細胞庫，TCHu138)，Detroit562(ATCC，CCL-138))，和一種不表達B7H3的陰性對照細胞系CHOK1(ATCC,CCL-61)。效應細胞(effector cell,E)是來自健康志願者的PBMC。將靶細胞接種在96孔板中，過夜培養，次日，每孔加入等量的新鮮抽提的PBMC和梯度稀釋的待測雙特異抗體(最高終濃度為300nM，1：3稀釋)，或PBS(對照(control)，有效應細胞和靶細胞，無抗體)。設置空白對照(blank，只有培養基，沒有細胞或抗體)，E：T的比例，對於A498，U87，Detroit562和CHOK1細胞，分別為10：1，5：1，5：1，5：1。培養48小時，用Cell Titer-glo檢測(參照說明書)，酶標儀讀取信號值，並最終換算成抑制率，用Graphpad Prism 5對數據進行處理分析。Four different target cells (T) are shared, including three tumor cell lines with different B7H3 expression levels (A498, U87 (Cell Bank of Chinese Academy of Sciences, TCHu138), Detroit562 (ATCC, CCL-138)), and one The negative control cell line CHOK1 (ATCC, CCL-61) expressing B7H3. Effector cells (E) are PBMCs from healthy volunteers. Inoculate the target cells in a 96-well plate and culture overnight. The next day, add an equal amount of freshly extracted PBMC and a serially diluted bispecific antibody to be tested (the highest final concentration is 300nM, 1:3 dilution), or PBS (control, with effector cells and target cells, no antibody). Set a blank control (blank, only medium, no cells or antibodies), the ratio of E:T, for A498, U87, Detroit562 and CHOK1 cells, are 10:1, 5:1, 5:1, 5:1, respectively. Incubate for 48 hours, use Cell Titer-glo to detect (refer to the instructions), read the signal value with a microplate reader, and finally convert it into an inhibition rate, and useGraphpad Prism 5 to process and analyze the data.

抑制率%(Inhibition %)=100%-(信號值_樣本-信號值_空白)/(信號值_對照-信號值_空白)Inhibition rate% (Inhibition %)=100%-(signal value_sample -signal value_blank )/(signal value_control -signal value_blank )

(Inhibition %=100%-(Signal_sample-Signal_blank)/(Signal_control-Signal_blank))(Inhibition %=100%-(Signal_sample -Signal_blank )/(Signal_control -Signal_blank ))

3.1 含不同親和力CD3抗原結合結構域的抗體比較3.1 Comparison of antibodies with different affinity CD3 antigen binding domains

使用不同親和力的CD3 scFv，構建的不同雙特異抗體，呈現出不同的體外靶細胞殺傷療效(見第3A圖和第3B圖)，以分別含有HRH5和HRH6的雙特異性抗體155、156、185和186的殺傷療效最弱，這點與Biacore親和力的檢測結果一致。Using CD3 scFv with different affinities to construct different bispecific antibodies, showing different in vitro target cell killing effects (see Figure 3A and Figure 3B) to containbispecific antibodies 155, 156, and 185 of HRH5 and HRH6, respectively And 186 has the weakest killing effect, which is consistent with the Biacore affinity test results.

3.2 B7H3單價和雙價雙特異性抗體的比較3.2 Comparison of B7H3 monovalent and bivalent bispecific antibodies

對於含有不同抗CD3抗體重鏈可變區的scFv構建成的雙特異性抗體，以AFF3(該結構示例性抗體為131和177)和AF3(該結構示例性抗體為181和187)結構比較為例(見第4A圖和第4B圖)，CD3-B7H3的AFF3結構B7H3雙價雙特異性抗體，比AF3結構的B7H3單價雙特異性抗體，在體外細胞殺傷活性上明顯增強。這一點適用於所有含不同CD3 VH的雙特異性抗體。For bispecific antibodies constructed from scFvs containing different heavy chain variable regions of anti-CD3 antibodies, the structure comparison of AFF3 (an exemplary antibody of this structure is 131 and 177) and AF3 (an exemplary antibody of this structure is 181 and 187) is For example (see Fig. 4A and Fig. 4B), the AFF3 structure B7H3 bivalent bispecific antibody of CD3-B7H3 is significantly stronger than the B7H3 monovalent bispecific antibody of AF3 structure in vitro cell killing activity. This applies to all bispecific antibodies containing different CD3 VHs.

3.3 B7H3雙價雙特異性抗體不同結構排序分子結構對腫瘤殺傷活性的影響3.3 The effect of different molecular structure of B7H3 bivalent bispecific antibody on tumor killing activity

對具有相同抗原結合結構域組件不同結構順序的B7H3雙價雙特異性抗體分子161、162、113和126(見第5A圖)，以及113與143(見第5B圖)的腫瘤細胞殺傷活性進行平行試驗，上述分子都使用HRH2作為CD3抗原結合結構域中的重鏈可變區。結果顯示，不同結構順序的B7H3雙價特異性抗體分子對A498細胞均有顯著的殺傷作用。其中161、162、113和126的殺傷活性與MGD009的殺傷活性相當或略優於MGD009。不同結構排列順序對B7H3雙價雙特異性抗體的腫瘤細胞殺傷活性影響不大。The tumor cell killing activity of B7H3 bivalentbispecific antibody molecules 161, 162, 113, and 126 (see Figure 5A), and 113 and 143 (see Figure 5B), which have the same antigen-binding domain components and different structural sequences In parallel experiments, the above molecules all use HRH2 as the heavy chain variable region in the CD3 antigen binding domain. The results showed that the B7H3 bivalent specific antibody molecules with different structural sequences had significant killing effects on A498 cells. Among them, the killing activity of 161, 162, 113 and 126 is equivalent to or slightly better than that of MGD009. The order of different structures has little effect on the tumor cell killing activity of the B7H3 bivalent bispecific antibody.

3.4 雙特異抗體對不同B7H3表達水平的腫瘤細胞系均有殺傷療效3.4 Bispecific antibodies have killing effects on tumor cell lines with different B7H3 expression levels

測試了三個待測雙特異抗體118、127和132在A498、U87和Detroit562腫瘤細胞系上的體外殺傷效果。殺傷療效與B7H3表達水平呈正相關，例如118在A498、U87和Detroit562上的EC50分別為0.34、2.4和14.5nM。三個抗體分子均顯示出這一趨勢。所有雙特異抗體對B7H3陰性的對照細胞系CHOK1均沒有殺傷，且陰性對照雙特異抗體NC1在所有靶細胞系上也沒有殺傷效果，這兩點共同說明細胞殺傷需要藉由雙特異抗體將效應細胞重定向到B7H3陽性的靶細胞，進行靶點特異性殺傷。The in vitro killing effects of three testbispecific antibodies 118, 127 and 132 on A498, U87 and Detroit562 tumor cell lines were tested. The killing effect is positively correlated with the expression level of B7H3. For example, the EC50 of 118 on A498, U87 and Detroit562 are 0.34, 2.4 and 14.5 nM, respectively. All three antibody molecules show this trend. All bispecific antibodies did not kill the B7H3-negative control cell line CHOK1, and the negative control bispecific antibody NC1 also had no killing effect on all target cell lines. These two points together indicate that cell killing requires the use of bispecific antibodies to kill effector cells. Redirect to B7H3 positive target cells for target-specific killing.

3.5 不同結構雙特異性抗體對A498細胞的殺傷比較3.5 Comparison of the killing effects of bispecific antibodies with different structures on A498 cells

測試了三個待測雙特異抗體127與201、202在A498腫瘤細胞系上的體外殺傷效果。結果顯示(見第5C圖)三個結構的雙特異性抗體均有腫瘤的殺傷活性，其中雙特異性抗體127的殺傷活性優於201和202。The in vitro killing effects of the threebispecific antibodies 127, 201, and 202 on the A498 tumor cell line were tested. The results show (see Figure 5C) that the bispecific antibodies of the three structures have tumor killing activity, and the killing activity ofbispecific antibody 127 is better than that of 201 and 202.

測試例4、體外T細胞活化實驗Test Example 4. In vitro T cell activation experiment

雙特異抗體對T細胞的活化功能的檢測，是利用Jurkat重組細胞系，在有或沒有A498腫瘤細胞系存在的情況下，檢測Jurkat被激活後NFAT驅動的螢光素酶報告基因(luciferase)的表達。The detection of the activation function of the bispecific antibody on T cells uses the Jurkat recombinant cell line to detect the NFAT-driven luciferase reporter gene (luciferase) after Jurkat is activated in the presence or absence of the A498 tumor cell line. expression.

接種A498細胞於96孔細胞培養板上(1×10⁵/ml，100μL/孔)，放置於37℃，5%CO₂培養箱培養20-24h。次日，移除細胞培養上清後，向每孔加入90μl的Jurkat重組細胞懸液(5.5×10⁵/ml)，和10μl梯度稀釋的待測雙特異抗體(最高500nM的終濃度，1：3梯度稀釋)，並設置陰性對照(對照(control)，有A498和Jurkat重組細胞，無抗體)和空白對照(blank，只有培養基，沒有細胞或抗體)，置於37℃，5%CO₂培養箱培養5-6h。非腫瘤細胞特異的Jurkat重組細胞活化，則是直接向空白96孔培養板中加入Jurkat重組細胞和待測抗體。共培養結束後，每孔中加入100μl的Bright-Glo Reagent(Bright-Glo^TM Luciferase Assay System，Promega,Cat#：E2620)，置於室溫放置5-10min，使用多功能酶標儀讀取化學發光信號值，螢光增強倍數(Fold increase)的計算公式為：增強倍數=((信號值_樣本-信號值_空白)/(信號值_對照-信號值_空白)Inoculate A498 cells on a 96-well cell culture plate (1×10⁵ /ml, 100 μL/well), and place them in a 37°C, 5% CO₂ incubator for 20-24 hours. On the next day, after removing the cell culture supernatant, add 90 μl of Jurkat recombinant cell suspension (5.5×10⁵ /ml) and 10 μl of the test bispecific antibody (up to 500 nM final concentration, 1: 3 gradient dilution), and set a negative control (control, A498 and Jurkat recombinant cells, no antibody) and blank control (blank, only medium, no cells or antibodies), and place them at 37℃, 5% CO₂ culture Incubate in a box for 5-6h. For activation of non-tumor cell-specific Jurkat recombinant cells, Jurkat recombinant cells and the antibody to be tested are directly added to the blank 96-well culture plate. After the co-cultivation, add 100μl of Bright-Glo Reagent (Bright-Glo^TM Luciferase Assay System, Promega, Cat#: E2620) to each well, leave it at room temperature for 5-10 minutes, and read the chemistry with a multifunctional microplate reader Luminous signal value, the calculation formula of fluorescence increase (Fold increase) is: enhancement factor=((signal value_sample -signal value_blank )/(signal value_control -signal value_blank )

(Fold increase=(Signal_sample-Signal_blank)/(Signal_control-Signal_blank))(Fold increase=(Signal_sample -Signal_blank )/(Signal_control -Signal_blank ))

4.1 不同排列順序的B7H3雙價分子均能有效激活T細胞4.1 B7H3 bivalent molecules in different arrangements can effectively activate T cells

將B7H3雙價雙特異性抗體118、127和132分別在A498存在或或不存在的情況下檢測Jurkat重組細胞的激活情況，以驗證雙特異性抗體對T細胞的特異性和非特異性激活效果。結果顯示，不同排列順序的B7H3雙價雙特異抗體118、127和132在腫瘤細胞系A498存在的情況下(見第6A圖)，都能有效地激活Jurkat重組細胞系，顯著地誘導螢光素酶的表達，因為陰性對照抗體NC1不能誘導螢光素酶的表達，可以證明Jurkat重組細胞的激活是B7H3靶點特異的。Jurkat重組細胞的激活，需要藉由雙特異抗體共募集表達CD3的Jurkat重組細胞和表達B7H3的腫瘤細胞才能實現，在只有Jurkat重組細胞，沒有A498細胞時(見第6B圖)，螢光素酶的表達非常低，只有在最高的幾個抗體濃度點能檢測到一些弱信號。The B7H3 bivalentbispecific antibodies 118, 127 and 132 were tested for the activation of Jurkat recombinant cells in the presence or absence of A498 to verify the specific and non-specific activation effects of the bispecific antibodies on T cells . The results show that the B7H3 bivalentbispecific antibodies 118, 127 and 132 in different sequences can effectively activate the Jurkat recombinant cell line and significantly induce luciferin in the presence of the tumor cell line A498 (see Figure 6A) The expression of the enzyme, because the negative control antibody NC1 cannot induce the expression of luciferase, it can prove that the activation of Jurkat recombinant cells is specific to the B7H3 target. The activation of Jurkat recombinant cells requires the co-recruitment of Jurkat recombinant cells expressing CD3 and tumor cells expressing B7H3 by a bispecific antibody. When only Jurkat recombinant cells are used, and A498 cells are not available (see Figure 6B), luciferase The expression of is very low, and only some weak signals can be detected at the highest antibody concentration points.

4.2 B7H3單價和雙價雙特異性抗體的比較4.2 Comparison of B7H3 monovalent and bivalent bispecific antibodies

雙價CD3-B7H3雙特異性抗體較B7H3單價的雙特異抗體，在靶點特異的T細胞激活明顯增強，這與測試例3中，B7H3雙價比B7H3單價分子的體外腫瘤殺傷能力增強相一致。與此同時，非靶點特異的T細胞激活保持不變。因此，B7H3雙價分子(131)比B7H3單價分子(181)具有更強的藥效(見第7A圖)，但因T細胞非特異激活而帶來的副作用並沒有增強(見第7B圖)。Compared with the B7H3 monovalent bispecific antibody, the bivalent CD3-B7H3 bispecific antibody has significantly enhanced target-specific T cell activation, which is consistent with the enhanced in vitro tumor killing ability of the B7H3 bivalent molecule than the B7H3 monovalent molecule in Test Example 3. . At the same time, non-target-specific T cell activation remains unchanged. Therefore, the B7H3 bivalent molecule (131) has stronger efficacy than the B7H3 monovalent molecule (181) (see Figure 7A), but the side effects caused by the non-specific activation of T cells are not enhanced (see Figure 7B) .

測試例5、體外細胞因子分泌實驗Test Example 5. In vitro cytokine secretion experiment

效應細胞在雙特異抗體的介導下重定向靶細胞，在殺傷靶細胞的同時，釋放細胞因子。其中細胞因子分泌是藉由ELISA的方法定量檢測細胞培養上清中的細胞因子含量，包括IL2、IFNγ、TNFα。The effector cells redirect the target cells under the mediation of the bispecific antibody, and release cytokines while killing the target cells. Among them, cytokine secretion is to quantitatively detect the cytokine content in the cell culture supernatant by ELISA, including IL2, IFNγ, and TNFα.

實驗設計和所用抗體與測試例4所描述相同，在體外殺傷實驗的終點收集細胞培養上清，至96孔板中(Corning#3795)，放置-20℃保存備用。ELISA實驗時，取出凍存的培養上清，室溫融化，3500rpm，離心10mins，收集上清用於ELISA實驗。ELISA步驟詳見試劑盒中的說明書(Human IL-2 ELISA kit、Human IFN-γ ELISA Kit、Human TNF-α ELISA kit、欣博盛，Cat # EHC003.96、EHC102g.96,EHC103a.96)。The experimental design and the antibodies used are the same as those described in Test Example 4. The cell culture supernatant was collected at the end of the in vitro killing experiment and placed in a 96-well plate (Corning#3795), and stored at -20°C for later use. In the ELISA experiment, the frozen culture supernatant was taken out, thawed at room temperature, centrifuged at 3500 rpm for 10 minutes, and the supernatant was collected for the ELISA experiment. For ELISA steps, please refer to the instructions in the kit (Human IL-2 ELISA kit, Human IFN-γ ELISA Kit, Human TNF-α ELISA kit, Xinbosheng, Cat # EHC003.96, EHC102g.96, EHC103a.96).

結果表明，測試雙特異抗體在PBMC和B7H3陽性的靶細胞A498共存在時(參見第8A圖至第8C圖)，均能有效誘導PBMC分泌IL2、IFNγ和TNFα，其中以MGD009和118所誘導的細胞因子分泌水平最高，其次為127和132，而陰性對照抗體NC1所誘導的細胞因子分泌不在檢測靈敏度範圍內。在PBMC和B7H3陰性細胞CHOK1共存在時(參見第9A圖至第9C圖)，MGD009在最高的三個濃度點能明顯地誘導IFNγ和TNFα的釋放，而待測的三個雙特異抗體，118、127和132則不能誘導IFNγ和TNFα的釋放，預示著三個待測雙特異抗體相比起MGD009，在非靶點特異的細胞因子分泌上具有更好的安全性。The results show that the test bispecific antibody can effectively induce PBMC to secrete IL2, IFNγ and TNFα when PBMC and B7H3-positive target cells A498 coexist (see Figure 8A to Figure 8C), among which are induced by MGD009 and 118 The level of cytokine secretion was the highest, followed by 127 and 132, while the cytokine secretion induced by the negative control antibody NC1 was not within the detection sensitivity range. When PBMC and B7H3-negative cells CHOK1 coexist (see Figure 9A to Figure 9C), MGD009 can significantly induce the release of IFNγ and TNFα at the three highest concentration points, while the three bispecific antibodies to be tested, 118 , 127 and 132 can not induce the release of IFNγ and TNFα, indicating that the three bispecific antibodies to be tested have better safety in the secretion of non-target-specific cytokines than MGD009.

測試例6、人PBMC重建的小鼠A498模型的藥效實驗Test Example 6. Pharmacodynamic experiment of mouse A498 model reconstructed by human PBMC

本測試例利用人PBMC重建的NOG小鼠(北京維通利華實驗動物有限公司)A498模型(ATCC)來評價本發明測試CD3-B7H3雙特異性抗體在小鼠體內的抗腫瘤療效。This test example uses the A498 model (ATCC) of NOG mice (Beijing Weitong Lihua Experimental Animal Co., Ltd.) reconstructed with human PBMC to evaluate the anti-tumor efficacy of the CD3-B7H3 bispecific antibody tested in the present invention in mice.

將A498細胞5×10⁶個細胞/小鼠/100μl(含50% matrigel)接種於NOG小鼠右肋部皮下，當荷瘤小鼠腫瘤體積達到130-150mm³左右時將小鼠隨機分組，每組5-6隻，並將分組當天定義為該實驗第0天。於第0天或第1天將新鮮抽提的2名志願者的PBMC以1：1比例混合，以5×10⁶個細胞/100μl注射到NOG小鼠腹腔，並開始腹腔注射各抗體，每週2次，共給藥6次，每週2次監測腫瘤體積、動物重量並記錄數據。Vehicle是以PBS緩衝液代替抗體給藥的陰性對照組。A498 cells 5×10⁶ cells/mouse/100μl (containing 50% matrigel) were inoculated subcutaneously on the right ribs of NOG mice. When the tumor volume of the tumor-bearing mice reached about 130-150mm³ , the mice were randomly divided into groups. Each group of 5-6 animals, and the grouping day is defined as the 0th day of the experiment. Onday 0 orday 1, the freshly extracted PBMCs of two volunteers were mixed at a ratio of 1:1 and injected into the abdominal cavity of NOG mice at a rate of 5×10⁶ cells/100 μl, and each antibody was injected into the abdominal cavity. Twice a week, a total of 6 administrations, 2 times a week to monitor tumor volume, animal weight and record data. Vehicle is a negative control group with PBS buffer instead of antibody administration.

抗體118和119在較低的劑量下(第10A圖)已經呈現出一定的抑瘤療效，且呈劑量依賴效應。其中抗體118在實驗終點時(第20天)，0.01mpk和0.03mpk劑量下的抑瘤率(TGI)分別為22.17%和60.39%。Antibodies 118 and 119 have shown certain anti-tumor effects at lower doses (Figure 10A), and are dose-dependent.Antibody 118 at the end of the experiment (day 20), the tumor inhibition rate (TGI) at the doses of 0.01mpk and 0.03mpk were 22.17% and 60.39%, respectively.

抗體113在第14天時，已經顯示出一定的抑瘤效果，0.6mpk和0.3mpk劑量組(第10B圖)抑瘤率分別達到70.05%(p<0.05)和60.78%(p<0.05)，至第20天時，抑瘤效果繼續增強且呈劑量依賴性，抑瘤率分別為大於100%(p<0.001)和77.92%(p<0.05)。Antibody 113 has shown a certain anti-tumor effect on the 14th day. The anti-tumor rate of the 0.6mpk and 0.3mpk dose groups (Figure 10B) reached 70.05% (p<0.05) and 60.78% (p<0.05), respectively. By the 20th day, the anti-tumor effect continued to increase and was dose-dependent. The anti-tumor rates were greater than 100% (p<0.001) and 77.92% (p<0.05), respectively.

在0.12mpk和0.36mpk劑量條件下(第10C圖)，抗體118在第12天時，0.12mpk和0.36mpk的劑量已經分別可以達到39.18%和57.44%(p<0.001)，至第21天時的抑瘤率分別增至81.72%(p<0.01)和大於100%(p<0.001)，其中0.36mpk劑量下，甚至有一隻小鼠的腫瘤完全消退(1/6)。Under the dosage conditions of 0.12mpk and 0.36mpk (Figure 10C),antibody 118 has reached 39.18% and 57.44% at the dose of 0.12mpk and 0.36mpk, respectively (p<0.001) on the 12th day, until the 21st day The tumor-inhibitory rates of the mice were increased to 81.72% (p<0.01) and greater than 100% (p<0.001), respectively. Among them, even at the dose of 0.36mpk, even one mouse had tumor regression (1/6).

在0.36mpk劑量條件下(第10D圖)，抗體126在第21天時抑瘤率達到47.78%(p<0.01)。抗體128在第19天已經顯示出顯著抑瘤效果(TGI=56.37%)，至Day21時，抑瘤率升至69.28%(p<0.001)。抗體127mpk，在第12天抑瘤率達到76.20%(p<0.001)，至第21天時，抑瘤效果繼續增強，抑瘤率大於100%(p<0.001)，5隻動物中，有3隻的腫瘤體積與分組時相比，發生了回縮，另外2隻的腫瘤完全消退。Under the condition of 0.36mpk dose (Figure 10D), the tumor inhibition rate ofantibody 126 reached 47.78% on day 21 (p<0.01). Antibody 128 has shown a significant anti-tumor effect (TGI=56.37%) on the 19th day. By Day21, the anti-tumor rate rose to 69.28% (p<0.001). Antibody 127mpk, the tumor inhibition rate reached 76.20% on the 12th day (p<0.001), by the 21st day, the tumor inhibition effect continued to increase, and the tumor inhibition rate was highAt 100% (p<0.001), 3 out of 5 animals had tumor volume shrinkage compared to the grouping time, and the other 2 tumors completely resolved.

抗體127的抗腫瘤活性，在另一次實驗中得到了重複(第10E圖)，第14天時的抑瘤率達到90.6%(p<0.001)，至第17天時抑瘤率增到95.80%(p<0.001)。127在較低劑量(0.12mpk)以及較低頻率給藥(每週1次，127-0.36mpk-qw)時仍舊有效，在第17天的抑瘤率分別達到51.37%(p<0.001)和96.20%(p<0.001)。The anti-tumor activity ofantibody 127 was repeated in another experiment (Figure 10E). The tumor inhibition rate reached 90.6% on day 14 (p<0.001), and the tumor inhibition rate increased to 95.80% on day 17 (p<0.001). 127 is still effective at lower doses (0.12mpk) and lower frequency administrations (once a week, 127-0.36mpk-qw), and the tumor inhibition rates on day 17 reached 51.37% (p<0.001) and 96.20% (p<0.001).

測試例7、hCD3 KI小鼠模型的藥效實驗Test Example 7. Pharmacodynamic experiment of hCD3 KI mouse model

本實驗在Balb/c-hCD3小鼠皮下接種CT26-hB7H3腫瘤細胞系(CT26細胞來源於中科院細胞庫，TCM37，經過表達hB7H3，獲得CT26-hB7H3細胞)，來評價本發明CD3-B7H3雙特異性抗體在小鼠體內對腫瘤生長的抑制作用。In this experiment, Balb/c-hCD3 mice were subcutaneously inoculated with CT26-hB7H3 tumor cell line (CT26 cells were derived from the cell bank of the Chinese Academy of Sciences, TCM37, after expressing hB7H3 to obtain CT26-hB7H3 cells) to evaluate the CD3-B7H3 bispecificity of the present invention Antibody's inhibitory effect on tumor growth in mice.

雌性hCD3E Balb/c轉基因小鼠，購自南大模式所(合格證編號201801374/5/6，許可證SCXK(蘇)2015-0001)。Female hCD3E Balb/c transgenic mice were purchased from Nanda Model Institute (certificate number 201801374/5/6, license SCXK (Su) 2015-0001).

將CT26-hB7H3細胞8×10⁵個細胞/小鼠/100μl接種於hCD3小鼠右肋部皮下。當荷瘤小鼠腫瘤體積達到約80-120mm³左右時將小鼠隨機分組，每組7隻。將分組當天定義為該實驗第0天，並開始腹腔註射各抗體，每週2次，共給藥5次，每週2次監測腫瘤體積、動物重量並記錄數據。Vehicle是以PBS緩衝液代替抗體給藥的陰性對照組。8×10⁵ cells/mouse/100 μl of CT26-hB7H3 cells were inoculated subcutaneously into the right ribs of hCD3 mice. When the tumor volume of the tumor-bearing mice reached about 80-120mm³ , the mice were randomly divided into groups of 7 mice. The day of grouping was defined as the 0th day of the experiment, and the intraperitoneal injection of each antibody was started, twice a week, for a total of 5 administrations. The tumor volume and animal weight were monitored twice a week and the data was recorded. Vehicle is a negative control group with PBS buffer instead of antibody administration.

結果顯示，抗體118在1mpk劑量下在初次給藥後即顯示出較強藥效(第11A圖)，第13天抑瘤率為38.34%(p<0.05)。The results showed thatantibody 118 showed strong efficacy after the initial administration at a dose of 1mpk (Figure 11A), and the tumor inhibition rate was 38.34% on day 13 (p<0.05).

抗體132在3.6mpk劑量下具有抑制腫瘤生長的趨勢(第11B圖)，第13天抑瘤率達到26.35%。Antibody 132 has a tendency to inhibit tumor growth at a dose of 3.6 mpk (Figure 11B), and the tumor inhibition rate reached 26.35% on the 13th day.

測試例8、大鼠體內PK實驗Test Example 8. PK experiment in rats

本實驗在SD大鼠尾靜脈注射CD3-B7H3雙特異性抗體，檢測不同時間點大鼠血清中的抗體濃度，以此來評價CD3-B7H3雙特異性抗體在SD大鼠體內的代謝。In this experiment, the CD3-B7H3 bispecific antibody was injected into the tail vein of SD rats to detect the antibody concentration in rat serum at different time points to evaluate the metabolism of CD3-B7H3 bispecific antibody in SD rats.

大鼠尾靜脈注射受試藥物3mg/kg，給藥體積為5mL/kg。於給藥前及給藥後5min、8h、1d、2d、4d、7d、10d、14d、21d、28d各時間點采血。採用ELISA方法檢測血清中的抗體濃度，分別使用兩種不同的ELISA方法，B7H3抗原(1μg/mL)或者CD3抗原(1μg/mL)鋪板，anti-human Fc-HRP(abcam,ab98624)作為二抗。採用Winnolin軟體計算受試藥物的藥動學參數，所得主要藥動學參數見表17。The test drug was injected into the tail vein of the rat with 3 mg/kg, and the dose was 5 mL/kg. Blood was collected before administration and 5min, 8h, 1d, 2d, 4d, 7d, 10d, 14d, 21d, 28d after administration. ELISA method was used to detect the antibody concentration in the serum. Two different ELISA methods were used, B7H3 antigen (1μg/mL) or CD3 antigen (1μg/mL) plated, and anti-human Fc-HRP (abcam, ab98624) was used as the secondary antibody . Winnolin software was used to calculate the pharmacokinetic parameters of the tested drug. The main pharmacokinetic parameters obtained are shown in Table 17.

抗體118、127和132在B7H3抗原結合區域的半衰期在4.9-8.1天，略長於MGD009，達到普通IgG抗體水平，CD3抗原結合區域的半衰期為3.2-5.6天。其中，抗體118在B7H3和CD3兩個不同抗原結合區域的動力學參數相差不大，說明該分子在體內的完整性較好，半衰期分別為4.9和4.4天。抗體127在B7H3和CD3兩個不同抗原結合區域的半衰期分別為4.9和3.2天，在暴露量和清除速率上相差較為明顯，以CD3部分較差，由於CD3部分在該分子結構的內部，更可能由於CD3部分結合功能變弱而不是分子斷裂造成。抗體132是在抗體127分子序列的基礎上，在B7H3 scFv內部加入了一對二硫鍵，此改造大大提高了分子的半衰期(65-75%)，在暴露量和清除速率上也有較大的改善。The half-life ofantibodies 118, 127 and 132 in the B7H3 antigen-binding region is 4.9-8.1 days, slightly longer than MGD009, reaching the level of ordinary IgG antibodies, and the half-life of CD3 antigen-binding region is 3.2-5.6 days. Among them, the kinetic parameters ofantibody 118 in the two different antigen binding regions of B7H3 and CD3 are not much different, indicating that the integrity of the molecule in the body is better, and the half-lives are 4.9 and 4.4 days, respectively. The half-life ofantibody 127 in the two different antigen-binding regions of B7H3 and CD3 is 4.9 and 3.2 days, respectively. The difference in exposure and clearance rate is more obvious. The CD3 part is worse. Because the CD3 part is inside the molecular structure, it is more likely to be due to The weakening of the binding function of CD3 is not caused by molecular breakage.Antibody 132 is based on the molecular sequence ofantibody 127, adding a pair of disulfide bonds inside the B7H3 scFv. This modification greatly increases the half-life of the molecule (65-75%), and has a greater exposure and clearance rate. improve.

<110> 江蘇恆瑞醫藥股份有限公司 上海恆瑞醫藥有限公司<110> Jiangsu Hengrui Pharmaceutical Co., Ltd. Shanghai Hengrui Pharmaceutical Co., Ltd.

<120> 多特異性蛋白分子<120> Multispecific protein molecules

<150> 201811491691.4<150> 201811491691.4

<151> 2018-12-7<151> 2018-12-7

<160> 104<160> 104

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 439<211> 439

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 檢測用人B7H3抗原<223> Human B7H3 antigen for detection

<400> 1<400> 1

<210> 2<210> 2

<211> 443<211> 443

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 檢測用猴B7H3抗原<223> Monkey B7H3 antigen for detection

<400> 2<400> 2

<210> 3<210> 3

<211> 222<211> 222

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 檢測用鼠B7H3抗原<223> Mouse B7H3 antigen for detection

<400> 3<400> 3

<210> 4<210> 4

<211> 534<211> 534

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 人B7H3全長胺基酸序列<223> Human B7H3 full-length amino acid sequence

<400> 4<400> 4

<210> 5<210> 5

<211> 534<211> 534

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 猴B7H3全長胺基酸序列<223> Monkey B7H3 full-length amino acid sequence

<400> 5<400> 5

<210> 6<210> 6

<211> 316<211> 316

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 鼠B7H3全長胺基酸序列<223> Mouse B7H3 full-length amino acid sequence

<400> 6<400> 6

<210> 7<210> 7

<211> 119<211> 119

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> h1702 VH<223> h1702 VH

<400> 7<400> 7

<210> 8<210> 8

<211> 110<211> 110

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> h1702 VL<223> h1702 VL

<400> 8<400> 8

<210> 9<210> 9

<211> 8<211> 8

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> h1702 HCDR1<223> h1702 HCDR1

<400> 9<400> 9

<210> 10<210> 10

<211> 8<211> 8

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> h1702 HCDR2<223> h1702 HCDR2

<400> 10<400> 10

<210> 11<210> 11

<211> 12<211> 12

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> h1702 HCDR3<223> h1702 HCDR3

<400> 11<400> 11

<210> 12<210> 12

<211> 9<211> 9

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> h1702 LCDR1<223> h1702 LCDR1

<400> 12<400> 12

<210> 13<210> 13

<211> 3<211> 3

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> h1702 LCDR2<223> h1702 LCDR2

<400> 13<400> 13

<210> 14<210> 14

<211> 10<211> 10

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> h1702 LCDR3<223> h1702 LCDR3

<400> 14<400> 14

<210> 15<210> 15

<211> 119<211> 119

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> B7H3 VH44C<223> B7H3 VH44C

<400> 15<400> 15

<210> 16<210> 16

<211> 110<211> 110

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> B7H3 VL103C<223> B7H3 VL103C

<400> 16<400> 16

<210> 17<210> 17

<211> 110<211> 110

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 人CD3 ε<223> Human CD3 ε

<400> 17<400> 17

<210> 18<210> 18

<211> 92<211> 92

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> 人CD3 δ<223> Human CD3 δ

<400> 18<400> 18

<210> 19<210> 19

<211> 102<211> 102

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 猴CD3 ε<223> Monkey CD3 ε

<400> 19<400> 19

<210> 20<210> 20

<211> 92<211> 92

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> 猴CD3 δ<223> Monkey CD3 δ

<400> 20<400> 20

<210> 21<210> 21

<211> 93<211> 93

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 鼠CD3 ε<223> Mouse CD3 ε

<400> 21<400> 21

<210> 22<210> 22

<211> 92<211> 92

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 鼠CD3 δ<223> Mouse CD3 δ

<400> 22<400> 22

<210> 23<210> 23

<211> 207<211> 207

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 人CD3 ε全長胺基酸序列<223> Human CD3 ε full-length amino acid sequence

<400> 23<400> 23

<210> 24<210> 24

<211> 171<211> 171

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 人CD3 δ全長胺基酸序列<223> Human CD3 δ full-length amino acid sequence

<400> 24<400> 24

<210> 25<210> 25

<211> 198<211> 198

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 猴CD3 ε全長胺基酸序列<223> Monkey CD3 ε full-length amino acid sequence

<400> 25<400> 25

<210> 26<210> 26

<211> 171<211> 171

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 猴CD3 δ全長胺基酸序列<223> Monkey CD3 δ full-length amino acid sequence

<400> 26<400> 26

<210> 27<210> 27

<211> 189<211> 189

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 鼠CD3 ε全長胺基酸序列<223> Mouse CD3 ε full-length amino acid sequence

<400> 27<400> 27

<210> 28<210> 28

<211> 173<211> 173

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 鼠CD3 δ全長胺基酸序列<223> Mouse CD3 δ full-length amino acid sequence

<400> 28<400> 28

<210> 29<210> 29

<211> 125<211> 125

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-1<223> HRH-1

<400> 29<400> 29

<210> 30<210> 30

<211> 125<211> 125

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-2<223> HRH-2

<400> 30<400> 30

<210> 31<210> 31

<211> 125<211> 125

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-3<223> HRH-3

<400> 31<400> 31

<210> 32<210> 32

<211> 125<211> 125

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-4<223> HRH-4

<400> 32<400> 32

<210> 33<210> 33

<211> 125<211> 125

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-5<223> HRH-5

<400> 33<400> 33

<210> 34<210> 34

<211> 125<211> 125

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-6<223> HRH-6

<400> 34<400> 34

<210> 35<210> 35

<211> 125<211> 125

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-7<223> HRH-7

<400> 35<400> 35

<210> 36<210> 36

<211> 109<211> 109

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRL<223> HRL

<400> 36<400> 36

<210> 37<210> 37

<211> 5<211> 5

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-1 HCDR1<223> HRH-1 HCDR1

<400> 37<400> 37

<210> 38<210> 38

<211> 19<211> 19

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-1 HCDR2<223> HRH-1 HCDR2

<400> 38<400> 38

<210> 39<210> 39

<211> 14<211> 14

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-1 HCDR3<223> HRH-1 HCDR3

<400> 39<400> 39

<210> 40<210> 40

<211> 19<211> 19

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-2 HCDR2<223> HRH-2 HCDR2

<400> 40<400> 40

<210> 41<210> 41

<211> 14<211> 14

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-2 HCDR3<223> HRH-2 HCDR3

<400> 41<400> 41

<210> 42<210> 42

<211> 14<211> 14

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-3 HCDR3<223> HRH-3 HCDR3

<400> 42<400> 42

<210> 43<210> 43

<211> 14<211> 14

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-4 HCDR3<223> HRH-4 HCDR3

<400> 43<400> 43

<210> 44<210> 44

<211> 5<211> 5

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-5 HCDR1<223> HRH-5 HCDR1

<400> 44<400> 44

<210> 45<210> 45

<211> 14<211> 14

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-5 HCDR3<223> HRH-5 HCDR3

<400> 45<400> 45

<210> 46<210> 46

<211> 19<211> 19

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-6 HCDR2<223> HRH-6 HCDR2

<400> 46<400> 46

<210> 47<210> 47

<211> 19<211> 19

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRH-7 HCDR2<223> HRH-7 HCDR2

<400> 47<400> 47

<210> 48<210> 48

<211> 14<211> 14

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRL LCDR1<223> HRL LCDR1

<400> 48<400> 48

<210> 49<210> 49

<211> 7<211> 7

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRL LCDR2<223> HRL LCDR2

<400> 49<400> 49

<210> 50<210> 50

<211> 9<211> 9

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 結構域<221> domain

<223> HRL LCDR3<223> HRL LCDR3

<400> 50<400> 50

<210> 51<210> 51

<211> 244<211> 244

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> B7H3-scFv1<223> B7H3-scFv1

<400> 51<400> 51

<210> 52<210> 52

<211> 244<211> 244

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> B7H3-scFv2<223> B7H3-scFv2

<400> 52<400> 52

<210> 53<210> 53

<211> 244<211> 244

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> B7H3-scFv3<223> B7H3-scFv3

<400> 53<400> 53

<210> 54<210> 54

<211> 244<211> 244

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> B7H3-scFv4<223> B7H3-scFv4

<400> 54<400> 54

<210> 55<210> 55

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv1H<223> CD3-scFv1H

<400> 55<400> 55

<210> 56<210> 56

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv2H<223> CD3-scFv2H

<400> 56<400> 56

<210> 57<210> 57

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv3H<223> CD3-scFv3H

<400> 57<400> 57

<210> 58<210> 58

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv4H<223> CD3-scFv4H

<400> 58<400> 58

<210> 59<210> 59

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv5H<223> CD3-scFv5H

<400> 59<400> 59

<210> 60<210> 60

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv6H<223> CD3-scFv6H

<400> 60<400> 60

<210> 61<210> 61

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv7H<223> CD3-scFv7H

<400> 61<400> 61

<210> 62<210> 62

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv1L<223> CD3-scFv1L

<400> 62<400> 62

<210> 63<210> 63

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv2L<223> CD3-scFv2L

<400> 63<400> 63

<210> 64<210> 64

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv3L<223> CD3-scFv3L

<400> 64<400> 64

<210> 65<210> 65

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv4L<223> CD3-scFv4L

<400> 65<400> 65

<210> 66<210> 66

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv5L<223> CD3-scFv5L

<400> 66<400> 66

<210> 67<210> 67

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv6L<223> CD3-scFv6L

<400> 67<400> 67

<210> 68<210> 68

<211> 249<211> 249

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> CD3-scFv7L<223> CD3-scFv7L

<400> 68<400> 68

<210> 69<210> 69

<211> 217<211> 217

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> knob-Fc<223> knob-Fc

<400> 69<400> 69

<210> 70<210> 70

<211> 217<211> 217

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> hole-Fc<223> hole-Fc

<400> 70<400> 70

<210> 71<210> 71

<211> 474<211> 474

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 113第二多肽鏈<223> 113 The second polypeptide chain

<400> 71<400> 71

<210> 72<210> 72

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 113第一多肽鏈<223> 113 The first polypeptide chain

<400> 72<400> 72

<210> 73<210> 73

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 118第一條多肽鏈<223> 118 The first polypeptide chain

<400> 73<400> 73

<210> 74<210> 74

<211> 732<211> 732

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 119第一多肽鏈<223> 119 The first polypeptide chain

<400> 74<400> 74

<210> 75<210> 75

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 126第一多肽<223> 126 first polypeptide

<400> 75<400> 75

<210> 76<210> 76

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 127第一多肽<223> 127 first polypeptide

<400> 76<400> 76

<210> 77<210> 77

<211> 739<211> 739

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 128第一多肽<223> 128 first polypeptide

<400> 77<400> 77

<210> 78<210> 78

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 132第一多肽<223> 132 first polypeptide

<400> 78<400> 78

<210> 79<210> 79

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 131第一多肽<223> 131 first polypeptide

<400> 79<400> 79

<210> 80<210> 80

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 154第一多肽<223> 154 first polypeptide

<400> 80<400> 80

<210> 81<210> 81

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 156第一多肽<223> 156 first polypeptide

<400> 81<400> 81

<210> 82<210> 82

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 155第一多肽<223> 155 first polypeptide

<400> 82<400> 82

<210> 83<210> 83

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 177第一多肽<223> 177 first polypeptide

<400> 83<400> 83

<210> 84<210> 84

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 172第一多肽<223> 172 First polypeptide

<400> 84<400> 84

<210> 85<210> 85

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 171第一多肽<223> 171 first polypeptide

<400> 85<400> 85

<210> 86<210> 86

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 161第一多肽<223> 161 first polypeptide

<400> 86<400> 86

<210> 87<210> 87

<211> 734<211> 734

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 162第一多肽<223> 162 first polypeptide

<400> 87<400> 87

<210> 88<210> 88

<211> 474<211> 474

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 142第二多肽<223> 142 second polypeptide

<400> 88<400> 88

<210> 89<210> 89

<211> 730<211> 730

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> NC1鏈1<223>NC1 chain 1

<400> 89<400> 89

<210> 90<210> 90

<211> 470<211> 470

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> NC1鏈2<223>NC1 chain 2

<400> 90<400> 90

<210> 91<210> 91

<211> 725<211> 725

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> NC2鏈1<223>NC2 chain 1

<400> 91<400> 91

<210> 92<210> 92

<211> 474<211> 474

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> NC2鏈2<223>NC2 chain 2

<400> 92<400> 92

<210> 93<210> 93

<211> 730<211> 730

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> NC3鏈1<223>NC3 chain 1

<400> 93<400> 93

<210> 94<210> 94

<211> 470<211> 470

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> NC3鏈2<223>NC3 chain 2

<400> 94<400> 94

<210> 95<210> 95

<211> 504<211> 504

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> MGD009鏈1<223>MGD009 chain 1

<400> 95<400> 95

<210> 96<210> 96

<211> 274<211> 274

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> MGD009鏈2<223>MGD009 chain 2

<400> 96<400> 96

<210> 97<210> 97

<211> 227<211> 227

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> MGD009鏈3<223>MGD009 chain 3

<400> 97<400> 97

<210> 98<210> 98

<211> 13<211> 13

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 肽<221> peptide

<223> 連接子<223> Linker

<400> 98<400> 98

<210> 99<210> 99

<211> 513<211> 513

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 鏈<221> chain

<223> 201鏈1<223> 201Chain 1

<400> 99<400> 99

<210> 100<210> 100

<211> 271<211> 271

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 鏈<221> chain

<223> 201鏈2<223> 201Chain 2

<400> 100<400> 100

<210> 101<210> 101

<211> 455<211> 455

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 鏈<221> chain

<223> 202鏈1<223> 202Chain 1

<400> 101<400> 101

<210> 102<210> 102

<211> 217<211> 217

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 鏈<221> chain

<223> 202鏈2<223> 202Chain 2

<400> 102<400> 102

<210> 103<210> 103

<211> 692<211> 692

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 鏈<221> chain

<223> 202鏈3<223> 202Chain 3

<400> 103<400> 103

<210> 104<210> 104

<211> 214<211> 214

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<220><220>

<221> 鏈<221> chain

<223> 202鏈4<223> 202Chain 4

<400> 104<400> 104

Claims (27)

Translated fromChinese

一種多特異性蛋白分子，其包含第一多肽鏈和第二多肽鏈，其中：A multispecific protein molecule comprising a first polypeptide chain and a second polypeptide chain, wherein:該第一多肽鏈從胺基末端至羧基末端依次包含針對第一靶抗原的第一結合區、針對第二靶抗原的第二結合區和第一Fc區，The first polypeptide chain includes a first binding region for a first target antigen, a second binding region for a second target antigen, and a first Fc region in sequence from the amino terminal to the carboxy terminal,該第二多肽鏈從胺基末端至羧基末端依次包含針對第三靶抗原的第三結合區和第二Fc區，The second polypeptide chain includes a third binding region and a second Fc region for the third target antigen in sequence from the amino terminal to the carboxy terminal,該第二結合區和/或第三結合區不包含抗體的恆定區結構域，The second binding region and/or the third binding region do not contain the constant region domain of an antibody,該第一多肽鏈和所述第二多肽鏈鏈內各區域由肽鍵和/或接頭連接。The regions in the first polypeptide chain and the second polypeptide chain are connected by peptide bonds and/or linkers.如申請專利範圍第1項所述的多特異性蛋白分子，其中，該第一結合區、和/或該第二結合區、和/或該第三結合區是單鏈抗體。The multispecific protein molecule according to claim 1, wherein the first binding region, and/or the second binding region, and/or the third binding region are single-chain antibodies.如申請專利範圍第1或2項所述的多特異性蛋白分子，其中，該第二靶抗原為CD3，且該第一靶抗原和第三靶抗原為相同或不同的腫瘤相關抗原；或The multispecific protein molecule according to item 1 or 2 of the scope of patent application, wherein the second target antigen is CD3, and the first target antigen and the third target antigen are the same or different tumor-associated antigens; or該第一靶抗原為CD3，且該第二靶抗原和第三靶抗原為相同或不同的腫瘤相關抗原。The first target antigen is CD3, and the second target antigen and the third target antigen are the same or different tumor-associated antigens.如申請專利範圍第1至3項任一項所述的多特異性蛋白分子，其中，該腫瘤相關抗原選自AFP、ALK、B7H3、BAGE蛋白質、BCMA、BIRC5、BIRC7、β-連環蛋白、brc-ab1、BRCA1、BORIS、CA9、CA125、碳酸酐酶IX、半胱天冬酶-8、CALR、CCR5、CD19、CD20、CD22、CD30、CD33、CD38、CD40、CD123、CD133、CD138、CDK4、CEA、Claudin 18.2、週期素-B1、CYP1B1、EGFR、EGFRvIII、ErbB2/Her2、ErbB3、ErbB4、ETV6-AML、EpCAM、EphA2、Fra-1、FOLR1、GAGE蛋白、GD2、GD3、GloboH、磷脂醯肌醇蛋白聚糖-3、GM3、gp100、Her2、HLA/B-raf、HLA/k-ras、HLA/MAGE-A3、hTERT、IL13Rα2、LMP2、κ-Light、LeY、MAGE蛋白、MART-1、間皮素、ML-IAP、MOv-γ、Muc1、Muc2、Muc3、Muc4、Muc5、CA-125、MUM1、NA17、NKG2D、NY-BR1、NY-BR62、NY-BR85、NY-ESO1、OX40、p15、p53、PAP、PAX3、PAX5、PCTA-1、PLAC1、PRLR、PRAME、PSMA、RAGE蛋白質、Ras、RGS5、Rho、ROR1、SART-1、SART-3、STEAP1、STEAP2、TAG-72、TGF-β、TMPRSS2、湯-諾氏抗原、TRP-1、TRP-2、酪胺酸酶、尿溶蛋白-3和5T4。The multispecific protein molecule according to any one of items 1 to 3 in the scope of the patent application, wherein the tumor-associated antigen is selected from AFP, ALK, B7H3, BAGE protein, BCMA, BIRC5, BIRC7, β-catenin, brc -ab1, BRCA1, BORIS, CA9, CA125, Carbonic Anhydrase IX, Caspase-8, CALR, CCR5, CD19, CD20, CD22, CD30, CD33, CD38, CD40, CD123, CD133, CD138, CDK4, CEA, Claudin 18.2, Cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein, GD2, GD3, GloboH, phospholipid muscle Glycan-3, GM3,gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, IL13Rα2, LMP2, κ-Light, LeY, MAGE protein, MART-1, Mesothelin, ML-IAP, MOv -γ, Muc1, Muc2, Muc3, Muc4, Muc5, CA-125, MUM1, NA17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5 , PCTA-1, PLAC1, PRLR, PRAME, PSMA, RAGE protein, Ras, RGS5, Rho, ROR1, SART-1, SART-3, STEP1, STEP2, TAG-72, TGF-β, TMPRSS2, Tang-Knot Antigen, TRP-1, TRP-2, Tyrosinase, Urinolytic Protein-3 and 5T4.如申請專利範圍第1至4項中任一項所述的多特異性蛋白分子，其中，該第一多肽鏈具有如下式I的結構：The multispecific protein molecule according to any one of items 1 to 4 in the scope of patent application, wherein the first polypeptide chain has the structure of formula I as follows:V_a1-L1-V_b1-L2-V_c2-L2-V_d2-L4-Fc1 式I，V_a 1-L1-V_b 1-L2-V_c 2-L2-V_d 2-L4-Fc1 Formula I,該第二多肽鏈具有如下式II的結構：The second polypeptide chain has the following structure of formula II:V_e3-L5-V_f3-L6-Fc2 式II，V_e 3-L5-V_f 3-L6-Fc2 formula II,該V_a1、V_b1、V_c2、V_d2、V_e3和V_f3為抗體的輕鏈可變區或重鏈可變區，且該V_a1和V_b1，該V_c2和V_d2，與該V_e3和V_f3分別不同時為輕鏈可變區或不同時為重鏈可變區。The V_a 1, V_b 1, V_c 2, V_d 2, V_e 3, and V_f 3 are the light chain variable region or the heavy chain variable region of the antibody, and the V_a 1 and V_b 1, the V_c 2 and V_d 2 are not the light chain variable region or the heavy chain variable region at the same time as the V_e 3 and V_f 3 respectively.如申請專利範圍第1至5項中任一項所述的多特異性蛋白分子，其中，該第一多肽鏈具有如下所示的結構：The multispecific protein molecule according to any one of items 1 to 5 in the scope of the patent application, wherein the first polypeptide chain has the following structure:VH_TAA-L1-VL_TAA-L2-VH_CD3-L3-VL_CD3-L4-Fc1，VH_TAA -L1-VL_TAA -L2-VH_CD3 -L3-VL_CD3 -L4-Fc1,VH_TAA-L1-VL_TAA-L2-VL_CD3-L3-VH_CD3-L4-Fc1，VH_TAA -L1-VL_TAA -L2-VL_CD3 -L3-VH_CD3 -L4-Fc1,VL_TAA-L1-VH_TAA-L2-VH_CD3-L3-VL_CD3-L4-Fc1，VL_TAA -L1-VH_TAA -L2-VH_CD3 -L3-VL_CD3 -L4-Fc1,VL_TAA-L1-VH_TAA-L2-VL_CD3-L3-VH_CD3-L4-Fc1，VL_TAA -L1-VH_TAA -L2-VL_CD3 -L3-VH_CD3 -L4-Fc1,VH_CD3-L1-VL_CD3-L2-VH_TAA-L3-VL_TAA-L4-Fc1，VH_CD3 -L1-VL_CD3 -L2-VH_TAA -L3-VL_TAA -L4-Fc1,VH_CD3-L1-VL_CD3-L2-VL_TAA-L3-VH_TAA-L4-Fc1，VH_CD3 -L1-VL_CD3 -L2-VL_TAA -L3-VH_TAA -L4-Fc1,VL_CD3-L1-VH_CD3-L2-VH_TAA-L3-VL_TAA-L4-Fc1或VL_CD3 -L1-VH_CD3 -L2-VH_TAA -L3-VL_TAA -L4-Fc1 orVL_CD3-L1-VH_CD3-L2-VL_TAA-L3-VH_TAA-L4-Fc1；VL_CD3 -L1-VH_CD3 -L2-VL_TAA -L3-VH_TAA -L4-Fc1;且該第二多肽鏈具有如下所示的結構：And the second polypeptide chain has the structure shown below:VL_TAA-L5-VH_TAA-L6-F_C2或VH_TAA-L5-VL_TAA-L6-F_C2。VL_TAA -L5-VH_TAA -L6-F_C 2 or VH_TAA -L5-VL_TAA -L6-F_C 2.如申請專利範圍第1至6項任一項所述的多特異性蛋白分子，其中，該第一Fc區和所述第二Fc區是相同的Fc或不同的Fc。The multispecific protein molecule according to any one of claims 1 to 6, wherein the first Fc region and the second Fc region are the same Fc or different Fc.如申請專利範圍第7項所述的多特異性蛋白分子，其中，該第一Fc區為knob-Fc，該第二Fc區為hole-Fc；或The multispecific protein molecule according to claim 7, wherein the first Fc region is knob-Fc, and the second Fc region is hole-Fc; or該第一Fc區為hole-Fc，該第二Fc區為knob-Fc。The first Fc region is hole-Fc, and the second Fc region is knob-Fc.如申請專利範圍第1至8項中任一項所述的多特異性蛋白分子，其中，該第一Fc區的羧基端連接His標簽或第二Fc區的羧基端連接His標簽。The multispecific protein molecule according to any one of items 1 to 8 in the scope of the patent application, wherein the carboxyl end of the first Fc region is connected to a His tag or the carboxyl end of the second Fc region is connected to a His tag.如申請專利範圍第1至9項中任一項所述的多特異性蛋白分子，其中，針對CD3的抗原結合區包含抗體輕鏈可變區和重鏈可變區，其中，該輕鏈可變區包含分別如SEQ ID NO：48、49和50所示的LCDR1、LCDR2和LCDR3，和該重鏈可變區為選自以下i)至v)任一項的重鏈可變區：The multispecific protein molecule according to any one of items 1 to 9 in the scope of patent application, wherein the antigen binding region against CD3 comprises an antibody light chain variable region and a heavy chain variable region, wherein the light chain can The variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 48, 49 and 50, respectively, and the heavy chain variable region is a heavy chain variable region selected from any one of i) to v) below:i)包含分別如SEQ ID NO：37、38和39所示的HCDR1、HCDR2和HCDR3的重鏈可變區；i) A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 37, 38 and 39, respectively;ii)包含分別如SEQ ID NO：37、40和41所示的HCDR1、HCDR2和HCDR3的重鏈可變區；ii) A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 37, 40 and 41, respectively;iii)包含分別如SEQ ID NO：37、40和42所示的HCDR1、HCDR2和HCDR3的重鏈可變區；iii) A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 37, 40 and 42, respectively;iv)包含分別如SEQ ID NO：37、40和43所示的HCDR1、HCDR2和HCDR3的重鏈可變區；或iv) A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 37, 40 and 43, respectively; orv)包含分別如SEQ ID NO：37、47和45所示的HCDR1、HCDR2和HCDR3的重鏈可變區。v) A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 37, 47 and 45, respectively.如申請專利範圍第10項所述的多特異性蛋白分子，其中，針對CD3的抗原結合區包含如SEQ ID NO：36所示的輕鏈可變區，和/或選自如SEQ ID NO：29、30、31、32和35中任一項所示的重鏈可變區。The multispecific protein molecule according to the tenth item of the scope of patent application, wherein the antigen binding region for CD3 comprises the light chain variable region as shown in SEQ ID NO: 36, and/or is selected from the group as shown in SEQ ID NO: 29 , 30, 31, 32, and 35.如申請專利範圍第11項所述的多特異性蛋白分子，其中，針對CD3的抗原結合區包含如SEQ ID NO：55、56、57、58、61、62、63、64、65或68所示的單鏈抗體。The multispecific protein molecule described in item 11 of the scope of patent application, wherein the antigen binding region for CD3 comprises as shown in SEQ ID NO: 55, 56, 57, 58, 61, 62, 63, 64, 65 or 68 Single chain antibody shown.如申請專利範圍第1至12項中任一項所述的多特異性蛋白分子，其中，該腫瘤相關抗原為B7H3，針對B7H3的抗原結合區包含抗體輕鏈可變區和重鏈可變區，其中：The multispecific protein molecule according to any one of items 1 to 12 in the scope of the patent application, wherein the tumor-associated antigen is B7H3, and the antigen binding region for B7H3 comprises an antibody light chain variable region and a heavy chain variable region ,among them:該輕鏈可變區包含分別如SEQ ID NO：12、13和14所示的LCDR1、LCDR2和LCDR3，和該重鏈可變區包含分別如SEQ ID NO：9、10和11所示的HCDR1、HCDR2和HCDR3。The light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 12, 13, and 14, respectively, and the heavy chain variable region includes HCDR1 as shown in SEQ ID NOs: 9, 10, and 11, respectively , HCDR2 and HCDR3.如申請專利範圍第13項所述的多特異性蛋白分子，其中，針對B7H3的抗原結合區包含：The multispecific protein molecule according to item 13 of the scope of patent application, wherein the antigen binding region for B7H3 includes:如SEQ ID NO：8所示的輕鏈可變區和/或如SEQ ID NO：7所示的重鏈可變區；The light chain variable region shown in SEQ ID NO: 8 and/or the heavy chain variable region shown in SEQ ID NO: 7;或如SEQ ID NO：16所示的輕鏈可變區和/或如SEQ ID NO：15所示的重鏈可變區。Or the light chain variable region shown in SEQ ID NO: 16 and/or the heavy chain variable region shown in SEQ ID NO: 15.如申請專利範圍第14項所述的多特異性蛋白分子，其中，針對B7H3的抗原結合區包含如SEQ ID NO：51、52、53或54所示的單鏈抗體。The multispecific protein molecule according to item 14 of the scope of patent application, wherein the antigen binding region for B7H3 comprises a single chain antibody as shown in SEQ ID NO: 51, 52, 53 or 54.如申請專利範圍第1至15項中任一項所述的多特異性蛋白分子，包含第一多肽鏈和第二多肽鏈，該第一多肽鏈選自胺基酸序列如SEQ ID NO：72、73、74、75、76、77、78、79、80、83、84、85、86或87所示的多肽，和/或該第二多肽鏈選自胺基酸序列如SEQ ID NO：71、88或70所示的多肽。The multispecific protein molecule according to any one of items 1 to 15 in the scope of the patent application, comprising a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain is selected from an amino acid sequence such as SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86, or 87 polypeptide, and/or the second polypeptide chain is selected from amino acid sequences such as A polypeptide represented by SEQ ID NO: 71, 88 or 70.一種多特異性蛋白分子，包含第一多肽鏈和第二多肽鏈，該第二多肽鏈的胺基酸序列如SEQ ID NO：71、88或70所示，和：A multispecific protein molecule comprising a first polypeptide chain and a second polypeptide chain, the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 71, 88 or 70, and:該第一多肽鏈的胺基酸序列如SEQ ID NO：72所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 72;該第一多肽鏈的胺基酸序列如SEQ ID NO：73所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 73;該第一多肽鏈的胺基酸序列如SEQ ID NO：74所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 74;該第一多肽鏈的胺基酸序列如SEQ ID NO：75所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 75;該第一多肽鏈的胺基酸序列如SEQ ID NO：76所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 76;該第一多肽鏈的胺基酸序列如SEQ ID NO：77所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 77;該第一多肽鏈的胺基酸序列如SEQ ID NO：78所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 78;該第一多肽鏈的胺基酸序列如SEQ ID NO：79所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 79;該第一多肽鏈的胺基酸序列如SEQ ID NO：80所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 80;該第一多肽鏈的胺基酸序列如SEQ ID NO：83所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 83;該第一多肽鏈的胺基酸序列如SEQ ID NO：84所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 84;該第一多肽鏈的胺基酸序列如SEQ ID NO：85所示；The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 85;該第一多肽鏈的胺基酸序列如SEQ ID NO：86所示；或者The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 86; or該第一多肽鏈的胺基酸序列如SEQ ID NO：87所示。The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO:87.一種醫藥組成物，其含有治療有效量的如申請專利範圍第1至17項中任一項所述的多特異性蛋白分子，以及一種或多種藥學上可接受的載體、稀釋劑、緩衝劑或賦形劑。A pharmaceutical composition, which contains a therapeutically effective amount of the multispecific protein molecule described in any one of items 1 to 17 in the scope of the patent application, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipient.一種分離的核酸分子，其編碼申請專利範圍第1至17項中任一項所述的多特異性蛋白分子。An isolated nucleic acid molecule that encodes the multispecific protein molecule described in any one of items 1 to 17 in the scope of the patent application.一種重組載體，其包含申請專利範圍第19項所述的分離的核酸分子。A recombinant vector comprising the isolated nucleic acid molecule described in item 19 of the scope of patent application.一種用如申請專利範圍第20項所述的重組載體轉化的宿主細胞，該宿主細胞選自原核細胞和真核細胞。A host cell transformed with the recombinant vector as described in item 20 of the scope of patent application, and the host cell is selected from prokaryotic cells and eukaryotic cells.如申請專利範圍第21項所述的宿主細胞，其中，該宿主細胞為真核細胞。The host cell according to claim 21, wherein the host cell is a eukaryotic cell.如申請專利範圍第22項所述的宿主細胞，其中，該真核細胞為哺乳動物細胞或昆蟲細胞。The host cell according to claim 22, wherein the eukaryotic cell is a mammalian cell or an insect cell.一種用於生產如申請專利範圍第1至17項中任一項所述的多特異性蛋白分子的方法，該方法包括將申請專利範圍第21項所述的宿主細胞在培養基中進行培養以形成並積累申請專利範圍第1至17項中任一項所述的多特異性蛋白分子，以及從培養物回收所述多特異性蛋白分子的步驟。A method for producing the multispecific protein molecule described in any one of the scope of the patent application item 1 to 17, the method comprising culturing the host cell described in the scope of the patent application item 21 in a culture medium to form And accumulate the multispecific protein molecules described in any one of items 1 to 17 in the scope of patent application, and the step of recovering the multispecific protein molecules from the culture.一種申請專利範圍第1至17項中任一項所述的多特異性蛋白分子或申請專利範圍第18項所述的醫藥組成物，或申請專利範圍第19項所述的分離的核酸分子的作為藥物的用途。A multispecific protein molecule according to any one of the scope of patent application 1 to 17, or a pharmaceutical composition according to clause 18 of the patent application, or an isolated nucleic acid molecule described in clause 19 of the patent application Use as medicine.如申請專利範圍第25項所述的用途，其中，該藥物為激活T細胞的藥物。The use according to item 25 of the scope of patent application, wherein the drug is a drug for activating T cells.如申請專利範圍第25項所述的用途，其中，該藥物為治療癌症、自身免疫性疾病或炎性疾病的藥物。The use according to item 25 of the scope of patent application, wherein the drug is a drug for treating cancer, autoimmune diseases or inflammatory diseases.

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