TW202430560A - Anti-il-18bp antibodies - Google Patents

Abstract

Provided are antibodies and antigen binding fragments thereof that bind to interleukin-18 binding protein (IL-18BP) and related compositions, which may be used in any of a variety of therapeutic or diagnostic methods, including the treatment or diagnosis of cancers and other diseases.

Description

Translated fromChinese

抗IL-18BP抗體Anti-IL-18BP antibody

介白素-18（IL-18）為具有抗腫瘤活性之免疫刺激細胞介素。其在聯繫發炎性免疫反應與腫瘤進展方面起關鍵作用。實際上，已將重組人類IL-18作為癌症免疫治療劑加以評估。然而，此方法至少部分由於人類中之回饋迴路而無效，其中投與IL-18會引起IL-18BP之產生增加，使得中和所投與之IL-18細胞介素（參見例如Robertson等人, Clinical Cancer Res. 12:4265-4273, 2006）。Interleukin-18 (IL-18) is an immunostimulatory interleukin with anti-tumor activity. It plays a key role in linking inflammatory immune responses to tumor progression. In fact, recombinant human IL-18 has been evaluated as a cancer immunotherapy. However, this approach has been ineffective due, at least in part, to a feedback loop in humans, in which administration of IL-18 leads to increased production of IL-18BP, which neutralizes the administered IL-18 interleukin (see, e.g., Robertson et al., Clinical Cancer Res. 12:4265-4273, 2006).

IL-18BP為在腫瘤中頻繁上調之高親和力IL-18誘餌受體。研究表明IL-18BP係分泌免疫檢查點且係IL-18免疫療法之障礙（參見例如Zhou等人, Nature. 583(7817):609-614, 2020）。IL-18BP藉由將IL-18螯合使其遠離細胞表面受體來抑制IL-18之促炎性活性。IL-18對於IL-18BP之親和力高於IL-18對於IL-18受體之親和力，且IL-18BP時常以超過IL-18之量存在，從而確保嚴格調節。亦已展示IL-18BP尤其會平衡Th1及Th2免疫反應，且在自體免疫疾病中起關鍵作用（參見例如Park等人, Biomedicines. 10(7): 1750, 2022）。因此，此項技術中需要特異性調節IL-18BP之活性的藥劑。IL-18BP is a high-affinity IL-18 decoy receptor that is frequently upregulated in tumors. Studies have shown that IL-18BP is a secretory immune checkpoint and a barrier to IL-18 immunotherapy (see, e.g., Zhou et al., Nature. 583(7817):609-614, 2020). IL-18BP inhibits the pro-inflammatory activity of IL-18 by sequestering IL-18 away from cell surface receptors. The affinity of IL-18 for IL-18BP is higher than the affinity of IL-18 for the IL-18 receptor, and IL-18BP is often present in excess of IL-18, ensuring tight regulation. It has also been shown that IL-18BP, in particular, balances Th1 and Th2 immune responses and plays a key role in autoimmune diseases (see, e.g., Park et al., Biomedicines. 10(7): 1750, 2022). Therefore, there is a need in the art for agents that specifically regulate the activity of IL-18BP.

本發明係關於與介白素-18結合蛋白（IL-18BP）結合之抗體及相關組合物，其可用於多種治療及診斷方法中之任一者中，包括癌症及其他疾病之治療或診斷。The present invention relates to antibodies and related compositions that bind to interleukin-18 binding protein (IL-18BP) and can be used in any of a variety of therapeutic and diagnostic methods, including the treatment or diagnosis of cancer and other diseases.

本發明之態樣包括一種經分離之抗體或其抗原結合片段，其與介白素-18結合蛋白（IL-18BP）結合，其中至少一種抗體或其抗原結合片段包含：重鏈可變區（V_H），其包含選自表A1之互補決定區V_HCDR1、V_HCDR2及V_HCDR3序列及其與IL-18BP特異性結合之變異體；以及輕鏈可變區（V_L），其包含選自表A1之互補決定區V_LCDR1、V_LCDR2及V_LCDR3序列及其與IL-18BP特異性結合之變異體。在某些實施例中： 該VHCDR1、VHCDR2及VHCDR3序列分別包含TFX₁X₂X₃X₄X₅H、IX₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅AQKFQG及X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂DY，且該VLCDR1、VLCDR2及VLCDR3序列分別包含X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀WX₃₁A、X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈及QX₃₉X₄₀X₄₁SFPYX₄₂（關於「X」殘基之定義，參見表E11）。 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 1-3，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 4-6； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 25-27，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 28-30； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 31-33，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 34-36； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 34-39，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 40-42； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 43-45，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 46-48； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 49-51，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 52-54； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 55-57，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 58-60； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 61-63，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 64-66； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 67-69，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 70-72； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 109-111，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 112-114； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 115-117，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 118-120； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 121-123，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 124-126； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 127-129，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 130-132； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 133-135，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 136-138； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 139-141，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 142-144； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 145-147，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 148-150； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 151-153，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 154-156； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 157-159，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 160-162； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 163-165，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 166-168； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 169-171，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 172-174； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 175-177，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 178-180； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 181-183，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 184-186； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 187-189，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 190-192； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 193-195，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 196-198； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 199-201，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 202-204； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 205-207，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 208-210； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 211-213，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 214-216； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 217-219，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 220-222； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 223-225，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 226-228； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 229-231，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 232-234； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 235-237，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 238-240； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 241-243，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 244-246； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 247-249，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 250-252； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 253-255，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 256-258； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 265-267，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 268-270；或 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 271-273，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 274-276。Aspects of the present invention include an isolated antibody or antigen-binding fragment thereof that binds to interleukin-18 binding protein (IL-18BP), wherein at least one antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (_VH ) comprising complementary determining region_VH CDR1,_VH CDR2 and_VH CDR3 sequences selected fromTableA1 and variants thereof that specifically bind to IL-18BP; and a light chain variable region (_VL ) comprising complementary determining region_VL CDR1,_VL CDR2 and_VL CDR3 sequences selected fromTableA1 and variants thereof that specifically bind to IL-18BP. In certain embodiments: the VHCDR1, VHCDR2 and VHCDR3 sequences comprise_TFX1 X₂ X₃ X₄ X₅ H, IX₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ AQKFQG and X₁₆ X 17 X₁₈ X₁₉ X₂₀ X_{21X 22DY} , respectively, and the VLCDR1, VLCDR2 and VLCDR3 sequences comprise X₂₃ X₂₄ X₂₅ X₂₆ X₂₇ X₂₈ X₂₉ X₃₀ WX₃₁ A, X₃₂ X₃₃ X₃₄ X₃₅ X₃₆ X₃₇ X₃₈ and QX₃₉ X₄₀ X₄₁ SFPYX₄₂ (For the definition of the “X” residue,see TableE11 ). the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 1-3, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 4-6, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 25-27, respectively, and the_VL CDR1,_VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 28-30, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 31-33, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 34-36, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 34-39, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 35-36 the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 40-42, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 43-45, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 46-48, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 49-51, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 52-54, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 55-57, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 58-60, respectively; the_VH CDR1,_VH CDR2 and_VH the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 109-111, and the_VL CDR1,_VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 112-114, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 115-117, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 118-119, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 120-121, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 122-123, respectively; the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 124-125, respectively; the_VH CDR1,_VH CDR2 and_VHCDR3 sequences comprise SEQ ID NOs: 125-126_, respectively; the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 118-120, respectively; the_VH CDR1,_VH CDR2 and VH CDR3 sequences comprise SEQ ID NOs: 121-123, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 124-126, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 127-129, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 130-132, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 133-135, respectively, and the_VL CDR1,_{VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 136-138, respectively; the VH CDR1, VH} CDR2 and_VHCDR3 sequences comprise SEQ ID NOs:_137-139 , respectively; the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 139-141, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 142-144, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 145-147, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 148-150, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 151-153, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 154-156, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 157-159, respectively, and the_VL the VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 160-162, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 163-165, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 166-168, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 169-171, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 172-174, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 175-177, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 193-195, and said_VL CDR1,_VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 196-198, respectively; said VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 197-200, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 198-201, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 199-202, respectively; said_VL CDR1,_VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 191-192, respectively; said VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 193-195, respectively; and said_VL CDR1, VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 196-198, respectively; said_VH CDR1,_VH CDR2 and_VH the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 211-213, and the_VL CDR1,_VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 214-216, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 217-219, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 220-221; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 221-222, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 223-224, respectively; the_VHCDR1 ,_VH CDR2 and_VHCDR3 sequences comprise SEQ ID NOs: 224-225, respectively; the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 220-222, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 223-225, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 226-228, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 229-231, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 232-234, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 235-237, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 238-240, respectively; the_VH the VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 241-243, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 244-246, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 247-249, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 250-252, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 253-255, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 256-258, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 265-267, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 268-270, respectively; or the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 271-273, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 274-276, respectively.

在一些實施例中，該V_H包含與選自表A2之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，視情況其中該V_H在構架區中具有1、2、3、4、5、6、7、8、9或10個改變。在一些實施例中，該V_L包含與選自表A2之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，視情況其中該V_L在構架區中具有1、2、3、4、5、6、7、8、9或10個改變。在具體實施例中： 該V_H包含與SEQ ID NO: 277至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 278至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 279至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 280至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 285至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 286至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 287至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 288至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 289至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 290至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 291至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 292至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 293至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 294至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 295至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 296至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 297至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 298至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 299至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 300至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 313至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 314至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 315至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 316至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 317至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 318至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 319至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 320至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 321至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 322至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 323至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 324至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 325至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 326至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 327至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 328至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 329至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 330至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 331至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 332至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 333至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 334至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 335至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 336至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 337至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 338至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 339至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 340至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 341至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 342至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 343至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 344至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 345至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 346至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 347至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 348至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 349至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 350至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 351至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 352至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 353至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 354至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 355至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 356至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 357至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 358至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 359至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 360至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 361至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 362至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 367至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 368至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列；或 該V_H包含與SEQ ID NO: 369至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 370至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列。In some embodiments, the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected fromTableA2 , optionally wherein the_VH has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations in the framework regions. In some embodiments, the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected fromTableA2 , optionally wherein the_VL has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations in the framework regions. In specific embodiments: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 277, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 278; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 279, and the VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 280; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 285, and_theVL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 286; the VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 287, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 288; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 289, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 290; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: : the_VH comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 293, and the VL comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 294; the_VH comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 295, and_theVL comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:₂₉₆ . the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 296; the VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 297, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 298; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 299, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 300; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 313 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 314, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 314; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 315, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 316; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 317, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 318; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 319, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 320; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 321, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 322; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 323 is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 324, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 324; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 325, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 326; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 327, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 328; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 329, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 330; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 331, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 332; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 333 is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 334, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 334; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 335, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 336; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 337, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 338; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 339, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 340; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 341, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 342; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 343 is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 344, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 344; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 345, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 346; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 347, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 348; the VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 349, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 350; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 351, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 352; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 353 is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 354, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 354; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 355, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 356; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 357, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 358; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 359, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 360; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 361, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 362; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: or the_VH comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 369 and_theVL comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 370.

本發明之態樣亦包括一種經分離之抗體或其抗原結合片段，其與抗原決定基結合，該抗原決定基包含根據UniProt：O95998之編號之I97及V153 （可替代地，如由SEQ ID NO: 1所定義之I95及V151；或如由SEQ ID NO: 2所定義之I67及V123）。在一些實施例中，經分離之抗體或其抗原結合片段在抗原決定基處與介白素-18結合蛋白（IL-18BP）結合，該抗原決定基包含根據UniProt：O95998之編號之I97及V153 （可替代地，如由SEQ ID NO: 1所定義之I95及V151；或如由SEQ ID NO: 2所定義之I67及V123）。Aspects of the invention also include an isolated antibody or antigen-binding fragment thereof that binds to an antigenic determinant comprising I97 and V153 numbered according to UniProt: 095998 (alternatively, I95 and V151 as defined by SEQ ID NO: 1; or I67 and V123 as defined by SEQ ID NO: 2). In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to interleukin-18 binding protein (IL-18BP) at an antigenic determinant comprising I97 and V153 numbered according to UniProt: 095998 (alternatively, I95 and V151 as defined by SEQ ID NO: 1; or I67 and V123 as defined by SEQ ID NO: 2).

在一些實施例中： 該VHCDR1、VHCDR2及VHCDR3序列分別包含TFX₁X₂X₃X₄X₅H、IX₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅AQKFQG及X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂DY，且該VLCDR1、VLCDR2及VLCDR3序列分別包含X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀WX₃₁A、X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈及QX₃₉X₄₀X₄₁SFPYX₄₂（關於「X」殘基之定義，參見表E11）。 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 1-3，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 4-6；或 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 265-267，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 268-270。In some embodiments: the VHCDR1, VHCDR2 and VHCDR3 sequences comprise_TFX1 X₂ X₃ X₄ X₅ H, IX₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ AQKFQG and X₁₆ X 17 X₁₈ X₁₉ X₂₀ X_{21X 22DY} , respectively, and the VLCDR1, VLCDR2 and VLCDR3 sequences comprise X₂₃ X₂₄ X₂₅ X₂₆ X₂₇ X₂₈ X₂₉ X₃₀ WX₃₁ A, X₃₂ X₃₃ X₃₄ X₃₅ X₃₆ X₃₇ X₃₈ and QX₃₉ X₄₀ X₄₁ SFPYX₄₂ (For the definition of "X" residue,seeTable E11 ). The_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 1-3, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 4-6, respectively; or the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 265-267, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 268-270, respectively.

在特定實施例中： 該V_H包含與SEQ ID NO: 277至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 278至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 279至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 280至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列；或 該V_H包含與SEQ ID NO: 367至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 368至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列。In certain embodiments: the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 277, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 278; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 279, and the VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 280; or the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 367, and_theVL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 368 sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical.

本發明之態樣亦包括一種經分離之抗體或其抗原結合片段，其中該抗體與介白素-18結合蛋白（IL-18BP）結合且其中該抗體與IL-18競爭結合IL-18BP。在另一態樣中，本發明提供一種抗體，其與IL-18BP結合且干擾IL-18與IL-18BP之結合。在另一態樣中，本發明提供一種抗體，其與IL-18BP結合且為IL-18BP拮抗劑，其拮抗IL-18BP與IL-18之間的結合活性。在一些實施例中，本發明提供一種與預形成之IL-18-IL-18BP複合物結合的抗體。在一些實施例中，本發明提供一種與游離IL-18BP結合之抗體。在一些實施例中，該抗體或其抗原結合片段與IL-18BP之構形抗原決定基結合。在一些實施例中，其構形抗原決定基包含選自由以下組成之群的兩個或更多個胺基酸殘基：SEQ ID NO: 372之K32、T40、S60、R61、S66、Y69、R91、R93、T96、K102、R131及H132。在一些實施例中，其構形抗原決定基包含SEQ ID NO: 372之K32、T40、S60、R61、S66、Y69、R91、R93、T96、K102、R131及H132之該等胺基酸殘基。在一些實施例中，該抗體或其抗原結合片段與IL-18BP之線性抗原決定基結合。在一些實施例中，該經分離之抗體或其抗原結合片段與IL-18與成熟形式之IL-18BP之間的結合界面結合。在一些實施例中，該經分離之抗體或其抗原結合片段結合SEQ ID NO: 372之該等胺基酸殘基R61、Y69及R131。在一些實施例中，該經分離之抗體或其抗原結合片段包含VH，其包含與SEQ ID NO: 333至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，及VL，其包含與SEQ ID NO: 334至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列。The present invention also includes an isolated antibody or antigen-binding fragment thereof, wherein the antibody binds to interleukin-18 binding protein (IL-18BP) and wherein the antibody competes with IL-18 for binding to IL-18BP. In another aspect, the present invention provides an antibody that binds to IL-18BP and interferes with the binding of IL-18 to IL-18BP. In another aspect, the present invention provides an antibody that binds to IL-18BP and is an IL-18BP antagonist that antagonizes the binding activity between IL-18BP and IL-18. In some embodiments, the present invention provides an antibody that binds to a preformed IL-18-IL-18BP complex. In some embodiments, the present invention provides an antibody that binds to free IL-18BP. In some embodiments, the antibody or antigen-binding fragment thereof binds to a conformational epitope of IL-18BP. In some embodiments, the conformational epitope comprises two or more amino acid residues selected from the group consisting of K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131, and H132 of SEQ ID NO: 372. In some embodiments, the conformational epitope comprises the amino acid residues K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131, and H132 of SEQ ID NO: 372. In some embodiments, the antibody or antigen-binding fragment thereof binds to a linear epitope of IL-18BP. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to the binding interface between IL-18 and the mature form of IL-18BP. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to the amino acid residues R61, Y69 and R131 of SEQ ID NO: 372. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a VH comprising a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 333, and a VL comprising a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 334.

在一些實施例中，該經分離之抗體或其抗原結合片段與人類IL-18BP及食蟹獼猴IL-18BP結合，但不與小鼠IL-18BP（特異性或實質上）結合。在一些實施例中，該經分離之抗體或其抗原結合片段與人類IL-18BP、食蟹獼猴IL-18BP及小鼠IL-18BP結合。在一些實施例中，該經分離之抗體或其抗原結合片段與IL-18與成熟形式之IL-18BP之間的結合界面結合。在具體實施例中，該經分離之抗體或其抗原結合片段以比IL-18與IL-18BP之間的結合親和力（K_D為約650 pM）更強的結合親和力，視情況以約1 pm至約650 pm，或約或小於約1、5、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200或300、400、500、600或650 pM之結合親和力與IL-18BP結合。在一些實施例中，該經分離之抗體或其抗原結合片段為IL-18BP拮抗劑，其拮抗IL-18BP與IL-18之間的結合活性。在一些實施例中，該經分離之抗體或其抗原結合片段阻斷IL-18BP對IL-18之抑制活性，且藉此增加IL-18介導之信號傳導，包括IFN-γ、CXCL10及/或TNFα之誘導。In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to human IL-18BP and cynomolgus macaque IL-18BP, but does not bind to mouse IL-18BP (specifically or substantially). In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to human IL-18BP, cynomolgus macaque IL-18BP, and mouse IL-18BP. In some embodiments, the isolated antibody or antigen-binding fragment thereof binds to the binding interface between IL-18 and the mature form of IL-18BP. In specific embodiments, the isolated antibody or antigen-binding fragment thereof binds to IL-18BP with a stronger binding affinity than the binding affinity between IL-18 and IL-18BP (_KD is about 650 pM), optionally with a binding affinity of about 1 pm to about 650 pm, or about or less than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 300, 400, 500, 600 or 650 pM. In some embodiments, the isolated antibody or antigen-binding fragment thereof is an IL-18BP antagonist that antagonizes the binding activity between IL-18BP and IL-18. In some embodiments, the isolated antibody or antigen-binding fragment thereof blocks the inhibitory activity of IL-18BP on IL-18, thereby increasing IL-18-mediated signaling, including the induction of IFN-γ, CXCL10 and/or TNFα.

在一些實施例中，該經分離之抗體或其抗原結合片段包含IgA（包括子類IgA1及IgA2）、IgD、IgE、IgG（包括子類IgG1、IgG2、IgG3及IgG4）或IgM Fc域，視情況人類Fc域或其雜合體及/或變異體。在一些實施例中，該經分離之抗體或其抗原結合片段包含在人類中具有高效應功能之IgG Fc域，視情況IgG1或IgG3 Fc域。在一些實施例中，該經分離之抗體或其抗原結合片段包含在人類中具有低效應功能之IgG Fc域，視情況IgG2或IgG4 Fc域。在一些實施例中，該經分離之抗體或其抗原結合片段為單株抗體。在一些實施例中，該經分離之抗體或其抗原結合片段為人源化抗體。在一些實施例中，該經分離之抗體或其抗原結合片段係選自Fv片段、單鏈Fv（scFv）多肽、纖連蛋白（adnectin）、抗運載蛋白（anticalin）、適體、高親和性多聚體（avimer）、駱駝科抗體、經設計之錨蛋白重複蛋白（DARPin）、微型抗體、奈米抗體及單抗體（unibody）。In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3 and IgG4) or IgM Fc domain, optionally a human Fc domain or a hybrid and/or variant thereof. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an IgG Fc domain with high efficacy in humans, optionally an IgG1 or IgG3 Fc domain. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an IgG Fc domain with low efficacy in humans, optionally an IgG2 or IgG4 Fc domain. In some embodiments, the isolated antibody or antigen-binding fragment thereof is a monoclonal antibody. In some embodiments, the isolated antibody or antigen-binding fragment thereof is a humanized antibody. In some embodiments, the isolated antibody or antigen-binding fragment thereof is selected from Fv fragments, single-chain Fv (scFv) polypeptides, adnectins, anticalins, aptamers, avimers, camelid antibodies, designed anchor protein repeat proteins (DARPins), minibodies, nanobodies, and unibodies.

亦包括經分離之聚核苷酸，其編碼本文所述之經分離之抗IL-18BP抗體或其抗原結合片段；包含該經分離之聚核苷酸的表現載體；及包含該載體之經分離之宿主細胞。亦提供一或多種經分離之聚核苷酸，其編碼本文所述之抗IL-18BP抗體。舉例而言，本文提供第一聚核苷酸，其編碼本文所揭示之抗體之V_H區；及第二聚核苷酸，其編碼本文所揭示之抗體之V_L區。Also included are isolated polynucleotides encoding an isolated anti-IL-18BP antibody or antigen-binding fragment thereof described herein; expression vectors comprising the isolated polynucleotides; and isolated host cells comprising the vectors. Also provided are one or more isolated polynucleotides encoding an anti-IL-18BP antibody described herein. For example, provided herein is a first polynucleotide encoding a_VH region of an antibody disclosed herein; and a second polynucleotide encoding a_VL region of an antibody disclosed herein.

某些實施例包括一種醫藥組合物，其包含本文所述之經分離之抗IL-18BP抗體或其抗原結合片段及醫藥學上可接受之載劑。在一些實施例中，該組合物具有就該至少一種抗體或抗原結合片段而言以蛋白質計至少約80%、85%、90%、95%、98%或99%之純度且實質上不含聚集體及內毒素。在一些實施例中，該組合物為視情況適合於靜脈內、肌肉內、皮下或腹膜內投與之無菌可注射溶液。Certain embodiments include a pharmaceutical composition comprising an isolated anti-IL-18BP antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier. In some embodiments, the composition has a purity of at least about 80%, 85%, 90%, 95%, 98% or 99% on a protein basis for the at least one antibody or antigen-binding fragment and is substantially free of aggregates and endotoxins. In some embodiments, the composition is a sterile injectable solution suitable for intravenous, intramuscular, subcutaneous or intraperitoneal administration, as appropriate.

亦包括治療有需要之個體的疾病或病狀的方法，其包含向該個體投與本文所述之醫藥組合物。在一些實施例中，該疾病或病狀為癌症或腫瘤或增生性疾病或病症，視情況選自以下之增生性疾病或病症：淋巴增生性病症、骨髓增生性病症、增生性腸炎、增生性糖尿病視網膜病變及增生性腎病。在一些實施例中，該癌症或腫瘤表現或過度表現IL-18BP及/或IL-18，或該增生性疾病或病症與IL-18BP及/或IL-18之表現增加相關。在一些實施例中，該癌症係選自以下中之一或多者：骨癌、前列腺癌、黑色素瘤（例如，轉移性黑色素瘤）、胰臟癌、小細胞肺癌、非小細胞肺癌（NSCLC）、間皮瘤、白血病（例如，淋巴球性白血病、慢性骨髓性白血病、急性骨髓性白血病、復發性急性骨髓性白血病、毛細胞白血病、急性淋巴母細胞性白血病）、淋巴瘤（例如，非何傑金氏淋巴瘤（non-Hodgkin's lymphoma）、何傑金氏淋巴瘤）、肝癌（肝細胞癌）、肉瘤、B細胞惡性病、乳癌、卵巢癌、結腸直腸癌、神經膠質瘤、多形性神經膠質母細胞瘤、腦膜瘤、垂體腺瘤、前庭神經鞘瘤、原發性CNS淋巴瘤、原始神經外胚層腫瘤（神經管胚細胞瘤）、腎癌（例如，腎細胞癌）、膀胱癌、子宮癌、尿道上皮癌、食道癌、腦癌、頭頸癌、子宮頸癌、睪丸癌、甲狀腺癌及胃癌。Also included are methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject a pharmaceutical composition described herein. In some embodiments, the disease or condition is cancer or a tumor or a proliferative disease or disorder, optionally selected from the following proliferative diseases or disorders: lymphoproliferative disorders, myeloproliferative disorders, proliferative enteritis, proliferative diabetic retinopathy, and proliferative nephropathy. In some embodiments, the cancer or tumor expresses or overexpresses IL-18BP and/or IL-18, or the proliferative disease or disorder is associated with increased expression of IL-18BP and/or IL-18. In some embodiments, the cancer is selected from one or more of the following: bone cancer, prostate cancer, melanoma (e.g., metastatic melanoma), pancreatic cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, hairy cell leukemia, acute lymphoblastic leukemia), lymphoma (e.g., non-Hodgkin's lymphoma lymphoma), Hodgkin's lymphoma), liver cancer (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, neuroglioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (neurotubular cell tumor), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, urothelial carcinoma, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.

某些實施例包含投與該醫藥組合物（具有抗IL18BP抗體或其抗原結合片段）與IL-18之組合。一些實施例包含投與該醫藥組合物（具有抗IL18BP抗體或其抗原結合片段）與免疫檢查點調節劑之組合，該免疫檢查點調節劑選自抑制性免疫檢查點分子之拮抗劑及刺激性免疫檢查點分子之促效劑。在一些實施例中，該免疫檢查點調節劑為多肽，視情況抗體或其抗原結合片段，或配位體或小分子。在一些實施例中，該抑制性免疫檢查點分子係選自以下中之一或多者：程式化死亡-配位體1（PD-L1）、程式化死亡1（PD-1）、程式化死亡-配位體2（PD-L2）、細胞毒性T淋巴球相關蛋白4（CTLA-4）、吲哚胺2,3-雙加氧酶（IDO）、色胺酸2,3-雙加氧酶（TDO）、T細胞免疫球蛋白域及黏蛋白域3（TIM-3）、淋巴球活化基因-3（LAG-3）、T細胞活化之V域Ig抑制因子（VISTA）、B及T淋巴球衰減因子（BTLA）、CD160、疱疹病毒侵入介體（HVEM）以及具有Ig及ITIM域之T細胞免疫受體（TIGIT）。Certain embodiments comprise administering the pharmaceutical composition (having an anti-IL18BP antibody or antigen-binding fragment thereof) in combination with IL-18. Some embodiments comprise administering the pharmaceutical composition (having an anti-IL18BP antibody or antigen-binding fragment thereof) in combination with an immune checkpoint modulator selected from an antagonist of an inhibitory immune checkpoint molecule and an agonist of a stimulatory immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is a polypeptide, optionally an antibody or antigen-binding fragment thereof, or a ligand or a small molecule. In some embodiments, the inhibitory immune checkpoint molecule is selected from one or more of the following: programmed death-ligand 1 (PD-L1), programmed death 1 (PD-1), programmed death-ligand 2 (PD-L2), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene-3 (LAG-3), V-domain Ig inhibitory factor of T cell activation (VISTA), B and T lymphocyte depletion factor (BTLA), CD160, herpes virus entry mediator (HVEM) and T cell immune receptor with Ig and ITIM domains (TIGIT).

在一些實施例中： 該拮抗劑為視情況選自以下中之一或多者的PD-L1及/或PD-L2拮抗劑：與PD-L1及/或PD-L2特異性結合之抗體或抗原結合片段或小分子、阿替利珠單抗（atezolizumab）（MPDL3280A）、阿維魯單抗（avelumab）（MSB0010718C）及度伐利尤單抗（durvalumab）（MEDI4736），視情況其中該癌症係選自以下中之一或多者：結腸直腸癌、黑色素瘤、乳癌、非小細胞肺癌、膀胱癌及腎細胞癌； 該拮抗劑為視情況選自以下中之一或多者的PD-1拮抗劑：與PD-1特異性結合之抗體或抗原結合片段或小分子、納武單抗（nivolumab）、帕博利珠單抗（pembrolizumab）、MK-3475、AMP-224、AMP-514PDR001及皮立珠單抗（pidilizumab），視情況其中該PD-1拮抗劑為納武單抗，且該癌症視情況選自以下中之一或多者：何傑金氏淋巴瘤、黑色素瘤、非小細胞肺癌、肝細胞癌、腎細胞癌及卵巢癌； 該PD-1拮抗劑為帕博利珠單抗，且該癌症視情況選自以下中之一或多者：黑色素瘤、非小細胞肺癌、小細胞肺癌、頭頸癌及尿道上皮癌； 該拮抗劑為視情況選自以下中之一或多者的CTLA-4拮抗劑：與CTLA-4特異性結合之抗體或抗原結合片段或小分子、伊匹單抗（ipilimumab）、曲美木單抗（tremelimumab），視情況其中該癌症係選自以下中之一或多者：黑色素瘤、前列腺癌、肺癌及膀胱癌； 該拮抗劑為視情況選自以下中之一或多者的IDO拮抗劑：與IDO特異性結合之抗體或抗原結合片段或小分子、因多莫得（indoximod）（NLG-8189）、1-甲基-色胺酸（1MT）、β-咔啉（去甲哈爾滿（norharmane）；9H-吡啶并[3,4-b]吲哚）、迷迭香酸及艾帕斯塔（epacadostat），且其中該癌症視情況選自以下中之一或多者：轉移性乳癌及腦癌、視情況多形性神經膠質母細胞瘤、神經膠質瘤、神經膠質肉瘤或惡性腦瘤； 該拮抗劑為視情況選自以下中之一或多者的TDO拮抗劑：與TDO特異性結合之抗體或抗原結合片段或小分子、680C91及LM10； 該拮抗劑為視情況選自與TIM-3特異性結合之抗體或抗原結合片段或小分子中之一或多者的TIM-3拮抗劑； 該拮抗劑為視情況選自以下中之一或多者的LAG-3拮抗劑：與LAG-3特異性結合之抗體或抗原結合片段或小分子,及BMS-986016； 該拮抗劑為視情況選自與VISTA特異性結合之抗體或抗原結合片段或小分子中之一或多者的VISTA拮抗劑； 該拮抗劑為視情況選自與BTLA、CD160及/或HVEM特異性結合之抗體或抗原結合片段或小分子中之一或多者的BTLA、CD160及/或HVEM拮抗劑； 該拮抗劑為視情況選自與TIGIT特異性結合之抗體或抗原結合片段或小分子中之一或多者的TIGIT拮抗劑。In some embodiments: The antagonist is a PD-L1 and/or PD-L2 antagonist selected from one or more of the following: antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L1 and/or PD-L2, atezolizumab (MPDL3280A), avelumab (MSB0010718C) and durvalumab (MEDI4736), wherein the cancer is selected from one or more of the following: colorectal cancer, melanoma, breast cancer, non-small cell lung cancer, bladder cancer and renal cell carcinoma; The antagonist is a PD-1 antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to PD-1, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514PDR001 and pidilizumab, wherein the PD-1 antagonist is nivolumab, and the cancer is selected from one or more of the following: Hodgkin's lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma and ovarian cancer;The PD-1 antagonist is pembrolizumab, and the cancer is selected from one or more of the following: melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial carcinoma;The antagonist is a CTLA-4 antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to CTLA-4, ipilimumab, tremelimumab, and the cancer is selected from one or more of the following: melanoma, prostate cancer, lung cancer, and bladder cancer;The antagonist is an IDO antagonist selected from one or more of the following: antibodies or antigen-binding fragments or small molecules that specifically bind to IDO, indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carbolines (norharmane; 9H-pyrido[3,4-b]indole), rosmarinic acid and epacadostat, and the cancer is selected from one or more of the following: metastatic breast cancer and brain cancer, glioblastoma multiforme, neuroglioma, neurosarcoma or malignant brain tumor;The antagonist is a TDO antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to TDO, 680C91 and LM10;The antagonist is a TIM-3 antagonist selected from one or more of the antibodies or antigen-binding fragments or small molecules that specifically bind to TIM-3;The antagonist is a LAG-3 antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to LAG-3, and BMS-986016;The antagonist is a VISTA antagonist selected from one or more of the antibodies or antigen-binding fragments or small molecules that specifically bind to VISTA;The antagonist is a BTLA, CD160 and/or HVEM antagonist selected from one or more of antibodies, antigen-binding fragments or small molecules that specifically bind to BTLA, CD160 and/or HVEM as appropriate;The antagonist is a TIGIT antagonist selected from one or more of antibodies, antigen-binding fragments or small molecules that specifically bind to TIGIT as appropriate.

在一些實施例中，該刺激性免疫檢查點分子係選自以下中之一或多者：OX40、CD40、糖皮質激素誘導之TNFR家族相關基因（GITR）、CD137（4-1BB）、CD27、CD28、CD226及疱疹病毒侵入介體（HVEM）。In some embodiments, the stimulatory immune checkpoint molecule is selected from one or more of the following: OX40, CD40, glucocorticoid-induced TNFR family-related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226 and herpes virus entry mediator (HVEM).

在某些實施例中： 該促效劑為視情況選自以下中之一或多者的OX40促效劑：與OX40特異性結合之抗體或抗原結合片段或小分子或配位體、OX86、Fc-OX40L及GSK3174998； 該促效劑為視情況選自以下中之一或多者的CD40促效劑：與CD40特異性結合之抗體或抗原結合片段或小分子或配位體、CP-870,893、達西組單抗（dacetuzumab）、Chi Lob 7/4、ADC-1013及rhCD40L，且其中該癌症視情況選自以下中之一或多者：黑色素瘤、胰臟癌、間皮瘤及血液癌，視情況淋巴瘤，諸如非何傑金氏淋巴瘤； 該促效劑為視情況選自以下中之一或多者的GITR促效劑：與GITR特異性結合之抗體或抗原結合片段或小分子或配位體、INCAGN01876、DTA-1及MEDI1873； 該促效劑為視情況選自以下中之一或多者的CD137促效劑：與CD137特異性結合之抗體或抗原結合片段或小分子或配位體、烏托米單抗（utomilumab）及4-1BB配位體； 該促效劑為視情況選自以下中之一或多者的CD27促效劑：與CD27特異性結合之抗體或抗原結合片段或小分子或配位體、瓦利魯單抗（varlilumab）及CDX-1127 (1F5)； 該促效劑為視情況選自以下中之一或多者的CD28促效劑：與CD28特異性結合之抗體或抗原結合片段或小分子或配位體，及TAB08；及/或 該促效劑為視情況選自與HVEM特異性結合之抗體或抗原結合片段或小分子或配位體中之一或多者的HVEM促效劑。In certain embodiments: The agonist is an OX40 agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to OX40, OX86, Fc-OX40L, and GSK3174998; The agonist is a CD40 agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD40, CP-870,893, dacetuzumab, Chi Lob 7/4, ADC-1013 and rhCD40L, and wherein the cancer is selected from one or more of the following: melanoma, pancreatic cancer, mesothelioma and blood cancer, and lymphoma, such as non-Hodgkin's lymphoma; The agonist is a GITR agonist selected from one or more of the following: an antibody or antigen binding fragment or small molecule or ligand that specifically binds to GITR, INCAGN01876, DTA-1 and MEDI1873; The agonist is a CD137 agonist selected from one or more of the following: an antibody or antigen binding fragment or small molecule or ligand that specifically binds to CD137, utomilumab and 4-1BB ligand; The agonist is a CD27 agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD27, varlilumab, and CDX-1127 (1F5);The agonist is a CD28 agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD28, and TAB08; and/orThe agonist is a HVEM agonist selected from one or more of the antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to HVEM.

特定實施例包含投與該醫藥組合物（具有抗IL18BP抗體或其抗原結合片段）與至少一種化學治療劑之組合。在一些實施例中，該至少一種化學治療劑係選自以下中之一或多者：烷基化劑、抗代謝物、細胞毒性抗生素、拓樸異構酶抑制劑（1型或II型）及抗微管劑。Particular embodiments comprise administering the pharmaceutical composition (having an anti-IL18BP antibody or antigen-binding fragment thereof) in combination with at least one chemotherapeutic agent. In some embodiments, the at least one chemotherapeutic agent is selected from one or more of the following: an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an anti-microtubule agent.

在一些實施例中： 該烷基化劑係選自以下中之一或多者：氮芥（nitrogen mustard）（視情況甲氮芥（mechlorethamine）、環磷醯胺、氮芥（mustine）、美法侖（melphalan）、苯丁酸氮芥（chlorambucil）、異環磷醯胺（ifosfamide）及硫酸布他卡因（busulfan））、亞硝基脲（視情況N-亞硝基-N-甲基脲（MNU）、卡莫司汀（carmustine）（BCNU）、洛莫司汀（lomustine）（CCNU）、司莫司汀（semustine）（MeCCNU）、福莫司汀（fotemustine）及鏈佐黴素（streptozotocin））、四（視情況達卡巴嗪（dacarbazine）、米托唑胺（mitozolomide）及替莫唑胺（temozolomide））、氮丙啶（視情況噻替派（thiotepa）、絲裂黴素（mytomycin）及地吖醌（diaziquone）（AZQ））、順鉑及其衍生物（視情況卡鉑（carboplatin）及奧沙利鉑（oxaliplatin））及非典型烷基化劑（視情況丙卡巴肼（procarbazine）及六甲三聚氰胺）； 該抗代謝物係選自以下中之一或多者：抗葉酸劑（視情況甲胺喋呤（methotrexate）及培美曲塞（pemetrexed））、氟嘧啶（視情況5-氟尿嘧啶及卡培他濱（capecitabine））、去氧核苷類似物（視情況安西他濱（ancitabine）、依諾他濱（enocitabine）、阿糖胞苷（cytarabine）、吉西他濱（gemcitabine）、地西他濱（decitabine）、阿紮胞苷（azacitidine）、氟達拉濱（fludarabine）、奈拉濱（nelarabine）、克拉屈濱（cladribine）、氯法拉濱（clofarabine）、氟達拉濱（fludarabine）及噴司他汀（pentostatin））及硫代嘌呤（視情況硫鳥嘌呤及巰基嘌呤）； 該細胞毒性抗生素係選自以下中之一或多者：蒽環黴素（視情況小紅莓（doxorubicin）、道諾黴素（daunorubicin）、表柔比星（epirubicin）、艾達黴素（idarubicin）、吡柔比星（pirarubicin）、阿克拉黴素（aclarubicin）及米托蒽醌（mitoxantrone））、博來黴素（bleomycin）、絲裂黴素（mitomycin）C、米托蒽醌及放線菌素（actinomycin）； 該拓樸異構酶抑制劑係選自以下中之一或多者：喜樹鹼（camptothecin）、伊立替康（irinotecan）、拓樸替康（topotecan）、依託泊苷（etoposide）、小紅莓、米托蒽醌、替尼泊苷（teniposide）、新生黴素（novobiocin）、麥爾巴隆（merbarone）及阿克拉黴素；及/或 該抗微管劑係選自以下中之一或多者：紫杉烷（視情況太平洋紫杉醇（paclitaxel）及多西他賽（docetaxel））及長春花生物鹼（視情況長春鹼（vinblastine）、長春新鹼（vincristine）、長春地辛（vindesine）、長春瑞濱（vinorelbine））。In some embodiments: the alkylating agent is selected from one or more of the following: nitrogen mustard (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosourea (optionally N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetracycline (tetrazolium chloride ... (dacarbazine, mitozolomide and temozolomide, as appropriate), aziridines (thiotepa, mytomycin and diaziquone (AZQ), as appropriate), cis-platinum and its derivatives (carboplatin and oxaliplatin, as appropriate) and atypical alkylating agents (procarbazine and hexamethonium, as appropriate); The anti-metabolite is selected from one or more of the following: antifolates (methotrexate and pemetrexed as appropriate), fluoropyrimidines (5-fluorouracil and capecitabine as appropriate), deoxynucleoside analogs (ancitabine, enocitabine, cytarabine, gemcitabine as appropriate), itabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine and pentostatin) and thiopurines (thioguanine and thioguanine as appropriate); The cytotoxic antibiotic is one or more selected from the group consisting of anthracyclines (depending on the situation, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone), bleomycin, mitomycin C, mitoxantrone and actinomycin; The topoisomerase inhibitor is selected from one or more of the following: camptothecin, irinotecan, topotecan, etoposide, cranberry, mitoxantrone, teniposide, novobiocin, merbarone and aclatomycin; and/or the anti-microtubule agent is selected from one or more of the following: taxanes (paclitaxel and docetaxel as appropriate) and vinca alkaloids (vinblastine, vincristine, vindesine, vinorelbine as appropriate).

在一些實施例中，該疾病或病狀為骨髓發育不良症候群（MDS）。在一些實施例中，該疾病或病狀為感染性疾病。在具體實施例中，該感染性疾病係選自病毒、細菌、真菌（視情況酵母）及原蟲感染。某些實施例包含投與該醫藥組合物（具有抗IL18BP抗體或其抗原結合片段）與IL-18之組合。In some embodiments, the disease or condition is myelodysplastic syndrome (MDS). In some embodiments, the disease or condition is an infectious disease. In specific embodiments, the infectious disease is selected from viral, bacterial, fungal (optionally yeast) and protozoan infections. Certain embodiments comprise administering a combination of the pharmaceutical composition (having an anti-IL18BP antibody or antigen-binding fragment thereof) and IL-18.

亦包括針對阻斷或抑制IL-18與IL-18BP之間的結合之能力篩選抗IL-18BP抗體或其抗原結合片段之方法，其包含 （a）     測定該抗體或其抗原結合片段對（i）單獨的IL-18BP及（ii）低IL-18融合蛋白之結合親和力，其中該低IL-18融合蛋白包含經由可撓性連接子（及其間的視情況存在之蛋白酶裂解位點）與IL-18BP融合的IL-18，其中該融合蛋白之IL-18部分結合至該融合蛋白之IL-18BP部分且空間阻斷該融合蛋白之該IL-18BP部分之IL-18結合位點； （b）     比較（i）之該結合親和力與（ii）之該結合親和力；及 （c）     若（i）之該結合親和力顯著強於（ii）之該結合親和力，則將該抗體或其抗原結合片段鑑別或選擇為能夠阻斷或抑制IL-18與IL-18BP之間的結合。Also included are methods for screening anti-IL-18BP antibodies or antigen-binding fragments thereof for their ability to block or inhibit binding between IL-18 and IL-18BP, comprising:(a)     determining the binding affinity of the antibody or antigen-binding fragment thereof to (i) IL-18BP alone and (ii) a low IL-18 fusion protein, wherein the low IL-18 fusion protein comprises IL-18 fused to IL-18BP via a flexible linker (and a protease cleavage site therebetween, if appropriate), wherein the IL-18 portion of the fusion protein binds to the IL-18BP portion of the fusion protein and sterically blocks the IL-18 binding site of the IL-18BP portion of the fusion protein;(b)     comparing the binding affinity of (i) with the binding affinity of (ii); and(c)    If the binding affinity of (i) is significantly stronger than the binding affinity of (ii), the antibody or antigen-binding fragment thereof is identified or selected as being capable of blocking or inhibiting the binding between IL-18 and IL-18BP.

在一些實施例中，該IL-18及該IL-18BP為小鼠IL-18及小鼠IL-18BP。在一些實施例中，該IL-18及該IL-18BP為人類IL-18及人類IL-18BP。在一些實施例中，該低IL-18融合蛋白在N端至C端定向上包含信號肽、IL-18、第一可撓性連接子、蛋白酶裂解位點（視情況TEV蛋白酶裂解位點）、可撓性連接子及IL-18BP。在一些實施例中，該低IL-18融合蛋白包含與來自表S1之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致之胺基酸序列。In some embodiments, the IL-18 and the IL-18BP are mouse IL-18 and mouse IL-18BP. In some embodiments, the IL-18 and the IL-18BP are human IL-18 and human IL-18BP. In some embodiments, the low IL-18 fusion protein comprises a signal peptide, IL-18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker, and IL-18BP in an N-terminal to C-terminal orientation. In some embodiments, the low IL-18 fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence fromTableS1 .

具體實施例包括一種經分離之低IL-18融合蛋白，其在N端至C端定向上包含信號肽、IL-18、第一可撓性連接子、蛋白酶裂解位點（視情況TEV蛋白酶裂解位點）、可撓性連接子及IL-18BP。在一些實施例中，該低IL-18融合蛋白包含與來自表S1之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的胺基酸序列，由其組成或基本上由其組成。Specific embodiments include an isolated low IL-18 fusion protein comprising a signal peptide, IL-18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker, and IL-18BP in an N-terminal to C-terminal orientation. In some embodiments, the low IL-18 fusion protein comprises, consists of, or consists essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a sequence fromTableS1 .

亦包括刺激有需要之個體之免疫反應的方法，其包含向該個體投與本文所述之醫藥組合物。在一些實施例中，該免疫反應為IL-18介導之免疫反應。在特定實施例中，該IL-18介導之免疫反應包含誘導有需要之個體中的IFN-γ、CXCL10及/或TNFα。Also included are methods of stimulating an immune response in an individual in need thereof, comprising administering to the individual a pharmaceutical composition described herein. In some embodiments, the immune response is an IL-18 mediated immune response. In particular embodiments, the IL-18 mediated immune response comprises inducing IFN-γ, CXCL10 and/or TNFα in an individual in need thereof.

相關申請案之交叉引用Cross-references to related applications

本申請案主張2023年1月6日申請之美國臨時專利申請案第63/437,526號及2023年10月13日申請之美國臨時專利申請案第63/590,348號之優先權，該等申請案之內容以全文引用之方式併入本文中。This application claims priority to U.S. Provisional Patent Application No. 63/437,526 filed on January 6, 2023 and U.S. Provisional Patent Application No. 63/590,348 filed on October 13, 2023, the contents of which are incorporated herein by reference in their entirety.

本發明係關於抗體及其抗原結合片段，其與介白素-18結合蛋白（IL-18BP），例如人類IL-18BP結合，尤其具有抗原決定基特異性及經改善之特徵的抗體。一些實施例包括特異性人源化抗體及其片段，其能夠與IL-18BP結合，阻斷或減少IL-18BP與其配位體IL-18之抑制性結合，且藉此增加IL-18介導之下游信號傳導。因此，在某些實施例中，抗IL-18BP抗體或其抗原結合片段為IL-18BP拮抗劑或抑制劑。The present invention relates to antibodies and antigen-binding fragments thereof, which bind to interleukin-18 binding protein (IL-18BP), such as human IL-18BP, and particularly antibodies with antigenic determinant specificity and improved characteristics. Some embodiments include specific humanized antibodies and fragments thereof, which are capable of binding to IL-18BP, blocking or reducing the inhibitory binding of IL-18BP to its ligand IL-18, and thereby increasing IL-18-mediated downstream signaling. Therefore, in certain embodiments, the anti-IL-18BP antibody or antigen-binding fragment thereof is an IL-18BP antagonist or inhibitor.

本文所述之IL-18BP拮抗劑抗體適用於治療及預防各種疾病及病狀，諸如癌症及其他疾病及病狀。一些實施例因此係關於抗IL-18BP抗體或其抗原結合片段之用途，其係用於診斷、評估及治療疾病及病狀，包括與IL-18及/或IL-18BP活性或其異常表現相關之疾病及病狀。The IL-18BP antagonist antibodies described herein are useful for treating and preventing a variety of diseases and conditions, such as cancer and other diseases and conditions. Some embodiments therefore relate to the use of anti-IL-18BP antibodies or antigen-binding fragments thereof for the diagnosis, assessment and treatment of diseases and conditions, including diseases and conditions associated with IL-18 and/or IL-18BP activity or abnormal expression thereof.

除非特定指示相反，否則本發明之實踐將採用此項技術之技能內的病毒學、免疫學、微生物學、分子生物學及重組DNA技術之習知方法，其中許多出於說明之目的而描述於下文中。此類技術於文獻中予以充分解釋。參見例如，Current Protocols in Molecular Biology或Current Protocols in Immunology, John Wiley & Sons, New York, N.Y.(2009)；Ausubel等人,Short Protocols in Molecular Biology, 第3版, Wiley & Sons, 1995；Sambrook及Russell,Molecular Cloning: A Laboratory Manual(第3版, 2001)；Maniatis等人Molecular Cloning: A Laboratory Manual(1982)；DNA Cloning: A Practical Approach, 第 I卷及第II卷(D. Glover編)；Oligonucleotide Synthesis(N. Gait編, 1984)；Nucleic Acid Hybridization(B. Hames & S. Higgins編, 1985)；Transcription and Translation(B. Hames & S. Higgins編, 1984)；Animal Cell Culture(R. Freshney編, 1986)；Perbal,A Practical Guide to Molecular Cloning(1984)及其他類似參考文獻。定義Unless specifically indicated to the contrary, the practice of the present invention will employ methods of virology, immunology, microbiology, molecular biology and recombinant DNA technology, which are within the skill of the art, many of which are described below for purposes of illustration. Such techniques are fully explained in the literature. See, e.g. ,Current Protocols in Molecular Biology orCurrent Protocols in Immunology , John Wiley & Sons, New York, NY (2009); Ausubelet al. ,Short Protocols in Molecular Biology , 3rd ed., Wiley & Sons, 1995; Sambrook and Russell,Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Maniatis etal., Molecular Cloning: A Laboratory Manual (1982);DNA Cloning: A Practical Approach , Vol. I and Vol. II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984);Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal,A Practical Guide to Molecular Cloning (1984) and other similar references.Definition

依本說明書及隨附申請專利範圍中所用，除非內容明確另外指示，否則單數形式「一（a）」、「一（an）」及「該（the）」包括複數個提及物。As used in this specification and the accompanying claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

「約」意謂相對於參考數量、水平、值、數目、頻率、百分比、尺寸、大小、量、重量或長度變化多至20%、15%、10%、9%、8%、7%、6%、5%、4%、3%、2%或1%之數量、水平、值、數目、頻率、百分比、尺寸、大小、量、重量或長度。“About” means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by up to 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% from a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

術語「抗原」係指能夠由選擇性結合劑（諸如抗體）結合，且另外能夠用於動物中以產生能夠結合於彼抗原之抗原決定基之抗體的分子或分子之一部分。抗原可具有一或多個抗原決定基。如本文所使用，術語「抗原」包括如下物質，其能夠在適當條件下誘發針對該物質之免疫反應且與免疫反應之產物反應。舉例而言，抗原可藉由抗體（體液免疫反應）或致敏T淋巴球（T輔助或細胞介導之免疫反應）或兩者來識別。抗原可為可溶性物質，諸如毒素及異體蛋白，或微粒，諸如細菌及組織細胞；然而，僅僅蛋白質或多醣分子之稱為抗原決定子（抗原決定基）之部分與抗體或淋巴球上之特定受體組合。更廣泛地，術語「抗原」包括抗體結合或抗體期望之任何物質，無論該物質是否具有免疫原性。對於此類抗原，抗體可藉由重組方法，不依賴於任何免疫反應來鑑別。The term "antigen" refers to a molecule or a portion of a molecule that can be bound by a selective binding agent (such as an antibody) and can additionally be used in an animal to produce an antibody that can bind to an antigenic determinant of that antigen. An antigen may have one or more antigenic determinants. As used herein, the term "antigen" includes a substance that is capable of inducing an immune response against the substance under appropriate conditions and reacting with the product of the immune response. For example, an antigen can be recognized by antibodies (humoral immune response) or sensitized T lymphocytes (T helper or cell-mediated immune response) or both. Antigens can be soluble substances, such as toxins and foreign proteins, or particles, such as bacteria and tissue cells; however, only the part of the protein or polysaccharide molecule called the antigenic determinant (antigenic determinant) binds to a specific receptor on an antibody or lymphocyte. More broadly, the term "antigen" includes any substance that an antibody binds or is expected to bind to, whether or not it is immunogenic. For such antigens, antibodies can be identified by recombinant methods, independent of any immune response.

「拮抗劑」係指干擾或以其他方式減少另一藥劑或分子之生理作用的藥劑（例如抗體）。在一些情況下，拮抗劑與另一藥劑或分子特異性結合。包括完全及部分拮抗劑。"Antagonist" refers to an agent (e.g., an antibody) that interferes with or otherwise reduces the physiological action of another agent or molecule. In some cases, an antagonist specifically binds to another agent or molecule. Both full and partial antagonists are included.

「促效劑」係指增加或增強另一藥劑或分子之生理作用的藥劑（例如抗體）。在一些情況下，促效劑與另一藥劑或分子特異性結合。包括完全及部分促效劑。"Aggressor" refers to an agent (e.g., an antibody) that increases or enhances the physiological effect of another agent or molecule. In some cases, an agonist specifically binds to another agent or molecule. This includes full and partial agonists.

如本文所用，術語「胺基酸」意欲意謂天然存在及非天然存在之胺基酸以及胺基酸類似物及模擬物。天然存在之胺基酸包括蛋白質生物合成期間所用之20種(L)-胺基酸以及其他胺基酸，諸如例如4-羥基脯胺酸、羥基離胺酸、鎖鏈素、異鎖鏈素、高半胱胺酸、瓜胺酸及鳥胺酸。非天然存在之胺基酸包括例如(D)-胺基酸、正白胺酸、正纈胺酸、對氟苯丙胺酸、乙硫胺酸及其類似物，其為熟習此項技術者所已知。胺基酸類似物包括天然及非天然存在之胺基酸的經修飾形式。此類修飾可包括例如胺基酸上化學基團及部分之取代或置換或藉由胺基酸衍生。胺基酸模擬物包括例如展現參考胺基酸所特有之功能相似特性，諸如電荷及電荷間距的有機結構。舉例而言，模擬精胺酸(Arg或R)之有機結構將具有位於相似分子空間中且移動性程度與天然存在之Arg胺基酸的側鏈之e胺基相同的正電荷部分。模擬物亦包括受限之結構，以便維持胺基酸或胺基酸官能基之最佳間距及電荷相互作用。熟習此項技術者已知或可確定何種結構構成功能等同之胺基酸類似物及胺基酸模擬物。As used herein, the term "amino acid" is intended to refer to naturally occurring and non-naturally occurring amino acids and amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids used during protein biosynthesis, as well as other amino acids, such as, for example, 4-hydroxyproline, hydroxylysine, lysine, isolating lysine, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, and analogs thereof, which are known to those skilled in the art. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications may include, for example, substitution or replacement of chemical groups and moieties on the amino acids or derivatization by the amino acids. Amino acid mimetics include, for example, organic structures that exhibit functionally similar properties, such as charge and charge distance, that are characteristic of a reference amino acid. For example, an organic structure that mimics arginine (Arg or R) would have a positively charged portion that is located in a similar molecular space and has the same degree of mobility as the e-amine group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures in order to maintain optimal distances and charge interactions of amino acids or amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

如本文所用，術語「抗體」不僅涵蓋完整多株或單株抗體，而且涵蓋其片段（諸如dAb、Fab、Fab'、F(ab')2、Fv）、單鏈（scFv）、其合成變異體、天然存在之變異體、包含具有具所需特異性之抗原結合片段之抗體部分的融合蛋白、人源化抗體、嵌合抗體及包含具有所需特異性之抗原結合位點或片段（抗原決定基識別位點）的免疫球蛋白分子之任何其他經修飾之組態。本文中更詳細地描述抗體（及其抗原結合片段）之某些特徵及特性。As used herein, the term "antibody" encompasses not only complete polyclonal or monoclonal antibodies, but also fragments thereof (such as dAb, Fab, Fab', F(ab')2, Fv), single chains (scFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion having an antigen-binding fragment with the desired specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen-binding site or fragment (antigenic determinant site) with the desired specificity. Certain features and properties of antibodies (and antigen-binding fragments thereof) are described in more detail herein.

抗體或抗原結合片段可基本上屬於任何類型。如此項技術中所熟知，抗體為能夠透過至少一個位於免疫球蛋白分子之可變區中的抗原決定基識別位點與諸如免疫檢查點分子之目標特異性結合的免疫球蛋白分子。The antibody or antigen-binding fragment may be of essentially any type. As is well known in the art, an antibody is an immunoglobulin molecule that is capable of specifically binding to a target, such as an immune checkpoint molecule, through at least one antigenic determinant site located in the variable region of the immunoglobulin molecule.

如本文中所用，術語「抗原結合片段」係指含有免疫球蛋白重鏈及/或輕鏈之與所關注抗原結合之至少一個CDR的多肽片段。就此而言，本文所述之抗體之抗原結合片段可包含來自與目標分子結合之抗體的V_H及V_L序列之1、2、3、4、5個或所有6個CDR。在特定實施例中，本發明之抗原結合片段包含本文所揭示之抗體之V_H及V_L序列的所有6個CDR。As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy chain and/or light chain that binds to an antigen of interest. In this regard, an antigen-binding fragment of an antibody described herein may include 1, 2, 3, 4, 5, or all 6 CDRs from the_VH and_VL sequences of an antibody that binds to a target molecule. In a specific embodiment, the antigen-binding fragment of the present invention includes all 6 CDRs of the_VH and_VL sequences of an antibody disclosed herein.

抗體及其抗原結合片段之結合特性可使用此項技術中熟知之方法定量（參見Davies等人, Annual Rev. Biochem. 59:439-473, 1990）。在一些實施例中，抗體或其抗原結合片段以約或範圍介於約≤10^-7M至約10^-8M之平衡解離常數與目標分子（例如，IL-18BP多肽或其抗原決定基或複合物）特異性結合。在一些實施例中，平衡解離常數為約或範圍介於約≤10^-9M至約≤10^-10M。在某些例示性實施例中，抗體或其抗原結合片段對目標分子（與該目標分子特異性結合）之親和力（K_D或EC₅₀）為約、至少約或小於約0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、40或50 nM。The binding properties of antibodies and antigen-binding fragments thereof can be quantified using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a target molecule (e.g., an IL-18BP polypeptide or an antigenic determinant or complex thereof) with an equilibrium dissociation constant of about or ranging from about^≤10-7 M to about^10-8 M. In some embodiments, the equilibrium dissociation constant is about or ranging from about^≤10-9 M to about^≤10-10 M. In certain exemplary embodiments, the antibody or antigen-binding fragment thereof has an affinity (_KD or_EC50 ) for a target molecule (specifically binds to the target molecule) of about, at least about, or less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM.

若諸如多肽或抗體之分子與特定細胞、物質或特定抗原決定基之反應或締合比其與替代細胞或物質或抗原決定基之反應或締合更頻繁、更快速、持續時間更長及/或親和力更大，則稱該分子為展現「特異性結合」或「優先結合」。若抗體與目標分子或抗原決定基之結合比其與其他物質或抗原決定基之結合親和力、親合力更大、更容易及/或持續時間更長，例如達到統計學上顯著之量，則該抗體與目標分子或抗原決定基「特異性結合」或「優先結合」。通常展現特異性結合之分子對之一個成員在其表面上具有一個區域或腔，該區域或腔與該分子對之另一成員之特定空間及/或極性組織特異性結合，且因此與該特定空間及/或極性組織互補。因此，該分子對之成員具有彼此特異性結合之特性。舉例而言，與特定抗原決定基特異性或優先結合之抗體為與特定抗原決定基之結合比其與其他抗原決定基之結合親和力、親合力更大、更容易及/或持續時間更長的抗體。藉由閱讀此定義亦應理解，例如與第一目標特異性或優先結合之抗體（或部分或抗原決定基）可或可不與第二目標特異性或優先結合。該術語亦適用於例如抗體對多種抗原所攜帶之特定抗原決定基具有特異性的情況，在此情況下攜帶抗原結合片段或域之特異性結合成員將能夠與攜帶該抗原決定基之多種抗原結合；例如其可與來自多個物種之享有共同抗原決定基之目標抗原之多種不同形式交叉反應。A molecule, such as a polypeptide or antibody, is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell, substance, or particular antigenic determinant more frequently, more rapidly, for a longer period of time, and/or with a greater affinity than it reacts or associates with alternative cells or substances or antigenic determinants. An antibody "specifically binds" or "preferentially binds" to a target molecule or antigenic determinant if it binds with greater affinity, avidity, more readily, and/or for a longer period of time than it binds to other substances or antigenic determinants, e.g., to a statistically significant amount. Typically one member of a pair of molecules that exhibit specific binding has an area or cavity on its surface that specifically binds to, and therefore complements, a specific spatial and/or polar organization of the other member of the pair. Thus, the members of the pair have the property of specifically binding to each other. For example, an antibody that specifically or preferentially binds to a particular antigenic determinant is one that binds to that particular antigenic determinant with greater affinity, avidity, more readily and/or longer duration than it binds to other antigenic determinants. By reading this definition it should also be understood that, for example, an antibody (or portion or antigenic determinant) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. The term also applies, for example, to situations where the antibody is specific for a particular antigenic determinant carried by multiple antigens, in which case the specific binding member carrying the antigen-binding fragment or domain will be able to bind to multiple antigens carrying that antigenic determinant; for example, it may cross-react with multiple different forms of the target antigen from multiple species that share a common antigenic determinant.

免疫結合一般係指免疫球蛋白分子與免疫球蛋白具有特異性之抗原之間出現的類型之非共價相互作用，例如（以說明而非限制之方式）由於靜電、離子、親水性及/或疏水性吸引或排斥、空間力、氫鍵結、凡得瓦爾力（van der Waals force）及其他相互作用實現。免疫結合相互作用之強度或親和力可以相互作用之解離常數（K_D）表示，其中較小的K_D表示較大的親和力。所選多肽之免疫結合特性可使用此項技術中熟知之方法定量。一種此類方法需要量測抗原結合位點/抗原複合物形成及解離之速率，其中彼等速率視複合搭配物之濃度、相互作用之親和力及同等影響兩個方向上之速率的幾何參數而定。因此，「締合速率常數」（Kon）及「解離速率常數」（Koff）可藉由計算濃度及締合與解離之實際速率來確定。Koff/Kon之比率能夠抵消所有與親和力無關之參數，且因此等於解離常數K_D。如本文所用，術語「親和力」包括兩種藥劑之可逆結合的平衡常數且表示為K_D或EC₅₀。結合蛋白與配位體之親和力，諸如抗體對抗原決定基之親和力可為例如約100奈莫耳（nM）至約0.1 nM、約100 nM至約1皮莫耳（pM）或約100 nM至約1飛莫耳（fM）。如本文所用，術語「親合力」係指兩種或更多種藥劑之複合物在稀釋後對解離之抗性。在一些實施例中，親和力以半最大有效濃度（EC₅₀）表示，其係指如本文所揭示之藥劑（諸如抗體或抗IL-18BP抗體）誘導基線與在指定暴露時間之後的最大值之間的半途的反應的濃度。EC₅₀通常用作抗體效力之量度。Immunobinding generally refers to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific, such as by way of illustration and not limitation, due to electrostatic, ionic, hydrophilic and/or hydrophobic attractions or repulsions, steric forces, hydrogen bonding, van der Waals forces, and other interactions. The strength or affinity of the immunobinding interaction can be expressed in terms of the dissociation constant (_KD ) of the interaction, where a smaller_KD indicates a greater affinity. The immunobinding properties of a selected polypeptide can be quantified using methods well known in the art. One such method entails measuring the rates of formation and dissociation of an antigen binding site/antigen complex, where those rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, the "association rate constant" (Kon) and "dissociation rate constant" (Koff) can be determined by calculating the concentration and the actual rates of association and dissociation. The ratio of Koff/Kon is able to cancel out all parameters not related to affinity and is therefore equal to the dissociation constant_KD . As used herein, the term "affinity" includes the equilibrium constant for the reversible binding of two agents and is expressed as_KD or_EC50 . The affinity of a binding protein to a ligand, such as the affinity of an antibody to an antigenic determinant, can be, for example, about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM). As used herein, the term "affinity" refers to the resistance of a complex of two or more agents to dissociate upon dilution. In some embodiments, affinity is expressed as half-maximal effective concentration (_EC50 ), which refers to the concentration of an agent (e.g., an antibody or anti-IL-18BP antibody) as disclosed herein that induces a response halfway between baseline and maximum after a specified exposure time._EC50 is often used as a measure of antibody potency.

抗體可藉由一般熟習此項技術者已知之多種技術中之任一者製備。參見例如Harlow及Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988。對所關注多肽具有特異性之單株抗體可例如使用以下之技術及其改良製備：Kohler及Milstein, Eur. J. Immunol. 6:511-519, 1976。亦包括利用諸如小鼠之轉殖基因動物表現人類抗體之方法。參見例如，Neuberger等人, Nature Biotechnology 14:826, 1996；Lonberg等人, Handbook of Experimental Pharmacology 113:49-101, 1994；及Lonberg等人, Internal Review of Immunology 13:65-93, 1995。特定實例包括REGENEREX®之VELOCIMMUNE®平台（參見例如美國專利第6,596,541號）。Antibodies can be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Monoclonal antibodies specific for a polypeptide of interest can be prepared, for example, using the following techniques and modifications thereof: Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976. Also included are methods of expressing human antibodies using transgenic animals such as mice. See, e.g., Neuberger et al., Nature Biotechnology 14:826, 1996; Lonberg et al., Handbook of Experimental Pharmacology 113:49-101, 1994; and Lonberg et al., Internal Review of Immunology 13:65-93, 1995. Specific examples include the VELOCIMMUNE® platform of REGENEREX® (see, e.g., U.S. Patent No. 6,596,541).

抗體亦可藉由使用噬菌體呈現庫或酵母呈現庫來產生或鑑別（參見例如美國專利第7,244,592號；Chao等人, Nature Protocols. 1:755-768, 2006）。可用庫之非限制性實例包括選殖或合成庫，諸如人類組合抗體庫（HuCAL），其中人類抗體譜系之結構多樣性由七個重鏈可變區基因及七個輕鏈可變區基因表示。此等基因之組合在主庫中產生49個構架。藉由在此等構架上疊加高度可變之基因卡匣（CDR=互補決定區），可複製大量的人類抗體譜系。亦包括設計成具有編碼輕鏈可變區、重鏈CDR-3之人類供體源片段、編碼重鏈CDR-1中之多樣性的合成DNA及編碼重鏈CDR-2中之多樣性之合成DNA的人類庫。其他適合使用之庫將為熟習此項技術者顯而易見的。Antibodies can also be produced or identified by using phage display libraries or yeast display libraries (see, e.g., U.S. Patent No. 7,244,592; Chao et al., Nature Protocols. 1:755-768, 2006). Non-limiting examples of available libraries include cloning or synthetic libraries, such as the Human Combinatorial Antibody Library (HuCAL), in which the structural diversity of the human antibody repertoire is represented by seven heavy chain variable region genes and seven light chain variable region genes. The combination of these genes produces 49 frameworks in the master library. By superimposing highly variable gene cassettes (CDR = complementary determining regions) on these frameworks, a large number of human antibody repertoires can be replicated. Also included are human libraries designed with human donor-derived fragments encoding light chain variable regions, heavy chain CDR-3, synthetic DNA encoding diversity in heavy chain CDR-1, and synthetic DNA encoding diversity in heavy chain CDR-2. Other libraries suitable for use will be apparent to those skilled in the art.

在某些實施例中，如本文所述之抗體及其抗原結合片段包括分別插入重鏈與輕鏈構架區（FR）集合之間的重鏈及輕鏈CDR集合，該重鏈及輕鏈構架區集合為CDR提供支撐且界定CDR相對於彼此之空間關係。如本文所用，術語「CDR集合」係指重鏈或輕鏈V區之三個高變區。自重鏈或輕鏈之N端開始，此等區域分別表示為「CDR1」、「CDR2」及「CDR3」。因此，抗原結合位點包括六個CDR，包含來自重鏈及輕鏈V區中之各者的CDR集合。包含單一CDR（例如CDR1、CDR2或CDR3）之多肽在本文中稱為「分子識別單元」。多種抗原-抗體複合物之結晶學分析已表明，CDR之胺基酸殘基與結合之抗原形成廣泛接觸，其中最廣泛抗原接觸係與重鏈CDR3。因此，分子識別單元主要導致抗原結合位點之特異性。In certain embodiments, antibodies and antigen-binding fragments thereof as described herein include heavy chain and light chain CDR sets inserted between heavy chain and light chain framework region (FR) sets, respectively, which provide support for the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of the heavy chain or light chain V region. Starting from the N-terminus of the heavy chain or light chain, these regions are respectively represented as "CDR1", "CDR2" and "CDR3". Therefore, the antigen binding site includes six CDRs, including CDR sets from each of the heavy chain and light chain V regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2 or CDR3) is referred to herein as a "molecular recognition unit". Crystallographic analysis of various antigen-antibody complexes has shown that the amino acid residues of CDRs make extensive contacts with the bound antigen, with the most extensive antigen contact being with heavy chain CDR3. Therefore, the molecular recognition unit is primarily responsible for the specificity of the antigen binding site.

如本文所用，術語「FR集合」係指四個側接胺基酸序列，其向重鏈或輕鏈V區之CDR集合中的CDR提供構架。一些FR殘基可接觸結合之抗原；然而，FR主要負責摺疊V區成為抗原結合位點，尤其與CDR直接相鄰之FR殘基。在FR內，某些胺基殘基及某些結構特徵極高度保守。就此而言，大部分V區序列含有大約90個胺基酸殘基之內部二硫環。當V區摺疊成為結合位點時，CDR呈現為形成抗原結合表面之突出環模體。一般認為存在FR保守結構區，其影響CDR環摺疊成某些「典型」結構之形狀，與精確CDR胺基酸序列無關。此外，已知某些FR殘基參與非共價域間接觸，該等接觸使抗體重鏈及輕鏈之相互作用穩定。As used herein, the term "FR set" refers to four flanking amino acid sequences that provide a framework for the CDRs in a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, the FR is primarily responsible for folding the V region into an antigen binding site, especially the FR residues directly adjacent to the CDR. Within the FR, certain amino acid residues and certain structural features are extremely highly conserved. In this regard, most V region sequences contain internal disulfide loops of approximately 90 amino acid residues. When the V region folds into a binding site, the CDR appears as a protruding loop motif that forms an antigen binding surface. It is generally believed that there are FR conserved structural regions that affect the shape of the CDR loop folding into certain "typical" structures, regardless of the exact CDR amino acid sequence. Furthermore, certain FR residues are known to participate in non-covalent interdomain contacts that stabilize the interactions of the antibody heavy and light chains.

免疫球蛋白可變域之結構及位置可參考Kabat, E. A.等人, Sequences of Proteins of Immunological Interest. 第4版. US Department of Health and Human Services. 1987及其更新版來確定。The structure and location of immunoglobulin variable domains can be determined by reference to Kabat, E. A. et al., Sequences of Proteins of Immunological Interest. 4th ed. US Department of Health and Human Services. 1987 and updated versions thereof.

亦包括「單株」抗體，其係指均質抗體群體，其中單株抗體包含參與抗原決定基之選擇性結合的胺基酸（天然存在之胺基酸及非天然存在之胺基酸）。術語「單株抗體」不僅涵蓋完整單株抗體及全長單株抗體，而且涵蓋其片段（諸如Fab、Fab'、F(ab')2、Fv）、單鏈（ScFv）、其變異體、包含抗原結合部分之融合蛋白、人源化單株抗體、嵌合單株抗體，及包含具有所需特異性且能夠與抗原決定基結合之抗原結合片段（抗原決定基識別位點）之免疫球蛋白分子的任何其他經修飾之組態。並不意欲限制抗體之來源或其製備方式（例如藉由融合瘤、噬菌體選擇、重組表現、轉殖基因動物）。該術語包括整個免疫球蛋白以及上文在「抗體」定義下所描述之片段等。"Monoclonal" antibodies are also included, which refers to a homogeneous antibody population, wherein the monoclonal antibody comprises amino acids (naturally occurring amino acids and non-naturally occurring amino acids) that participate in the selective binding of the antigenic determinant. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chains (ScFv), variants thereof, fusion proteins comprising antigen-binding portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of immunoglobulin molecules comprising antigen-binding fragments (antigenic determinant recognition sites) having the desired specificity and capable of binding to antigenic determinants. It is not intended to limit the source of the antibody or the manner in which it is prepared (e.g., by fusion tumors, phage selection, recombinant expression, transgenic animals). The term includes whole immunoglobulins as well as fragments thereof as described above under the definition of "antibody".

蛋白水解酶木瓜蛋白酶優先使IgG分子裂解，以產生若干片段，其中兩個片段（F(ab)片段）各自構成包括完整抗原結合位點之共價雜二聚體。酶胃蛋白酶能夠使IgG分子裂解，以提供若干片段，包括包含兩個抗原結合位點之F(ab')2片段。根據某些實施例使用之Fv片段可藉由優先蛋白水解裂解IgM且極偶爾地蛋白水解裂解IgG或IgA免疫球蛋白分子來產生。然而，Fv片段更通常使用此項技術中已知之重組技術得到。Fv片段包括非共價VH::VL雜二聚體，包括保持原生抗體分子之大部分抗原識別及結合能力的抗原結合位點（Inbar等人, PNAS USA. 69:2659-2662, 1972；Hochman等人, Biochem. 15:2706-2710, 1976；及Ehrlich等人, Biochem. 19:4091-4096, 1980）。在一些實施例中，Fv藉由其他方式，例如併入至少一個二硫鍵而穩定化（Wörn及Pluckthun, J. Mol. Biol. 305, 989-1010, 2001）。The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (the F(ab) fragments) each constitute a covalent heterodimer that includes an intact antigen binding site. The enzyme pepsin is capable of cleaving IgG molecules to provide several fragments, including a F(ab')2 fragment that includes two antigen binding sites. The Fv fragments used according to certain embodiments can be produced by preferentially proteolytically cleaving IgM and, very occasionally, IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly obtained using recombinant techniques known in the art. Fv fragments include non-covalent VH::VL heterodimers, including an antigen binding site that retains most of the antigen recognition and binding capabilities of the native antibody molecule (Inbar et al., PNAS USA. 69:2659-2662, 1972; Hochman et al., Biochem. 15:2706-2710, 1976; and Ehrlich et al., Biochem. 19:4091-4096, 1980). In some embodiments, Fv is stabilized by other means, such as by incorporation of at least one disulfide bond (Wörn and Pluckthun, J. Mol. Biol. 305, 989-1010, 2001).

在某些實施例中，涵蓋單鏈Fv（scFV）抗體。舉例而言，κ體（Ill等人, Prot. Eng. 10:949-57, 1997）；微型抗體（Martin等人, EMBO J 13:5305-9, 1994）；雙功能抗體（Holliger等人, PNAS 90: 6444-8, 1993）；或Janusin（Traunecker等人, EMBO J 10: 3655-59, 1991；及Traunecker等人, Int. J. Cancer增刊7:51-52, 1992）可使用標準分子生物學技術根據本申請案關於選擇具有所需特異性之抗體之教示來製備。In certain embodiments, single-chain Fv (scFV) antibodies are contemplated. For example, kappa bodies (Ill et al., Prot. Eng. 10:949-57, 1997); minibodies (Martin et al., EMBO J 13:5305-9, 1994); bifunctional antibodies (Holliger et al., PNAS 90: 6444-8, 1993); or Janusins (Traunecker et al., EMBO J 10: 3655-59, 1991; and Traunecker et al., Int. J. Cancer Suppl. 7:51-52, 1992) can be prepared using standard molecular biology techniques according to the teachings of the present application for selecting antibodies with desired specificity.

單鏈Fv（scFv）多肽為共價連接之VH::VL雜二聚體，其自包括由編碼肽之連接子連接的VH及VL編碼基因之基因融合物表現。Huston等人, (PNAS USA. 85(16):5879-5883, 1988)。已描述多種方法來辨別用於使來自抗體V區之天然聚集但經化學分離之輕多肽鏈及重多肽鏈轉化成為scFv分子的化學結構，該scFv分子將摺疊成實質上類似於抗原結合位點之結構的三維結構。參見例如Huston等人之美國專利第5,091,513號及第5,132,405號；及Ladner等人之美國專利第4,946,778號。Single-chain Fv (scFv) polypeptides are covalently linked VH::VL heterodimers expressed from gene fusions comprising VH and VL encoding genes linked by a linker encoding a peptide. Huston et al., (PNAS USA. 85(16):5879-5883, 1988). Various methods have been described to identify chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that will fold into a three-dimensional structure substantially similar to that of the antigen binding site. See, e.g., U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Patent No. 4,946,778 to Ladner et al.

在某些實施例中，本文所述之抗體或抗原結合片段呈「雙功能抗體」形式。雙功能抗體為多肽之多聚體，各多肽包含有包含免疫球蛋白輕鏈之結合區的第一域及包含免疫球蛋白重鏈之結合區的第二域，該等兩個域經連接(例如藉由肽連接子)但不能彼此結合以形成抗原結合位點：抗原結合位點係藉由多聚體內之一個多肽之第一域與多聚體內之另一多肽之第二域結合而形成（WO94/13804）。抗體之dAb片段係由VH域組成（Ward等人, Nature 341:544-546, 1989）。雙功能抗體及其他多價或多特異性片段可例如藉由基因融合構築（參見WO94/13804；及Holliger等人, PNAS USA. 90:6444-6448, 1993）。In certain embodiments, the antibodies or antigen-binding fragments described herein are in the form of "bifunctional antibodies". Bifunctional antibodies are polymers of polypeptides, each polypeptide comprising a first domain comprising a binding region for an immunoglobulin light chain and a second domain comprising a binding region for an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but not binding to each other to form an antigen binding site: the antigen binding site is formed by the binding of the first domain of one polypeptide in the multimer to the second domain of another polypeptide in the multimer (WO94/13804). The dAb fragment of an antibody is composed of a VH domain (Ward et al., Nature 341:544-546, 1989). Bifunctional antibodies and other multivalent or multispecific fragments can be constructed, for example, by gene fusion (see WO 94/13804; and Holliger et al., PNAS USA. 90:6444-6448, 1993).

亦包括包含接合至CH3域之scFv的微型抗體（參見Hu等人, Cancer Res. 56:3055-3061, 1996）。亦參見Ward等人, Nature. 341:544-546, 1989；Bird等人, Science. 242:423-426, 1988；Huston等人, PNAS USA. 85:5879-5883, 1988；PCT/US92/09965；WO94/13804；及Reiter等人, Nature Biotech. 14:1239-1245, 1996。Also included are minibodies comprising scFvs joined to a CH3 domain (see Hu et al., Cancer Res. 56:3055-3061, 1996). See also Ward et al., Nature. 341:544-546, 1989; Bird et al., Science. 242:423-426, 1988; Huston et al., PNAS USA. 85:5879-5883, 1988; PCT/US92/09965; WO94/13804; and Reiter et al., Nature Biotech. 14:1239-1245, 1996.

在使用雙特異性抗體之情況下，此等抗體可為習知雙特異性抗體，其可以多種方式製造（Holliger及Winter, Current Opinion Biotechnol. 4:446-449, 1993），例如以化學方式製備或自雜交融合瘤製備，或可為上文所提及之雙特異性抗體片段中之任一者。Where bispecific antibodies are used, these may be known bispecific antibodies, which can be produced in a variety of ways (Holliger and Winter, Current Opinion Biotechnol. 4:446-449, 1993), for example chemically prepared or prepared from hybridomas, or may be any of the bispecific antibody fragments mentioned above.

與雙特異性完全抗體相對比，雙特異性雙功能抗體亦可尤其適用，因為其可容易地構築且在大腸桿菌（E. coli）中表現。可使用噬菌體呈現（WO94/13804）自庫容易地選擇具有適當結合特異性之雙功能抗體（及許多其他多肽，諸如抗體片段）。若雙功能抗體之一個臂保持恆定，例如具有針對抗原X之特異性，則可製備其中另一個臂變化之庫且選擇具有適當特異性之抗體。雙特異性完整抗體可藉由多種方法（Brinkman及Kontermann, mAbs 9:182-212, 2017）製備，包括臼包杵工程改造（Ridgeway等人, Protein Eng. 9:616-621, 1996）。Bispecific bifunctional antibodies may also be particularly useful, as they can be easily constructed and expressed in E. coli, as opposed to bispecific complete antibodies. Bifunctional antibodies (and many other polypeptides, such as antibody fragments) can be easily selected from libraries with appropriate binding specificity using phage display (WO94/13804). If one arm of the bifunctional antibody remains constant, for example with specificity for antigen X, a library can be prepared in which the other arm is varied and antibodies with appropriate specificity selected. Bispecific intact antibodies can be prepared by a variety of methods (Brinkman and Kontermann, mAbs 9:182-212, 2017), including mortar-in-pestle engineering (Ridgeway et al., Protein Eng. 9:616-621, 1996).

在某些實施例中，本文所述之抗體或抗原結合片段呈UniBody®形式。UniBody®為移除鉸鏈區之IgG4抗體（參見GenMab Utrecht, The Netherlands；亦參見，例如US20090226421）。此抗體技術產生預期治療窗比當前之小抗體型式長之穩定的較小抗體型式。IgG4抗體視為惰性的，且因此不與免疫系統相互作用。完全人類IgG4抗體可藉由消除抗體之鉸鏈區修飾，以獲得相對於對應完整IgG4（GenMab，Utrecht）具有明顯穩定性特性之半分子片段。將IgG4分子切半僅在UniBody®上保留一個可與同源抗原（例如疾病目標）結合之區域，且因此使UniBody®僅與目標細胞上之一個位點單價結合。對於某些癌細胞表面抗原而言，此單價結合可能不如使用具有相同抗原特異性之二價抗體可見般地刺激癌細胞生長，且因此UniBody®技術可為難以用習知抗體治療之一些類型之癌症提供治療選項。當治療一些形式之癌症時，小尺寸之UniBody®可具有極大益處，使得分子較佳分佈於較大實體腫瘤之上且可能增加功效。In certain embodiments, the antibodies or antigen-binding fragments described herein are in the form of UniBody®. UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, e.g., US20090226421). This antibody technology produces a stable, smaller antibody format with a longer therapeutic window than current small antibody formats. IgG4 antibodies are considered inert and therefore do not interact with the immune system. Fully human IgG4 antibodies can be modified by eliminating the hinge region of the antibody to obtain half-molecule fragments with significant stability properties relative to the corresponding full IgG4 (GenMab, Utrecht). Chopping the IgG4 molecule in half leaves only one region on the UniBody® that can bind to a cognate antigen (e.g., a disease target), and thus allows the UniBody® to bind monovalently to only one site on the target cell. For some cancer cell surface antigens, this monovalent binding may not stimulate cancer cell growth as much as would be seen with a bivalent antibody of the same antigenic specificity, and thus the UniBody® technology may provide a treatment option for some types of cancer that are difficult to treat with conventional antibodies. The small size of the UniBody® may be of great benefit when treating some forms of cancer, allowing for better distribution of the molecule over larger solid tumors and potentially increasing efficacy.

在某些實施例中，本文所述之抗體及抗原結合片段呈奈米抗體形式。奈米抗體係由單一基因編碼且高效地產生於幾乎所有的原核及真核宿主中，例如大腸桿菌（參見美國專利第6,765,087號）、黴菌（例如麴黴菌屬（Aspergillus）或木黴屬（Trichoderma））及酵母（例如酵母菌（Saccharomyces）、克魯維酵母屬（Kluyveromyces）、漢遜酵母屬（Hansenula）或畢赤酵母屬（Pichia））（參見美國專利第6,838,254號）。生產製程可放大且已生產數公斤量的奈米抗體。奈米抗體可調配為具有長存放期的即用型溶液。奈米選殖（Nanoclone）方法（參見WO 06/079372）為基於B細胞之自動化高通量選擇，產生針對所需目標之奈米抗體的專有方法。In certain embodiments, the antibodies and antigen-binding fragments described herein are in the form of nanobodies. Nanobodies are encoded by a single gene and are efficiently produced in almost all prokaryotic and eukaryotic hosts, such as Escherichia coli (see U.S. Patent No. 6,765,087), molds (such as Aspergillus or Trichoderma) and yeasts (such as Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see U.S. Patent No. 6,838,254). The production process can be scaled up and nanobodies have been produced in kilogram quantities. Nanobodies can be formulated as ready-to-use solutions with long shelf life. The Nanoclone method (see WO 06/079372) is a proprietary method based on automated high-throughput selection of B cells to generate nanobodies against desired targets.

在一些實施例中，本文所述之抗體或抗原結合片段呈適體形式（參見例如Ellington等人, Nature. 346, 818-22, 1990；及Tuerk等人, Science. 249, 505-10, 1990，該等文獻以引用之方式併入）。適體之實例包括核酸適體（例如DNA適體、RNA適體）及肽適體。核酸適體一般係指已透過重複數輪活體外選擇或等效方法加以工程改造的核酸物種，諸如SELEX（藉由指數級富集達成的配位體系統性演化），以結合各種分子目標，諸如小分子、蛋白質、核酸及甚至細胞、組織及生物體。參見例如美國專利第6,376,190號及第6,387,620號，其以引用之方式併入。In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of aptamers (see, e.g., Ellington et al., Nature. 346, 818-22, 1990; and Tuerk et al., Science. 249, 505-10, 1990, which are incorporated by reference). Examples of aptamers include nucleic acid aptamers (e.g., DNA aptamers, RNA aptamers) and peptide aptamers. Nucleic acid aptamers generally refer to nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalent methods, such as SELEX (systematic evolution of ligands by exponential enrichment), to bind to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. See, e.g., U.S. Patent Nos. 6,376,190 and 6,387,620, which are incorporated by reference.

肽適體通常包括附接在蛋白質骨架兩端之可變肽環，此為雙重結構約束，通常使肽適體之結合親和力增加至與抗體之結合親和力相當的水平（例如奈莫耳濃度範圍內）。在某些實施例中，可變環長度可由約10-20個胺基酸（包括兩者之間的所有整數）構成，且骨架可包括具有良好溶解度及緊湊度特性之任何蛋白質。某些例示性實施例利用細菌蛋白質硫氧還蛋白-A作為骨架蛋白質，可變環插入還原活性位點內（野生蛋白質中之-Cys-Gly-Pro-Cys-環），其中兩個半胱胺酸側鏈能夠形成二硫橋鍵。用於鑑別肽適體之方法描述於例如以引用方式併入之美國申請案第2003/0108532號中。肽適體選擇可使用此項技術中已知之不同系統，包括酵母雙雜交系統來進行。Peptide aptamers typically include variable peptide loops attached to both ends of a protein backbone, which is a double structural constraint that typically increases the binding affinity of the peptide aptamer to a level comparable to that of an antibody (e.g., in the nanomolar concentration range). In certain embodiments, the variable loop length may be composed of about 10-20 amino acids (including all integers in between), and the backbone may include any protein with good solubility and compactness properties. Certain exemplary embodiments utilize the bacterial protein thioredoxin-A as a backbone protein, with the variable loop inserted into the reducing active site (-Cys-Gly-Pro-Cys-loop in the wild protein), where the two cysteine side chains are capable of forming a disulfide bridge. Methods for identifying peptide aptamers are described, for example, in U.S. Application No. 2003/0108532, which is incorporated by reference. Peptide aptamer selection can be performed using various systems known in the art, including the yeast two-hybrid system.

在一些實施例中，本文所述之抗體或抗原結合片段呈高親和性多聚體形式。高親和性多聚體係指使用活體外外顯子改組及噬菌體呈現加以工程改造之多聚體結合蛋白或肽。多個結合域連接，使得親和力及特異性比單一抗原決定基免疫球蛋白域大。參見例如，Silverman等人, Nature Biotechnology. 23:1556-1561, 2005；美國專利第7,166,697號；及美國申請案第2004/0175756號、第2005/0048512號、第2005/0053973號、第2005/0089932號及第2005/0221384號，該等文獻以引用之方式併入。In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of high-affinity multimers. High-affinity multimers refer to multimeric binding proteins or peptides that are engineered using in vitro exon shuffling and phage display. Multiple binding domains are linked so that the affinity and specificity are greater than a single antigenic determinant immunoglobulin domain. See, e.g., Silverman et al., Nature Biotechnology. 23:1556-1561, 2005; U.S. Patent No. 7,166,697; and U.S. Application Nos. 2004/0175756, 2005/0048512, 2005/0053973, 2005/0089932, and 2005/0221384, which are incorporated by reference.

在一些實施例中，本文所述之抗體或抗原結合片段呈纖連蛋白形式。纖連蛋白係指一類來源於人類纖維結合蛋白之靶向生物製劑，纖維結合蛋白為與其他蛋白質天然結合之豐富細胞外蛋白質。參見例如美國申請案第2007/0082365號、第2008/0139791號及第2008/0220049號，該等文獻以引用之方式併入。纖連蛋白典型地由天然纖維結合蛋白骨幹以及人類纖維結合蛋白之特定部分的多個靶向域組成。靶向域可經工程改造以使纖連蛋白能夠特異性識別IL-18BP多肽或其抗原決定基。In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of fibronectin. Fibronectin refers to a class of targeted biological agents derived from human fibronectin, which is an abundant extracellular protein that naturally binds to other proteins. See, for example, U.S. Application Nos. 2007/0082365, 2008/0139791, and 2008/0220049, which are incorporated by reference. Fibronectin typically consists of multiple targeting domains of a specific portion of a natural fibronectin backbone and human fibronectin. The targeting domain can be engineered to enable fibronectin to specifically recognize an IL-18BP polypeptide or its antigenic determinant.

在一些實施例中，本文所述之抗體或抗原結合片段呈抗運載蛋白形式。抗運載蛋白係指通常由人類脂質運載蛋白合成之一類抗體模擬物，脂質運載蛋白為具有由結構剛性構架支撐之高變環區的結合蛋白家族。參見例如美國申請案第2006/0058510號。抗運載蛋白典型地具有約20 kDa之尺寸。抗運載蛋白之特徵可在於由八個反平行β股形成之桶狀結構（穩定β桶狀骨架），該等β股由四個肽環及所連接之α螺旋成對連接。在某些態樣中，在高變環區中進行構形偏差以實現特異性結合。參見例如Skerra, FEBS J. 275:2677-83, 2008，其以引用之方式併入。In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of anticalins. Anticalins refer to a class of antibody mimetics that are typically synthesized from human lipocalin, a family of binding proteins having hypervariable loop regions supported by a structurally rigid framework. See, e.g., U.S. Application No. 2006/0058510. Anticalins typically have a size of about 20 kDa. Anticalins can be characterized by a barrel structure (stable β-barrel backbone) formed by eight antiparallel β strands connected in pairs by four peptide rings and the α-helices connected thereto. In certain aspects, conformational deviations are made in the hypervariable loop regions to achieve specific binding. See, e.g., Skerra, FEBS J. 275:2677-83, 2008, which is incorporated by reference.

在一些實施例中，本文所述之抗體或抗原結合片段呈經設計之錨蛋白重複蛋白（DARPin）形式。錨蛋白重複蛋白包括在藥物發現及藥物開發中在目標結合方面優於抗體的一類非免疫球蛋白。在其他用途中，錨蛋白重複蛋白由於其有利的分子特性（包括小尺寸及高穩定性）而理想地適合於活體內成像或遞送毒素或其他治療有效負載。在細菌中之低成本生產及多種目標特異性錨蛋白重複蛋白之快速產生使得錨蛋白重複蛋白方法適用於藥物發現。另外，錨蛋白重複蛋白可容易以多特異性型式產生，從而提供使效應子錨蛋白重複蛋白靶向特定器官的可能性或以由若干個錨蛋白重複蛋白構成之一個分子靶向多個受體的可能性。參見例如，Stumpp等人, Curr Opin Drug Discov Devel. 10:153-159, 2007；美國申請案第2009/0082274號；及PCT/EP2001/10454，該等文獻以引用之方式併入。In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of designed DARPins. DARPins include a class of non-immunoglobulins that are superior to antibodies in terms of target binding in drug discovery and drug development. Among other uses, DARPins are ideally suited for in vivo imaging or delivery of toxins or other therapeutic payloads due to their favorable molecular properties, including small size and high stability. Low-cost production in bacteria and rapid production of multiple target-specific DARPins make the DARPin method applicable to drug discovery. In addition, DARPins can be easily produced in multispecific forms, thereby providing the possibility of targeting effector DARPins to specific organs or the possibility of targeting multiple receptors with one molecule composed of several DARPins. See, e.g., Stumpp et al., Curr Opin Drug Discov Devel. 10:153-159, 2007; U.S. Application No. 2009/0082274; and PCT/EP2001/10454, which are incorporated by reference.

亦包括重鏈二聚體，諸如來自駱駝科及鯊魚之抗體。駱駝科及鯊魚抗體包含V樣及C樣域之兩條鏈的同源二聚體對（均無輕鏈）。由於駱駝科之重鏈二聚體IgG的V_H不必與輕鏈發生疏水相互作用，因此與輕鏈正常接觸之重鏈區域變成駱駝科中之親水性胺基酸殘基。重鏈二聚體IgG之VH域稱為VHH域。鯊魚Ig-NAR包含一個可變域（稱為V-NAR域）及五個C樣恆定域（C-NAR域）之均二聚體。Heavy chain dimers are also included, such as antibodies from camel family and shark. Camel family and shark antibodies contain homodimeric pairs of two chains of V-like and C-like domains (both without light chains). Since the_VH of the heavy chain dimer IgG of camel family does not have to undergo hydrophobic interactions with the light chain, the heavy chain region that normally contacts the light chain becomes a hydrophilic amino acid residue in camel family. The VH domain of the heavy chain dimer IgG is called the VHH domain. Shark Ig-NAR contains a homodimer of one variable domain (called the V-NAR domain) and five C-like constant domains (C-NAR domains).

在駱駝科中，抗體譜系之多樣性由VH或VHH區中之互補決定區（CDR）1、2及3決定。駱駝科VHH區中之CDR3的特徵在於其相對較長之長度，平均16個胺基酸（Muyldermans等人, 1994, Protein Engineering 7(9): 1129）。此與許多其他物種之抗體之CDR3區形成對比。舉例而言，小鼠VH之CDR3具有平均9個胺基酸。可藉由例如美國專利申請案第20050037421號中所揭示之方法製得駱駝科衍生之抗體可變區之庫，其維持駱駝科之可變區的活體內多樣性，該申請案公開於2005年2月17日。In Camelidae, the diversity of the antibody repertoire is determined by the complementary determining regions (CDRs) 1, 2, and 3 in the VH or VHH regions. The CDR3 in the Camelidae VHH region is characterized by its relatively long length, averaging 16 amino acids (Muyldermans et al., 1994, Protein Engineering 7(9): 1129). This is in contrast to the CDR3 regions of antibodies from many other species. For example, the CDR3 of mouse VH has an average of 9 amino acids. A library of Camelidae-derived antibody variable regions that maintains the in vivo diversity of Camelidae variable regions can be prepared, for example, by the method disclosed in U.S. Patent Application No. 20050037421, which was published on February 17, 2005.

在某些實施例中，抗體或其抗原結合片段經人源化。此等實施例係關於一種嵌合分子，其一般使用重組技術來製備，具有來源於來自非人類物種之免疫球蛋白的抗原結合位點且分子之剩餘免疫球蛋白結構基於人類免疫球蛋白之結構及/或序列。抗原結合位點可包含與恆定域融合的完整可變域或僅包含移植至可變域中之適當構架區上的CDR（全部或一部分）。抗原決定基結合位點可為野生型或藉由一或多個胺基酸取代來修飾。此消除作為人類個體中之免疫原的恆定區，但保留對外來可變區之免疫反應之可能性（LoBuglio等人, PNAS USA 86:4220-4224, 1989；Queen等人, PNAS USA. 86:10029-10033, 1988；Riechmann等人, Nature. 332:323-327, 1988）。用於抗體人源化之說明性方法包括美國專利第7,462,697號中所述之方法。In certain embodiments, the antibody or antigen-binding fragment thereof is humanized. These embodiments relate to a chimeric molecule, which is generally prepared using recombinant technology, having an antigen binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule is based on the structure and/or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain or only CDRs (all or part) grafted onto appropriate framework regions in a variable domain. The antigen determinant binding site may be wild type or modified by one or more amino acid substitutions. This eliminates the constant regions that serve as immunogens in human subjects, but retains the possibility of immune responses to foreign variable regions (LoBuglio et al., PNAS USA 86:4220-4224, 1989; Queen et al., PNAS USA. 86:10029-10033, 1988; Riechmann et al., Nature. 332:323-327, 1988). Illustrative methods for antibody humanization include those described in U.S. Patent No. 7,462,697.

另一方法不僅聚焦於提供人類來源之恆定區，且亦修飾可變區，從而將其重塑成儘可能接近人類形式。已知重鏈與輕鏈之可變區含有三個互補決定區（CDR），該三個互補決定區回應於相關抗原決定基而變化且決定結合能力，由在既定物種中相對保守且假定為CDR提供骨架之四個構架區（FR）側接。當關於特定抗原決定基製備非人類抗體時，可藉由將來源於非人類抗體之CDR移植於待修飾之人類抗體中所存在之FR上而將可變區「重塑」或「人源化」。此方法對於各種抗體的應用已報導於Sato等人, Cancer Res. 53:851-856, 1993；Riechmann等人, Nature 332:323-327, 1988；Verhoeyen等人, Science 239:1534-1536, 1988；Kettleborough等人, Protein Engineering. 4:773-3783, 1991；Maeda等人, Human Antibodies Hybridoma 2:124-134, 1991；Gorman等人, PNAS USA. 88:4181-4185, 1991；Tempest等人, Bio/Technology 9:266-271, 1991；Co等人, PNAS USA. 88:2869-2873, 1991；Carter等人, PNAS USA. 89:4285-4289, 1992；及Co等人, J Immunol. 148:1149-1154, 1992。在一些實施例中，人源化抗體保留所有CDR序列（例如，含有來自小鼠抗體之所有六個CDR的人源化小鼠抗體）。在一些實施例中，僅一些CDR序列自非人類抗體移植（Bowers等人, J. Biol. Chem. 288:7688-7696, 2013）。在某些實施例中，人源化抗體具有一或多個相對於原始抗體改變之CDR（一、二、三、四、五、六個），其亦稱為「來源於」一或多個來自原始抗體之CDR的一或多個CDR。Another approach focuses not only on providing constant regions of human origin, but also on modifying the variable regions, thereby remodeling them as close to human form as possible. It is known that the variable regions of the heavy and light chains contain three complementary determining regions (CDRs) that vary in response to the relevant antigenic determinant and determine the binding ability, flanked by four framework regions (FRs) that are relatively conserved in a given species and are assumed to provide the backbone for the CDRs. When non-human antibodies are prepared for a specific antigenic determinant, the variable regions can be "remodeled" or "humanized" by grafting CDRs from the non-human antibody onto the FRs present in the human antibody to be modified. The application of this method to various antibodies has been reported in Sato et al., Cancer Res. 53:851-856, 1993; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988; Kettleborough et al., Protein Engineering. 4:773-3783, 1991; Maeda et al., Human Antibodies Hybridoma 2:124-134, 1991; Gorman et al., PNAS USA. 88:4181-4185, 1991; Tempest et al., Bio/Technology 9:266-271, 1991; Co et al., PNAS USA. 88:2869-2873, 1991; Carter et al., PNAS USA. 89:4285-4289, 1992; and Co et al., J Immunol. 148:1149-1154, 1992. In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In some embodiments, only some CDR sequences are transplanted from non-human antibodies (Bowers et al., J. Biol. Chem. 288:7688-7696, 2013). In certain embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, six) that are changed relative to the original antibody, which is also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

在某些實施例中，抗體為「嵌合」抗體。就此而言，嵌合抗體包含與不同抗體之異源Fc部分可操作地連接或以其他方式融合的抗體之抗原結合片段。在某些實施例中，Fc域或異源Fc域來源於人類。在某些實施例中，Fc域或異源Fc域來源於小鼠。在其他實施例中，異源Fc域可來自親本抗體之不同Ig類別，包括IgA（包括子類IgA1及IgA2）、IgD、IgE、IgG（包括子類IgG1、IgG2、IgG3及IgG4）及IgM。在其他實施例中，異源Fc域可包含來自不同Ig類別中之一或多者的CH2域及CH3域。如上文關於人源化抗體所指出，嵌合抗體之抗原結合片段可僅包含本文所述之抗體之CDR中之一或多者（例如本文所述之抗體之1、2、3、4、5或6個CDR），或可包含整個可變域（VL、VH或兩者）。In certain embodiments, the antibody is a "chimeric" antibody. In this regard, a chimeric antibody comprises an antigen-binding fragment of an antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the Fc domain or heterologous Fc domain is of human origin. In certain embodiments, the Fc domain or heterologous Fc domain is of mouse origin. In other embodiments, the heterologous Fc domain may be from different Ig classes of the parent antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In other embodiments, the heterologous Fc domain may include a CH2 domain and a CH3 domain from one or more of the different Ig classes. As noted above with respect to humanized antibodies, the antigen-binding fragment of a chimeric antibody may comprise only one or more of the CDRs of an antibody described herein (e.g., 1, 2, 3, 4, 5, or 6 CDRs of an antibody described herein), or may comprise an entire variable domain (VL, VH, or both).

術語「結合」係指兩個分子之間由於例如共價、靜電、疏水性及離子性及/或氫鍵相互作用，包括諸如鹽橋及水橋之相互作用而產生的直接締合。The term "binding" refers to the direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic and ionic and/or hydrogen bonding interactions, including interactions such as salt bridges and water bridges.

「編碼序列」意謂促成基因之多肽產物之編碼的任何核酸序列。相比之下，術語「非編碼序列」係指不直接促成基因之多肽產物之編碼的任何核酸序列。"Coding sequence" means any nucleic acid sequence that contributes to the coding of the polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not directly contribute to the coding of the polypeptide product of a gene.

在通篇本說明書中，除非上下文另有要求，否則字組「包含（comprise）」或諸如「包含（comprises）」或「包含（comprising）」之變化形式應理解為暗示包括所述元素或整數、或元素或整數群組，但不排除任何其他元素或整數、或元素或整數群組。Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of stated elements or integers, or groups of elements or integers, but not the exclusion of any other elements or integers, or groups of elements or integers.

「由……組成」意欲包括且限於片語「由……組成」中間的任何事物。因此，片語「由……組成」指示所列元素為所需或必選的，且不可存在其他元素。「基本上由……組成」意謂包括該片語中間所列之任何元素，且限於不干擾或促進所列元素在本發明中所指定之活性或作用的其他元素。因此，片語「基本上由……組成」指示所列元素為所需或必選的，但其他元素為視情況存在的且視其是否實質上影響所列元素之活性或作用而定可存在或可不存在。"Consisting of is intended to include and be limited to anything in between the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and no other elements may be present. "Consisting essentially of is meant to include any of the elements listed in the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action of the listed elements as specified in the present invention. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but other elements are present as appropriate and may or may not be present depending on whether or not they materially affect the activity or action of the listed elements.

在抗體之背景下，術語「效應功能」或「ADCC效應功能」係指抗體與免疫系統之其他臂接合之能力，包括例如活化經典補體路徑或通過接合Fc受體。補體依賴性路徑主要由C1q與具有叢集抗體Fc域之C1複合物的相互作用驅動。抗體依賴性細胞毒性（ADCC）主要由效應細胞（自然殺手細胞、巨噬細胞、單核球及嗜伊紅血球）之表面上的Fc受體（FcR）結合於本身結合至目標細胞之IgG之Fc區的相互作用而驅動。Fc受體（FcR）為將抗體介導之（體液）免疫反應與細胞效應功能相連接的關鍵免疫調節受體。已鑑別用於全部類別之免疫球蛋白之受體，包括FcγR（IgG）、FcεRI（IgE）、FcαRI（IgA）、FcμR（IgM）及FcδR（IgD）。在白血球上發現至少三種類別之用於人類IgG的受體：CD64（FcγRI）、CD32（FcγRIIa、FcγRIIb及FcγRIIc）及CD16（FcγRIIIa及FcγRIIIb）。FcγRI分類為高親和力受體（奈莫耳濃度範圍K_D），而FcγRII及FcγRIII為低至中等親和力（微莫耳濃度範圍K_D）。Fc結合後，觸發信號傳導路徑，其引起諸如溶解酶、穿孔蛋白、顆粒酶及腫瘤壞死因子之各種物質之分泌，此介導目標細胞之破壞。ADCC效應功能水平視人類IgG亞型而變。儘管此依賴於同種異型及特定FcvR，但簡言之，人類IgG1及IgG3之ADCC效應功能「高」，且IgG2及IgG4之ADCC效應功能「低」。In the context of antibodies, the term "effector function" or "ADCC effector function" refers to the ability of an antibody to engage other arms of the immune system, including, for example, activation of the classical complement pathway or by engaging Fc receptors. The complement-dependent pathway is primarily driven by the interaction of C1q with the C1 complex with the Fc domain of clustered antibodies. Antibody-dependent cytotoxicity (ADCC) is primarily driven by the interaction of Fc receptors (FcRs) on the surface of effector cells (natural killer cells, macrophages, monocytes and eosinophils) bound to the Fc region of IgG that is itself bound to the target cell. Fc receptors (FcRs) are key immunoregulatory receptors that link antibody-mediated (humoral) immune responses to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcγR (IgG), FcεRI (IgE), FcαRI (IgA), FcμR (IgM), and FcδR (IgD). At least three classes of receptors for human IgG are found on leukocytes: CD64 (FcγRI), CD32 (FcγRIIa, FcγRIIb, and FcγRIIc), and CD16 (FcγRIIIa and FcγRIIIb). FcγRI is classified as a high-affinity receptor (nanomolar range_KD ), while FcγRII and FcγRIII are low to intermediate affinity (micromolar range_KD ). Upon Fc binding, signaling pathways are triggered that result in the secretion of various substances such as lytic enzymes, perforins, granzymes, and tumor necrosis factor, which mediate the destruction of target cells. The level of ADCC effector function varies depending on the human IgG subtype. Although this depends on the allotype and the specific FcvR, in short, the ADCC effector function of human IgG1 and IgG3 is "high", and the ADCC effector function of IgG2 and IgG4 is "low".

術語「不含內毒素」或「實質上不含內毒素」一般係指組合物、溶劑及/或容器含有至多痕量（例如對個體無臨床上有害生理作用的量）之內毒素且較佳不可偵測量之內毒素。內毒素為與某些微生物（諸如細菌，典型地為革蘭氏陰性細菌）相關之毒素，但內毒素可發現於革蘭氏陽性細菌中，諸如單核球增多性李氏菌（Listeria monocytogenes）。最普遍之內毒素為在各種革蘭氏陰性細菌之外膜中發現的脂多醣（LPS）或脂寡醣（LOS），且其代表使得此等細菌能夠致病之重要病原性特徵。少量內毒素在人類中可引起發熱，降低血壓且活化發炎及凝血以及其他不良生理作用。The term "endotoxin-free" or "substantially endotoxin-free" generally refers to compositions, solvents, and/or containers that contain no more than trace amounts (e.g., amounts that have no clinically deleterious physiological effects on an individual) of endotoxins, and preferably no detectable amounts of endotoxins. Endotoxins are toxins associated with certain microorganisms, such as bacteria, typically gram-negative bacteria, but endotoxins can be found in gram-positive bacteria, such asListeria monocytogenes . The most common endotoxins are lipopolysaccharides (LPS) or lipolyoligosaccharides (LOS) found in the outer membrane of various gram-negative bacteria and represent important pathogenic characteristics that enable these bacteria to cause disease. Small amounts of endotoxins can cause fever, lower blood pressure, and activate inflammation and coagulation, as well as other adverse physiological effects in humans.

因此，在醫藥生產中，常常需要自藥品及/或藥物容器中移除大部分或所有痕量內毒素，因為即使少量亦可能在人類中產生有害影響。去熱原烘箱可用於達成此目的，因為典型地需要超過300℃之溫度來使大部分內毒素分解。舉例而言，基於諸如注射器或小瓶之主要包裝材料，250℃之玻璃溫度與30分鐘之保存時間的組合往往足以達成內毒素水平下降3 log。涵蓋其他去除內毒素之方法，包括例如層析法及過濾法，依本文所描述及此項技術中已知。Therefore, in pharmaceutical production, it is often necessary to remove most or all trace amounts of endotoxins from a drug product and/or drug container, since even small amounts may produce deleterious effects in humans. A depyrogenation oven may be used to achieve this purpose, since temperatures in excess of 300°C are typically required to decompose most endotoxins. For example, based on primary packaging materials such as syringes or vials, a glass temperature of 250°C combined with a 30 minute hold time is often sufficient to achieve a 3 log reduction in endotoxin levels. Other methods of removing endotoxins are contemplated, including, for example, chromatography and filtration, as described herein and known in the art.

可使用此項技術中已知之常規技術偵測內毒素。舉例而言，鱟（Limulus）變形細胞溶胞物分析利用來自鱟（horseshoe crab）之血液，該分析為一種非常靈敏的用於偵測內毒素之存在的分析。在此測試中，極低水平之LPS可引起鱟溶胞物之可偵測凝集，此係因為強大的酶級聯將此反應放大。內毒素亦可藉由酶聯免疫吸附分析（ELISA）來定量。為了實現實質上不含內毒素，每毫克活性化合物之內毒素含量可小於約0.001、0.005、0.01、0.02、0.03、0.04、0.05、0.06、0.08、0.09、0.1、0.5、1.0、1.5、2、2.5、3、4、5、6、7、8、9或10 EU。通常，1 ng脂多醣（LPS）對應於約1-10 EU。Endotoxins can be detected using conventional techniques known in the art. For example, the Limulus morpholysate assay, which utilizes blood from horseshoe crabs, is a very sensitive assay for detecting the presence of endotoxins. In this test, very low levels of LPS can cause detectable agglutination of the horseshoe crab lysate because a powerful enzyme cascade amplifies this reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). To achieve substantial absence of endotoxin, the endotoxin content per mg of active compound may be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9 or 10 EU. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to about 1-10 EU.

術語「抗原決定基」包括能夠與免疫球蛋白或T細胞受體特異性結合之任何決定子，較佳為多肽決定子。抗原決定基包括由抗體結合之抗原區。在某些實施例中，抗原決定基決定子包括分子之化學活性表面群組（諸如胺基酸、糖側鏈、磷醯基或磺醯基），且在某些實施例中，其可具有特定三維結構特徵及/或特定電荷特徵。抗原決定基可與抗原之一級結構，例如IL-18BP多肽相鄰或不相鄰。在特定實施例中，抗原決定基包含、由以下組成或基本上由以下組成：本文所述之參考序列（參見例如表B1）或目標分子之約、至少約或不超過約3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20個連續胺基酸（亦即線性抗原決定基）或非連續胺基酸（亦即構形抗原決定基）。The term "antigenic determinant" includes any determinant, preferably a polypeptide determinant, that is capable of specifically binding to an immunoglobulin or T cell receptor. Antigenic determinants include regions of an antigen that are bound by an antibody. In certain embodiments, an antigenic determinant includes a chemically active surface group of a molecule (such as an amino acid, a sugar side chain, a phospho group, or a sulfonyl group), and in certain embodiments, it may have a specific three-dimensional structural characteristic and/or a specific charge characteristic. An antigenic determinant may be adjacent or non-adjacent to the primary structure of an antigen, such as an IL-18BP polypeptide. Incertain embodiments, an antigenic determinant comprises, consists of, or consists essentially of about, at least about, or no more than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids (i.e., a linear antigenicdeterminant ) or non-consecutive amino acids (i.e., a conformational antigenic determinant) of a reference sequence described herein (see, e.g., Table B1) or a target molecule.

「抗原決定基」包括抗原或其他大分子之能夠形成結合相互作用之部分，其與結合蛋白之可變區結合袋相互作用。此類結合相互作用可體現為與CDR之一或多個胺基酸殘基之分子間接觸。抗原結合可涉及CDR3或CDR3對。抗原決定基可為線性肽序列（亦即「連續」）或可由非相鄰胺基酸序列（亦即「構形」或「不連續」）構成。結合蛋白可識別一或多個胺基酸序列；因此，抗原決定基可界定超過一種不同胺基酸序列。由結合蛋白識別之抗原決定基可藉由熟習此項技術者熟知之肽定位及序列分析技術來確定。「隱藏抗原決定基」或「隱藏結合位點」係蛋白質序列之一種抗原決定基或結合位點，其未暴露或實質上在未經修飾之多肽內避免被識別，但能夠由變性或經蛋白質水解之多肽之結合蛋白識別。在未經修飾之多肽結構中未暴露或僅僅部分暴露之胺基酸序列為潛在的隱藏抗原決定基。若抗原決定基未暴露或僅僅部分暴露，則可能其埋在多肽內部。候選的隱藏抗原決定基可例如藉由檢查未經修飾之多肽之三維結構來鑑別。"Antigenic determinants" include portions of antigens or other macromolecules that are capable of forming binding interactions that interact with the variable region binding pocket of a binding protein. Such binding interactions may be manifested as intermolecular contacts with one or more amino acid residues of a CDR. Antigen binding may involve a CDR3 or a CDR3 pair. An antigenic determinant may be a linear peptide sequence (i.e., "continuous") or may be composed of non-adjacent amino acid sequences (i.e., "conformational" or "discontinuous"). A binding protein may recognize one or more amino acid sequences; therefore, an antigenic determinant may define more than one different amino acid sequence. The antigenic determinant recognized by a binding protein may be determined by peptide mapping and sequence analysis techniques well known to those skilled in the art. A "hidden antigenic determinant" or "hidden binding site" is an antigenic determinant or binding site in a protein sequence that is not exposed or substantially avoids recognition in an unmodified polypeptide, but can be recognized by binding proteins of a denatured or proteolytic polypeptide. Amino acid sequences that are not exposed or only partially exposed in the unmodified polypeptide structure are potential hidden antigenic determinants. If an antigenic determinant is not exposed or only partially exposed, it is likely buried within the polypeptide. Candidate hidden antigenic determinants can be identified, for example, by examining the three-dimensional structure of the unmodified polypeptide.

術語「半最大有效濃度」或「EC₅₀」係指如本文所述之藥劑（例如抗體）之濃度，在此濃度下該藥劑誘導基線與在一定指定暴露時間之後的最大值之間的半途的反應；分級劑量反應曲線之EC₅₀因此表示觀測到其最大效應之50%的化合物濃度。EC50亦表示獲得50%活體內最大效應所需的血漿濃度。類似地，「EC₉₀」係指觀測到90%其最大效應之藥劑或組合物的濃度。「EC₉₀」可由「EC₅₀」及希爾斜率（Hill slope）計算，或其可使用此項技術中之常識直接自資料確定。在一些實施例中，藥劑（例如抗體）之EC₅₀小於約0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、40、50、60、70、80、90、100、200或500 nM。在一些實施例中，藥劑將具有約1 nM或更小之EC₅0值。The term "half maximal effective concentration" or "_EC50 " refers to the concentration of an agent (e.g., an antibody) as described herein, at which the agent induces a response halfway between baseline and maximum after a certain specified exposure time; the_EC50 of a graded dose-response curve therefore represents the concentration of the compound at which 50% of its maximal effect is observed. The EC50 also represents the plasma concentration required to obtain 50% of the maximal effect in vivo. Similarly, the "_EC90 " refers to the concentration of an agent or composition at which 90% of its maximal effect is observed. The "_EC90 " can be calculated from the "_EC50 " and the Hill slope, or it can be determined directly from the data using common sense in the art. In some embodiments, the_EC50 of an agent (e.g., an antibody) is less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 nM. In some embodiments, an agent will have an_EC50 value of about 1 nM or less.

「免疫反應」意謂起源於免疫系統之任何免疫反應，包括來自細胞及體液、先天性及適應性免疫系統之反應。例示性細胞免疫細胞包括例如淋巴球、巨噬細胞、T細胞、B細胞、NK細胞、嗜中性球、嗜伊紅血球、樹突狀細胞、肥大細胞、單核球及其所有子集。細胞反應包括例如效應功能、細胞介素釋放、吞噬作用、胞葬作用、易位、遷移、增殖、分化、活化、抑制、細胞-細胞相互作用、細胞凋亡等。體液反應包括例如IgG、IgM、IgA、IgE、反應及其對應效應功能。"Immune response" means any immune response originating from the immune system, including responses from cellular and humoral, innate and adaptive immune systems. Exemplary cellular immune cells include, for example, lymphocytes, macrophages, T cells, B cells, NK cells, neutrophils, eosinophils, dendritic cells, mast cells, monocytes, and all subsets thereof. Cellular responses include, for example, effector functions, interleukin release, phagocytosis, efferocytosis, translocation, migration, proliferation, differentiation, activation, inhibition, cell-cell interactions, apoptosis, and the like. Humoral responses include, for example, IgG, IgM, IgA, IgE, responses, and their corresponding effector functions.

諸如抗體之藥劑之「半衰期」可指相對於投與至生物體之血清或組織中時之藥理活性、生理活性或其他活性，或相對於任何其他界定之時間點，藥劑喪失其此類活性之一半所耗費之時間。「半衰期」亦可指相對於投與至生物體之血清或組織中時的量或濃度，或相對於任何其他界定之時間點，藥劑之量或濃度減少達投與至生物體之血清或組織中之起始量的一半時所耗費的時間。半衰期可在血清及/或任一或多個所選組織中量測。The "half-life" of an agent such as an antibody may refer to the time it takes for an agent to lose half of its pharmacological, physiological or other activity relative to the amount or concentration when administered to the serum or tissue of an organism, or relative to any other defined time point. "Half-life" may also refer to the time it takes for the amount or concentration of an agent to decrease to half of the initial amount in the serum or tissue of an organism, relative to the amount or concentration when administered to the serum or tissue of an organism, or relative to any other defined time point. Half-life may be measured in serum and/or any one or more selected tissues.

術語「調節」及「改變」包括相對於對照，通常統計學上顯著或生理學上顯著之量或程度的「增加」、「增強」或「刺激」以及「降低」、「減少」或「抑制」。「增加」、「刺激」或「增強」之量通常為「統計學上顯著」之量，且可包括由無組合物（例如不存在藥劑）或對照組合物產生之量的1.1、1.2、1.5、2、3、4、5、6、7、8、9、10、15、20、30、40、50、60、70、80、90、100或更多倍（例如500、1000倍）（包括其間的所有整數及範圍，例如1.5、1.6、1.7、1.8倍等）增加。「降低」或「減少」或「抑制」之量通常為「統計學上顯著」之量，且可包括由無組合物（例如不存在藥劑）或對照組合物產生之量的1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或100%降低（包括其間的所有整數及範圍）。本文描述比較及「統計學上顯著」之量的實例。The terms "modulate" and "alter" include "increases", "enhancements" or "stimulations" as well as "decreases", "reductions" or "inhibitions" relative to a control, usually by a statistically significant or physiologically significant amount or degree. The amount of "increase", "stimulation" or "enhancement" is usually a "statistically significant" amount and can include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more times (e.g., 500, 1000 times) (including all integers and ranges therebetween, e.g., 1.5, 1.6, 1.7, 1.8 times, etc.) over the amount produced by no composition (e.g., the absence of the agent) or a control composition. An amount that is "reduced" or "reduced" or "inhibited" is generally a "statistically significant" amount and may include 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% reduction (including all integers and ranges therebetween) of the amount produced by no composition (e.g., in the absence of the agent) or a control composition. Examples of comparisons and "statistically significant" amounts are described herein.

術語「多肽」、「蛋白質」及「肽」可互換使用，且意謂不限於任何特定長度之胺基酸聚合物。術語「酶」包括多肽或蛋白質催化劑。該等術語包括修飾，諸如豆蔻醯化、硫酸化、糖基化、磷酸化及信號序列之添加或缺失。術語「多肽」或「蛋白質」意謂一或多個胺基酸鏈，其中各鏈包含由肽鍵共價連接之胺基酸，且其中該多肽或蛋白質可包含複數個由肽鍵非共價及/或共價連接在一起之鏈，其具有原生蛋白質（亦即藉由天然存在且特別是非重組細胞產生之蛋白質）或由基因工程改造或重組細胞產生之蛋白質的序列，且包含具有原生蛋白質之胺基酸序列的分子，或對原生序列之一或多個胺基酸進行缺失、添加及/或取代而得之分子。在某些實施例中，多肽為「重組」多肽，由包含一或多個重組DNA分子之重組細胞產生，該等重組DNA分子通常由異源聚核苷酸序列或聚核苷酸序列之組合（其不應另外發現於細胞中）製成。The terms "polypeptide", "protein" and "peptide" are used interchangeably and are not limited to amino acid polymers of any particular length. The term "enzyme" includes polypeptide or protein catalysts. These terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The term "polypeptide" or "protein" means one or more amino acid chains, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein the polypeptide or protein may comprise a plurality of chains non-covalently and/or covalently linked by peptide bonds, having the sequence of a native protein (i.e., a protein produced by naturally occurring and particularly non-recombinant cells) or a protein produced by genetic engineering or recombinant cells, and including molecules having the amino acid sequence of a native protein, or molecules resulting from deletions, additions and/or substitutions of one or more amino acids in the native sequence. In certain embodiments, the polypeptide is a "recombinant" polypeptide, produced by a recombinant cell comprising one or more recombinant DNA molecules, which are typically made from a heterologous polynucleotide sequence or combination of polynucleotide sequences that should not otherwise be found in the cell.

術語「聚核苷酸」及「核酸」包括mRNA、RNA、cRNA、cDNA及DNA。該術語通常係指具有至少10個鹼基長度之核苷酸聚合物形式（核糖核苷酸或去氧核苷酸）或任一種類型的核苷酸之經修飾形式。該術語包括DNA之單股及雙股形式。術語「經分離之DNA」及「經分離之聚核苷酸」及「經分離之核酸」係指經分離而不含特定物種之全基因體DNA的分子。因此，編碼多肽之經分離之DNA區段係指含有一或多個編碼序列但實質上自DNA區段所獲自之物種之全基因體DNA分離出來或經純化而不含全基因體DNA的DNA區段。亦包括非編碼聚核苷酸（例如引子、探針、寡核苷酸），其不編碼多肽。亦包括重組載體，包括例如表現載體、病毒載體、質體、黏質體、噬菌粒、噬菌體、病毒及其類似物。The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA and DNA. The terms generally refer to a polymeric form of nucleotides (ribonucleotides or deoxynucleotides) of at least 10 bases in length or a modified form of either type of nucleotide. The terms include single-stranded and double-stranded forms of DNA. The terms "isolated DNA" and "isolated polynucleotide" and "isolated nucleic acid" refer to molecules that have been isolated and are free of the whole genome DNA of a particular species. Thus, an isolated DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences but has been isolated or purified from substantially the whole genome DNA of the species from which the DNA segment was obtained. Non-coding polynucleotides (e.g., primers, probes, oligonucleotides) are also included, which do not encode a polypeptide. Also included are recombinant vectors, including, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, bacteriophages, viruses, and the like.

額外編碼或非編碼序列可（但未必）存在於本文所述之聚核苷酸內，且聚核苷酸可（但未必）連接至其他分子及/或支撐材料。因此，聚核苷酸或可表現之聚核苷酸無論編碼序列本身之長度如何，均可與例如表現控制序列之其他序列組合。Additional coding or non-coding sequences may (but need not) be present in the polynucleotides described herein, and polynucleotides may (but need not) be linked to other molecules and/or support materials. Thus, a polynucleotide or an expressible polynucleotide, regardless of the length of the coding sequence itself, may be combined with other sequences, such as expression control sequences.

「表現控制序列」包括核酸或對應胺基酸之調節序列，諸如啟動子、前導子、強化子、內含子、RNA或DNA結合蛋白之識別模體、聚腺苷酸化信號、終止子、內部核糖體進入位點（IRES）、分泌信號、亞細胞定位信號及其類似物，該等調節序列能夠影響編碼序列在宿主細胞中之轉錄或轉譯或者亞細胞或細胞位置。例示性表現控制序列描述於Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)。"Expression control sequences" include nucleic acid or corresponding amino acid regulatory sequences, such as promoters, leaders, enhancers, introns, recognition motifs for RNA or DNA binding proteins, polyadenylation signals, terminators, internal ribosome entry sites (IRES), secretion signals, subcellular localization signals and the like, which are capable of affecting the transcription or translation of a coding sequence in a host cell or the subcellular or cellular location. Exemplary expression control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

「啟動子」為能夠結合細胞中之RNA聚合酶且起始下游（3'方向）編碼序列之轉錄的DNA調節區。如本文所用，啟動子序列由轉錄起始位點限定在其3'端，且在上游（5'方向）延伸，以包括起始超過背景之可偵測水平之轉錄所需的最小數目之鹼基或元件。轉錄起始位點（宜藉由用核酸酶S1定位而界定）可見於啟動子序列內，以及蛋白質結合域（共同序列）負責RNA聚合酶之結合。真核啟動子可經常但未必始終含有「TATA」盒及「CAT」盒。除-10及-35共同序列之外，原核生物啟動子亦含有夏因-達爾加諾序列（Shine-Dalgarno sequence）。A "promoter" is a DNA regulatory region that is capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. As used herein, a promoter sequence is defined at its 3' end by a transcription start site and extends upstream (5' direction) to include the minimum number of bases or elements required to initiate transcription at detectable levels above background. A transcription start site (preferably defined by localization with nuclease S1) can be found within the promoter sequence, as well as a protein binding domain (consensus sequence) responsible for the binding of RNA polymerase. Eukaryotic promoters may often, but not always, contain a "TATA" box and a "CAT" box. In addition to the -10 and -35 consensus sequences, prokaryotic promoters also contain a Shine-Dalgarno sequence.

來自多種不同來源的大量啟動子，包括組成型、誘導型和阻遏型啟動子為此項技術中所熟知。代表性來源包括例如病毒、哺乳動物、昆蟲、植物、酵母及細菌細胞類型，且來自此等來源之適合啟動子容易獲得，或可基於可在線上或例如自諸如ATCC之寄存處以及其他商業或個人來源公開獲得之序列以合成方式製得。啟動子可為單向（亦即在一個方向上起始轉錄）或雙向（亦即在3'或5'方向上起始轉錄）。啟動子之非限制性實例包括例如T7細菌表現系統、pBAD（araA）細菌表現系統、巨細胞病毒（CMV）啟動子、SV40啟動子、RSV啟動子。誘導型啟動子包括：Tet系統（美國專利5,464,758及5,814,618）；蛻皮激素誘導型系統（No等人, Proc. Natl. Acad. Sci. (1996) 93 (8): 3346-3351；T-RExTM系統（Invitrogen Carlsbad。CA）、LacSwitch®（Stratagene（San Diego，CA）及Cre-ERT他莫昔芬誘導型重組酶系統（Indra等人, Nuc. Acid. Res. (1999) 27 (22): 4324-4327；Nuc. Acid. Res. (2000) 28 (23): e99；美國專利第7,112,715號；以及Kramer及Fussenegger, Methods Mol. Biol. (2005) 308: 123-144）或此項技術中已知的適合於表現於所需細胞中之任何啟動子。A large number of promoters from a variety of different sources, including constitutive, inducible and repressible promoters are well known in the art. Representative sources include, for example, viral, mammalian, insect, plant, yeast and bacterial cell types, and suitable promoters from these sources are readily available or can be made synthetically based on sequences that are publicly available online or, for example, from depositories such as the ATCC and other commercial or personal sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in the 3' or 5' direction). Non-limiting examples of promoters include, e.g., T7 bacterial expression system, pBAD (araA) bacterial expression system, cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter. Inducible promoters include: the Tet system (U.S. Patents 5,464,758 and 5,814,618); the epidermal growth factor-induced system (No et al., Proc. Natl. Acad. Sci. (1996) 93 (8): 3346-3351; the T-RExTM system (Invitrogen Carlsbad, CA), LacSwitch® (Stratagene (San Diego, CA) and the Cre-ERT tamoxifen-induced recombinase system (Indra et al., Nuc. Acid. Res. (1999) 27 (22): 4324-4327; Nuc. Acid. Res. (2000) 28 (23): e99; U.S. Patent No. 7,112,715; and Kramer and Fussenegger, Methods Mol. Biol. (2005) 308: 123-144) or any promoter known in the art suitable for expression in the desired cells.

「可表現聚核苷酸」包括cDNA、RNA、mRNA或包含至少一個編碼序列且視情況包含至少一個表現控制序列，例如轉錄及/或轉譯調控元件且在引入至細胞（例如個體中之細胞）中時可表現編碼多肽之其他聚核苷酸。"Expressible polynucleotides" include cDNA, RNA, mRNA, or other polynucleotides that comprise at least one coding sequence and, optionally, at least one expression control sequence, such as transcriptional and/or translational regulatory elements, and that can express a polypeptide encoding a polypeptide when introduced into a cell (e.g., a cell in a subject).

可用於遞送可表現聚核苷酸之多種病毒載體包括腺病毒載體、疱疹病毒載體、痘瘡病毒載體、腺相關病毒（AAV）載體及反轉錄病毒載體。在一些情況下，反轉錄病毒載體為鼠類或禽類反轉錄病毒之衍生物，或為慢病毒載體。可插入單個外源基因之反轉錄病毒載體之實例包括但不限於：莫洛尼鼠類白血病病毒（Moloney murine leukemia virus，MoMuLV）、哈維鼠類肉瘤病毒（Harvey murine sarcoma virus，HaMuSV）、鼠類乳房腫瘤病毒（murine mammary tumor virus，MuMTV）、SIV、BIV、HIV及勞氏肉瘤病毒（Rous Sarcoma Virus，RSV）。多種額外反轉錄病毒載體可併入有多種基因。所有此等載體可轉移或併入針對可選擇標記之基因以使得可鑑別及產生經轉導細胞。例如藉由將相關多肽序列以及編碼特定目標細胞上之受體之配位體的另一基因插入至病毒載體中，可使載體對目標具有特異性。可藉由插入例如編碼蛋白質之聚核苷酸而使反轉錄病毒載體對目標具有特異性。說明性靶向可藉由使用靶向反轉錄病毒載體之抗體來實現。熟習此項技術者將已知或可容易在不進行過多實驗之情況下確定可插入至反轉錄病毒基因體中以允許反轉錄病毒載體之目標特異性遞送的特定聚核苷酸序列。A variety of viral vectors that can be used to deliver expressible polynucleotides include adenoviral vectors, herpes virus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, and retroviral vectors. In some cases, the retroviral vector is a derivative of a murine or avian retrovirus, or is a lentiviral vector. Examples of retroviral vectors that can be inserted with a single foreign gene include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous Sarcoma Virus (RSV). A variety of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker to allow identification and generation of transduced cells. The vector can be made specific for a target, for example, by inserting into the viral vector a polypeptide sequence of interest and another gene encoding a ligand for a receptor on a specific target cell. Retroviral vectors can be made specific for a target by inserting, for example, a polynucleotide encoding a protein. Illustrative targeting can be achieved by the use of antibodies that target retroviral vectors. Those skilled in the art will know or can readily determine without undue experimentation the specific polynucleotide sequences that can be inserted into the retroviral genome to allow target-specific delivery of a retroviral vector.

在特定實施例中，可表現聚核苷酸為經修飾之RNA或經修飾之mRNA聚核苷酸，例如非天然存在之RNA類似物。在某些實施例中，經修飾之RNA或mRNA多肽包含一或多個經修飾之或非天然鹼基，例如除腺嘌呤（A）、鳥嘌呤（G）、胞嘧啶（C）、胸腺嘧啶（T）及/或尿嘧啶（U）外之核苷酸鹼基。在一些實施例中，經修飾之mRNA包含一或多個經修飾或非天然之核苷酸間鍵。用於遞送所編碼之治療多肽的可表現RNA聚核苷酸描述於例如以下中：Kormann等人, Nat Biotechnol. 29:154-7, 2011；以及美國申請案第2015/0111248號、第2014/0243399號、第2014/0147454號及第2013/0245104號，該等文獻以全文引用之方式併入。In certain embodiments, the polynucleotide may be a modified RNA or modified mRNA polynucleotide, such as a non-naturally occurring RNA analog. In certain embodiments, the modified RNA or mRNA polypeptide comprises one or more modified or non-natural bases, such as nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T) and/or uracil (U). In some embodiments, the modified mRNA comprises one or more modified or non-natural internucleotide bonds. Expressible RNA polynucleotides for delivery of encoded therapeutic polypeptides are described, for example, in Kormann et al., Nat Biotechnol. 29:154-7, 2011; and U.S. Application Nos. 2015/0111248, 2014/0243399, 2014/0147454, and 2013/0245104, which are incorporated by reference in their entirety.

本文中所提及之術語「經分離之」多肽或蛋白質意謂本發明蛋白質（1）不含通常在自然界中與之一起存在之至少一些其他蛋白質，（2）基本上不含來自同一來源（例如同一物種）之其他蛋白質，（3）由來自不同物種之細胞表現，（4）已自至少約50百分比的聚核苷酸、脂質、碳水化合物或在自然界中與之結合之其他物質分離，（5）不（藉由共價或非共價相互作用）與蛋白質之部分結合，「經分離之蛋白質」在自然界中與蛋白質之部分結合，（6）可（藉由共價或非共價相互作用）與不在自然界中與之結合的多肽操作地結合，或（7）在自然界中不存在。此類經分離之蛋白質可由基因體DNA、cDNA、mRNA或其他RNA編碼，或可為合成來源，或其任何組合。在某些實施例中，經分離之蛋白質實質上不含其天然環境中發現的將干擾其使用（治療、診斷、預防、研究或其他）的蛋白質或多肽或其他污染物。As used herein, the term "isolated" polypeptide or protein means that the protein of the invention (1) is free of at least some other proteins with which it is normally found in nature, (2) is substantially free of other proteins from the same source (e.g., the same species), (3) is expressed by cells from a different species, (4) has been separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other substances with which it is associated in nature, (5) is not associated (by covalent or non-covalent interactions) with a portion of a protein, an "isolated protein" is associated with a portion of a protein in nature, (6) can be operatively associated (by covalent or non-covalent interactions) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA or other RNA, or may be of synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, preventive, research or otherwise).

在某些實施例中，可限定任何給定藥劑（例如抗體）在組合物中之「純度」。舉例而言，某些組合物可包含如例如且決不限於藉由高效液相層析（HPLC）量測以蛋白質或重量-重量計至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%純（包括其間所有小數及範圍）的藥劑，HPLC為一種在生物化學及分析化學中常用於分離、鑑別及定量化合物之熟知管柱層析形式。In certain embodiments, the "purity" of any given agent (e.g., an antibody) in a composition can be defined. For example, certain compositions can include an agent that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure (including all decimals and ranges therebetween) on a protein or weight-weight basis as measured, for example and without limitation, by high performance liquid chromatography (HPLC), a well-known form of column analysis commonly used in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

術語「參考序列」一般係指與另一序列進行比較之核酸編碼序列或胺基酸序列。以參考序列之形式包括本文所述之所有多肽及聚核苷酸序列，包括藉由名稱所描述之彼等序列及描述於表及序列表中之彼等序列。The term "reference sequence" generally refers to a nucleic acid coding sequence or an amino acid sequence to which another sequence is compared. All polypeptide and polynucleotide sequences described herein are included in the form of reference sequences, including those sequences described by name and those sequences described in the Tables and Sequence Listings.

某些實施例包括本文所述之多肽（例如抗體）之生物活性「變異體」及「片段」以及編碼其之聚核苷酸。「變異體」相對於參考多肽或聚核苷酸（參見例如表及序列表）含有一或多個取代、添加、缺失及/或插入。變異多肽或聚核苷酸包含與如本文所述之參考序列具有至少約50%、55%、60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更大序列一致性或相似性或同源性的胺基酸或核苷酸序列，且實質上保持彼參考序列之活性。亦包括由參考序列組成或與參考序列之不同之處在於1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、30、40、50、60、70、80、90、100、110、120、130、140、150或更多個胺基酸或核苷酸添加、缺失、插入或取代且實質上保留彼參考序列之活性的序列。在某些實施例中，添加或缺失包括C端及/或N端添加及/或缺失。Certain embodiments include biologically active "variants" and "fragments" of the polypeptides (e.g., antibodies) described herein and polynucleotides encoding the same. "Variants" contain one or more substitutions, additions, deletions, and/or insertions relative to a reference polypeptide or polynucleotide (see, e.g., Tables and Sequence Listings). Variant polypeptides or polynucleotides comprise amino acid or nucleotide sequences having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or similarity or homology to a reference sequence as described herein, and substantially retain the activity of that reference sequence. Also included are sequences consisting of a reference sequence or differing from a reference sequence in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acids or nucleotides added, deleted, inserted or substituted and substantially retaining the activity of the reference sequence. In certain embodiments, additions or deletions include C-terminal and/or N-terminal additions and/or deletions.

如本文所用，術語「序列一致性」或例如包含「與……至少50%一致的序列」係指序列在比較窗內以逐核苷酸計或以逐胺基酸計一致的程度。因此，「序列一致性百分比」可藉由以下來計算：在比較窗內比較兩個最佳比對序列，確定在兩個序列中出現一致核酸鹼基（例如A、T、C、G、I）或一致胺基酸殘基（例如Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys及Met）的位置數以得到匹配位置數，用匹配位置數除以比較窗中之位置總數（亦即窗大小），及將結果乘以100以得到序列一致性百分比。用於比對比較窗之最佳序列比對可藉由電腦化實施演算法（Wisconsin Genetics套裝軟體7.0版中之GAP、BESTFIT、FASTA及TFASTA，Genetics Computer Group，575 Science Drive Madison, Wis., USA）或藉由進行檢驗及由所選各種方法中之任一者產生之最佳比對（亦即在比較窗內產生最高同源性百分比）來進行。亦可參考BLAST程式族，如例如Altschul等人, Nucl. Acids Res. 25:3389, 1997所揭示。As used herein, the term "sequence identity" or, for example, "a sequence that is at least 50% identical to..." refers to the degree to which a sequence is identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis within a comparison window. Thus, "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences within a comparison window, determining the number of positions where identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appear in the two sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The best sequence alignment for comparing the comparison window can be performed by computerized implementation algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics software package version 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by testing and the best alignment (i.e., producing the highest homology percentage in the comparison window) produced by any one of the selected various methods. Also reference can be made to the BLAST family of programs, such as disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

術語「溶解度」係指本文提供之藥劑（例如抗體）溶解在液體溶劑中且形成均質溶液之特性。溶解度通常表示為濃度，以每單位體積溶劑之溶質質量（每公斤溶劑之溶質公克數、g/dL（100 mL）、mg/ml等）、莫耳濃度、重量莫耳濃度、莫耳分數或濃度之其他類似描述計。在包括溫度、壓力、pH值及溶劑性質之規定條件下，每份量之溶劑可溶解之溶質的最大平衡量為該溶質在該溶劑中之溶解度。在某些實施例中，在生理pH或其他pH下，例如在pH 5.0、pH 6.0、pH 7.0、pH 7.4、pH 7.6、pH 7.8或pH 8.0（例如約pH 5-8）下量測溶解度。在某些實施例中，在水或諸如PBS或NaCl（有或無NaPO4之情況下）之生理緩衝液中量測溶解度。在具體實施例中，在相對較低pH（例如pH 6.0）及相對較高濃度鹽（例如500 mM NaCl及10 mM NaPO4）下量測溶解度。在某些實施例中，在諸如血液或血清之生物流體（溶劑）中量測溶解度。在某些實施例中，溫度可為約室溫（例如約20℃、21℃、22℃、23℃、24℃、25℃）或約體溫（37℃）。在某些實施例中，藥劑在室溫下或在37℃下之溶解度為至少約0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、40、50、60、70、80、90或100 mg/ml。The term "solubility" refers to the property of an agent (e.g., an antibody) provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is usually expressed as concentration, in terms of mass of solute per unit volume of solvent (grams of solute per kilogram of solvent, g/dL (100 mL), mg/ml, etc.), molar concentration, weight molar concentration, molar fraction, or other similar descriptions of concentration. Under specified conditions including temperature, pressure, pH, and solvent properties, the maximum equilibrium amount of solute that can be dissolved per amount of solvent is the solubility of the solute in the solvent. In some embodiments, solubility is measured at physiological pH or other pH, such as pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5-8). In some embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaPO4). In specific embodiments, solubility is measured at a relatively low pH (e.g., pH 6.0) and a relatively high salt concentration (e.g., 500 mM NaCl and 10 mM NaPO4). In some embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In some embodiments, the temperature can be about room temperature (e.g., about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C.) or about body temperature (37° C.). In some embodiments, the solubility of the agent at room temperature or at 37° C. is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg/ml.

「個體」或「有需要之個體」或「患者」或「有需要之患者」包括諸如人類個體之哺乳動物個體。"Individual" or "individual in need" or "patient" or "patient in need" includes mammalian individuals such as human individuals.

「實質上」或「基本上」」意謂幾乎全部或完全，例如一些給出量之95%、96%、97%、98%、99%或更大。"Substantially" or "essentially" means nearly all or completely, such as 95%, 96%, 97%, 98%, 99% or more of some given amount.

「在統計學上顯著」意謂結果不太可能偶然出現。統計顯著性可藉由此項技術中已知之任何方法來加以確定。顯著性之常用量度包括p值，其為虛無假設為真之情況下，所觀測事件將發生之頻率或機率。若所獲得之p值小於顯著性水準，則駁回虛無假設。在簡單情況下，顯著性水準定義為p值為0.05或更小。"Statistically significant" means that the results are unlikely to occur by chance. Statistical significance can be determined by any method known in the art. Common measures of significance include the p-value, which is the frequency or probability that the observed event would occur if the null hypothesis were true. If the p-value obtained is less than the significance level, the null hypothesis is refuted. In simple cases, the significance level is defined as a p-value of 0.05 or less.

「治療反應」係指基於一或多種治療劑之投與改善症狀（無論是否持續）。"Therapeutic response" refers to improvement in symptoms (whether or not lasting) based on administration of one or more therapeutic agents.

如本文所用，術語「治療有效量」、「治療劑量」、「預防有效量」或「診斷有效量」為投與後引起所需生物反應所需的藥劑（例如抗IL-18BP抗體、免疫治療劑）之量。As used herein, the term "therapeutically effective amount", "therapeutic dose", "prophylactically effective amount" or "diagnostically effective amount" is the amount of an agent (e.g., anti-IL-18BP antibody, immunotherapeutic agent) required to elicit a desired biological response after administration.

如本文所用，個體（例如哺乳動物，諸如人類）或細胞之「治療」為嘗試改變疾病之自然病程所用的任何類型之干預。治療包括但不限於投與醫藥組合物，且可以預防性執行或在病理性事件起始或接觸病原體之後執行。亦包括「預防性」治療，其可針對降低所治療疾病或病狀之進展速率，延緩彼疾病或病狀之發作或降低其發作之嚴重程度。「治療」或「預防」不一定指示疾病或病狀或其相關症狀之完全根除、治癒或預防。As used herein, "treatment" of an individual (e.g., a mammal, such as a human) or cell is any type of intervention used to attempt to alter the natural course of a disease. Treatment includes, but is not limited to, the administration of a pharmaceutical composition, and may be performed prophylactically or after the initiation of a pathological event or exposure to a pathogen. Also included is "preventive" treatment, which may be directed to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily indicate complete eradication, cure, or prevention of a disease or condition or its associated symptoms.

術語「野生型」係指最常在群體中觀測到且因此任意設計為該基因之「普通」或「野生型」形式的基因或基因產物（例如多肽）。The term "wild-type" refers to a gene or gene product (eg, a polypeptide) that is most commonly observed in a population and is therefore arbitrarily designated as the "normal" or "wild-type" form of the gene.

除非另外明確說明，否則本說明書中之各實施例在加以必要修正後應用於其他每一實施例。 抗IL-18BP抗體Unless otherwise expressly stated, each embodiment in this specification applies to each other embodiment with necessary modifications.Anti-IL-18BP Antibody

某些實施例包括抗體及其抗原結合片段，其與IL-18BP結合。在一些實施例中，抗體或其抗原結合片段調節（例如干擾、拮抗、抑制）IL-18BP與其配位體介白素18（IL-18）之結合。在某些實施例中，抗體或其抗原結合片段之特徵在於或包含重鏈可變區（VH），其包含互補決定區VHCDR1、VHCDR2及VHCDR3序列；以及輕鏈可變區（VL），其包含互補決定區VLCDR1、VLCDR2及VLCDR3序列。例示性VH，VHCDR1、VHCDR2、VHCDR3；VL，VLCDR1、VLCDR2及VLCDR3序列提供於下表A1及表A2中。表A1：例示性CDR序列描述序列SEQ ID NO:SA01aV_HCDR1TNYYIH1V_HCDR2IINPNGGSTSYAQKFQG2V_HCDR3GIAMALFDY3V_LCDR1RTSQGISSWLA4V_LCDR2AASSLQS5V_LCDR3QQANSFPYT6SA08aV_HCDR1TSYGIS25V_HCDR2WISAYNGNTNYAQKLQG26V_HCDR3EGMTGTYFYYYLDV27V_LCDR1KSSQSVLFSSNIKNYLA28V_LCDR2WASTRES29V_LCDR3QQYYSTPT30SA09aV_HCDR1SSNWWT31V_HCDR2DIYHSESTNYNPSLKS32V_HCDR3VPPPYFMDV33V_LCDR1RASQSVSSSNLA34V_LCDR2GASRRAI35V_LCDR3QQFGSSMYT36SA10aV_HCDR1DDYGMN37V_HCDR2GINWNGGSTGYVDSLKG38V_HCDR3NLGRGLYYYYYYMDV39V_LCDR1RASQSVSSYLA40V_LCDR2DASNRAT41V_LCDR3QQRSNSIT42SA12aV_HCDR1SDHYMS43V_HCDR2YISIIGTTVYYADSVKG44V_HCDR3GVGRRAFDY45V_LCDR1RTSQEISSWLA46V_LCDR2VASSLQS47V_LCDR3QQANSFPWT48SA13aV_HCDR1TNYGIN49V_HCDR2WINSYNGNTNYAQKLQG50V_HCDR3EGRGTVMYEYFYYYMDV51V_LCDR1KSSQSVLFSSNNKNYLA52V_LCDR2WSSTRES53V_LCDR3QQYYTTYT54SA14aV_HCDR1TSYGIS55V_HCDR2WINVYNGNINYAQKLQG56V_HCDR3DGYCSRTSCFPTWLDT57V_LCDR1RASQSVSSYLT58V_LCDR2DASNRAT59V_LCDR3QQRRSWPRT60SA16aV_HCDR1TSYGIS61V_HCDR2WISAYNGNTNYAQKLQG62V_HCDR3NFPYCSSTRCYERDYYSYYMAV63V_LCDR1RASQSVSGYLA64V_LCDR2DASNRAT65V_LCDR3QQRSNWPIT66SA17aV_HCDR1SGYSWN67V_HCDR2EINHSGSTNYNPSLKS68V_HCDR3PIGSTGFDY69V_LCDR1RASQGISRWLA70V_LCDR2AASNLQS71V_LCDR3QQASNFPLT72SA45aV_HCDR1TNYYIH109V_HCDR2IINPNGGSTSYAQKFQG110V_HCDR3GLAMALFDY111V_LCDR1RTSQGISSWLA112V_LCDR2AASSLQS113V_LCDR3QQAYSFPYT114SA54aV_HCDR1TNYFIH115V_HCDR2IINPEGGSTSYAQKFQG116V_HCDR3GLAMSLFDY117V_LCDR1RTSQGISSWLA118V_LCDR2AASSLQS119V_LCDR3QQAYSFPYT120SA55aV_HCDR1TNYFIH121V_HCDR2IINVEGGSTSYAQKFQG122V_HCDR3GLAMALFDY123V_LCDR1RTSQGISSWLA124V_LCDR2AASSLQS125V_LCDR3QQAYSFPYT126SA56aV_HCDR1TNYYIH127V_HCDR2IINPEGGSTSYAQKFQG128V_HCDR3GLAMALFDY129V_LCDR1RTSQGISSWLA130V_LCDR2AASSLQS131V_LCDR3QQAYSFPYT132SA57aV_HCDR1TNYFIH133V_HCDR2IINPEGGSTSYAQKFQG134V_HCDR3GLAMALFDY135V_LCDR1RTSQGISSWLA136V_LCDR2AASSLQS137V_LCDR3QQAYSFPYT138SA59aV_HCDR1TNYFIH139V_HCDR2IINVEGGSTSYAQKFQG140V_HCDR3GLAMSLFDY141V_LCDR1RTSQGISSWLA142V_LCDR2AASSLQS143V_LCDR3QQAYSFPYT144SA60aV_HCDR1TNYYIH145V_HCDR2IINPNGGSTSYAQKFQG146V_HCDR3GIAMALFDY147V_LCDR1RTSQGISSWLA148V_LCDR2AASSLQS149V_LCDR3QQAYSFPYT150SA61aV_HCDR1TNYYIH151V_HCDR2IINPEGGSTSYAQKFQG152V_HCDR3GIAMALFDY153V_LCDR1RTSQGISSWLA154V_LCDR2AASSLQS155V_LCDR3QQAYSFPYT156SA62aV_HCDR1TNYYIH157V_HCDR2IINVEGGSTSYAQKFQG158V_HCDR3GIAMALFDY159V_LCDR1RTSQGISSWLA160V_LCDR2AASSLQS161V_LCDR3QQAYSFPYT162SA63aV_HCDR1TNYFIH163V_HCDR2IINPEGGSTSYAQKFQG164V_HCDR3GIAMALFDY165V_LCDR1RTSQGISSWLA166V_LCDR2AASSLQS167V_LCDR3QQAYSFPYT168SA64aV_HCDR1TNYFIH169V_HCDR2IINVEGGSTSYAQKFQG170V_HCDR3GLAMSLFDY171V_LCDR1RTSQGISSWTA172V_LCDR2AASSLQS173V_LCDR3QQAYSFPYT174SA65aV_HCDR1TNYFIH175V_HCDR2IINVEGGSTSYAQKFQG176V_HCDR3GLAMSLFDY177V_LCDR1RMSQGISSWLA178V_LCDR2AASSLQS179V_LCDR3QQAYSFPYT180SA66aV_HCDR1TNYFIH181V_HCDR2IINVEGGSTSYAQKFQG182V_HCDR3GLAMSLFDY183V_LCDR1DTSQGISSWLA184V_LCDR2AASSLQS185V_LCDR3QQAYSFPYT186SA73aV_HCDR1TNYYIH187V_HCDR2IINVEGGSTSYAQKFQG188V_HCDR3GLAMSLFDY189V_LCDR1DTSQGISSWTA190V_LCDR2AASSLQS191V_LCDR3QQAYSFPYT192SA77aV_HCDR1TNYYIH193V_HCDR2IINPEGGSTSYAQKFQG194V_HCDR3GLAMSLFDY195V_LCDR1DTSQGISSWTA196V_LCDR2AASSLQS197V_LCDR3QQAYSFPYT198SA59a-R24QV_HCDR1TNYFIH199V_HCDR2IINVEGGSTSYAQKFQG200V_HCDR3GLAMSLFDY201V_LCDR1QTSQGISSWLA202V_LCDR2AASSLQS203V_LCDR3QQAYSFPYT204SA59a-S26IV_HCDR1TNYFIH205V_HCDR2IINVEGGSTSYAQKFQG206V_HCDR3GLAMSLFDY207V_LCDR1RTIQGISSWLA208V_LCDR2AASSLQS209V_LCDR3QQAYSFPYT210SA59a-S26RV_HCDR1TNYFIH211V_HCDR2IINVEGGSTSYAQKFQG212V_HCDR3GLAMSLFDY213V_LCDR1RTRQGISSWLA214V_LCDR2AASSLQS215V_LCDR3QQAYSFPYT216SA59a-G28QV_HCDR1TNYFIH217V_HCDR2IINVEGGSTSYAQKFQG218V_HCDR3GLAMSLFDY219V_LCDR1RTSQQISSWLA220V_LCDR2AASSLQS221V_LCDR3QQAYSFPYT222SA55a-R24DV_HCDR1TNYFIH223V_HCDR2IINVEGGSTSYAQKFQG224V_HCDR3GLAMALFDY225V_LCDR1DTSQGISSWLA226V_LCDR2AASSLQS227V_LCDR3QQAYSFPYT228SA55a-T25MV_HCDR1TNYFIH229V_HCDR2IINVEGGSTSYAQKFQG230V_HCDR3GLAMALFDY231V_LCDR1RMSQGISSWLA232V_LCDR2AASSLQS233V_LCDR3QQAYSFPYT234SA55a-S26IV_HCDR1TNYFIH235V_HCDR2IINVEGGSTSYAQKFQG236V_HCDR3GLAMALFDY237V_LCDR1RTIQGISSWLA238V_LCDR2AASSLQS239V_LCDR3QQAYSFPYT240SA55a-S26RV_HCDR1TNYFIH241V_HCDR2IINVEGGSTSYAQKFQG242V_HCDR3GLAMALFDY243V_LCDR1RTRQGISSWLA244V_LCDR2AASSLQS245V_LCDR3QQAYSFPYT246SA55a-G28QV_HCDR1TNYFIH247V_HCDR2IINVEGGSTSYAQKFQG248V_HCDR3GLAMALFDY249V_LCDR1RTSQQISSWLA250V_LCDR2AASSLQS251V_LCDR3QQAYSFPYT252SA55a-L33TV_HCDR1TNYFIH253V_HCDR2IINVEGGSTSYAQKFQG254V_HCDR3GLAMALFDY255V_LCDR1RTSQGISSWTA256V_LCDR2AASSLQS257V_LCDR3QQAYSFPYT258SA51d（抗小鼠）V_HCDR1TFTNEYIH265V_HCDR2IINPNYGSTSYAQKFQG266V_HCDR3GLAMHLFDY267V_LCDR1RTSQGISSWLA268V_LCDR2AASSLQS269V_LCDR3QQAYSFPYT270SA52d（抗小鼠）V_HCDR1TFTNEYIH271V_HCDR2IINPEGGSTSYAQKFQG272V_HCDR3GLAMHLFDY273V_LCDR1RTSQGISSWLA274V_LCDR2AASSLQS275V_LCDR3QQAYSFPYT276Certain embodiments include antibodies and antigen-binding fragments thereof that bind to IL-18BP. In some embodiments, the antibody or antigen-binding fragment thereof modulates (e.g., interferes with, antagonizes, inhibits) the binding of IL-18BP to its ligand interleukin 18 (IL-18). In certain embodiments, the antibody or antigen-binding fragment thereof is characterized by or comprises a heavy chain variable region (VH) comprising complementary determining regions VHCDR1, VHCDR2, and VHCDR3 sequences; and a light chain variable region (VL) comprising complementary determining regions VLCDR1, VLCDR2, and VLCDR3 sequences. Exemplary VH, VHCDR1, VHCDR2, VHCDR3; VL, VLCDR1, VLCDR2, and VLCDR3 sequences are provided in Table A1 and Table A2 below.TableA1: ExemplaryCDRsequencesdescribesequenceSEQ ID NO:SA01a_VH CDR1 TNYYI 1_VH CDR2 IINPNGGSTSYAQKFQG 2_VH CDR3 GIAMALFDY 3 V_L CDR1 RTSQGISSWLA 4 V_L CDR2 AASSLQS 5 V_L CDR3 QQANSFPYT 6SA08a_VH CDR1 TSYGIS 25_VH CDR2 WISAYNGNTNYAQKLQG 26_VH CDR3 EGMTGTYFYYYLDV 27 V_L CDR1 KSSQSVLFSSNIKNYLA 28 V_L CDR2 WASTRES 29 V_L CDR3 QQYYSTPT 30SA09a_VH CDR1 SSNWWT 31_VH CDR2 DIYHSESTNYNPSLKS 32_VH CDR3 VPPPYFMDV 33 V_L CDR1 RASQSVSSSNLA 34 V_L CDR2 GASRRAI 35 V_L CDR3 QQFGSSMYT 36SA10a_VH CDR1 DDYGMN 37_VH CDR2 GINWNGGSTGYVDSLKG 38_VH CDR3 NLGRGLYYYYYYMDV 39 V_L CDR1 RASQSVSSYLA 40 V_L CDR2 DASNRAT 41 V_L CDR3 QQRSNSIT 42SA12a_VH CDR1 SDHYMS 43_VH CDR2 YISIIGTTVYYADSVKG 44_VH CDR3 GVGRRAFDY 45 V_L CDR1 RTSQEISSWLA 46 V_L CDR2 VASSLQS 47 V_L CDR3 QQANSFPWT 48SA13a_VH CDR1 TNYGIN 49_VH CDR2 WINSYNGNTNYAQKLQG 50_VH CDR3 EGRGTVMYEYFYYYMDV 51 V_L CDR1 KSSQSVLFSSNNKNYLA 52 V_L CDR2 WSSTRES 53 V_L CDR3 QQYYTTYT 54SA14a_VH CDR1 TSYGIS 55_VH CDR2 WINVYNGNINYAQKLQG 56_VH CDR3 DGYCSRTSCFPTWLDT 57 V_L CDR1 RASQSVSSYLT 58 V_L CDR2 DASNRAT 59 V_L CDR3 QQRRSWPRT 60SA16a_VH CDR1 TSYGIS 61_VH CDR2 WISAYNGNTNYAQKLQG 62_VH CDR3 NFPYCSSTRCYERDYYSYYMAV 63 V_L CDR1 RASQSVSGYLA 64 V_L CDR2 DASNRAT 65 V_L CDR3 QQRSNWPIT 66SA17a_VH CDR1 SGYSWN 67_VH CDR2 EINHSGSTNYNPSLKS 68_VH CDR3 PIGSTGFDY 69 V_L CDR1 RASQGISRWLA 70 V_L CDR2 AASNLQS 71 V_L CDR3 QQASNFPLT 72SA45a_VH CDR1 TNYYI 109_VH CDR2 IINPNGGSTSYAQKFQG 110_VH CDR3 GLAMALFDY 111 V_L CDR1 RTSQGISSWLA 112 V_L CDR2 AASSLQS 113 V_L CDR3 QQAYSFPYT 114SA54a_VH CDR1 TNYFIH 115_VH CDR2 IINPEGGSTSYAQKFQG 116_VH CDR3 GLAMSLFDY 117 V_L CDR1 RTSQGISSWLA 118 V_L CDR2 AASSLQS 119 V_L CDR3 QQAYSFPYT 120SA55a_VH CDR1 TNYFIH 121_VH CDR2 IINVEGGSTSYAQKFQG 122_VH CDR3 GLAMALFDY 123 V_L CDR1 RTSQGISSWLA 124 V_L CDR2 AASSLQS 125 V_L CDR3 QQAYSFPYT 126SA56a_VH CDR1 TNYYI 127_VH CDR2 IINPEGGSTSYAQKFQG 128_VH CDR3 GLAMALFDY 129 V_L CDR1 RTSQGISSWLA 130 V_L CDR2 AASSLQS 131 V_L CDR3 QQAYSFPYT 132SA57a_VH CDR1 TNYFIH 133_VH CDR2 IINPEGGSTSYAQKFQG 134_VH CDR3 GLAMALFDY 135 V_L CDR1 RTSQGISSWLA 136 V_L CDR2 AASSLQS 137 V_L CDR3 QQAYSFPYT 138SA59a_VH CDR1 TNYFIH 139_VH CDR2 IINVEGGSTSYAQKFQG 140_VH CDR3 GLAMSLFDY 141 V_L CDR1 RTSQGISSWLA 142 V_L CDR2 AASSLQS 143 V_L CDR3 QQAYSFPYT 144SA60a_VH CDR1 TNYYI 145_VH CDR2 IINPNGGSTSYAQKFQG 146_VH CDR3 GIAMALFDY 147 V_L CDR1 RTSQGISSWLA 148 V_L CDR2 AASSLQS 149 V_L CDR3 QQAYSFPYT 150SA61a_VH CDR1 TNYYI 151_VH CDR2 IINPEGGSTSYAQKFQG 152_VH CDR3 GIAMALFDY 153 V_L CDR1 RTSQGISSWLA 154 V_L CDR2 AASSLQS 155 V_L CDR3 QQAYSFPYT 156SA62a_VH CDR1 TNYYI 157_VH CDR2 IINVEGGSTSYAQKFQG 158_VH CDR3 GIAMALFDY 159 V_L CDR1 RTSQGISSWLA 160 V_L CDR2 AASSLQS 161 V_L CDR3 QQAYSFPYT 162SA63a_VH CDR1 TNYFIH 163_VH CDR2 IINPEGGSTSYAQKFQG 164_VH CDR3 GIAMALFDY 165 V_L CDR1 RTSQGISSWLA 166 V_L CDR2 AASSLQS 167 V_L CDR3 QQAYSFPYT 168SA64a_VH CDR1 TNYFIH 169_VH CDR2 IINVEGGSTSYAQKFQG 170_VH CDR3 GLAMSLFDY 171 V_L CDR1 RTSQGISSWTA 172 V_L CDR2 AASSLQS 173 V_L CDR3 QQAYSFPYT 174SA65a_VH CDR1 TNYFIH 175_VH CDR2 IINVEGGSTSYAQKFQG 176_VH CDR3 GLAMSLFDY 177 V_L CDR1 RMSQGISSWLA 178 V_L CDR2 AASSLQS 179 V_L CDR3 QQAYSFPYT 180SA66a_VH CDR1 TNYFIH 181_VH CDR2 IINVEGGSTSYAQKFQG 182_VH CDR3 GLAMSLFDY 183 V_L CDR1 DTSQGISSWLA 184 V_L CDR2 AASSLQS 185 V_L CDR3 QQAYSFPYT 186SA73a_VH CDR1 TNYYI 187_VH CDR2 IINVEGGSTSYAQKFQG 188_VH CDR3 GLAMSLFDY 189 V_L CDR1 DTSQGISSWTA 190 V_L CDR2 AASSLQS 191 V_L CDR3 QQAYSFPYT 192SA77a_VH CDR1 TNYYI 193_VH CDR2 IINPEGGSTSYAQKFQG 194_VH CDR3 GLAMSLFDY 195 V_L CDR1 DTSQGISSWTA 196 V_L CDR2 AASSLQS 197 V_L CDR3 QQAYSFPYT 198SA59a-R24Q_VH CDR1 TNYFIH 199_VH CDR2 IINVEGGSTSYAQKFQG 200_VH CDR3 GLAMSLFDY 201 V_L CDR1 QTSQGISSWLA 202 V_L CDR2 AASSLQS 203 V_L CDR3 QQAYSFPYT 204SA59a-S26I_VH CDR1 TNYFIH 205_VH CDR2 IINVEGGSTSYAQKFQG 206_VH CDR3 GLAMSLFDY 207 V_L CDR1 RTIQGISSWLA 208 V_L CDR2 AASSLQS 209 V_L CDR3 QQAYSFPYT 210SA59a-S26R_VH CDR1 TNYFIH 211_VH CDR2 IINVEGGSTSYAQKFQG 212_VH CDR3 GLAMSLFDY 213 V_L CDR1 RTRQGISSWLA 214 V_L CDR2 AASSLQS 215 V_L CDR3 QQAYSFPYT 216SA59a-G28Q_VH CDR1 TNYFIH 217_VH CDR2 IINVEGGSTSYAQKFQG 218_VH CDR3 GLAMSLFDY 219 V_L CDR1 RTSQQISSWLA 220 V_L CDR2 AASSLQS 221 V_L CDR3 QQAYSFPYT 222SA55a-R24D_VH CDR1 TNYFIH 223_VH CDR2 IINVEGGSTSYAQKFQG 224_VH CDR3 GLAMALFDY 225 V_L CDR1 DTSQGISSWLA 226 V_L CDR2 AASSLQS 227 V_L CDR3 QQAYSFPYT 228SA55a-T25M_VH CDR1 TNYFIH 229_VH CDR2 IINVEGGSTSYAQKFQG 230_VH CDR3 GLAMALFDY 231 V_L CDR1 RMSQGISSWLA 232 V_L CDR2 AASSLQS 233 V_L CDR3 QQAYSFPYT 234SA55a-S26I_VH CDR1 TNYFIH 235_VH CDR2 IINVEGGSTSYAQKFQG 236_VH CDR3 GLAMALFDY 237 V_L CDR1 RTIQGISSWLA 238 V_L CDR2 AASSLQS 239 V_L CDR3 QQAYSFPYT 240SA55a-S26R_VH CDR1 TNYFIH 241_VH CDR2 IINVEGGSTSYAQKFQG 242_VH CDR3 GLAMALFDY 243 V_L CDR1 RTRQGISSWLA 244 V_L CDR2 AASSLQS 245 V_L CDR3 QQAYSFPYT 246SA55a-G28Q_VH CDR1 TNYFIH 247_VH CDR2 IINVEGGSTSYAQKFQG 248_VH CDR3 GLAMALFDY 249 V_L CDR1 RTSQQISSWLA 250 V_L CDR2 AASSLQS 251 V_L CDR3 QQAYSFPYT 252SA55a-L33T_VH CDR1 TNYFIH 253_VH CDR2 IINVEGGSTSYAQKFQG 254_VH CDR3 GLAMALFDY 255 V_L CDR1 RTSQGISSWTA 256 V_L CDR2 AASSLQS 257 V_L CDR3 QQAYSFPYT 258SA51d(anti-mouse)_VH CDR1 TFTNEYIH 265_VH CDR2 IINPNYGSTSYAQKFQG 266_VH CDR3 GLAMHLFDY 267 V_L CDR1 RTSQGISSWLA 268 V_L CDR2 AASSLQS 269 V_L CDR3 QQAYSFPYT 270SA52d(anti-mouse)_VH CDR1 TFTNEYIH 271_VH CDR2 IINPEGGSTSYAQKFQG 272_VH CDR3 GLAMHLFDY 273 V_L CDR1 RTSQGISSWLA 274 V_L CDR2 AASSLQS 275 V_L CDR3 QQAYSFPYT 276

因此，在某些實施例中，抗體或其抗原結合片段包含V_H序列，其包含選自表A1之互補決定區V_HCDR1、V_HCDR2及V_HCDR3序列及其與IL-18BP結合之變異體；以及V_L序列，其包含選自表A1之互補決定區V_LCDR1、V_LCDR2及V_LCDR3序列及其與IL-18BP結合之變異體。在特定實施例中，抗體包含V_H序列，其包含V_HCDR1、V_HCDR2及V_HCDR3序列；以及V_L序列，其包含V_LCDR1、V_LCDR2及V_LCDR3序列，其中所有CDR序列來自表A1中之單命名抗體（例如，SA01a）。Thus, in certain embodiments, the antibody or antigen-binding fragment thereof comprises a_VH sequence comprising complementary determining region_VH CDR1,_VH CDR2 and_VH CDR3 sequences selected fromTableA1 and variants thereof that bind to IL-18BP; and a_VL sequence comprising complementary determining region_VL CDR1,_VL CDR2 and_VL CDR3 sequences selected fromTableA1 and variants thereof that bind to IL-18BP. In specific embodiments, the antibody comprises a_VH sequence comprising_VH CDR1,_VH CDR2 and_VH CDR3 sequences; and a_VL sequence comprising_VL CDR1,_VL CDR2 and_VL CDR3 sequences, wherein all CDR sequences are from a single named antibody inTableA1 (e.g., SAO1a).

在某些實施例中，CDR序列如下： 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 1-3，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 4-6； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 25-27，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 28-30； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 31-33，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 34-36； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 37-39，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 40-42； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 43-45，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 46-48； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 49-51，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 52-54； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 55-57，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 58-60； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 61-63，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 64-66； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 67-69，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 70-72； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 109-111，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 112-114； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 115-117，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 118-120； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 121-123，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 124-126； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 127-129，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 130-132； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 133-135，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 136-138； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 139-141，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 142-144； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 145-147，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 148-150； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 151-153，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 154-156； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 157-159，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 160-162； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 163-165，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 166-168； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 169-171，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 172-174； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 175-177，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 178-180； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 181-183，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 184-186； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 187-189，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 190-192； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 193-195，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 196-198； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 199-201，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 202-204； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 205-207，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 208-210； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 211-213，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 214-216； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 217-219，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 220-222； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 223-225，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 226-228； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 229-231，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 232-234； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 235-237，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 238-240； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 241-243，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 244-246； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 247-249，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 250-252； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 253-255，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 256-258； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 265-267，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 268-270；或 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 271-273，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 274-276。In certain embodiments, the CDR sequences are as follows: the_VH CDR1,_VH CDR2, and_VH CDR3 sequences comprise SEQ ID NOs: 1-3, respectively, and the_VL CDR1,_VL CDR2, and_VL CDR3 sequences comprise SEQ ID NOs: 4-6, respectively; the_VH CDR1,_VH CDR2, and_VH CDR3 sequences comprise SEQ ID NOs: 25-27, respectively, and the_VL CDR1,_VL CDR2, and_VL CDR3 sequences comprise SEQ ID NOs: 28-30, respectively; the_VH CDR1,_VH CDR2, and_VH CDR3 sequences comprise SEQ ID NOs: 31-33, respectively, and the_VL CDR1,_VL CDR2, and_VL CDR3 sequences comprise SEQ ID NOs: 34-36, respectively; the_VH CDR1,_VH CDR2, and_VH CDR3 sequences comprise SEQ ID NOs: said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 49-51, and said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 52-54, respectively; said_VHCDR1 ,_VHCDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 55-57_, and_saidVLCDR1 ,_VLCDR2 and_VLCDR3 sequences comprise SEQ ID NOs: 58-60_, respectively_; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 61-63, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 64-66, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 67-69, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 70-72, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 109-111, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 112-114, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 127-129, and said_VL CDR1,_VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 130-132, respectively; said VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 133-135, respectively; said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 136-137, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 137-138, respectively; said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 139-140, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 141-142, respectively; said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 143-144, respectively; the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 136-138, respectively; the_VH CDR1,_VH CDR2 and VH CDR3 sequences comprise SEQ ID NOs: 139-141, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 142-144, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 145-147, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 148-150, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 151-153, respectively, and the_VL CDR1,_{VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 154-156, respectively; the VH CDR1, VH} CDR2 and_VHCDR3 sequences comprise SEQ ID NOs:_157-158 , respectively the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 157-159, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 160-162, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 163-165, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 166-168, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 169-171, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 172-174, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 175-177, respectively, and the_VL the VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 181-183, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 184-186, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 187-189, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 190-192, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 193-195, and_theVL CDR1,_VLCDR2 and_VLCDR3 sequences comprise SEQ ID NOs: said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 196-198; said_{VH CDR1, VH CDR2 and VH CDR3 sequences comprise SEQ ID NOs: 199-201, respectively, and said VL CDR1, VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 202-204, respectively; said VHCDR1,VHCDR2andVH} CDR3 sequences comprise SEQ ID NOs: 205-207, respectively, and said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 208-210, respectively; said_VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 211-213, respectively, and said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 214-216, respectively; said_VH CDR1,_VH CDR2 and_VH the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 229-231, and the_VL CDR1,_VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 232-234, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 235-237, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 236-238, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 237-239, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs_: 240-241, respectively; the_VH CDR1,_VH CDR2 and_VHCDR3 sequences comprise SEQ ID NOs: 242-243, respectively; the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 238-240, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 241-243, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 244-246, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 247-249, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 250-252, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 253-255, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 256-258, respectively; the_VH The VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 265-267, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 268-270, respectively; or the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 271-273, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 274-276, respectively.

在某些實施例中，抗體或其抗原結合片段（例如，SA01a抗體或其抗原結合片段之變異體）包含CDR共同序列，例如其中VHCDR1、VHCDR2及VHCDR3序列分別包含TFX₁X₂X₃X₄X₅H、IX₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅AQKFQG及X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂DY，且VLCDR1、VLCDR2及VLCDR3序列分別包含X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀WX₃₁A、X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈及QX₃₉X₄₀X₄₁SFPYX₄₂（關於「X」殘基之定義，參見表E11）。In certain embodiments, the antibody or antigen-binding fragment thereof (e.g., a variant of the SA01a antibody or antigen-binding fragment thereof) comprises a CDR consensus sequence, for example, wherein the VHCDR1, VHCDR2, and VHCDR3 sequences comprise_TFX1 X₂ X₃ X₄ X₅ H, IX₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ AQKFQG, and X₁₆ X₁₇ X₁₈ X₁₉ X₂₀ X₂₁ X₂₂ DY, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise X₂₃ X₂₄ X₂₅ X₂₆ X₂₇ X₂₈ X₂₉ X₃₀ WX₃₁ A, X₃₂ X₃₃ X₃₄ X₃₅ X₃₆ X₃₇ X₃₈ , and Q₃₉ X₄₀ X₄₁ SFPYX₄₂ (For the definition of the “X” residue,see TableE11 ).

亦包括前述CDR之微小變異體。例示性變異體與IL-18BP結合且在個別CDR中之任一或多者（例如本文所述之V_HCDR1、V_HCDR2、V_HCDR3、V_LCDR1、V_LCDR2及/或V_LCDR3序列中之任一或多者）中具有1、2或3個總改變。例示性「改變」包括胺基酸取代、添加及缺失。Minor variants of the aforementioned CDRs are also included. Exemplary variants bind to IL-18BP and have 1, 2, or 3 total alterations in any one or more of the individual CDRs (e.g., any one or more of the_VH CDR1,_VH CDR2,_VH CDR3,_VL CDR1,_VL CDR2, and/or_VL CDR3 sequences described herein). Exemplary "alterations" include amino acid substitutions, additions, and deletions.

下表A2中提供例示性V_H及V_L序列。表A2：例示性多肽序列描述序列SEQ ID NO:SA01a變異體共同序列重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFX₁X₂X₃X₄X₅HWVRQAPGQGLEWMGIX₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARX₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂DYWGQG 關於「X」殘基之定義，參見表E11277輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCX₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀WX₃₁AWYQQKPGKAPKLLIYX₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQX₃₉X₄₀X₄₁SFPYX₄₂FGQGTKLEIK 關於「X」殘基之定義，參見表E11278SA01a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGIINPNGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIAMALFDYWGQGTLVTVSS279輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPYTFGQGTKLEIK280SA08a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREGMTGTYFYYYLDVWGKGTTVTVSS285輕鏈可變區 (V_L)DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNIKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPTLGQGTKVEIK286SA09a重鏈可變區 (V_H)QVQLQESGPGLVKPSGTLSLTCVVSGGSIISSNWWTWVRQPPGKGLEWVGDIYHSESTNYNPSLKSRVTISIDKSKNHFSLILSSVTAADTAVYYCARVPPPYFMDVWGKGTTVTVSS287輕鏈可變區 (V_L)EIVLTQSPATLSLSPGERATLSCRASQSVSSSNLAWYQQKPGQAPRLLMYGASRRAIGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFGSSMYTFGQGTKLEIK288SA10a重鏈可變區 (V_H)EVQLVESGGGVVRPGGSLRLSCAASGFNFDDYGMNWVRLAPGKGLEWVSGINWNGGSTGYVDSLKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARNLGRGLYYYYYYMDVWGKGTTVTVSS289輕鏈可變區 (V_L)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFSLTISSLEPEDFAVYFCQQRSNSITFGQGTRLEIK290SA12a重鏈可變區 (V_H)EVQLVESGGGLVKPGGSLRLSCAVSGFTFSDHYMSWIRQAPGKGLKWISYISIIGTTVYYADSVKGRFTISRDDAKNSLYLQMDSLRAEDTAVYYCARGVGRRAFDYWGQGTLVTVSS291輕鏈可變區（V_L）DIQMTQSPSSLSASVGDRVTITCRTSQEISSWLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPGDFATYYCQQANSFPWTFGQGTKVEIK292SA13a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQAPGQGLEWMGWINSYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCTREGRGTVMYEYFYYYMDVWGKGTTVTVSS293輕鏈可變區 (V_L)DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNKNYLAWYQQKPGQPPKLLIYWSSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTYTFGQGTKLEIK294SA14a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKTSGYTFTSYGISWLRQVPGQGLEWMAWINVYNGNINYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGYCSRTSCFPTWLDTWGQGTLVTVSS295輕鏈可變區 (V_L)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLTWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRRSWPRTFGQGTKVEIK296SA16a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARNFPYCSSTRCYERDYYSYYMAVWGKGTTVTVSS297輕鏈可變區 (V_L)EIVLTQSPATLSLSPGERATLSCRASQSVSGYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGPGTKVDIK298SA17a重鏈可變區 (V_H)QVQLQESGPGLLKPSETLSLTCAVYGGSFSGYSWNWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNHFSLKLTSVTAADTALYYCARPIGSTGFDYWGQGTLVIVSS299輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPGKAPNLLIFAASNLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQASNFPLTFGGGTNVEIK300SA45a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGIINPNGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDYWGQGTLVTVSS313輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK314SA54a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINPEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDYWGQGTLVTVSS315輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK316SA55a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDYWGQGTLVTVSS317輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK318SA56a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGIINPEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDYWGQGTLVTVSS319輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK320SA57a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINPEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDYWGQGTLVTVSS321輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK322SA59a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDYWGQGTLVTVSS323輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK324SA60a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGIINPNGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIAMALFDYWGQGTLVTVSS325輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK326SA61a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGIINPEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIAMALFDYWGQGTLVTVSS327輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK328SA62a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIAMALFDYWGQGTLVTVSS329輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK330SA63a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINPEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIAMALFDYWGQGTLVTVSS331輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK332SA64a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDYWGQGTLVTVSS333輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWTAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK334SA65a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDYWGQGTLVTVSS335輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRMSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK336SA66a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDYWGQGTLVTVSS337輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCDTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK338SA73a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDYWGQGTLVTVSS339輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCDTSQGISSWTAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK340SA77a重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGIINPEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDYWGQGTLVTVSS341輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCDTSQGISSWTAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK342SA59a-R24Q重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDYWGQGTLVTVSS343輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCQTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK344SA59a-S26I重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDYWGQGTLVTVSS345輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTIQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK346SA59a-S26R重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDYWGQGTLVTVSS347輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTRQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK348SA59a-G28Q重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDYWGQGTLVTVSS349輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQQISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK350SA55a-R24D重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDYWGQGTLVTVSS351輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCDTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK352SA55a-T25M重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDYWGQGTLVTVSS353輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRMSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK354SA55a-S26I重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDYWGQGTLVTVSS355輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTIQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK356SA55a-S26R重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDYWGQGTLVTVSS357輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTRQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK358SA55a-G28Q重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDYWGQGTLVTVSS359輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQQISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK360SA55a-L33T重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQGLEWMGIINVEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDYWGQGTLVTVSS361輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWTAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK362SA51d（抗小鼠）重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNEYIHWVRQAPGQGLEWMGIINPNYGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMHLFDYWGQGTLVTVSS367輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK368SA52d（抗小鼠）重鏈可變區 (V_H)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNEYIHWVRQAPGQGLEWMGIINPEGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMHLFDYWGQGTLVTVSS369輕鏈可變區 (V_L)DIQMTQSPSSLSASVGDRVTITCRTSQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYTFGQGTKLEIK370Exemplary_VH and_VL sequences are provided inTableA2 below.TableA2: Exemplary polypeptide sequencesdescribesequenceSEQ ID NO:SA01avariant consensus sequence Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFX₁ X₂ X₃ X₄ X₅ H WVRQAPGQGLEWMGIX₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ AQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARX₁₆ X₁₇ X₁₈ X₁₉ X₂₀ X₂₁ X₂₂ DY WGQG For the definition of “X” residue,see TableE11 277 Light chain variable region (V_L ) DIQMTQSPSSLSASVGDRVTITCX₂₃ X₂₄ X₂₅ X₂₆ X₂₇ X₂₈ X₂₉ X₃₀ WX₃₁ A WYQQKPGKAPKLLIYX₃₂ X_{33 X 34X35} X₃₆ X₃₇ X₃₈ GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQX₃₉ X₄₀ X₄₁ SFPYX₄₂ FGQGTKLEIK For the definition of residual “X”,see TableE11 278SA01a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIH WVRQAPGQGLEWMGIINPNGGSTSYAQKFQG RVTTMRDTSTSTVYMELSSLRSEDTAVYYCARGIAMALFDY WGQGTLVTVSS 279 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPYT FGQGTKLEIK 280SA08a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGIS WVRQAPGQGLEWMGWISAYNGNTNYAQKLQG RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREGMTGTYFYYYLDV WGKGTTVTVSS 285 Light chain variable region (V_L ) DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNIKNYLA WYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPT LGQGTKVEIK 286SA09a Heavy chain variable region (V_H ) QVQLQESGPGLVKPSGTLSLTCVVSGGSIISSNWWT WVRQPPGKGLEWVGDIYHSESTNYNPSLKS RVTISIDKSKNHFSLILSSVTAADTAVYYCARVPPPYFMDV WGKGTTVTVSS 287 Light chain variable region (V_L ) EIVLTQSPATLSLSPGERATLSCRASQSVSSSNLA WYQQKPGQAPRLLMYGASRRAI GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFGSSMYT FGQGTKLEIK 288SA10a Heavy chain variable region (V_H ) EVQLVESGGGVVRPGGSLRLSCAASGFNFDDYGMN WVRLAPGKGLEWVSGINWNGGSTGYVDSLKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARNLGRGLYYYYYYMDV WGKGTTVTVSS 289 Light chain variable region (V_L ) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLA WYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFSLTISSLEPEDFAVYFCQQRSNSIT FGQGTRLEIK 290SA12a Heavy chain variable region (V_H ) EVQLVESGGGLVKPGGSLRLSCAVSGFTFSDHYMS WIRQAPGKGLKWISYISIIGTTVYYADSVKG RFTISRDDAKNSLYLQMDSLRAEDTAVYYCARGVGRRAFDY WGQGTLVTVSS 291 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQEISSWLA WYQQKPGKAPKLLIYVASSLQS GVPSRFSGSGSGTDFTLTISSLQPGDFATYYCQQANSFPWT FGQGTKVEIK 292SA13a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGIN WVRQAPGQGLEWMGWINSYNGNTNYAQKLQG RVTMTTDTSTSTAYMELRSLRSDDTAVYYCTREGRGTVMYEYEYFYYYMDV WGKGTTVTVSS 293 Light chain variable region (V_L ) DIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNNKNYLA WYQQKPGQPPKLLIYWSSTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTYT FGQGTKLEIK 294SA14a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKTSGYTFTSYGIS WLRQVPGQGLEWMAWINVYNGNINYAQKLQG RVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGYCSRTSCFPTWLDT WGQGTLVTVSS 295 Light chain variable region (V_L ) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLT WYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRRSWPRT FGQGTKVEIK 296SA16a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGIS WVRQAPGQGLEWMGWISAYNGNTNYAQKLQG RVTMTTDTSTSTAYMELRSLRSDDTAVYYCARNFPYCSSTRCYERDYYSYYMAV WGKGTTVTVSS 297 Light chain variable region (V_L ) EIVLTQSPATLSLSPGERATLSCRASQSVSGYLA WYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPIT FGPGTKVDIK 298SA17a Heavy chain variable region (V_H ) QVQLQESGPGLLKPSETLSLTCAVYGGSFSGYSWN WIRQPPGKGLEWIGEINHSGSTNYNPSLKS RVTISVDTSKNHFSLKLTSVTAADTALYYCARPIGSTGFDY WGQGTLVIVSS 299 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRASQGISRWLA WYQQKPGKAPNLLIFAASNLQS GVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQASNFPLT FGGGTNVEIK 300SA45a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIH WVRQAPGQGLEWMGIINPNGGSTSYAQKFQG RVTTMRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDY WGQGTLVTVSS 313 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 314SA54a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINGEGGSTSYAQKFQG RVTTMRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDY WGQGTLVTVSS 315 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 316SA55a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDY WGQGTLVTVSS 317 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 318SA56a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIH WVRQAPGQGLEWMGIINPEGGSTSYAQKFQG RVTTMRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDY WGQGTLVTVSS 319 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 320SA57a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINGEGGSTSYAQKFQG RVTTMRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDY WGQGTLVTVSS 321 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 322SA59a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDY WGQGTLVTVSS 323 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 324SA60a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIH WVRQAPGQGLEWMGIINPNGGSTSYAQKFQG RVTTMRDTSTSTVYMELSSLRSEDTAVYYCARGIAMALFDY WGQGTLVTVSS 325 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 326SA61a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIH WVRQAPGQGLEWMGIINPEGGSTSYAQKFQG RVTTMRDTSTSTVYMELSSLRSEDTAVYYCARGIAMALFDY WGQGTLVTVSS 327 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 328SA62a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTTMRDTSTSTVYMELSSLRSEDTAVYYCARGIAMALFDY WGQGTLVTVSS 329 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 330SA63a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINGEGGSTSYAQKFQG RVTTMRDTSTSTVYMELSSLRSEDTAVYYCARGIAMALFDY WGQGTLVTVSS 331 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 332SA64a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDY WGQGTLVTVSS 333 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWTA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 334SA65a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDY WGQGTLVTVSS 335 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRMSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 336SA66a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDY WGQGTLVTVSS 337 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCDTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 338SA73a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDY WGQGTLVTVSS 339 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCDTSQGISSWTA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 340SA77a Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIH WVRQAPGQGLEWMGIINPEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDY WGQGTLVTVSS 341 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCDTSQGISSWTA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 342SA59a-R24Q Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDY WGQGTLVTVSS 343 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCQTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 344SA59a-S26I Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDY WGQGTLVTVSS 345 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTIQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 346SA59a-S26R Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDY WGQGTLVTVSS 347 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTRQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 348SA59a-G28Q Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMSLFDY WGQGTLVTVSS 349 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQQISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 350SA55a-R24D Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDY WGQGTLVTVSS 351 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCDTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 352SA55a-T25M Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDY WGQGTLVTVSS 353 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRMSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 354SA55a-S26I Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDY WGQGTLVTVSS 355 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTIQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 356SA55a-S26R Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDY WGQGTLVTVSS 357 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTRQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 358SA55a-G28Q Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDY WGQGTLVTVSS 359 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQQISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 360SA55a-L33T Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIH WVRQAPGQGLEWMGIINVEGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLAMALFDY WGQGTLVTVSS 361 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWTA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 362SA51d(anti-mouse) Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNEYIH WVRQAPGQGLEWMGIINPNYGSTSYAQKFQG RVTTMRDTSTSTVYMELSSLRSEDTAVYYCARGLAMHLFDY WGQGTLVTVSS 367 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 368SA52d(anti-mouse) Heavy chain variable region (V_H ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNEYIH WVRQAPGQGLEWMGIINPEGGSTSYAQKFQG RVTTMRDTSTSTVYMELSSLRSEDTAVYYCARGLAMHLFDY WGQGTLVTVSS 369 Light chain variable region (V_L ) DIQMTQSPSSSLSASVGDRVTITCRTSQGISSWLA WYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPYT FGQGTKLEIK 370

因此，在某些實施例中，抗體或其抗原結合片段與IL-18BP結合且包含選自表A2之V_H序列及對應V_L序列。在某些實施例中，V_H包含與選自表A2之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，包括例如其中V_H在一或多個構架區中具有1、2、3、4、5、6、7、8、9或10個改變。在一些實施例中，V_L包含與選自表A2之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，包括例如其中V_L在一或多個構架區中具有1、2、3、4、5、6、7、8、9或10個改變。在特定實施例中，V_H包含與選自表A2之序列至少70%、75%、80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且V_L包含與選自表A2之序列至少70、75、80、85、90、95、97、98、99或100%一致的序列且與V_H區來自相同的單命名抗體（例如，SA01a）。在特定實施例中，V_H包含與選自表A2之序列至少70%、75%、80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且V_L包含與選自表A2之序列至少70%、75%、80%、85%、90%、95%、97%、98%、99%或100%一致的序列且與VH區來自相同的單命名抗體（例如，SA01a），其中CDR中未發現任何改變，如表A2中加下劃線的所示。因此，抗體可包含V_H及V_L序列，其與來自表A2中之單命名抗體（例如，SA01a）之相應序列至少70%、75%、80%、85%、90%、95%、97%、98%、99%或100%一致，其中該抗體包含如表A1中所列舉的該單命名抗體（例如，SA01a）之CDR。Thus, in certain embodiments, the antibody or antigen-binding fragment thereof binds to IL-18BP and comprises a_VH sequence selected fromTableA2 and a corresponding_VL sequence. In certain embodiments, the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected fromTableA2 , including, for example, wherein the_VH has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 changes in one or more framework regions. In some embodiments, the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected fromTableA2 , including, for example, wherein_{the VL} has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 changes in one or more framework regions. In specific embodiments,_VH comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected fromTableA2 , and_VL comprises a sequence that is at least 70, 75, 80, 85, 90, 95, 97, 98, 99 or 100% identical to a sequence selected fromTableA2 and is from the same single-named antibody (e.g., SAO1a) as the_VH region. In a specific embodiment, the_VH comprises a sequencethat is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected fromTable A2, and the_VL comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected fromTableA2 and is from the same single-named antibody (e.g., SAO1a) as the VH region, wherein no changes are found in the CDRs, as indicated by underlining inTableA2 . Thus, the antibody may comprise_VH and_VL sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the corresponding sequencesfrom asingle -named antibody in Table A2 (e.g., SAO1a), wherein the antibody comprises the CDRs of the single-named antibody (e.g., SAO1a) as listed inTableA1 .

在一些實施例中，抗體或抗原結合片段之V_H及V_L如下： 該V_H包含與SEQ ID NO: 277至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 278至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 279至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 280至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 285至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 286至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 287至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 288至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 289至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 290至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 291至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 292至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 293至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 294至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 295至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 296至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 297至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 298至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 299至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 300至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 313至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 314至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 315至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 316至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 317至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 318至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 319至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 320至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 321至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 322至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 323至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 324至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 325至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 326至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 327至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 328至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 329至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 330至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 331至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 332至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 333至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 334至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 335至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 336至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 337至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 338至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 339至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 340至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 341至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 342至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 343至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 344至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 345至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 346至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 347至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 348至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 349至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 350至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 351至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 352至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 353至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 354至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 355至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 356至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 357至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 358至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 359至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 360至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 361至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 362至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 367至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 368至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列；或 該V_H包含與SEQ ID NO: 369至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 370至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列。In some embodiments, the_VH and_VL of the antibody or antigen-binding fragment are as follows: the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 277, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 278; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 279, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 280; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 285, and the V_{The L} comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 286; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 287, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 288; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 289, and the V_{L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 290; the V H} comprises a sequence that is at least 80%, 85%, 90%, 95_% , 97%, 98%, 99% or 100% identical to SEQ ID NO: : the_VH comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 293, and the VL comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 294; the_VH comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 295, and_theVL comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:₂₉₆ . the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 296; the VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 297, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 298; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 299, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 300; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 313 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 314, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 314; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 315, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 316; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 317, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 318; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 319, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 320; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 321, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 322; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 323 is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 324, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 324; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 325, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 326; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 327, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 328; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 329, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 330; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 331, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 332; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 333 is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 334, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 334; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 335, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 336; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 337, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 338; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 339, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 340; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 341, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 342; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 343 is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 344, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 344; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 345, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 346; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 347, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 348; the VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 349, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 350; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 351, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 352; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 353 is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 354, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 354; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 355, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 356; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 357, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 358; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 359, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 360; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 361, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 362; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: or the_VH comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 369 and_theVL comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 370.

亦包括其與IL-18BP結合之變異體，例如在前述V_H及/或V_L序列中之任一或多者之一或多個構架區中具有1、2、3、4、5、6、7、8、9或10個改變之變異體。例示性「改變」包括胺基酸取代、添加及缺失。Also included are variants thereof that bind to IL-18BP, such as variants having 1,₂ , 3, 4, 5, 6, 7, 8, 9 or 10 alterations in one or more framework regions of any one or more of the aforementioned VH and/or_VL sequences. Exemplary "alterations" include amino acid substitutions, additions and deletions.

如上文所指出，本文所述之抗體或其抗原結合片段與IL-18BP結合。在某些實施例中，抗體或其抗原結合片段與人類IL-18BP、食蟹獼猴IL-18BP及/或小鼠IL-18BP或其區域或片段或抗原決定基結合。As noted above, the antibodies or antigen-binding fragments thereof described herein bind to IL-18BP. In certain embodiments, the antibodies or antigen-binding fragments thereof bind to human IL-18BP, cynomolgus macaque IL-18BP and/or mouse IL-18BP or a region or fragment or antigenic determinant thereof.

人類介白素-18結合蛋白或IL-18BP由IL18BP基因（參見Gene ID: 10068；及UniProt：O95998）編碼且具有至少三種同功異型物。在一些實施例中，本發明之抗體與IL-18BP之同功異型物A結合。在一些實施例中，本發明之抗體與IL-18BP之同功異型物B結合。在一些實施例中，本發明之抗體與IL-18BP之同功異型物A及同功異型物C結合。在一些實施例中，本發明之抗體與IL-18BP之所有同功異型物結合。其為早期Th1細胞介素反應及促炎性細胞介素IL-18之抑制劑。舉例而言，IL-18BP與IL-18結合，抑制IL-18與其受體之結合，且藉此抑制IL-18誘導之IFN-γ產生，以及其他IL-18信號傳導活性。人類、食蟹獼猴及小鼠IL-18BP同功異型物之胺基酸序列提供於下表B1中（關於比對，亦參見圖2）。表B1.例示性IL-18BP序列名稱序列SEQ ID NO:全長人類同功異型物AMRHNWTPDLSPLWVLLLCAHVVTLLVRATPVSQTTTAATASVRSTKDPCPSQPPVFPAAKQCPALEVTWPEVEVPLNGTLSLSCVACSRFPNFSILYWLGNGSFIEHLPGRLWEGSTSRERGSTGTQLCKALVLEQLTPALHSTNFSCVLVDPEQVVQRHVVLAQLWAGLRATLPPTQEALPSSHSSPQQQG371成熟人類同功異型物ATPVSQTTTAATASVRSTKDPCPSQPPVFPAAKQCPALEVTWPEVEVPLNGTLSLSCVACSRFPNFSILYWLGNGSFIEHLPGRLWEGSTSRERGSTGTQLCKALVLEQLTPALHSTNFSCVLVDPEQVVQRHVVLAQLWAGLRATLPPTQEALPSSHSSPQQQG372全長食蟹獼猴MRHNWTPDLSFLWVLLCAHIITLLVRAtpvsqtttaatassrstkdpcpsqppvfpaakqcpalevtwpevemplngtltlsctacsrfpnfsmlywlgngsfiehlpgqlwegstsrehgstgtrlykalvleqltpalhstnfscvlmdpeqvvqrhvilaqlwaglrttlpptqealpsshstgpqqptaaglrlstgpaaarp373成熟食蟹獼猴tpvsqtttaatassrstkdpcpsqppvfpaakqcpalevtwpevemplngtltlsctacsrfpnfsmlywlgngsfiehlpgqlwegstsrehgstgtrlykalvleqltpalhstnfscvlmdpeqvvqrhvilaqlwaglrttlpptqealpsshstgpqqptaaglrlstgpaaarp374全長小鼠MRHCWTAGPSSWWVLLLYVHVILARATSAPQTTATVLTGSSKDPCSSWSPAVPTKQYPALDVIWPEKEVPLNGTLTLSCTACSRFPYFSILYWLGNGSFIEHLPGRLKEGHTSREHRNTSTWLHRALVLEELSPTLRSTNFSCLFVDPGQVAQYHIILAQLWDGLKTAPSPSQETLSSHSPVSRSAGPGVA375成熟小鼠TSAPQTTATVLTGSSKDPCSSWSPAVPTKQYPALDVIWPEKEVPLNGTLTLSCTACSRFPYFSILYWLGNGSFIEHLPGRLKEGHTSREHRNTSTWLHRALVLEELSPTLRSTNFSCLFVDPGQVAQYHIILAQLWDGLKTAPSPSQETLSSHSPVSRSAGPGVA376Human interleukin-18 binding protein or IL-18BP is encoded by theIL18BP gene (see Gene ID: 10068; and UniProt: O95998) and has at least three isoforms. In some embodiments, the antibodies of the present invention bind to isoform A of IL-18BP. In some embodiments, the antibodies of the present invention bind to isoform B of IL-18BP. In some embodiments, the antibodies of the present invention bind to isoform A and isoform C of IL-18BP. In some embodiments, the antibodies of the present invention bind to all isoforms of IL-18BP. It is an inhibitor of early Th1 interleukin response and proinflammatory interleukin IL-18. For example, IL-18BP binds to IL-18, inhibits the binding of IL-18 to its receptor, and thereby inhibits IL-18-induced IFN-γ production, as well as other IL-18 signaling activities. The amino acid sequences of human, cynomolgus monkey, and mouse IL-18BP isoforms are provided inTableB1 below (see alsoFigure2 for alignment).TableB1.ExemplaryIL-18BPsequences Name sequence SEQ ID NO: Full-length human isoform AMRHNWTPDLSPLWVLLLCAHVVTLLVRATPVSQTTTAATASVRSTKDPCPSQPPVFPAAKQCPALEVTWPEVEVPLNGTLSLSCVACSRFPNFSI LYWLGNGSFIEHLPGRLWEGSTSRERGSTGTQLCKALVLEQLTPALHSTNFSCVL 371 Mature human isomorph A TPVSQTTTAATASVRSTKDPPCSQPPVFPAAKQCPALEVTWPEVEVPLNGTLSLSCVACSRFPNFSI LYWLGNGSFIEHLPGRLWEGSTSRERGSTGTQLCKALVLEQLTPALHSTNFSCVLV DPEQVVQRHVVLAQLWAGLRATLPPTQEALPSSHSSPQQQG 372 Full-length crab-eating macaqueMRHNWTPDLSFLWVLLCAHIITLLVRA tpvsqtttaatassrstkdpcpsqppvfpaakqcpalevtwpevemplngtltlsctacsrfpnfsmlywlgngsfiehlpgqlwegstsrehgstgtrlykalvleqltpalhstnfscvlmdpeqvvqrhvilaqlwaglrttlpptqealpsshstgpqqp taaglrlstgpaaarp 373 Mature crab-eating macaque tpvsqtttaatassrstkdpcpsqppvfpaakqcpalevtwpevemplngtltlsctacsrfpnfsmlywlgngsfiehlpgqlwegstsrehgstgtrlykalvleqltpalhstnfscvlmdpeqvvqrhvilaqlwaglrttlpptqealpsshstgpqqptaaglrlstgpaaarp 374 Full-length mouseMRHCWTAGPSSWWVLLLYVHVILARA TSAPQTTATVLTGSSKDPCSSWSPAVPTKQYPALDVIWPEKEVPLNGTLTLSCTACSRFPYFSILYWLGNGSFIEHLPGRLKEGHTSREHRNTSTWLHRALVLEELSPPTLRSTNFSCLFVDPGQVAQYHIILAQLWDGLKTAPSPSQETLSSHSPVSRSAGPGVA 375 Mature mice TSAPQTTATVLTGSSKDPCSSWSPAVPTKQYPALDVIWPEKEVPLNGTLTLSCTACSRFPYFSILYWLGNGSFIEHLPGRLKEGHTSREHRNTSTWLHRALVLEELSPPTLRSTNFSCLFVDPGQVAQYHIILAQLWDGLKTAPSPSQETLSSHSPVSRSAGPGVA 376

因此，在某些實施例中，抗體或其抗原結合片段例如在不包括信號肽（加下劃線）之區域處與表B1中之成熟IL-18BP序列結合。在具體實施例中，抗體或其抗原結合片段與包含成熟形式之IL-18BP之IL-18結合界面的抗原決定基結合。在一些實施例中，抗體或其抗原結合片段與抗原決定基結合，該抗原決定基包含根據UniProt：O95998之編號之I97及V153 （可替代地，如由SEQ ID NO: 1所定義之I95及V151；或如由SEQ ID NO: 2所定義之I67及V123）。Thus, in certain embodiments, the antibody or antigen-binding fragment thereof binds to a mature IL-18BP sequence in Table B1, e.g., at a region that does not include the signal peptide (underlined). In specific embodiments, the antibody or antigen-binding fragment thereof binds to an antigenic determinant comprising the IL-18 binding interface of the mature form of IL-18BP. In some embodiments, the antibody or antigen-binding fragment thereof binds to an antigenic determinant comprising I97 and V153 numbered according to UniProt: 095998 (alternatively, I95 and V151 as defined by SEQ ID NO: 1; or I67 and V123 as defined by SEQ ID NO: 2).

在某些實施例中，抗體與SEQ ID NO: 372之成熟IL-18BP序列（成熟人類同功異型物A）之構形抗原決定基結合。在例示性實施例中，本發明之抗體與選自由以下組成之群的至少兩個殘基結合：SEQ ID NO: 372之K32、T40、S60、R61、S66、Y69、R91、R93、T96、K102、R131及H132。在例示性實施例中，本發明之抗體與SEQ ID NO: 372之殘基K32、T40、S60、R61、S66、Y69、R91、R93、T96、K102、R131及H132結合。在例示性實施例中，此類抗體包含序列VH，其包含與SEQ ID NO: 333之至少70%、75%、80%、85%、90%、95%、97%、98%、99%或100%一致性，及序列VL，其包含與SEQ ID NO: 334之至少70%、75%、80%、85%、90%、95%、97%、98%、99%或100%一致性。在例示性實施例中，VHCDR1、VHCDR2及VHCDR3序列分別包含SEQ ID NO: 169-171，且VLCDR1、VLCDR2及VLCDR3序列分別包含SEQ ID NO: 172-174。In certain embodiments, the antibody binds to a conformational epitope of the mature IL-18BP sequence (mature human isoform A) of SEQ ID NO: 372. In exemplary embodiments, the antibody of the present invention binds to at least two residues selected from the group consisting of K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131, and H132 of SEQ ID NO: 372. In exemplary embodiments, the antibody of the present invention binds to residues K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131, and H132 of SEQ ID NO: 372. In exemplary embodiments, such antibodies comprise a sequence VH comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 333, and a sequence VL comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 334. In exemplary embodiments, the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 169-171, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 172-174, respectively.

在某些實施例中，抗體與包含成熟形式之IL-18BP之IL-18結合界面的抗原決定基結合。IL-18BP上與IL-18相互作用之殘基鑑別為：R61、Y69、S75、H79、T116、S119及R131。在例示性實施例中，本發明之抗體與亦由IL-18識別之殘基R61、Y69及R131結合。在例示性實施例中，序列VH包含與SEQ ID NO: 333之至少70%、75%、80%、85%、90%、95%、97%、98%、99%或100%一致性，且序列VL包含與SEQ ID NO: 334之至少70%、75%、80%、85%、90%、95%、97%、98%、99%或100%一致性。在例示性實施例中，此類抗體包含：VHCDR1、VHCDR2及VHCDR3序列，其分別包含SEQ ID NO: 169-171；以及VLCDR1、VLCDR2及VLCDR3序列，其分別包含SEQ ID NO: 172-174。In certain embodiments, the antibody binds to an antigenic determinant of the IL-18 binding interface comprising the mature form of IL-18BP. Residues on IL-18BP that interact with IL-18 are identified as: R61, Y69, S75, H79, T116, S119, and R131. In exemplary embodiments, the antibodies of the invention bind to residues R61, Y69, and R131 that are also recognized by IL-18. In exemplary embodiments, the sequence VH comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 333, and the sequence VL comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 334. In exemplary embodiments, such antibodies comprise: VHCDR1, VHCDR2, and VHCDR3 sequences comprising SEQ ID NOs: 169-171, respectively; and VLCDR1, VLCDR2, and VLCDR3 sequences comprising SEQ ID NOs: 172-174, respectively.

在某些實施例中，抗體與SEQ ID NO: 372之成熟IL-18BP序列（成熟人類同功異型物A）之線性抗原決定基結合。在某些實施例中，抗體對IL-18BP具有直系同源物特異性或直系同源物交叉反應性。舉例而言，在某些實施例中，抗體與人類IL-18BP及食蟹獼猴IL-18BP結合，但不與小鼠IL-18BP（特異性或實質上）結合。在一些實施例中，抗體與人類IL-18BP、食蟹獼猴IL-18BP及小鼠IL-18BP結合。In certain embodiments, the antibody binds to a linear epitope of the mature IL-18BP sequence (mature human isoform A) of SEQ ID NO: 372. In certain embodiments, the antibody has ortholog specificity or ortholog cross-reactivity to IL-18BP. For example, in certain embodiments, the antibody binds to human IL-18BP and cynomolgus macaque IL-18BP, but not to mouse IL-18BP (specifically or substantially). In some embodiments, the antibody binds to human IL-18BP, cynomolgus macaque IL-18BP, and mouse IL-18BP.

在某些實施例中，抗體或其抗原結合片段對IL-18BP具有直系同源物特異性或直系同源物交叉反應性。舉例而言，在某些實施例中，抗體或其抗原結合片段與人類IL-18BP及食蟹獼猴IL-18BP結合，但不與小鼠IL-18BP（特異性或實質上）結合。在一些實施例中，抗體或其抗原結合片段與人類IL-18BP、食蟹獼猴IL-18BP及小鼠IL-18BP結合（例如SA01a、SA51d、SA45a、SA54a、SA55a、SA56a、SA57a、SA59a、SA60a、SA61a、SA62a、SA63a、SA65a、SA73a、SA77a、SA64a、SA66a）。In certain embodiments, the antibody or antigen-binding fragment thereof has ortholog specificity or ortholog cross-reactivity to IL-18BP. For example, in certain embodiments, the antibody or antigen-binding fragment thereof binds to human IL-18BP and cynomolgus macaque IL-18BP, but does not bind to mouse IL-18BP (specifically or substantially). In some embodiments, the antibody or antigen-binding fragment thereof binds to human IL-18BP, cynomolgus macaque IL-18BP, and mouse IL-18BP (e.g., SA01a, SA51d, SA45a, SA54a, SA55a, SA56a, SA57a, SA59a, SA60a, SA61a, SA62a, SA63a, SA65a, SA73a, SA77a, SA64a, SA66a).

在一些實施例中，抗體或其抗原結合片段以比IL-18與IL-18BP之間的結合親和力（對人類IL-18與IL-18BP之KD為約650 pM；或特異性量測為655 ± 136 pM）更強的結合親和力與人類IL-18BP結合。在一些情況下，抗體或其抗原結合片段以以下結合親和力與人類IL-18BP結合：約1 pM至約10 pM至約600 pM或65 pM、或約、至少約或小於約1、5、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200或300、400、500、600或650 pM之結合親和力，或視情況以範圍介於約1 pM至約600 pM、1 pM至約500 pM、1 pM 至約400 pM、1 pM至約300 pM、約1 pM至約200 pM、約1 pM至約100 pM、約1 pM至約50 pM、約1 pM至約40 pM、約1 pM至約30 pM、約1 pM至約20 pM、約1 pM至約10 pM、約1 pM至約5 pM、約5 pM至約600 pM、約5 pM至約500 pM、約5 pM至約400 pM、約5 pM至約300 pM、約5 pM至約200 pM、約5 pM至約100 pM、約5 pM至約50 pM、約5 pM至約40 pM、約5 pM至約30 pM、約5 pM至約20 pM、約5 pM至約10 pM、約10 pM至約600 pM、約10 pM至約500 pM、約10 pM至約400 pM、約10 pM至約300 pM、約10 pM至約200 pM、約10 pM至約100 pM、約10 pM至約50 pM、約10 pM至約40 pM、約10 pM至約30 pM、約10 pM至約20 pM或約20 pM至約600 pM、約20 pM至約500 pM、約20 pM至約400 pM、約20 pM至約300 pM、約20 pM至約200 pM、約20 pM至約100 pM、約20 pM至約50 pM、約20 pM至約40 pM、約20 pM至約30 pM或約30 pM至約600 pM、約30 pM至約500 pM、約30 pM至約400 pM、約30 pM至約300 pM、約30 pM至約200 pM、約30 pM至約100 pM、約30 pM至約50 pM或約30 pM至約40 pM之親和力。KD可藉由本文所述之生物膜干涉技術（BLI）分析來確定。舉例而言，藉由將mAb裝載至抗人類恆定域（AHC）生物感測器（FortéBio）於由含有0.1% BSA、0.02% Tween 20之PBS組成之10×動力學緩衝液中90-120 s以達到0.8至1.2 nm之間的光譜移位值來在Fortébio（現為Sartorius）Octet RED96e儀器上獲取結合動力學量測值。接著可在2倍稀釋系列之hIL-18BP存在下進行締合且使其進行90-120 s。可量測解離300至1200 s。對於較弱變異體，稀釋系列可以100 nM開始，或對於最強效的mAb，則可以10 nM開始。In some embodiments, the antibody or antigen-binding fragment thereof binds to human IL-18BP with a stronger binding affinity than the binding affinity between IL-18 and IL-18BP (KD for human IL-18 and IL-18BP is about 650 pM; or specificity is measured as 655 ± 136 pM). In some cases, the antibody or antigen-binding fragment thereof binds to human IL-18BP with a binding affinity of about 1 pM to about 10 pM to about 600 pM or 65 pM, or about, at least about, or less than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 300, 400, 500, 600, or 650 pM, or, as the case may be, with a binding affinity ranging from about 1 pM to about 600 pM, 1 pM to about 500 pM, 1 pM to about 400 pM, 1 pM to about 300 pM, about 1 pM to about 200 pM, about 1 pM to about 100 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 5 pM to about 600 pM, about 5 pM to about 500 pM, about 5 pM to about 400 pM, about 5 pM to about 300 pM, about 5 pM to about 200 pM, about 5 pM to about 100 pM, about 5 pM to about 50 pM, about 5 pM to about 40 pM, about 5 pM to about 30 pM, about 5 pM to about 20 pM, about 5 pM to about 10 pM, about 10 pM to about 600 pM, about 10 pM to about 500 pM, about 10 pM to about 400 pM, about 10 pM to about 300 pM, about 10 pM to about 200 pM, about 10 pM to about 100 pM, about 10 pM to about 50 pM, about 10 pM to about 40 pM, about 10 pM to about 30 pM, about 10 pM to about 20 pM, or about 20 pM to about 600 pM, about 20 pM to about 500 pM, about 20 pM to about 400 pM, about 20 pM to about 300 pM, about 20 pM to about 200 pM, about 20 pM to about 100 pM, about 20 pM to about 50 pM, about 20 pM to about 40 pM, about 20 pM to about 30 pM, or about 30 pM to about 600 pM, about 30 pM to about 500 pM, about 30 pM to about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200 pM pM to about 200 pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM, or about 30 pM to about 40 pM. KD can be determined by biofilm interferometry (BLI) analysis as described herein. For example, binding kinetics measurements are obtained on a Fortébio (now Sartorius) Octet RED96e instrument by loading mAb onto anti-human constant domain (AHC) biosensors (FortéBio) in 10× kinetic buffer consisting of PBS containing 0.1% BSA, 0.02% Tween 20 for 90-120 s to achieve spectral shift values between 0.8 and 1.2 nm. Binding can then be performed in the presence of a 2-fold dilution series of hIL-18BP and allowed to proceed for 90-120 s. Dissociation can be measured for 300 to 1200 s. The dilution series can start at 100 nM for weaker variants or 10 nM for the most potent mAb.

在一些實施例中，抗體或其抗原結合片段為IL-18BP拮抗劑。在一些情況下，抗體或其抗原結合片段拮抗IL-18BP與其配位體IL-18之間的結合及/或信號傳導活性。在一些實施例中，抗體或其抗原結合片段例如在基於細胞之分析中拮抗或降低IL-18BP與IL-18之間的結合及/或信號傳導活性約或至少約10%-1000%（例如約20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、90%、100%、200%、300%、400%、500%、600%、700%、800%、900%、1000%或更大）。在一些情況下，拮抗性抗IL-18BP抗體或其抗原結合片段阻斷IL-18BP對IL-18之抑制活性，且藉此增加IL-18介導之信號傳導，例如IL-18介導之對IFN-γ、CXCL10及TNFα的誘導。此等功能活性可藉由本文所揭示之分析量測。舉例而言，抗體可與IL-18BP（例如人類IL-18BP）一起培育，之後添加IL-18（例如，重組人類IL-18）。接著可將所得溶液添加至IL-18報導HEK 293細胞中，該等細胞藉由表現NF-κB/AP-1誘導型分泌胚胎鹼性磷酸酶（SEAP）報導基因而對外部添加之IL-18作出反應。接著可藉由相較於適合之對照（諸如同型對照抗體）對報導細胞之作用來分析抗體之作用。其他細節揭示於本文中之材料及方法部分中。另一可能分析涉及量測在KG-1細胞中由抗IL-18BP mAb對IFNγ表現的除抑制作用。簡言之，IL-18BP可用抗體連續稀釋液來預阻斷，接著可將IL-18添加至混合物中，且可將此混合物添加至KG-1細胞中且進行培育。接著可根據習知手段（諸如ELISA）量測分泌的IFN-γ，且將測試抗體之效應與適合對照（諸如同型對照抗體）之效應相比。其他細節揭示於本文中之材料及方法部分中。又一可能分析涉及將PBMC與測試抗體、IL-12及IL-18一起培育及藉由習知手段量測IFNγ及/或CCL2。可將測試抗體之效應與適合對照（諸如同型對照抗體）之效應相比。其他細節揭示於本文中之材料及方法部分中。另一分析涉及培育NK細胞及預複合之hIL-18/hIL-18BP，之後添加IL-12，且接著添加測試抗體之連續稀釋液。將測試抗體之效應與適合對照（諸如同型對照抗體）之效應相比。其他細節揭示於本文中之材料及方法部分中。In some embodiments, the antibody or antigen-binding fragment thereof is an IL-18BP antagonist. In some cases, the antibody or antigen-binding fragment thereof antagonizes the binding and/or signaling activity between IL-18BP and its ligand IL-18. In some embodiments, the antibody or antigen-binding fragment thereof antagonizes or reduces the binding and/or signaling activity between IL-18BP and IL-18 by about or at least about 10%-1000% (e.g., about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more), for example, in a cell-based assay. In some cases, the antagonist anti-IL-18BP antibody or antigen-binding fragment thereof blocks the inhibitory activity of IL-18BP on IL-18, and thereby increases IL-18-mediated signaling, such as IL-18-mediated induction of IFN-γ, CXCL10, and TNFα. Such functional activities can be measured by the assays disclosed herein. For example, the antibody can be incubated with IL-18BP (e.g., human IL-18BP), followed by the addition of IL-18 (e.g., recombinant human IL-18). The resulting solution can then be added to IL-18 reporter HEK 293 cells, which respond to exogenously added IL-18 by expressing the NF-κB/AP-1-induced secreted embryonic alkaline phosphatase (SEAP) reporter gene. The effect of the antibody can then be analyzed by comparing the effect of a suitable control (such as isotype control antibodies) on reporter cells. Other details are disclosed in the Materials and Methods section herein. Another possible analysis involves measuring the inhibitory effect of anti-IL-18BP mAb on IFNγ expression in KG-1 cells. In brief, IL-18BP can be pre-blocked with serial dilutions of the antibody, IL-18 can then be added to the mixture, and this mixture can be added to KG-1 cells and incubated. Secreted IFN-γ can then be measured according to known means (such as ELISA), and the effect of the test antibody can be compared with the effect of a suitable control (such as isotype control antibodies). Other details are disclosed in the Materials and Methods section herein. Another possible analysis involves culturing PBMC with test antibodies, IL-12 and IL-18 and measuring IFNγ and/or CCL2 by known means. The effect of the test antibody can be compared to the effect of a suitable control (such as isotype control antibodies). Other details are disclosed in the Materials and Methods section herein. Another analysis involves culturing NK cells and pre-complexed hIL-18/hIL-18BP, followed by the addition of IL-12, and then adding serial dilutions of the test antibody. The effect of the test antibody is compared to the effect of a suitable control (such as isotype control antibodies). Other details are disclosed in the Materials and Methods section herein.

某些實施例包括針對阻斷或抑制IL-18與IL-18BP之間的結合之能力篩選抗IL-18BP抗體或其抗原結合片段之方法，其包含（a）測定該抗體或其抗原結合片段對（i）單獨的IL-18BP及（ii）低IL-18融合蛋白之結合親和力，其中該低IL-18融合蛋白包含經由可撓性連接子（及其間的視情況存在之蛋白酶裂解位點）與IL-18BP融合的IL-18，其中該融合蛋白之IL-18部分結合至該融合蛋白之IL-18BP部分且空間阻斷該融合蛋白之該IL-18BP部分之IL-18結合位點；（b）比較（i）之結合親和力與（ii）之結合親和力；及（c）若（i）之結合親和力顯著強於（ii）之結合親和力，則將該抗體或其抗原結合片段鑑別或選擇為能夠阻斷或抑制IL-18與IL-18BP之間的結合。某些實施例包含（c）若（i）之結合親和力為（ii）之結合親和力強度的約或至少約2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、20倍、30倍、40倍、50倍、60倍、70倍、80倍、90倍、100倍、200倍、300倍、400倍、500倍、600倍、700倍、800倍、900倍或1000倍或更大倍數，則將該抗體或其抗原結合片段鑑別或選擇為能夠阻斷或抑制IL-18與IL-18BP之間的結合。在某些實施例中，該IL-18及該IL-18BP為小鼠IL-18及小鼠IL-18BP。在一些實施例中，該IL-18及該IL-18BP為人類IL-18及人類IL-18BP。在一些實施例中，該低IL-18融合蛋白在N端至C端定向上包含信號肽、IL-18、第一可撓性連接子、蛋白酶裂解位點（視情況TEV蛋白酶裂解位點）、可撓性連接子及IL-18BP。在具體實施例中，低IL-18融合蛋白包含與來自表S1之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的胺基酸序列。Certain embodiments include methods of screening an anti-IL-18BP antibody or antigen-binding fragment thereof for its ability to block or inhibit the binding between IL-18 and IL-18BP, comprising (a) determining the binding affinity of the antibody or antigen-binding fragment thereof to (i) IL-18BP alone and (ii) a low IL-18 fusion protein, wherein the low IL-18 fusion protein comprises IL-18BP fused to IL-18BP via a flexible linker (and optionally a protease cleavage site therebetween); -18, wherein the IL-18 portion of the fusion protein binds to the IL-18BP portion of the fusion protein and sterically blocks the IL-18 binding site of the IL-18BP portion of the fusion protein; (b) comparing the binding affinity of (i) with the binding affinity of (ii); and (c) if the binding affinity of (i) is significantly stronger than the binding affinity of (ii), identifying or selecting the antibody or antigen-binding fragment thereof as being capable of blocking or inhibiting the binding between IL-18 and IL-18BP. In some embodiments, the antibody or antigen-binding fragment thereof is identified or selected as being capable of blocking or inhibiting the binding between IL-18 and IL-18BP if the binding affinity of (i) is about or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times or more as strong as the binding affinity of (ii). In some embodiments, the IL-18 and the IL-18BP are mouse IL-18 and mouse IL-18BP. In some embodiments, the IL-18 and the IL-18BP are human IL-18 and human IL-18BP. In some embodiments, the low IL-18 fusion protein comprises a signal peptide, IL-18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker, and IL-18BP in an N-terminal to C-terminal orientation. In specific embodiments, the low IL-18 fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence from Table S1.

僅出於說明性目的，可使用各種常規方法，包括Biacore®分析（例如，用適當標記之可溶性試劑，結合至感測器晶片）、利用在細胞表面（原生或重組）上表現IL-18BPr之細胞進行的FACS分析、免疫分析、螢光染色分析、ELISA分析及諸如等溫滴定量熱法（Isothermal Titration Calorimetry，ITC）之微量量熱法方法，來偵測及定量本文所述之IL-18BP、IL-18（例如，低IL-18）及/或抗IL-18BP抗體或其抗原結合片段的任何組合之間的結合相互作用（例如結合親和力），或IL-18BP與IL-18之間的結合/信號傳導。類似地，抗IL-18BP抗體之功能特性可使用熟習此項技術者已知之多種方法評估：親和力/結合分析（例如，表面電漿子共振、競爭性抑制分析）；細胞毒性分析、細胞存活率分析、細胞增殖或分化分析、使用活體外或活體內模型之癌細胞及/或腫瘤生長抑制。其他分析可測試本文所述之抗體調節（例如抑制）IL-18BP及/或IL-18介導之反應的能力。亦可針對活體外及活體內功效測試本文所述之抗體。此類分析可使用熟習此項技術者已知之公認方案（參見例如Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY)；Current Protocols in Immunology (編輯：John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY）；或市售套組進行。For illustrative purposes only, binding interactions (e.g., binding affinity) between any combination of IL-18BP, IL-18 (e.g., low IL-18) and/or anti-IL-18BP antibodies or antigen-binding fragments thereof described herein, or binding/signaling between IL-18BP and IL-18 can be detected and quantified using a variety of conventional methods, including Biacore® analysis (e.g., with appropriately labeled soluble reagents bound to a sensor chip), FACS analysis using cells expressing IL-18BPr on the cell surface (native or recombinant), immunoassays, fluorescent staining analysis, ELISA analysis, and microcalorimetric methods such as Isothermal Titration Calorimetry (ITC). Similarly, the functional properties of anti-IL-18BP antibodies can be evaluated using a variety of methods known to those skilled in the art: affinity/binding assays (e.g., surface plasmon resonance, competitive inhibition assays); cytotoxicity assays, cell viability assays, cell proliferation or differentiation assays, inhibition of cancer cell and/or tumor growth using in vitro or in vivo models. Other assays can test the ability of the antibodies described herein to modulate (e.g., inhibit) IL-18BP and/or IL-18 mediated responses. The antibodies described herein can also be tested for efficacy in vitro and in vivo. Such analyses can be performed using recognized protocols known to those skilled in the art (see, e.g., Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY); Current Protocols in Immunology (Editors: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); or commercially available kits.

在特定實施例中，抗體或其抗原結合片段之Fc區包含以下、由以下組成或基本上由以下組成：IgA（包括子類IgA1及IgA2）、IgD、IgE、IgG（包括子類IgG1、IgG2、IgG3及IgG4）或IgM Fc域，視情況人類Fc域或其雜合體及/或變異體。在特定實施例中，Fc區包含以下、由以下組成或基本上由以下組成：來自人類IgG1或IgG4之Fc（參見例如Allberse及Schuurman, Immunology. 105:9-19, 2002）或其片段或變異體。In certain embodiments, the Fc region of the antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of an IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), or IgM Fc domain, optionally a human Fc domain, or a hybrid and/or variant thereof. In certain embodiments, the Fc region comprises, consists of, or consists essentially of an Fc from human IgG1 or IgG4 (see, e.g., Allberse and Schuurman, Immunology. 105:9-19, 2002), or a fragment or variant thereof.

在某些實施例中，抗體或其抗原結合片段包含變異體或以其他方式修飾之Fc區，包括相對於野生型Fc區具有改變之特性或生物活性之彼等。經修飾之Fc區的實例包括具有突變序列之彼等，例如相對於野生型序列藉由取代、插入、缺失或截斷一或多個胺基酸；由來自不同免疫球蛋白類別/子類之域構成的雜合Fc多肽；具有改變之糖基化/唾液酸化模式的Fc多肽；及例如藉由生物素化（參見例如美國申請案第2010/0209424號）、磷酸化、硫酸化等或前述之任何組合修飾或衍生化的Fc多肽。相對於抗體或其抗原結合片段之對應野生型Fc序列，此類修飾可用於改變（例如，提高、降低）Fc區對一或多種特定FcR（例如，FcγRI、FcγRIIa、FcγRIIb、FcγRIIc、FcγRIIIa、FcγRIIIb、FcRn）之結合特性、其藥物動力學特性（例如，穩定性或半衰期、生物可用性、組織分佈、分佈體積、濃度、消除速率常數、消除速率、曲線下面積（AUC）、清除率、Cmax、Tmax、Cmin、波動）、其免疫原性、其補體結合或活化及/或Fc區之CDC/ADCC/ADCP相關活性以及本文所述之其他特性。包括人類及/或小鼠來源之經修飾之Fc區。In certain embodiments, the antibodies or antigen-binding fragments thereof comprise variant or otherwise modified Fc regions, including those having altered properties or biological activities relative to wild-type Fc regions. Examples of modified Fc regions include those having mutant sequences, e.g., by substitution, insertion, deletion, or truncation of one or more amino acids relative to the wild-type sequence; hybrid Fc polypeptides composed of domains from different immunoglobulin classes/subclasses; Fc polypeptides having altered glycosylation/sialylation patterns; and Fc polypeptides modified or derivatized, e.g., by biotinylation (see, e.g., U.S. Application No. 2010/0209424), phosphorylation, sulfation, etc., or any combination of the foregoing. Such modifications can be used to alter (e.g., increase, decrease) the binding properties of the Fc region to one or more specific FcRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, FcRn), its pharmacokinetic properties (e.g., stability or half-life, bioavailability, tissue distribution, distribution volume, concentration, elimination rate constant, elimination rate, area under the curve (AUC), clearance, Cmax, Tmax, Cmin, fluctuation), its immunogenicity, its complement binding or activation and/or CDC/ADCC/ADCP-related activities of the Fc region, and other properties described herein, relative to the corresponding wild-type Fc sequence of the antibody or antigen-binding fragment thereof. Modified Fc regions of human and/or mouse origin are included.

在某些實施例中，抗體或其抗原結合片段包含雜合Fc區，例如包含來自不同物種（例如人類、小鼠）、不同Ig類別及/或不同Ig子類之免疫球蛋白之Fc域（例如，鉸鏈、CH2、CH3、CH4）之組合的Fc區。亦包括包含衍生化或以其他方式修飾之Fc區的抗體或其抗原結合片段。在某些態樣中，Fc區藉由例如相對於野生型或天然存在之Fc區進行磷酸化、硫酸化、丙烯醯化、糖基化、甲基化、法呢基化、乙醯化、醯胺化及其類似方式來修飾。在某些實施例中，Fc區包含野生型或天然糖基化模式，或可替代地，其包含相對於天然形式增加之糖基化、相對於天然形式減少之糖基化，或其完全去糖基化。作為經修飾之Fc糖型之一個實例，Fc區之糖基化減少可減少與第一補體組分C1之C1q區的結合、降低ADCC相關活性及/或降低CDC相關活性。因此，某些實施例採用去糖基化或非糖基化之Fc區。關於例示性非糖基化之Fc區之產生，參見例如WO 2005/047337。Fc區糖型之另一實例係根據Kabat等人之編號系統，藉由用半胱胺酸殘基取代Q295位置產生（參見例如美國申請案第2010/0080794號）。某些實施例包括如下Fc區，其中Fc區中約80%-100%之醣蛋白包含缺乏岩藻糖之成熟核心碳水化合物結構（參見例如美國申請案第2010/0255013號）。一些實施例包括如下Fc區：其藉由取代或缺失而最佳化以降低岩藻糖基化水平，例如增加對FcγRI、FcγRIa或FcγRIIIa之親和力，及/或改善由表現FcγRIIa之細胞的吞噬作用（參見美國申請案第2010/0249382號及第2007/0148170號）。In certain embodiments, the antibody or antigen-binding fragment thereof comprises a hybrid Fc region, for example, an Fc region comprising a combination of Fc domains (e.g., hinge, CH2, CH3, CH4) of immunoglobulins from different species (e.g., human, mouse), different Ig classes, and/or different Ig subclasses. Antibodies or antigen-binding fragments thereof comprising a derivatized or otherwise modified Fc region are also included. In certain aspects, the Fc region is modified by, for example, phosphorylation, sulfation, acrylylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like relative to a wild-type or naturally occurring Fc region. In certain embodiments, the Fc region comprises a wild-type or native glycosylation pattern, or alternatively, it comprises increased glycosylation relative to the native form, reduced glycosylation relative to the native form, or it is completely deglycosylated. As an example of a modified Fc glycoform, reduced glycosylation of the Fc region can reduce binding to the C1q region of the first complement component C1, reduce ADCC-related activity, and/or reduce CDC-related activity. Therefore, certain embodiments employ a deglycosylated or non-glycosylated Fc region. For the generation of exemplary non-glycosylated Fc regions, see, e.g., WO 2005/047337. Another example of an Fc region glycoform is generated according to the numbering system of Kabat et al. by replacing the Q295 position with a cysteine residue (see, e.g., U.S. Application No. 2010/0080794). Certain embodiments include an Fc region in which about 80%-100% of the glycoprotein in the Fc region comprises a mature core carbohydrate structure lacking fucose (see, e.g., U.S. Application No. 2010/0255013). Some embodiments include an Fc region that is optimized by substitution or deletion to reduce the level of fucosylation, e.g., to increase affinity for FcγRI, FcγRIa, or FcγRIIIa, and/or to improve phagocytosis by cells expressing FcγRIIa (see, e.g., U.S. Application Nos. 2010/0249382 and 2007/0148170).

作為經修飾之Fc糖型的另一實例，抗體或其抗原結合片段之Fc區可包含寡甘露糖型N-聚醣，且相對於含有複合型N-聚醣之對應Fc區，視情況具有以下中之一或多者：增加的ADCC效應活性、增加的對FcγRIIIA（及某些其他FcR）之結合親和力、類似或增加的對IL-18BP多肽之目標的結合特異性、類似或更高的對IL-18BP多肽之目標的結合親和力及/或類似或更低的對甘露糖受體的結合親和力（參見例如美國申請案第2007/0092521號及美國專利第7,700,321號）。作為另一實例，Fc區對FcγR增強的親和力已使用工程化糖型達成，該等工程化糖型係藉由在工程化或變異細胞株中表現抗體來產生（參見例如，Umana等人, Nat Biotechnol. 17:176-180, 1999；Davies等人, Biotechnol Bioeng. 74:288-294, 2001；Shields等人, J Biol Chem. 277:26733-26740, 2002；Shinkawa等人, 2003, J Biol Chem. 278:3466-3473, 2003；以及美國申請案第2007/0111281號）。某些Fc區糖型包含增加比例之N-醣苷鍵型複合糖鏈，在糖鏈之還原端岩藻糖之1位未結合至N-乙醯基葡糖胺之6位（參見例如美國申請案第2010/0092997號）。特定實施例可包括經藉由α-2,6鍵連接至各別末端唾液酸部分之至少一個半乳糖部分而糖基化的IgG Fc區，視情況其中Fc區相對於對應野生型Fc區具有較高的抗發炎活性（參見美國申請案第2008/0206246號）。某些此等及相關改變之糖基化方法已顯著增強Fc區選擇性地結合諸如FcγRIII之FcR、介導ADCC及改變如本文所述之Fc區之其他特性的能力。As another example of a modified Fc glycoform, the Fc region of an antibody or antigen-binding fragment thereof may comprise oligomannose-type N-glycans and, relative to a corresponding Fc region containing complex N-glycans, optionally have one or more of the following: increased ADCC effector activity, increased binding affinity for FcγRIIIA (and certain other FcRs), similar or increased binding specificity for a target of the IL-18BP polypeptide, similar or higher binding affinity for a target of the IL-18BP polypeptide, and/or similar or lower binding affinity for a mannose receptor (see, e.g., U.S. Application No. 2007/0092521 and U.S. Patent No. 7,700,321). As another example, enhanced affinity of the Fc region for FcγRs has been achieved using engineered glycoforms produced by expressing the antibody in engineered or mutant cell lines (see, e.g., Umana et al., Nat Biotechnol. 17:176-180, 1999; Davies et al., Biotechnol Bioeng. 74:288-294, 2001; Shields et al., J Biol Chem. 277:26733-26740, 2002; Shinkawa et al., 2003, J Biol Chem. 278:3466-3473, 2003; and U.S. Appl. No. 2007/0111281). Certain Fc region glycoforms comprise an increased proportion of N-glycosidically bonded complex sugar chains, with the 1-position of the reducing end fucose not bound to the 6-position of N-acetylglucosamine of the sugar chain (see, e.g., U.S. Application No. 2010/0092997). Specific embodiments may include IgG Fc regions glycosylated with at least one galactose moiety linked to a respective terminal sialic acid moiety via an α-2,6 bond, optionally wherein the Fc region has a higher anti-inflammatory activity relative to a corresponding wild-type Fc region (see, e.g., U.S. Application No. 2008/0206246). Certain of these and related altered glycosylation methods have significantly enhanced the ability of the Fc region to selectively bind to FcRs such as FcγRIII, mediate ADCC, and alter other properties of the Fc region as described herein.

相對於對應野生型Fc序列（例如相同物種、相同Ig類別、相同Ig子類），抗體或其抗原結合片段之某些變異體、片段、雜合體或以其他方式修飾之Fc區可具有改變之與一或多種FcR的結合，及/或效應功能可發生相應變化。舉例而言，相對於對應野生型Fc序列，此類Fc區可具有增加之與Fcγ受體、Fcα受體、Fcε受體及/或新生兒Fc受體中之一或多者的結合。在其他實施例中，相對於對應野生型Fc序列，變異體、片段、雜合體或經修飾之Fc區可具有減少之與Fcγ受體、Fcα受體、Fcε受體及/或新生兒Fc受體中之一或多者的結合。本文中其他地方描述特定FcR。Certain variants, fragments, hybrids, or otherwise modified Fc regions of antibodies or antigen-binding fragments thereof may have altered binding to one or more FcRs, and/or corresponding changes in effector function, relative to a corresponding wild-type Fc sequence (e.g., same species, same Ig class, same Ig subclass). For example, such Fc regions may have increased binding to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and/or a neonatal Fc receptor, relative to a corresponding wild-type Fc sequence. In other embodiments, the variant, fragment, hybrid, or modified Fc region may have decreased binding to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and/or a neonatal Fc receptor, relative to a corresponding wild-type Fc sequence. Specific FcRs are described elsewhere herein.

在一些實施例中，抗體包含Fc域，相對於對應野生型Fc序列，該Fc域包含一或多個突變以增加與Fcγ受體、Fcα受體、Fcε受體及/或新生兒Fc受體中之一或多者的結合。在一些實施例中，抗體包含IgG1或IgG3 Fc域，相對於對應野生型Fc序列，該Fc域包含一或多個突變以增加與Fcγ受體、Fcα受體、Fcε受體及/或新生兒Fc受體中之一或多者的結合。在一些實施例中，抗體包含有包含一或多個突變以增加效應功能的Fc域。在一些實施例中，抗體包含選自人類IgG1及IgG3的包含一或多個突變以增加效應功能的Fc域。In some embodiments, the antibody comprises an Fc domain comprising one or more mutations relative to a corresponding wild-type Fc sequence to increase binding to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and/or a neonatal Fc receptor. In some embodiments, the antibody comprises an IgG1 or IgG3 Fc domain comprising one or more mutations relative to a corresponding wild-type Fc sequence to increase binding to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and/or a neonatal Fc receptor. In some embodiments, the antibody comprises an Fc domain comprising one or more mutations to increase effector function. In some embodiments, the antibody comprises an Fc domain selected from human IgG1 and IgG3 comprising one or more mutations to increase effector function.

在一些實施例中，抗體為包含具有高效應子活性之Fc域的阻斷抗體。在一些實施例中，阻斷抗體包含選自人類IgG1及IgG3的包含一或多個突變以增加效應功能的Fc域。在一些實施例中，抗體為包含具有高效應子活性之Fc域的部分阻斷抗體。在一些實施例中，部分阻斷抗體包含選自人類IgG1及IgG3的包含一或多個突變以增加效應功能的Fc域。在一些實施例中，抗體為包含具有高效應子活性之Fc域的非阻斷抗體。在一些實施例中，非阻斷抗體包含選自人類IgG1或IgG3的包含一或多個突變以增加效應功能的Fc域。In some embodiments, the antibody is a blocking antibody comprising an Fc domain with high-efficiency antigenic activity. In some embodiments, the blocking antibody comprises an Fc domain selected from human IgG1 and IgG3 comprising one or more mutations to increase effector function. In some embodiments, the antibody is a partial blocking antibody comprising an Fc domain with high-efficiency antigenic activity. In some embodiments, the partial blocking antibody comprises an Fc domain selected from human IgG1 and IgG3 comprising one or more mutations to increase effector function. In some embodiments, the antibody is a non-blocking antibody comprising an Fc domain with high-efficiency antigenic activity. In some embodiments, the non-blocking antibody comprises an Fc domain selected from human IgG1 or IgG3 comprising one or more mutations to increase effector function.

在一些實施例中，抗體包含Fc域，相對於對應野生型Fc序列，該Fc域包含一或多個突變以減少與Fcγ受體、Fcα受體、Fcε受體及/或新生兒Fc受體中之一或多者的結合。在一些實施例中，抗體包含IgG1或IgG3 Fc域，相對於對應野生型Fc序列，該Fc域包含一或多個突變以減少與Fcγ受體、Fcα受體、Fcε受體及/或新生兒Fc受體中之一或多者的結合。在一些實施例中，抗體包含有包含一或多個突變以減少效應功能之Fc域。在一些實施例中，抗體包含選自人類IgG2及IgG4的包含一或多個突變以減少效應功能的Fc域。In some embodiments, the antibody comprises an Fc domain comprising one or more mutations relative to a corresponding wild-type Fc sequence to reduce binding to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and/or a neonatal Fc receptor. In some embodiments, the antibody comprises an IgG1 or IgG3 Fc domain comprising one or more mutations relative to a corresponding wild-type Fc sequence to reduce binding to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and/or a neonatal Fc receptor. In some embodiments, the antibody comprises an Fc domain comprising one or more mutations to reduce effector function. In some embodiments, the antibody comprises an Fc domain selected from human IgG2 and IgG4 comprising one or more mutations to reduce effector function.

在一些實施例中，抗體為包含具有低效應子活性之Fc域的阻斷抗體。在一些實施例中，阻斷抗體包含選自人類IgG2及IgG4的包含一或多個突變以減少效應功能的Fc域。在一些實施例中，抗體為包含具有低效應子活性之Fc域的部分阻斷抗體。在一些實施例中，部分阻斷抗體包含選自人類IgG2及IgG4的包含一或多個突變以減少效應功能的Fc域。在一些實施例中，抗體為包含具有低效應子活性之Fc域的非阻斷抗體。在一些實施例中，非阻斷抗體包含選自人類IgG2及IgG4的包含一或多個突變以減少效應功能的Fc域。In some embodiments, the antibody is a blocking antibody comprising an Fc domain with low effector activity. In some embodiments, the blocking antibody comprises an Fc domain selected from human IgG2 and IgG4 comprising one or more mutations to reduce effector function. In some embodiments, the antibody is a partial blocking antibody comprising an Fc domain with low effector activity. In some embodiments, the partial blocking antibody comprises an Fc domain selected from human IgG2 and IgG4 comprising one or more mutations to reduce effector function. In some embodiments, the antibody is a non-blocking antibody comprising an Fc domain with low effector activity. In some embodiments, the non-blocking antibody comprises an Fc domain selected from human IgG2 and IgG4 comprising one or more mutations to reduce effector function.

具有改變（例如增加、減少）之效應功能/FcR結合的Fc變異體之特定實例可見於例如美國專利第5,624,821號及第7,425,619號；美國申請案第2009/0017023號、第2009/0010921號及第2010/0203046號；以及WO 2000/42072及WO 2004/016750中。某些實例包括在位置298、333及/或334處具有一或多個取代，例如S298A、E333A及/或K334A（基於Kabat等人之EU索引之編號）的人類Fc區，已顯示其增加與活化受體FcγRIIIa之結合且減少與抑制受體FcγRIIb之結合。此等突變可加以組合以獲得與FcR之結合進一步改善的雙重及三重突變變異體。某些實施例包括S298A/E333A/K334A三重突變體，其增加與FcγRIIIa的結合，減少與FcγRIIb的結合且增加ADCC（參見例如Shields等人, J Biol Chem. 276:6591-6604, 2001；以及Presta等人, Biochem Soc Trans. 30:487-490, 2002）。亦參見如Umana等人, 見上文；及美國專利第7,662,925號中所揭示的具有增加之與FcR之結合的經工程改造之Fc糖型。一些實施例包括Fc區，基於Kabat等人之EU索引，該等Fc區包含一或多個選自434S、252Y/428L、252Y/434S及428L/434S之取代（參見美國申請案第2009/0163699號及第20060173170號）。一些實施例包括Fc區，基於Kabat等人之EU索引，該等Fc區包含一或多個選自L234A、L235A及G237A之取代（參見美國申請案第17/779,425號）。一些實施例包括Fc區，基於Kabat等人之EU索引，該等Fc區包含在L234A及L235A處之取代。一些實施例包括Fc區，基於Kabat等人之EU索引，該等Fc區包含在L234A及G237A處之取代。一些實施例包括Fc區，基於Kabat等人之EU索引，該等Fc區包含在L235A及G237A處之取代。一些實施例包括Fc區，基於Kabat等人之EU索引，該等Fc區包含在L234A、L235A及G237A處之取代。一些實施例包括Fc區，基於Kabat等人之EU索引，該等Fc區包含一或多個選自M252Y、S254T及T256E之取代。一些實施例包括Fc區，基於Kabat等人之EU索引，該等Fc區包含一或多個選自M428L及N434S之取代。在一些實施例中，上文提及的Fc取代係針對選自人類IgG1、IgG2、IgG3及IgG4之Fc域。在一些實施例中，上文提及的Fc取代係針對人類IgG1 Fc域。在一些實施例中，上文提及的Fc取代係針對人類IgG2 Fc域。在一些實施例中，上文提及的Fc取代係針對人類IgG3 Fc域。在一些實施例中，上文提及的Fc取代係針對人類IgG4 Fc域。在一些實施例中，本發明之抗體包含本文所揭示之Fc取代以及VH及VL序列，其與來自表A2中之單一命名抗體（例如SA01a）之相應序列至少70%、75%、80%、85%、90%、95%、97%、98%、99%或100%一致，其中該抗體包含如表A1中所列舉的該單一命名抗體（例如SA01a）之CDR。Specific examples of Fc variants with altered (e.g., increased, decreased) effector function/FcR binding can be found in, e.g., U.S. Patent Nos. 5,624,821 and 7,425,619; U.S. Application Nos. 2009/0017023, 2009/0010921, and 2010/0203046; and WO 2000/42072 and WO 2004/016750. Certain examples include human Fc regions with one or more substitutions at positions 298, 333, and/or 334, such as S298A, E333A, and/or K334A (numbering based on the EU index of Kabat et al.), which have been shown to increase binding to the activating receptor FcγRIIIa and decrease binding to the inhibitory receptor FcγRIIb. These mutations can be combined to obtain double and triple mutant variants with further improved binding to FcRs. Certain embodiments include the S298A/E333A/K334A triple mutant, which increases binding to FcγRIIIa, decreases binding to FcγRIIb and increases ADCC (see, e.g., Shields et al., J Biol Chem. 276:6591-6604, 2001; and Presta et al., Biochem Soc Trans. 30:487-490, 2002). See also the engineered Fc glycoforms with increased binding to FcR disclosed in Umana et al., supra; and U.S. Pat. No. 7,662,925. Some embodiments include Fc regions comprising one or more substitutions selected from 434S, 252Y/428L, 252Y/434S, and 428L/434S, based on the EU index of Kabat et al. (see U.S. Application Nos. 2009/0163699 and 20060173170). Some embodiments include Fc regions comprising one or more substitutions selected from L234A, L235A, and G237A, based on the EU index of Kabat et al. (see U.S. Application No. 17/779,425). Some embodiments include Fc regions comprising substitutions at L234A and L235A, based on the EU index of Kabat et al. Some embodiments include Fc regions comprising substitutions at L234A and G237A based on the EU index of Kabat et al. Some embodiments include Fc regions comprising substitutions at L235A and G237A based on the EU index of Kabat et al. Some embodiments include Fc regions comprising substitutions at L234A, L235A and G237A based on the EU index of Kabat et al. Some embodiments include Fc regions comprising substitutions at L234A, L235A and G237A based on the EU index of Kabat et al. Some embodiments include Fc regions comprising one or more substitutions selected from M252Y, S254T and T256E based on the EU index of Kabat et al. Some embodiments include Fc regions comprising one or more substitutions selected from M428L and N434S based on the EU index of Kabat et al. In some embodiments, the Fc substitutions mentioned above are for an Fc domain selected from human IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc substitutions mentioned above are for human IgG1 Fc domains. In some embodiments, the Fc substitutions mentioned above are for human IgG2 Fc domains. In some embodiments, the Fc substitutions mentioned above are for human IgG3 Fc domains. In some embodiments, the Fc substitutions mentioned above are for human IgG4 Fc domains. In some embodiments, the antibodies of the invention comprise Fc substitutions and VH and VL sequences disclosed herein that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the corresponding sequences from a single named antibody in Table A2 (e.g., SA01a), wherein the antibody comprises the CDRs of the single named antibody (e.g., SA01a) as listed in Table A1.

相對於對應野生型Fc序列，某些變異體、片段、雜合物或經修飾之Fc區可具有改變之效應功能。舉例而言，相對於對應野生型Fc序列，此類Fc區可具有增加之補體結合或活化、增加之Clq結合親和力、增加之CDC相關活性、增加之ADCC相關活性及/或增加之ADCP相關活性。在其他實施例中，相對於對應野生型Fc序列，此類Fc區可具有減少的補體結合或活化、減少的Clq結合親和力、減少的CDC相關活性、減少的ADCC相關活性及/或減少的ADCP相關活性。僅僅作為一個說明性實例，Fc區可包含諸如C1q結合位點之補體結合位點中的缺失或取代，及/或ADCC位點中之缺失或取代。此類缺失/取代之實例描述於例如美國專利第7,030,226號中。可根據此項技術中之常規技術分析諸如ADCC的許多Fc效應功能。（參見例如Zuckerman等人, CRC Crit Rev Microbiol. 7:1-26, 1978）。用於此類分析之適用效應細胞包括但不限於自然殺手（NK）細胞、巨噬細胞及其他周邊血液單核細胞（PBMC）。替代地或另外，可活體內評估某些Fc效應功能，例如藉由採用以下文獻中所述的動物模型：Clynes等人, PNAS. 95:652-656, 1998。Certain variants, fragments, hybrids or modified Fc regions may have altered effector functions relative to the corresponding wild-type Fc sequence. For example, such Fc regions may have increased complement binding or activation, increased Clq binding affinity, increased CDC-related activity, increased ADCC-related activity and/or increased ADCP-related activity relative to the corresponding wild-type Fc sequence. In other embodiments, such Fc regions may have reduced complement binding or activation, reduced Clq binding affinity, reduced CDC-related activity, reduced ADCC-related activity and/or reduced ADCP-related activity relative to the corresponding wild-type Fc sequence. As an illustrative example only, the Fc region may include deletions or substitutions in a complement binding site, such as a C1q binding site, and/or deletions or substitutions in an ADCC site. Examples of such deletions/substitutions are described, for example, in U.S. Patent No. 7,030,226. Many Fc effector functions, such as ADCC, can be analyzed according to routine techniques in the art. (See, for example, Zuckerman et al., CRC Crit Rev Microbiol. 7:1-26, 1978). Suitable effector cells for such analysis include, but are not limited to, natural killer (NK) cells, macrophages, and other peripheral blood mononuclear cells (PBMCs). Alternatively or additionally, certain Fc effector functions can be assessed in vivo, for example by employing an animal model as described in Clynes et al., PNAS. 95:652-656, 1998.

相對於對應野生型Fc序列，某些變異型雜合體或經修飾之Fc區可具有改變的穩定性或半衰期。在某些實施例中，相對於對應野生型Fc序列，此類Fc區可具有增加之半衰期。在其他實施例中，相對於對應野生型Fc序列，變異型雜合體或經修飾之Fc區可具有減少之半衰期。可根據此項技術中之常規技術，諸如放射性標記、ELISA或其他方法，在活體外（例如在生理條件下）或在活體內量測半衰期。穩定性或半衰期的活體內量測值可在包括血液、血清、血漿、尿液或腦脊髓液之一或多種體液或諸如肝臟、腎臟、肌肉、中樞神經系統組織、骨骼等指定組織中量測。作為一個實例，改變Fc區結合FcRn之能力的對Fc區之修飾可在活體內改變其半衰期。用於量測活體內藥物動力學特性（例如活體內平均消除半衰期）的分析及改變其與FcRn之結合的Fc修飾之非限制性實例描述於例如美國專利第7,217,797號及第7,732,570號；以及美國申請案第US 2010/0143254號及第2010/0143254號中。Certain variant hybrids or modified Fc regions may have altered stability or half-life relative to the corresponding wild-type Fc sequence. In certain embodiments, such Fc regions may have increased half-life relative to the corresponding wild-type Fc sequence. In other embodiments, variant hybrids or modified Fc regions may have decreased half-life relative to the corresponding wild-type Fc sequence. Half-life may be measured in vitro (e.g., under physiological conditions) or in vivo according to conventional techniques in the art, such as radiolabeling, ELISA or other methods. In vivo measurements of stability or half-life can be measured in one or more body fluids including blood, serum, plasma, urine, or cerebrospinal fluid, or in specified tissues such as liver, kidney, muscle, central nervous system tissue, bone, etc. As an example, modifications to the Fc region that alter the ability of the Fc region to bind to FcRn can alter its half-life in vivo. Non-limiting examples of assays for measuring in vivo pharmacokinetic properties (e.g., in vivo mean elimination half-life) and Fc modifications that alter their binding to FcRn are described, for example, in U.S. Patent Nos. 7,217,797 and 7,732,570; and U.S. Application Nos. US 2010/0143254 and 2010/0143254.

改變穩定性或半衰期之修飾的其他非限制性實例包括根據Kabat等人之編號系統，選自CH2域中之251-256、285-290及308-314以及CH3域中之385-389及428-436之一或多個胺基酸殘基的取代/缺失。參見美國申請案第2003/0190311號。特定實例包括在位置251處經白胺酸取代；在位置252處經酪胺酸、色胺酸或苯丙胺酸取代；在位置254處經蘇胺酸或絲胺酸取代；在位置255處經精胺酸取代；在位置256處經麩醯胺酸、精胺酸、絲胺酸、蘇胺酸或麩胺酸取代；在位置308處經蘇胺酸取代；在位置309處經脯胺酸取代；在位置311處經絲胺酸取代；在位置312處經天冬胺酸取代；在位置314處經白胺酸取代；在位置385處經精胺酸、天冬胺酸或絲胺酸取代；在位置386處經蘇胺酸或脯胺酸取代；在位置387處經精胺酸或脯胺酸取代；在位置389處經脯胺酸、天冬醯胺或絲胺酸取代；在位置428處經甲硫胺酸或蘇胺酸取代；在位置434處經酪胺酸或苯丙胺酸取代；在位置433處經組胺酸、精胺酸、離胺酸或絲胺酸取代；及/或在位置436處經組胺酸、酪胺酸、精胺酸或蘇胺酸取代，包括其任何組合。相對於對應野生型Fc區，此類修飾視情況增加Fc區對FcRn之親和力，且從而增加半衰期。Other non-limiting examples of modifications that alter stability or half-life include substitution/deletion of one or more amino acid residues selected from 251-256, 285-290 and 308-314 in the CH2 domain and 385-389 and 428-436 in the CH3 domain according to the numbering system of Kabat et al. See U.S. Application No. 2003/0190311. Specific examples include substitution at position 251 with leucine; substitution at position 252 with tyrosine, tryptophan, or phenylalanine; substitution at position 254 with threonine or serine; substitution at position 255 with arginine; substitution at position 256 with glutamine, arginine, serine, threonine, or glutamine; substitution at position 308 with threonine; substitution at position 309 with proline; substitution at position 311 with serine; substitution at position 312 with aspartic acid; substitution at position 314 with leucine; substitution at position at position 428 with methionine or threonine; at position 434 with tyrosine or phenylalanine; at position 433 with histidine, arginine, lysine or serine; and/or at position 436 with histidine, tyrosine, arginine or threonine, including any combination thereof. Such modifications optionally increase the affinity of the Fc region for FcRn relative to a corresponding wild-type Fc region, and thereby increase the half-life.

相對於對應野生型Fc序列，某些變異雜合體或經修飾之Fc區可具有改變之溶解度。在某些實施例中，相對於對應野生型Fc序列，此類Fc區可具有增加之溶解度。在其他實施例中，相對於對應野生型Fc序列，變異雜合體或經修飾之Fc區可具有減少之溶解度。可根據此項技術中之常規技術例如活體外（例如在生理條件下）量測溶解度。本文中其他地方描述例示性溶解度量測結果。Certain variant hybrids or modified Fc regions may have altered solubility relative to the corresponding wild-type Fc sequence. In certain embodiments, such Fc regions may have increased solubility relative to the corresponding wild-type Fc sequence. In other embodiments, variant hybrids or modified Fc regions may have decreased solubility relative to the corresponding wild-type Fc sequence. Solubility may be measured according to conventional techniques in the art, such as in vitro (e.g., under physiological conditions). Exemplary solubility measurement results are described elsewhere herein.

變異Fc區亦可具有如例如美國申請案第2003/0118592號中所描述的一或多個突變鉸鏈區。舉例而言，鉸鏈區中之一或多個半胱胺酸可缺失或經不同胺基酸取代。突變鉸鏈區可不包含半胱胺酸殘基，或其可包含比對應野生型鉸鏈區少1、2或3個半胱胺酸殘基。在一些實施例中，具有此類型之突變鉸鏈區的Fc區展現相對於野生型Ig鉸鏈區降低之二聚合能力。Variant Fc regions may also have one or more mutant hinge regions as described, for example, in U.S. Application No. 2003/0118592. For example, one or more cysteines in the hinge region may be deleted or substituted with different amino acids. A mutant hinge region may contain no cysteine residues, or it may contain 1, 2, or 3 fewer cysteine residues than the corresponding wild-type hinge region. In some embodiments, an Fc region having a mutant hinge region of this type exhibits reduced dimerization ability relative to a wild-type Ig hinge region.

在特定實施例中，抗體或其抗原結合片段在約pH 7.4下，在約生理pH下，在約25℃或室溫下及/或在約37℃或人體溫度（例如活體內，血清中，給定組織中，諸如大鼠、小鼠、猴或人類之給定物種中）下的生物半衰期為約或至少約30分鐘、約1小時、約2小時、約3小時、約4小時、約5小時、約6小時、約12小時、約18小時、約20小時、約24小時、約30小時、約36小時、約40小時、約48小時、約50小時、約60小時、約70小時、約72小時、約80小時、約84小時、約90小時、約96小時、約120小時或約144小時或更長時間，或者約1週、或約2週、或約3週、或約4週、或約5週、或約6週或更長時間、或任何介於其間之半衰期，包括其間所有範圍。In certain embodiments, the antibody or antigen-binding fragment thereof has a biological half-life of about or at least about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 20 hours, about 24 ... The half-life of the drug may be about 0 hours, about 36 hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours, about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90 hours, about 96 hours, about 120 hours, or about 144 hours or more, or about 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks or more, or any half-life therebetween, including all ranges therebetween.

在一些實施例中，抗體或其抗原結合片段之Tm為約或至少約60、61、62、63、64、65、66、67、68、69、70、71、72、73、74或75℃。在一些實施例中，抗體或其抗原結合片段例如在磷酸鹽緩衝鹽水（PBS）中之Tm為約65℃或更大。In some embodiments, the antibody or antigen-binding fragment thereof has a Tm of about or at least about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75° C. In some embodiments, the antibody or antigen-binding fragment thereof has a Tm of about 65° C. or greater, for example in phosphate buffered saline (PBS).

在一些實施例中，抗體或其抗原結合片段與一或多種細胞毒性劑或化學治療劑結合。細胞毒性劑或化學治療劑之一般實例包括但不限於烷基化劑、抗代謝物、蒽環黴素、抗腫瘤抗生素、鉑類、I型拓樸異構酶抑制劑、II型拓樸異構酶抑制劑、長春花生物鹼及紫杉烷。細胞毒性劑或化學治療劑之特定實例包括但不限於環磷醯胺、西侖吉肽（cilengitide）、洛莫司汀（CCNU）、美法侖、丙卡巴肼、卡莫司汀（BCNU）、恩紮妥林（enzastaurin）、硫酸布他卡因、道諾黴素、小紅莓、吉非替尼（gefitinib）、厄洛替尼（erlotinib）、艾達黴素、替莫唑胺、表柔比星、米托蒽醌、博萊黴素、順鉑、卡鉑、奧沙利鉑、喜樹鹼、伊立替康、拓樸替康、安吖啶（amsacrine）、依託泊苷、磷酸依託泊苷（etoposide phosphate）、替尼泊苷、坦羅莫司（temsirolimus）、依維莫司（everolimus）、長春新鹼、長春鹼、長春瑞濱、長春地辛、CT52923、太平洋紫杉醇、伊馬替尼（imatinib）、達沙替尼（dasatinib）、索拉非尼（sorafenib）、帕唑帕尼（pazopanib）、舒尼替尼（sunitnib）、瓦他拉尼（vatalanib）、吉非替尼（geftinib）、厄洛替尼（erlotinib）、AEE-788、二氯乙酸鹽、他莫昔芬（tamoxifen）、法舒地爾（fasudil）、SB-681323、司馬沙尼（semaxanib）、多奈哌齊（donepizil）、加蘭他敏（galantamine）、美金剛（memantine）、雷斯替明（rivastigmine）、他可林（tacrine）、雷沙吉蘭（rasigiline）、納曲酮（naltrexone）、魯比前列酮（lubiprostone）、沙芬醯胺（safinamide）、伊曲茶鹼（istradefylline）、匹莫范色林（pimavanserin）、必托里賽（pitolisant）、伊拉地平（isradipine）、普多比啶（pridopidine）（ACR16）、四苯那嗪（tetrabenazine）、貝瑟羅汀（bexarotene）、乙酸格拉替雷（glatirimer acetate）、芬戈莫德（fingolimod）及米托蒽醌，包括其醫藥學上可接受之鹽及酸。細胞毒性劑或化學治療劑之其他實例包括：烷基化劑，諸如噻替派（thiotepa）、環磷醯胺（CYTOXAN™）；磺酸烷基酯，諸如硫酸布他卡因、英丙舒凡（improsulfan）及哌泊舒凡（piposulfan）；氮丙啶，諸如苯唑多巴（benzodopa）、卡波醌（carboquone）、米特多巴（meturedopa）及尤利多巴（uredopa）；伸乙亞胺及甲基三聚氰胺，包括六甲蜜胺（altretamine）、曲他胺（triethylenemelamine）、三伸乙基磷醯胺、三伸乙基硫代磷醯胺及三甲基三聚氰胺；氮芥，諸如苯丁酸氮芥、萘氮芥、氯磷醯胺、雌氮芥（estramustine）、異環磷醯胺（ifosfamide）、甲氮芥、氧化甲氮芥鹽酸鹽、美法侖、新氮芥（novembichin）、苯芥膽甾醇（phenesterine）、潑尼莫司汀（prednimustine）、曲磷胺（trofosfamide）、尿嘧啶氮芥；亞硝基脲，諸如卡莫司汀、氯脲黴素（chlorozotocin）、福莫司汀、洛莫司汀、尼莫司汀、雷莫司汀；抗生素，諸如阿克拉黴素（aclacinomysin）、放線菌素、安麴黴素、偶氮絲胺酸、博來黴素、放線菌素C、卡奇黴素、卡拉比辛（carabicin）、洋紅黴素（carminomycin）、嗜癌菌素（carzinophilin）、色黴素（chromomycin）、放線菌素D（dactinomycin）、道諾黴素、地托比星（detorubicin）、6-重氮-5-側氧基-L-正白胺酸、小紅莓、表柔比星、依索比星、艾達黴素、麻西羅黴素（marcellomycin）、絲裂黴素、黴酚酸（mycophenolic acid）、諾加黴素（nogalamycin）、橄欖黴素（olivomycin）、培洛黴素（peplomycin）、潑非黴素（potfiromycin）、嘌呤黴素（puromycin）、奎那黴素（quelamycin）、羅多比星（rodorubicin）、鏈黑菌素（streptonigrin）、鏈脲菌素（streptozocin）、殺結核菌素（tubercidin）、烏苯美司（ubenimex）、淨司他丁（zinostatin）、左柔比星（zorubicin）；抗代謝物，諸如甲胺喋呤（methotrexate）及5-氟尿嘧啶（5-FU）；葉酸類似物，諸如迪諾特寧（denopterin）、甲胺喋呤、蝶羅呤（pteropterin）、曲美沙特（trimetrexate）；嘌呤類似物，諸如氟達拉濱（fludarabine）、6-巰基嘌呤、硫咪嘌呤（thiamiprine）、硫鳥嘌呤；嘧啶類似物，諸如安西他濱（ancitabine）、阿紮胞苷、6-氮雜尿苷（6-azauridine）、卡莫氟（carmofur）、阿糖胞苷（cytarabine）、二去氧尿苷（dideoxyuridine）、去氧氟尿苷（doxifluridine）、依諾他濱（enocitabine）、氟尿苷（floxuridine）、5-FU；雄激素，諸如卡魯睾酮（calusterone）、丙酸屈他雄酮（dromostanolone propionate）、環硫雄醇（epitiostanol）、美雄烷（mepitiostane）、睾內酯（testolactone）；抗腎上腺，諸如胺麩精（aminoglutethimide）、米托坦（mitotane）、曲洛司坦（trilostane）；葉酸補充劑，諸如亞葉酸；乙醯葡醛酯（aceglatone）；醛磷醯胺醣苷（aldophosphamide glycoside）；胺基乙醯丙酸（aminolevulinic acid）；安吖啶（amsacrine）；貝斯布西（bestrabucil）；比生群（bisantrene）；依達曲沙（edatraxate）；得弗伐胺（defofamine）；地美可辛（demecolcine）；地吖醌；艾福米辛（elformithine）；依利醋銨（elliptinium acetate）；依託格魯（etoglucid）；硝酸鎵；羥基脲；磨菇多糖（lentinan）；氯尼達明（lonidamine）；丙脒腙（mitoguazone）；米托蒽醌；莫哌達醇（mopidamol）；二胺硝吖啶（nitracrine）；噴司他丁；苯來美特（phenamet）；吡柔比星（pirarubicin）；鬼臼酸；2-乙基醯肼；丙卡巴肼（procarbazine）；PSK；雷佐生（razoxane）；西索菲蘭（sizofiran）；鍺螺胺（spirogermanium）；細交鏈孢菌酮酸（tenuazonic acid）；三亞胺醌（triaziquone）；2,2',2''-三氯三乙胺；尿烷（urethan）；長春地辛；達卡巴嗪；甘露醇氮芥；二溴甘露醇（mitobronitol）；二溴衛矛醇（mitolactol）；哌泊溴烷（pipobroman）；加西托星（gacytosine）；阿拉伯糖苷（「Ara-C」）；環磷醯胺；噻替派；紫杉烷類，例如太平洋紫杉醇（TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.）及多西他賽（TAXOTERE®, Rhne-Poulenc Rorer, Antony, France）；苯丁酸氮芥；吉西他濱；6-硫代鳥嘌呤；巰基嘌呤；甲胺喋呤；鉑類似物，諸如順鉑及卡鉑；長春鹼；鉑；依託泊苷（VP-16）；異環磷醯胺；絲裂黴素C；米托蒽醌；長春新鹼；長春瑞濱；溫諾平（navelbine）；米托蒽醌；替尼泊苷；道諾黴素（daunomycin）；胺基喋呤（aminopterin）；截瘤達（xeloda）；伊班膦酸鹽（ibandronate）；CPT-11；拓樸異構酶抑制劑RFS 2000；二氟甲基鳥胺酸（DMFO）；視黃酸衍生物，諸如Targretin™（貝瑟羅汀）、Panretin™（亞利崔托寧（alitretinoin））；ONTAK™（地尼介白素迪夫托斯（denileukin diftitox））；埃斯波黴素（esperamicin）；卡培他濱（capecitabine）；以及以上中之任一者的醫藥學上可接受之鹽、酸或衍生物。In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to one or more cytotoxic or chemotherapeutic agents. General examples of cytotoxic or chemotherapeutic agents include, but are not limited to, alkylating agents, anti-metabolites, anthracyclines, anti-tumor antibiotics, platinums, type I topoisomerase inhibitors, type II topoisomerase inhibitors, vinca alkaloids, and taxanes. Specific examples of cytotoxic agents or chemotherapeutic agents include, but are not limited to, cyclophosphamide, cilengitide, lomustine (CCNU), melphalan, procarbazine, carmustine (BCNU), enzastaurin, butacaine sulfate, daunorubicin, cranberries, gefitinib, erlotinib, idarucizumab, temozolomide, epirubicin, mitoxantrone, bleomycin, cisplatin, carboplatin, oxaliplatin, camptothecin, irinotecan, toponotecan, amsacrine, etoposide, etoposide phosphate, phosphate), teniposide, temsirolimus, everolimus, vincristine, vinblastine, vinorelbine, vindesine, CT52923, paclitaxel, imatinib, dasatinib, sorafenib, pazopanib, sunitnib, vatalanib, geftinib, erlotinib, AEE-788, dichloroacetate, tamoxifen, fasudil, SB-681323, semaxanib, donepezil il), galantamine, memantine, rivastigmine, tacrine, rasigiline, naltrexone, lubiprostone, safinamide, istradefylline, pimavanserin, pitolisant, isradipine, pridopidine (ACR16), tetrabenazine, bexarotene, glatirimer acetate, fingolimod and mitoxantrone, including their pharmaceutically acceptable salts and acids. Other examples of cytotoxic or chemotherapeutic agents include: alkylating agents such as thiotepa and CYTOXAN™; alkyl sulfonates such as butacaine sulfate, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimine and Methylmelamines, including altretamine, triethylenemelamine, triethylphosphatamide, triethylphosphatamide and trimethylmelamine; nitrogen mustards, such as chlorambucil, naphthyl mustard, chlorphosphatamide, estramustine, ifosfamide, mefenamic acid, mefenamic acid hydrochloride, melphalan, novembichin, phenergol sterine), prednimustine, trofosfamide, uracil nitrogen mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysin, actinomycin, ancinomycin, azoserine, bleomycin, actinomycin C, kacinomycin, carabicin ), carminomycin, carzinophilin, chromomycin, dactinomycin, daunomycin, detorubicin, 6-diazo-5-oxo-L-nor-leucine, cranberries, epirubicin, esorubicin, idarucidin, marcellomycin, mitomycin, mycophenolic acid acid), nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-hydroxypurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calusterone, dromostanolone propionate propionate, epitiostanol, mepitiostane, testolactone; adrenal antidotes, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diazocone; elformithine; elliptinium acetate); etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllic acid; 2-ethylhydrazine; procarbazine; PSK; razoxane; sizofiran; spirogermanium; tenuazonic acid acid); triaziquone; 2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannitol mustard; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; oxalopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; VP-16; isocyclic phosphamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; mitoxantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid derivatives, such as Targretin™ (besarotin), Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); esperamicin; capecitabine; and any pharmaceutically acceptable salts, acids or derivatives thereof.

在一些實施例中，本文所揭示之抗體結合或可操作地連接至放射性同位素以形成適用於結合放射性金屬離子之放射性結合物及/或巨環螯合劑。多種放射性同位素可用於產生放射性結合物抗體。實例包括但不限於90Y、123I、125I、131I、186Re、188Re、211At及212Bi。在某些實施例中，巨環螯合劑為1,4,7,10-四氮雜環十二烷-N,N',N'',N'''-四乙酸（DOTA），其可經由連接子分子附接至抗體。此類連接子分子為此項技術中通常已知的且描述於Denardo等人, 1998, Clin Cancer Res. 4:2483-90；Peterson等人, 1999, Bioconjug. Chem. 10:553；及Zimmerman等人, 1999, Nucl. Med. Biol. 26:943-50。In some embodiments, the antibodies disclosed herein are conjugated or operably linked to a radioactive isotope to form a radioactive conjugate and/or macrocyclic chelator suitable for binding radioactive metal ions. A variety of radioactive isotopes can be used to produce radioactive conjugate antibodies. Examples include, but are not limited to, 90Y, 123I, 125I, 131I, 186Re, 188Re, 211At, and 212Bi. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which can be attached to the antibody via a linker molecule. Such linker molecules are generally known in the art and are described in Denardo et al., 1998, Clin Cancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug. Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943-50.

本文中亦考慮本發明之抗體（及多肽）之其他修飾。舉例而言，在一些實施例中，抗體連接至多種非蛋白質聚合物中之一者，例如聚乙二醇、聚丙二醇、聚氧化烯或聚乙二醇與聚丙二醇之共聚物。在一些實施例中，抗體包覆於例如藉由凝聚技術或藉由界面聚合製備之微膠囊（例如分別為羥甲基纖維素或明膠微膠囊及聚(甲基丙烯酸甲酯)微膠囊）中、膠態藥物遞送系統（例如脂質體、白蛋白微球體、微乳液、奈米粒子及奈米膠囊）中或巨乳液中。此類技術揭示於Remington's Pharmaceutical Sciences, 第16版, Oslo, A.編輯(1980)中。Other modifications of the antibodies (and polypeptides) of the present invention are also contemplated herein. For example, in some embodiments, the antibody is linked to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene or a copolymer of polyethylene glycol and polypropylene glycol. In some embodiments, the antibody is encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. ed. (1980).

抗體或其抗原結合片段可用於本文所述之組合物、方法及/或套組中之任一者中，且與本文所述之額外藥劑中之一或多者組合。使用方法及醫藥組合物The antibodies or antigen-binding fragments thereof can be used in any of the compositions, methods and/or kits described herein, and in combination with one or more of the additional agents described herein.Methods of Use and Pharmaceutical Compositions

某些實施例係關於治療有需要之個體之疾病或病狀、改善該疾病或病狀之症狀及/或減少該疾病或病狀之進展的方法，其包含向該個體投與如本文所述之與IL-18BP結合之抗體或其抗原結合片段或包含其之醫藥組合物。亦包括刺激有需要之個體之免疫反應（例如，IL-18介導之免疫反應）的方法，其包含向該個體投與本文所述之醫藥組合物。在一些情況下，抗體或其抗原結合片段拮抗IL-18BP與其配位體IL-18之間的結合/信號傳導活性，且藉此增加IL-18介導之信號傳導或活性（例如IFN-γ、CXCL10及/或TNFα之誘導增加）。在一些實施例中，如上文所指出，疾病或病狀為癌症或腫瘤，或感染性疾病。在一些實施例中，疾病為免疫系統活化可為有益的任何疾病。Certain embodiments relate to methods of treating a disease or condition in a subject in need thereof, ameliorating symptoms of the disease or condition, and/or reducing the progression of the disease or condition, comprising administering to the subject an antibody or antigen-binding fragment thereof that binds to IL-18BP as described herein, or a pharmaceutical composition comprising the same. Also included are methods of stimulating an immune response (e.g., an IL-18-mediated immune response) in a subject in need thereof, comprising administering to the subject a pharmaceutical composition as described herein. In some cases, the antibody or antigen-binding fragment thereof antagonizes the binding/signaling activity between IL-18BP and its ligand IL-18, and thereby increases IL-18-mediated signaling or activity (e.g., increased induction of IFN-γ, CXCL10, and/or TNFα). In some embodiments, as noted above, the disease or condition is cancer or a tumor, or an infectious disease. In some embodiments, the disease is any disease in which activation of the immune system would be beneficial.

在一些實施例中，如上文所指出，疾病或病狀為癌症或腫瘤或其他增生性疾病或病症，諸如淋巴增生性病症、骨髓增生性病症、增生性腸炎、增生性糖尿病視網膜病變或增生性腎病。在一些情況下，癌症或腫瘤表現或過度表現IL-18BP、IL-18或兩者。在一些情況下，增生性疾病或病症與IL-18BP、IL-18或兩者之表現增加相關。在一些情況下，癌症為原發性癌症。在一些情況下，癌症為轉移性癌症。某些實施例因此包括治療有需要之患者之癌症、降低癌症之嚴重程度或預防癌症的方法，其包含向該患者投與本文所述之組合物，包括其中該抗體或其抗原結合片段為IL-18BP拮抗劑，藉此治療該癌症、降低該癌症之嚴重程度或預防該癌症。In some embodiments, as noted above, the disease or condition is cancer or a tumor or other proliferative disease or disorder, such as a lymphoproliferative disorder, a myeloproliferative disorder, a proliferative enteritis, a proliferative diabetic retinopathy, or a proliferative nephropathy. In some cases, the cancer or tumor expresses or overexpresses IL-18BP, IL-18, or both. In some cases, the proliferative disease or disorder is associated with increased expression of IL-18BP, IL-18, or both. In some cases, the cancer is a primary cancer. In some cases, the cancer is a metastatic cancer. Certain embodiments therefore include methods of treating, reducing the severity of, or preventing cancer in a patient in need thereof, comprising administering to the patient a composition described herein, including wherein the antibody or antigen-binding fragment thereof is an IL-18BP antagonist, thereby treating, reducing the severity of, or preventing the cancer.

例示性癌症包括但不限於：骨癌、前列腺癌、黑色素瘤（例如，轉移性黑色素瘤）、胰臟癌、小細胞肺癌、非小細胞肺癌（NSCLC）、間皮瘤、白血病（例如，淋巴球性白血病、慢性骨髓性白血病、急性骨髓性白血病、復發性急性骨髓性白血病、毛細胞白血病、急性淋巴母細胞性白血病）、淋巴瘤（例如，非何傑金氏淋巴瘤、何傑金氏淋巴瘤）、肝癌（肝細胞癌）、肉瘤、B細胞惡性病、乳癌、卵巢癌、結腸直腸癌、神經膠質瘤、多形性神經膠質母細胞瘤、腦膜瘤、垂體腺瘤、前庭神經鞘瘤、原發性CNS淋巴瘤、原始神經外胚層腫瘤（神經管胚細胞瘤）、腎癌（例如，腎細胞癌）、膀胱癌、子宮癌、食道癌、腦癌、頭頸癌、子宮頸癌、睪丸癌、甲狀腺癌及胃癌。在具體實施例中，癌症為例如已轉移至骨骼之轉移性癌症。Exemplary cancers include, but are not limited to, bone cancer, prostate cancer, melanoma (e.g., metastatic melanoma), pancreatic cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, hairy cell leukemia, acute lymphoblastic leukemia), lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), Lymphoma), liver cancer (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, neuroglioma, multiform neuroglioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (neurotubercline tumor), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer. In specific embodiments, the cancer is a metastatic cancer, such as one that has metastasized to the bones.

亦提供一種本發明之抗體、其抗原結合片段或醫藥組合物，其用作藥物。本發明之抗體、其片段或醫藥組合物可用於本文所揭示之任何治療方法中。在特定實施例中，本發明之抗體、其片段或醫藥組合物可用於治療本文所揭示之任何疾病或病症（諸如癌症或腫瘤或其他增生性疾病或病症，諸如淋巴增生性病症、骨髓增生性病症、增生性腸炎、增生性糖尿病視網膜病變或增生性腎病）、改善其症狀及/或降低其進展的方法中。Also provided is an antibody, an antigen-binding fragment thereof, or a pharmaceutical composition of the present invention for use as a medicament. The antibody, fragment thereof, or pharmaceutical composition of the present invention can be used in any therapeutic method disclosed herein. In a specific embodiment, the antibody, fragment thereof, or pharmaceutical composition of the present invention can be used in a method for treating any disease or condition disclosed herein (such as cancer or tumor or other proliferative disease or condition, such as lymphoproliferative disorder, myeloproliferative disorder, proliferative enteritis, proliferative diabetic retinopathy, or proliferative nephropathy), improving its symptoms, and/or reducing its progression.

某些實施例包括組合療法，例如其包含投與本文所述之醫藥組合物（包含抗IL-18BP抗體或其抗原結合片段）與一或多種額外治療劑（例如免疫刺激劑、免疫檢查點調節劑及/或化學治療劑）之組合。在一些實施例中，額外治療劑包含IL-18，包括人類IL-18（或其功能變異體或片段）。Certain embodiments include combination therapies, e.g., comprising administering a pharmaceutical composition described herein (comprising an anti-IL-18BP antibody or antigen-binding fragment thereof) in combination with one or more additional therapeutic agents (e.g., an immunostimulant, an immune checkpoint modulator, and/or a chemotherapeutic agent). In some embodiments, the additional therapeutic agent comprises IL-18, including human IL-18 (or a functional variant or fragment thereof).

在一些實施例中，額外治療劑包含免疫檢查點調節劑。免疫檢查點調節劑之特定實例包括一或多種抑制性免疫檢查點分子之「拮抗劑」或「抑制劑」，及一或多種刺激性免疫檢查點分子之「促效劑」。一般而言，免疫檢查點分子為免疫系統之增強信號（協同刺激分子）或降低信號之組分，其靶向具有癌症治療潛能，因為癌細胞可干擾免疫檢查點分子之天然功能（參見例如Sharma及Allison, Science. 348:56-61, 2015；Topalian等人, Cancer Cell. 27:450-461, 2015；Pardoll, Nature Reviews Cancer. 12:252-264, 2012）。在一些實施例中，免疫檢查點調節劑（例如拮抗劑、促效劑）與一或多個免疫檢查點分子「結合」或「特異性結合」，如本文所描述。In some embodiments, the additional therapeutic agent comprises an immune checkpoint modulator. Specific examples of immune checkpoint modulators include "antagonists" or "inhibitors" of one or more inhibitory immune checkpoint molecules, and "agonists" of one or more stimulatory immune checkpoint molecules. In general, immune checkpoint molecules are components of the immune system that enhance signaling (co-stimulatory molecules) or reduce signaling, and their targeting has cancer therapeutic potential because cancer cells can interfere with the natural function of immune checkpoint molecules (see, e.g., Sharma and Allison, Science. 348:56-61, 2015; Topalian et al., Cancer Cell. 27:450-461, 2015; Pardoll, Nature Reviews Cancer. 12:252-264, 2012). In some embodiments, an immune checkpoint modulator (e.g., antagonist, agonist) "binds" or "specifically binds" to one or more immune checkpoint molecules, as described herein.

在特定實施例中，免疫檢查點調節劑為多肽或肽。術語「肽」及「多肽」在本文中可互換使用，然而，在某些情況下，術語「肽」可指較短多肽，例如由約2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、35、40、45或50個胺基酸（包括其間所有整數及範圍（例如，5-10、8-12、10-15個））組成之多肽。多肽及肽可由如本文所描述之天然存在之胺基酸及/或非天然存在之胺基酸構成。In certain embodiments, the immune checkpoint modulator is a polypeptide or peptide. The terms "peptide" and "polypeptide" are used interchangeably herein, however, in some cases, the term "peptide" may refer to a shorter polypeptide, such as a polypeptide consisting of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 amino acids (including all integers and ranges therebetween (e.g., 5-10, 8-12, 10-15)). Polypeptides and peptides may be composed of naturally occurring amino acids and/or non-naturally occurring amino acids as described herein.

亦包括抗體作為多肽。因此，在一些實施例中，免疫檢查點調節多肽藥劑為如本文所描述之「抗體或其抗原結合片段」。Antibodies are also included as polypeptides. Therefore, in some embodiments, the immune checkpoint modulating polypeptide agent is an "antibody or antigen-binding fragment thereof" as described herein.

在一些實施例中，藥劑為或包含免疫檢查點分子之「配位體」，例如天然配位體。「配位體」一般係指與目標分子（例如生物分子）形成複合物以供生物學用途之物質或分子，且包括一般藉由與目標分子或目標蛋白上之位點結合而產生信號的「蛋白質配位體」。因此，某些藥劑為在自然界中與免疫檢查點分子結合且產生信號之蛋白質配位體。亦包括「經修飾之配位體」，例如與例如來源於免疫球蛋白之Fc區的藥物動力學調節劑融合的蛋白質配位體。In some embodiments, the agent is or comprises a "ligand" of an immune checkpoint molecule, such as a natural ligand. A "ligand" generally refers to a substance or molecule that forms a complex with a target molecule (e.g., a biomolecule) for biological purposes, and includes "protein ligands" that generally generate a signal by binding to a site on a target molecule or target protein. Thus, certain agents are protein ligands that bind to immune checkpoint molecules in nature and generate a signal. Also included are "modified ligands," such as protein ligands fused to a pharmacokinetic modulator, such as that derived from the Fc region of an immunoglobulin.

多肽之結合特性可使用此項技術中熟知之方法定量（參見Davies等人, Annual Rev. Biochem. 59:439-473, 1990）。在一些實施例中，多肽以約或範圍介於約≤10-7至約10-8 M之平衡解離常數與目標分子（例如，免疫檢查點分子或其抗原決定基）特異性結合。在一些實施例中，平衡解離常數為約或範圍介於約≤10-9 M至約≤10-10 M。在某些例示性實施例中，多肽對本文所述之目標（與該目標特異性結合）之親和力（Kd或EC50）為約、至少約或小於約0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、40或50 nM。The binding properties of the polypeptide can be quantified using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, the polypeptide specifically binds to a target molecule (e.g., an immune checkpoint molecule or an antigenic determinant thereof) with an equilibrium dissociation constant of about or ranging from about ≤10-7 to about 10-8 M. In some embodiments, the equilibrium dissociation constant is about or ranging from about ≤10-9 M to about ≤10-10 M. In certain exemplary embodiments, the polypeptide has an affinity (Kd or EC50) for (specifically binds to) a target described herein of about, at least about, or less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM.

在一些實施例中，藥劑為「小分子」，其係指合成或生物來源（生物分子）之有機化合物，但通常不為聚合物。有機化合物係指分子含有碳之一大類化合物，通常排除僅含有碳酸酯之化合物、簡單碳氧化物或氰化物。「生物分子」一般係指由活有機體產生之有機分子，亦包括大聚合物分子（生物聚合物），諸如肽、多醣及核酸，以及小分子，諸如主要次級代謝物、脂質、磷脂、糖脂、固醇、甘油脂、維生素及激素。「聚合物」一般係指由通常由共價化學鍵連接之重複結構單元構成之大分子或巨分子。In some embodiments, the agent is a "small molecule," which refers to an organic compound of synthetic or biological origin (biomolecule), but generally not a polymer. Organic compounds refer to a broad class of compounds whose molecules contain carbon, generally excluding compounds containing only carbonates, simple carbon oxides, or cyanides. "Biomolecules" generally refer to organic molecules produced by living organisms, and also include large polymer molecules (biopolymers), such as peptides, polysaccharides, and nucleic acids, as well as small molecules, such as major secondary metabolites, lipids, phospholipids, glycolipids, sterols, glycerolipids, vitamins, and hormones. "Polymers" generally refer to large or macromolecules composed of repeating structural units that are usually linked by covalent chemical bonds.

在某些實施例中，小分子之分子量為約或小於約1000-2000道爾頓，通常在約300與700道爾頓之間，且包括約或小於約50、100、150、200、250、300、350、400、450、500、550、500、650、600、750、700、850、800、950、1000或2000道爾頓。In certain embodiments, the molecular weight of a small molecule is about or less than about 1000-2000 daltons, typically between about 300 and 700 daltons, and includes about or less than about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 500, 650, 600, 750, 700, 850, 800, 950, 1000, or 2000 daltons.

某些小分子可具有本文中關於諸如抗體之多肽所描述的「特異性結合」特徵。舉例而言，在一些實施例中，小分子以約、至少約或小於約0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、40或50 nM之結合親和力（Kd或EC50）與目標（例如，免疫檢查點分子）特異性結合。Certain small molecules may have the "specific binding" characteristics described herein with respect to polypeptides such as antibodies. For example, in some embodiments, the small molecule specifically binds to a target (e.g., an immune checkpoint molecule) with a binding affinity (Kd or EC50) of about, at least about, or less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM.

在一些實施例中，免疫檢查點調節劑為一或多種抑制性免疫檢查點分子之拮抗劑或抑制劑。例示性抑制性免疫檢查點分子包括程式化死亡-配位體1（PD-L1）、程式化死亡-配位體2（PD-L2）、程式化死亡1（PD-1）、細胞毒性T淋巴球相關蛋白4（CTLA-4）、吲哚胺2,3-雙加氧酶（IDO）、色胺酸2,3-雙加氧酶（TDO）、T細胞免疫球蛋白域及黏蛋白域3（TIM-3）、淋巴球活化基因-3（LAG-3）、T細胞活化之V域Ig抑制因子（VISTA）、B及T淋巴球衰減因子（BTLA）、CD160以及具有Ig及ITIM域之T細胞免疫受體（TIGIT）。In some embodiments, the immune checkpoint modulator is an antagonist or inhibitor of one or more inhibitory immune checkpoint molecules. Exemplary inhibitory immune checkpoint molecules include programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), programmed death 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene-3 (LAG-3), V domain Ig inhibitor of T cell activation (VISTA), B and T lymphocyte depletion factor (BTLA), CD160, and T cell immune receptor with Ig and ITIM domains (TIGIT).

在某些實施例中，藥劑為PD-1（受體）拮抗劑或抑制劑，已顯示其靶向恢復腫瘤環境中之免疫功能（參見例如Phillips等人, Int Immunol. 27:39-46, 2015）。PD-1為屬於免疫球蛋白超家族且在T細胞及祖B細胞上表現之細胞表面受體。PD-1與兩種配位體PD-L1及PD-L2相互作用。PD-1例如藉由降低或防止T細胞之活化，繼而降低自體免疫性且促進自體耐受性來充當抑制性免疫檢查點分子。PD-1之抑制作用係至少部分通過促進淋巴結中之抗原特異性T細胞之細胞凋亡且同時亦減少調節性T細胞（抑制T細胞）之細胞凋亡的雙重機制來實現。PD-1拮抗劑或抑制劑之一些實例包括與PD-1特異性結合且降低其免疫抑制活性中之一或多者，例如其下游信號傳導或其與PD-L1之相互作用的抗體或抗原結合片段或小分子。PD-1拮抗劑或抑制劑之特定實例包括抗體納武單抗、帕博利珠單抗、PDR001、MK-3475、AMP-224、AMP-514及皮立珠單抗，及其抗原結合片段（參見例如美國專利第8,008,449號、第8,993,731號、第9,073,994號、第9,084,776號、第9,102,727號、第9,102,728號、第9,181,342號、第9,217,034號、第9,387,247號、第9,492,539號、第9,492,540號；及美國申請案第2012/0039906號、第2015/0203579號）。In certain embodiments, the agent is a PD-1 (receptor) antagonist or inhibitor, which has been shown to target restoration of immune function in the tumor environment (see, e.g., Phillips et al., Int Immunol. 27:39-46, 2015). PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and progenitor B cells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 acts as an inhibitory immune checkpoint molecule, for example, by reducing or preventing the activation of T cells, thereby reducing autoimmunity and promoting autotolerance. The inhibitory effect of PD-1 is achieved at least in part through a dual mechanism of promoting apoptosis of antigen-specific T cells in lymph nodes and simultaneously reducing apoptosis of regulatory T cells (suppressor T cells). Some examples of PD-1 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-1 and reduce one or more of its immunosuppressive activities, such as its downstream signaling or its interaction with PD-L1. Specific examples of PD-1 antagonists or inhibitors include the antibodies nivolumab, pembrolizumab, PDR001, MK-3475, AMP-224, AMP-514, and pilizumab, and antigen-binding fragments thereof (see, e.g., U.S. Patent Nos. 8,008,449, 8,993,731, 9,073,994, 9,084,776, 9,102,727, 9,102,728, 9,181,342, 9,217,034, 9,387,247, 9,492,539, 9,492,540; and U.S. Application Nos. 2012/0039906, 2015/0203579).

在一些實施例中，藥劑為PD-L1拮抗劑或抑制劑。如上文所指出，PD-L1為PD-1受體之天然配位體之一。PD-L1拮抗劑或抑制劑之一般實例包括與PD-L1特異性結合且降低其免疫抑制活性中之一或多者，例如其與PD-1受體之結合的抗體或抗原結合片段或小分子。PD-L1拮抗劑之特定實例包括抗體阿替利珠單抗（MPDL3280A）、阿維魯單抗（MSB0010718C）及度伐利尤單抗（MEDI4736）以及其抗原結合片段（參見例如美國專利第9,102,725號、第9,393,301號、第9,402,899號、第9,439,962號）。In some embodiments, the agent is a PD-L1 antagonist or inhibitor. As noted above, PD-L1 is one of the natural ligands of the PD-1 receptor. General examples of PD-L1 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L1 and reduce one or more of its immunosuppressive activities, such as its binding to the PD-1 receptor. Specific examples of PD-L1 antagonists include the antibodies atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), and antigen-binding fragments thereof (see, e.g., U.S. Patent Nos. 9,102,725, 9,393,301, 9,402,899, 9,439,962).

在一些實施例中，藥劑為PD-L2拮抗劑或抑制劑。如上文所指出，PD-L2為PD-1受體之天然配位體之一。PD-L2拮抗劑或抑制劑之一般實例包括與PD-L2特異性結合且降低其免疫抑制活性中之一或多者，例如其與PD-1受體之結合的抗體或抗原結合片段或小分子。In some embodiments, the agent is a PD-L2 antagonist or inhibitor. As noted above, PD-L2 is one of the natural ligands of the PD-1 receptor. General examples of PD-L2 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L2 and reduce one or more of its immunosuppressive activities, such as its binding to the PD-1 receptor.

在一些實施例中，藥劑為CTLA-4拮抗劑或抑制劑。細胞毒性T淋巴球相關蛋白4（CTLA4或CTLA-4）亦稱為分化簇152（CD152），為一種例如藉由在其結合至抗原呈遞細胞之表面上之CD80或CD86時傳輸抑制信號至T細胞，而充當抑制性免疫檢查點分子的蛋白質受體。CTLA-4拮抗劑或抑制劑之一般實例包括與CTLA-4特異性結合之抗體或抗原結合片段或小分子。特定實例包括抗體伊匹單抗及曲美木單抗以及其抗原結合片段。咸信伊匹單抗之至少一些活性係由抗體依賴性細胞介導之細胞毒性（ADCC）殺死表現CTLA-4之抑制Treg來介導。In some embodiments, the agent is a CTLA-4 antagonist or inhibitor. Cytotoxic T lymphocyte-associated protein 4 (CTLA4 or CTLA-4), also known as cluster of differentiation 152 (CD152), is a protein receptor that acts as an inhibitory immune checkpoint molecule, for example, by transmitting inhibitory signals to T cells when it binds to CD80 or CD86 on the surface of antigen-presenting cells. General examples of CTLA-4 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to CTLA-4. Specific examples include the antibodies ipilimumab and tremelimumab and antigen-binding fragments thereof. It is believed that at least some of the activity of ipilimumab is mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) killing of inhibitory Tregs expressing CTLA-4.

在一些實施例中，藥劑為IDO拮抗劑或抑制劑，或TDO拮抗劑或抑制劑。IDO及TDO為具有免疫抑制特性之色胺酸分解代謝酶。舉例而言，已知IDO抑制T細胞及NK細胞，產生及活化Treg及骨髓衍生之抑制細胞，且促進腫瘤血管生成。IDO及TDO拮抗劑或抑制劑之一般實例包括與IDO或TDO特異性結合（參見例如Platten等人, Front Immunol. 5: 673, 2014）且降低或抑制一或多種免疫抑制活性之抗體或抗原結合片段或小分子。IDO拮抗劑或抑制劑之特定實例包括因多莫得（NLG-8189）、1-甲基-色胺酸（1MT）、β-咔啉（去甲哈爾滿；9H-吡啶并[3,4-b]吲哚）、迷迭香酸及艾帕斯塔（參見例如Sheridan, Nature Biotechnology. 33:321-322, 2015）。TDO拮抗劑或抑制劑之特定實例包括680C91及LM10（參見例如Pilotte等人, PNAS USA. 109:2497-2502, 2012）。In some embodiments, the agent is an IDO antagonist or inhibitor, or a TDO antagonist or inhibitor. IDO and TDO are tryptophan metabolizing enzymes with immunosuppressive properties. For example, IDO is known to inhibit T cells and NK cells, generate and activate Treg and bone marrow-derived suppressor cells, and promote tumor angiogenesis. General examples of IDO and TDO antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to IDO or TDO (see, e.g., Platten et al., Front Immunol. 5: 673, 2014) and reduce or inhibit one or more immunosuppressive activities. Specific examples of IDO antagonists or inhibitors include indomod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norhalman; 9H-pyrido [3,4-b] indole), rosmarinic acid and epasta (see, e.g., Sheridan, Nature Biotechnology. 33:321-322, 2015). Specific examples of TDO antagonists or inhibitors include 680C91 and LM10 (see, e.g., Pilotte et al., PNAS USA. 109:2497-2502, 2012).

在一些實施例中，藥劑為TIM-3拮抗劑或抑制劑。T細胞免疫球蛋白域及黏蛋白域3（TIM-3）表現於活化之人類CD4+ T細胞上且調節Th1及Th17細胞介素。TIM-3亦藉由在與其配位體半乳糖凝集素-9相互作用時觸發細胞死亡來充當Th1/Tc1功能之負調節因子。TIM-3促成抑制腫瘤微環境，且其過度表現與多種癌症之不良預後相關（參見例如Li等人., Acta Oncol. 54:1706-13, 2015）。TIM-3拮抗劑或抑制劑之一般實例包括與TIM-3特異性結合且降低或抑制其免疫抑制活性中之一或多者的抗體或抗原結合片段或小分子。In some embodiments, the agent is a TIM-3 antagonist or inhibitor. T cell immunoglobulin domain and mucin domain 3 (TIM-3) is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 also acts as a negative regulator of Th1/Tc1 function by triggering cell death upon interaction with its ligand galectin-9. TIM-3 contributes to the suppression of the tumor microenvironment, and its overexpression is associated with poor prognosis in a variety of cancers (see, e.g., Li et al., Acta Oncol. 54:1706-13, 2015). General examples of TIM-3 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to TIM-3 and reduce or inhibit one or more of its immunosuppressive activities.

在一些實施例中，藥劑為LAG-3拮抗劑或抑制劑。淋巴球活化基因-3（LAG-3）在活化之T細胞、自然殺手細胞、B細胞及漿細胞樣樹突狀細胞上表現。其以類似於CTLA-4及PD-1之方式負調節T細胞之細胞增殖、活化及穩態（參見例如Workman及Vignali. European Journal of Immun. 33: 970-9, 2003；以及Workman等人, Journal of Immun. 172: 5450-5, 2004），且已報導在Treg抑制功能中發揮作用（參見例如Huang等人, Immunity. 21: 503-13, 2004）。LAG3亦將CD8+ T細胞維持於耐受性狀態且與PD-1組合以維持CD8 T細胞耗竭。LAG-3拮抗劑或抑制劑之一般實例包括與LAG-3特異性結合且抑制其免疫抑制活性中之一或多者的抗體或抗原結合片段或小分子。特定實例包括抗體BMS-986016及其抗原結合片段。In some embodiments, the agent is a LAG-3 antagonist or inhibitor. Lymphocyte activation gene-3 (LAG-3) is expressed on activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. It negatively regulates cell proliferation, activation, and homeostasis of T cells in a manner similar to CTLA-4 and PD-1 (see, e.g., Workman and Vignali. European Journal of Immun. 33: 970-9, 2003; and Workman et al., Journal of Immun. 172: 5450-5, 2004), and has been reported to play a role in Treg suppressive function (see, e.g., Huang et al., Immunity. 21: 503-13, 2004). LAG3 also maintains CD8+ T cells in a state of tolerance and combines with PD-1 to maintain CD8 T cell exhaustion. General examples of LAG-3 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to LAG-3 and inhibit one or more of its immunosuppressive activities. Specific examples include the antibody BMS-986016 and its antigen-binding fragments.

在一些實施例中，藥劑為VISTA拮抗劑或抑制劑。T細胞活化之V域Ig抑制因子（VISTA）主要表現於造血細胞上，且為抑制T細胞活化、誘導Foxp3表現且高度表現於其抑制抗腫瘤T細胞反應之腫瘤微環境內的抑制性免疫檢查點調節因子（參見例如Lines等人, Cancer Res. 74:1924-32, 2014）。VISTA拮抗劑或抑制劑之一般實例包括與VISTA特異性結合且降低其免疫抑制活性中之一或多者的抗體或抗原結合片段或小分子。In some embodiments, the agent is a VISTA antagonist or inhibitor. V-domain Ig inhibitor of T cell activation (VISTA) is primarily expressed on hematopoietic cells and is an inhibitory immune checkpoint regulator that inhibits T cell activation, induces Foxp3 expression, and is highly expressed in the tumor microenvironment where it inhibits anti-tumor T cell responses (see, e.g., Lines et al., Cancer Res. 74:1924-32, 2014). General examples of VISTA antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to VISTA and reduce one or more of its immunosuppressive activities.

在一些實施例中，藥劑為BTLA拮抗劑或抑制劑。在T細胞活化期間誘發B及T淋巴球衰減因子（BTLA；CD272）之表現，且其經由與腫瘤壞死家族受體（TNF-R）及B7家族細胞表面受體之相互作用而抑制T細胞。BTLA為腫瘤壞死因子（受體）超家族成員14（TNFRSF14）之配位體，亦稱為疱疹病毒侵入介體（HVEM）。BTLA-HVEM複合物例如藉由抑制人類CD8+癌症特異性T細胞之功能來負調節T細胞免疫反應（參見例如Derré等人, J Clin Invest 120:157-67, 2009）。BTLA拮抗劑或抑制劑之一般實例包括與BTLA-4特異性結合且降低其免疫抑制活性中之一或多者的抗體或抗原結合片段或小分子。In some embodiments, the agent is a BTLA antagonist or inhibitor. The expression of B and T lymphocyte depletion factor (BTLA; CD272) is induced during T cell activation, and it inhibits T cells through interaction with tumor necrosis family receptor (TNF-R) and B7 family cell surface receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM). BTLA-HVEM complexes negatively regulate T cell immune responses, for example, by inhibiting the function of human CD8+ cancer-specific T cells (see, e.g., Derré et al., J Clin Invest 120:157-67, 2009). General examples of BTLA antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to BTLA-4 and reduce one or more of its immunosuppressive activities.

在一些實施例中，藥劑為HVEM拮抗劑或抑制劑，例如與HVEM特異性結合且干擾其與BTLA或CD160之相互作用的拮抗劑或抑制劑。HVEM拮抗劑或抑制劑之一般實例包括與HVEM特異性結合，視情況減少HVEM/BTLA及/或HVEM/CD160相互作用，且從而降低HVEM之免疫抑制活性中之一或多者的抗體或抗原結合片段或小分子。In some embodiments, the agent is a HVEM antagonist or inhibitor, such as an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLA or CD 160. General examples of HVEM antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to HVEM, reduce HVEM/BTLA and/or HVEM/CD160 interactions, and thereby reduce one or more of the immunosuppressive activities of HVEM.

在一些實施例中，藥劑為CD160拮抗劑或抑制劑，例如與CD160特異性結合且干擾其與HVEM之相互作用的拮抗劑或抑制劑。CD160拮抗劑或抑制劑之一般實例包括與CD160特異性結合，視情況減少CD160/HVEM相互作用，且從而降低或抑制其免疫抑制活性中之一或多者的抗體或抗原結合片段或小分子。In some embodiments, the agent is a CD160 antagonist or inhibitor, such as an antagonist or inhibitor that specifically binds to CD160 and interferes with its interaction with HVEM. General examples of CD160 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to CD160, reduce CD160/HVEM interactions, and thereby reduce or inhibit one or more of its immunosuppressive activities.

在一些實施例中，藥劑為TIGIT拮抗劑或抑制劑。T細胞Ig及ITIM域（TIGIT）為發現於多種淋巴細胞表面上，且例如經由Treg抑制抗腫瘤免疫性的共抑制受體（Kurtulus等人, J Clin Invest. 125:4053-4062, 2015）。TIGIT拮抗劑或抑制劑之一般實例包括與TIGIT特異性結合且降低其免疫抑制活性中之一或多者的抗體或抗原結合片段或小分子（參見例如Johnston等人, Cancer Cell. 26:923-37, 2014）。In some embodiments, the agent is a TIGIT antagonist or inhibitor. T cell Ig and ITIM domain (TIGIT) is a co-inhibitory receptor found on the surface of a variety of lymphocytes and inhibits anti-tumor immunity, for example, via Tregs (Kurtulus et al., J Clin Invest. 125:4053-4062, 2015). General examples of TIGIT antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to TIGIT and reduce one or more of its immunosuppressive activities (see, e.g., Johnston et al., Cancer Cell. 26:923-37, 2014).

在某些實施例中，免疫檢查點調節劑為一或多種刺激性免疫檢查點分子之促效劑。例示性刺激性免疫檢查點分子包括OX40、CD40、糖皮質激素誘導之TNFR家族相關基因（GITR）、CD137（4-1BB）、CD27、CD28、CD226及疱疹病毒侵入介體（HVEM）。In certain embodiments, the immune checkpoint modulator is an agonist of one or more stimulatory immune checkpoint molecules. Exemplary stimulatory immune checkpoint molecules include OX40, CD40, glucocorticoid-induced TNFR family-related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpes virus entry mediator (HVEM).

在一些實施例中，藥劑為OX40促效劑。OX40（CD134）促進效應T細胞及記憶T細胞之擴增，且抑制T調節細胞之分化及活性（參見例如Croft等人, Immunol Rev. 229:173-91, 2009）。其配位體為OX40L（CD252）。因為OX40信號傳導影響T細胞活化及存活二者，所以其在引發淋巴結中之抗腫瘤免疫反應中及維持腫瘤微環境中之抗腫瘤免疫反應中發揮關鍵作用。OX40促效劑之一般實例包括與OX40特異性結合且提高其免疫刺激活性中之一或多者的抗體或抗原結合片段或小分子或配位體。特定實例包括OX86、OX-40L、Fc-OX40L、GSK3174998、MEDI0562（人源化OX40促效劑）、MEDI6469（鼠類OX4促效劑）及MEDI6383（OX40促效劑）以及其抗原結合片段。In some embodiments, the agent is an OX40 agonist. OX40 (CD134) promotes the expansion of effector T cells and memory T cells, and inhibits the differentiation and activity of T regulatory cells (see, e.g., Croft et al., Immunol Rev. 229:173-91, 2009). Its ligand is OX40L (CD252). Because OX40 signaling affects both T cell activation and survival, it plays a key role in initiating anti-tumor immune responses in lymph nodes and in maintaining anti-tumor immune responses in the tumor microenvironment. General examples of OX40 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to OX40 and enhance one or more of its immunostimulatory activities. Specific examples include OX86, OX-40L, Fc-OX40L, GSK3174998, MEDI0562 (humanized OX40 agonist), MEDI6469 (murine OX4 agonist), and MEDI6383 (OX40 agonist), and antigen-binding fragments thereof.

在一些實施例中，藥劑為CD40促效劑。CD40表現於抗原呈遞細胞（APC）及一些惡性腫瘤上。其配位體為CD40L（CD154）。在APC上，接合引起共刺激分子之上調，在抗腫瘤免疫反應中可能無需T細胞輔助。CD40促效劑療法在APC成熟及其自腫瘤至淋巴結之遷移中發揮重要作用，引起抗原呈遞升高及T細胞活化。抗CD40促效劑抗體在動物模型中產生顯著反應及持久抗癌免疫性，此為至少部分藉由細胞毒性T細胞介導之作用（參見例如Johnson等人, Clin Cancer Res. 21: 1321-1328, 2015；以及Vonderheide及Glennie, Clin Cancer Res. 19:1035-43, 2013）。CD40促效劑之一般實例包括與CD40特異性結合且提高其免疫刺激活性中之一或多者的抗體或抗原結合片段或小分子或配位體。特定實例包括索替加利單抗（sotigalilmab）、CP-870,893、達西組單抗、Chi Lob 7/4、ADC-1013、CD40L、rhCD40L及其抗原結合片段。In some embodiments, the agent is a CD40 agonist. CD40 is expressed on antigen presenting cells (APCs) and some malignant tumors. Its ligand is CD40L (CD154). On APCs, engagement causes upregulation of co-stimulatory molecules, which may not require T cell assistance in anti-tumor immune responses. CD40 agonist therapy plays an important role in APC maturation and its migration from tumors to lymph nodes, leading to increased antigen presentation and T cell activation. Anti-CD40 agonist antibodies produce dramatic responses and long-lasting anti-cancer immunity in animal models, an effect mediated at least in part by cytotoxic T cells (see, e.g., Johnson et al., Clin Cancer Res. 21: 1321-1328, 2015; and Vonderheide and Glennie, Clin Cancer Res. 19: 1035-43, 2013). General examples of CD40 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD40 and enhance one or more of its immunostimulatory activities. Specific examples include sotigalilmab, CP-870,893, darilimab, Chi Lob 7/4, ADC-1013, CD40L, rhCD40L, and antigen-binding fragments thereof.

在一些實施例中，藥劑為GITR促效劑。糖皮質激素誘導之TNFR家族相關基因（GITR）增加T細胞擴增，抑制Treg之抑制活性，且延長T效應細胞之存活。GITR促效劑已展示經由損失Treg譜系穩定性促進抗腫瘤反應（參見例如Schaer等人, Cancer Immunol Res. 1:320-31, 2013）。此等不同機制說明GITR在引發淋巴結中之免疫反應中及維持腫瘤組織中之免疫反應中發揮重要作用。其配位體為GITRL。GITR促效劑之一般實例包括與GITR特異性結合且提高其免疫刺激活性中之一或多者的抗體或抗原結合片段或小分子或配位體。特定實例包括GITRL、INCAGN01876、DTA-1、MEDI1873及其抗原結合片段。In some embodiments, the agent is a GITR agonist. Glucocorticoid-induced TNFR family-related genes (GITR) increase T cell expansion, inhibit the suppressive activity of Tregs, and prolong the survival of T effector cells. GITR agonists have been shown to promote anti-tumor responses by impairing Treg lineage stability (see, e.g., Schaer et al., Cancer Immunol Res. 1:320-31, 2013). These different mechanisms suggest that GITR plays an important role in initiating immune responses in lymph nodes and maintaining immune responses in tumor tissues. Its ligand is GITRL. General examples of GITR agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to GITR and enhance one or more of its immunostimulatory activities. Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873, and antigen-binding fragments thereof.

在一些實施例中，藥劑為CD137促效劑。CD137（4-1BB）為腫瘤壞死因子（TNF）受體家族之成員，且CD137之交聯增強T細胞增殖、IL-2分泌、存活及細胞溶解活性。CD137介導之信號傳導亦保護諸如CD8+ T細胞之T細胞避免活化誘導之細胞死亡。CD137促效劑之一般實例包括與CD137特異性結合且提高其免疫刺激活性中之一或多者的抗體或抗原結合片段或小分子或配位體。特定實例包括CD137（或4-1BB）配位體（參見例如Shao及Schwarz, J Leukoc Biol. 89:21-9, 2011）及抗體烏托米單抗（utomilumab），包括其抗原結合片段。In some embodiments, the agent is a CD137 agonist. CD137 (4-1BB) is a member of the tumor necrosis factor (TNF) receptor family, and cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. CD137-mediated signaling also protects T cells such as CD8+ T cells from activation-induced cell death. General examples of CD137 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD137 and enhance one or more of its immunostimulatory activities. Specific examples include CD137 (or 4-1BB) ligands (see, e.g., Shao and Schwarz, J Leukoc Biol. 89:21-9, 2011) and the antibody utomilumab, including antigen-binding fragments thereof.

在一些實施例中，藥劑為CD27促效劑。CD27之刺激增加初始T細胞之抗原特異性擴增，且有助於T細胞記憶及T細胞免疫性之長期維持。其配位體為CD70。促效劑抗體靶向人類CD27刺激T細胞活化及抗腫瘤免疫性（參見例如Thomas等人, Oncoimmunology. 2014;3:e27255. doi:10.4161/onci.27255；及He等人, J Immunol. 191:4174-83, 2013）。CD27促效劑之一般實例包括與CD27特異性結合且提高其免疫刺激活性中之一或多者的抗體或抗原結合片段或小分子或配位體。特定實例包括CD70及抗體瓦利魯單抗及CDX-1127 (1F5)，包括其抗原結合片段。In some embodiments, the agent is a CD27 agonist. Stimulation of CD27 increases the antigen-specific expansion of naive T cells and contributes to the long-term maintenance of T cell memory and T cell immunity. Its ligand is CD70. Agonist antibodies target human CD27 to stimulate T cell activation and anti-tumor immunity (see, e.g., Thomas et al., Oncoimmunology. 2014;3:e27255. doi:10.4161/onci.27255; and He et al., J Immunol. 191:4174-83, 2013). General examples of CD27 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD27 and enhance one or more of its immunostimulatory activities. Specific examples include CD70 and the antibodies valirumab and CDX-1127 (1F5), including antigen-binding fragments thereof.

在一些實施例中，藥劑為CD28促效劑。CD28由CD4+ T細胞、一些CD8+ T細胞組成性表現。其配位體包括CD80及CD86，且其刺激增加T細胞擴增。CD28促效劑之一般實例包括與CD28特異性結合且提高其免疫刺激活性中之一或多者的抗體或抗原結合片段或小分子或配位體。特定實例包括CD80、CD86、抗體TAB08及其抗原結合片段。In some embodiments, the agent is a CD28 agonist. CD28 is constitutively expressed by CD4+ T cells, some CD8+ T cells. Its ligands include CD80 and CD86, and its stimulation increases T cell expansion. General examples of CD28 agonists include antibodies or antigen binding fragments or small molecules or ligands that specifically bind to CD28 and enhance one or more of its immunostimulatory activities. Specific examples include CD80, CD86, antibody TAB08 and antigen binding fragments thereof.

在一些實施例中，藥劑為CD226促效劑。CD226為與TIGIT共有配位體之刺激受體，且與TIGIT相反，CD226之接合增強T細胞活化（參見例如Kurtulus等人, J Clin Invest. 125:4053-4062, 2015；Bottino等人, J Exp Med. 1984:557-567, 2003；及Tahara-Hanaoka等人, Int Immunol. 16:533-538, 2004）。CD226促效劑之一般實例包括與CD226特異性結合且提高其免疫刺激活性中之一或多者的抗體或抗原結合片段或小分子或配位體（例如，CD112、CD155）。In some embodiments, the agent is a CD226 agonist. CD226 is a stimulatory receptor that shares a ligand with TIGIT, and in contrast to TIGIT, engagement of CD226 enhances T cell activation (see, e.g., Kurtulus et al., J Clin Invest. 125:4053-4062, 2015; Bottino et al., J Exp Med. 1984:557-567, 2003; and Tahara-Hanaoka et al., Int Immunol. 16:533-538, 2004). General examples of CD226 agonists include antibodies or antigen binding fragments or small molecules or ligands that specifically bind to CD226 and enhance one or more of its immunostimulatory activities (e.g., CD112, CD155).

在一些實施例中，藥劑為HVEM促效劑。疱疹病毒侵入介體（HVEM），亦稱為腫瘤壞死因子受體超家族成員14（TNFRSF14），為TNF受體超家族之人類細胞表面受體。HVEM發現於包括T細胞、APC及其他免疫細胞之多種細胞上。不同於其他受體，HVEM在靜息T細胞上以高水平表現且在活化時下調。已展示HVEM信號傳導在T細胞活化早期及在淋巴結中之腫瘤特異性淋巴球群體擴增期間發揮關鍵作用。HVEM促效劑之一般實例包括與HVEM特異性結合且提高其免疫刺激活性中之一或多者的抗體或抗原結合片段或小分子或配位體。In some embodiments, the agent is a HVEM agonist. Herpes virus invasion mediator (HVEM), also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is a human cell surface receptor of the TNF receptor superfamily. HVEM is found on a variety of cells including T cells, APCs and other immune cells. Unlike other receptors, HVEM is expressed at high levels on resting T cells and is downregulated upon activation. HVEM signaling has been shown to play a key role in the early stages of T cell activation and during the expansion of tumor-specific lymphocyte populations in lymph nodes. General examples of HVEM agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to HVEM and enhance one or more of its immunostimulatory activities.

在一些實施例中，額外治療劑包含化學治療劑，例如小分子化學治療劑。化學治療劑之非限制性實例包括烷基化劑、抗代謝物、細胞毒性抗生素、拓樸異構酶抑制劑（1型或II型）及抗微管劑等。In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent, such as a small molecule chemotherapeutic agent. Non-limiting examples of chemotherapeutic agents include alkylating agents, anti-metabolites, cytotoxic antibiotics, topoisomerase inhibitors (type 1 or type II), and anti-microtubule agents, etc.

烷基化劑之實例包括氮芥（例如，甲氮芥、環磷醯胺、氮芥、美法侖、苯丁酸氮芥、異環磷醯胺及硫酸布他卡因）、亞硝基脲（例如，N-亞硝基-N-甲基脲（MNU）、卡莫司汀（BCNU）、洛莫司汀（CCNU）、司莫司汀（MeCCNU）、福莫司汀及鏈佐黴素）、四（例如，達卡巴嗪、米托唑胺及替莫唑胺）、氮丙啶（例如，噻替派、絲裂黴素及地吖醌（AZQ））、順鉑及其衍生物（例如，卡鉑及奧沙利鉑）及非典型烷基化劑（視情況丙卡巴肼及六甲三聚氰胺）。Examples of alkylating agents include nitrogen mustards (e.g., chlorambucil, cyclophosphamide, mechlorethamine, melphalan, chlorambucil, isocyclophosphamide, and butacaine sulfate), nitrosoureas (e.g., N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozocin), tetrazobactam (e.g., tetrazobactam, tetrazobactam, and tetrazobactam ... (e.g., dacarbazine, mitozolomide, and temozolomide), aziridines (e.g., thiotepa, mitomycin, and diazocone (AZQ)), cis-platinum and its derivatives (e.g., carboplatin and oxaliplatin), and atypical alkylating agents (optionally, procarbazine and hexamethonium).

抗代謝物之實例包括抗葉酸劑（例如，甲胺喋呤及培美曲塞）、氟嘧啶（例如，5-氟尿嘧啶及卡培他濱）、去氧核苷類似物（例如，安西他濱、依諾他濱、阿糖胞苷、吉西他濱、地西他濱、阿紮胞苷、氟達拉濱、奈拉濱、克拉屈濱、氯法拉濱、氟達拉濱及噴司他汀）及硫代嘌呤（例如硫鳥嘌呤及巰基嘌呤）。Examples of anti-metabolites include antifolates (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogs (e.g., ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and thioguanine).

細胞毒性抗生素之實例包括蒽環黴素（例如，小紅莓、道諾黴素、表柔比星、艾達黴素、吡柔比星、阿克拉黴素及米托蒽醌）、博來黴素、絲裂黴素C、米托蒽醌及放線菌素。拓樸異構酶抑制劑之實例包括喜樹鹼、伊立替康、拓樸替康、依託泊苷、小紅莓、米托蒽醌、替尼泊苷、新生黴素、麥爾巴隆及阿克拉黴素。Examples of cytotoxic antibiotics include anthracyclines (e.g., cyprodinil, daunorubicin, epirubicin, idarucizumab, pirarubicin, aclatomycin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, toponotecan, etoposide, cyprodinil, mitoxantrone, teniposide, neomycin, melbourne, and aclatomycin.

抗微管劑之實例包括紫杉烷（例如，太平洋紫杉醇及多西他賽）及長春花生物鹼（例如，長春鹼、長春新鹼、長春地辛、長春瑞濱）。Examples of anti-microtubule agents include taxanes (eg, paclitaxel and docetaxel) and vinca alkaloids (eg, vinblastine, vincristine, vindesine, vinorelbine).

在某些實施例中，本文所述之方法及組合物足以引起腫瘤消退，如由以下所指示：活腫瘤量之統計顯著減小，例如腫瘤質量減小至少10%、20%、30%、40%、50%或更大，或掃描尺寸改變（例如隨統計顯著性而減小）。在一些實施例中，相對於未經處理之對照，本文所述之方法及組合物使癌症之生長速率（例如，活體內或活體外，包括自生檢或其他樣品分離且活體外生長的癌細胞）降低約或至少約5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、60%、70%、80%、90%、100%、200%、300%、400%、500%、600%、700%、800%、900%、1000%、2000%或更大。在一些情況下，相對於未經處理之對照，本文所述之方法及組合物使癌細胞起始、遷移、黏附、侵襲性及/或轉移降低約或至少約5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、60%、70%、80%、90%、100%、200%、300%、400%、500%、600%、700%、800%、900%、1000%、2000%或更大。在一些情況下，相對於未經處理之對照，本文所述之方法及組合物使腫瘤環境中之血管生成減少約或至少約5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、60%、70%、80%、90%、100%、200%、300%、400%、500%、600%、700%、800%、900%、1000%、2000%或更大。In certain embodiments, the methods and compositions described herein are sufficient to cause tumor regression as indicated by a statistically significant decrease in the amount of viable tumor, such as a decrease in tumor mass of at least 10%, 20%, 30%, 40%, 50% or greater, or a change in scan size (e.g., a decrease with statistical significance). In some embodiments, the methods and compositions described herein reduce the growth rate of a cancer (e.g., in vivo or in vitro, including cancer cells isolated from a biopsy or other sample and grown ex vivo) by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more relative to an untreated control. In some cases, the methods and compositions described herein reduce cancer cell initiation, migration, adhesion, invasiveness and/or metastasis by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more relative to untreated controls. In some cases, the methods and compositions described herein reduce angiogenesis in a tumor environment by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more relative to untreated controls.

在某些實施例中，該疾病或病狀為骨髓發育不良症候群（MDS）（參見例如Wang等人, Blood. 140 (增刊1): 12297, 2022），舉例而言，對於該疾病或病狀，拮抗IL-18BP代表可行方法。MDS係指一組癌症，其中骨髓中之不成熟血細胞並不成熟，且因此不發展成健康血細胞。某些實施例因此包括治療有需要之患者之MDS、降低MDS嚴重程度或預防MDS之方法，其包含向該患者投與本文所述之組合物，包括其中該抗體或其抗原結合片段為IL-18BP拮抗劑，藉此治療MDS、降低MDS之嚴重程度或預防MDS。In certain embodiments, the disease or condition is myelodysplastic syndrome (MDS) (see, e.g., Wang et al., Blood. 140 (Suppl 1): 12297, 2022), for which, for example, antagonizing IL-18BP represents a viable approach. MDS refers to a group of cancers in which immature blood cells in the bone marrow do not mature and therefore do not develop into healthy blood cells. Certain embodiments therefore include methods of treating, reducing the severity of, or preventing MDS in a patient in need thereof, comprising administering to the patient a composition described herein, including wherein the antibody or antigen-binding fragment thereof is an IL-18BP antagonist, thereby treating, reducing the severity of, or preventing MDS.

在一些實施例中，該疾病或病狀為感染性疾病。舉例而言，在某些實施例中，感染性疾病係選自病毒（參見例如Vecchie等人, J Cell Physiol. 236(3): 1638-1657, 2021）、細菌（參見例如Kinoshita等人, Ann Surg. 240(2): 313-20, 2004）、真菌（例如酵母）及原蟲感染。一些實施例因此包括治療有需要之患者之感染性疾病、降低感染性疾病之嚴重程度或預防感染性疾病的方法，其包含向該患者投與本文所述之組合物，包括其中該抗體或其抗原結合片段為IL-18BP拮抗劑，藉此治療該感染性疾病、降低該感染性疾病之嚴重程度或預防該感染性疾病。In some embodiments, the disease or condition is an infectious disease. For example, in certain embodiments, the infectious disease is selected from viral (see, e.g., Vecchie et al., J Cell Physiol. 236(3): 1638-1657, 2021), bacterial (see, e.g., Kinoshita et al., Ann Surg. 240(2): 313-20, 2004), fungal (e.g., yeast) and protozoan infections. Some embodiments therefore include methods of treating an infectious disease in a patient in need thereof, reducing the severity of an infectious disease, or preventing an infectious disease, comprising administering to the patient a composition described herein, including wherein the antibody or antigen-binding fragment thereof is an IL-18BP antagonist, thereby treating the infectious disease, reducing the severity of the infectious disease, or preventing the infectious disease.

在一些實施例中，本文所述之方法及組合物使個體之中值存活時間增加4週、5週、6週、7週、8週、9週、10週、15週、20週、25週、30週、40週或更長時間。在某些實施例中，本文所述之方法及組合物使個體之中值存活時間增加1年、2年、3年或更長時間。在一些實施例中，本文所述之方法及組合物使無進展存活期增加2週、3週、4週、5週、6週、7週、8週、9週、10週或更長時間。在某些實施例中，本文所述之方法及組合物使無進展存活期增加1年、2年、3年或更長時間。In some embodiments, the methods and compositions described herein increase the median survival of a subject by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or more. In certain embodiments, the methods and compositions described herein increase the median survival of a subject by 1 year, 2 years, 3 years, or more. In some embodiments, the methods and compositions described herein increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more. In certain embodiments, the methods and compositions described herein increase progression-free survival by 1 year, 2 years, 3 years, or more.

在某些實施例中，本文所述之方法及組合物足以引起疾病穩定。在某些實施例中，本文所述之方法及組合物足以引起熟練臨床醫師已知之特定疾病適應症之症狀在臨床上相關減輕。In certain embodiments, the methods and compositions described herein are sufficient to cause disease stabilization. In certain embodiments, the methods and compositions described herein are sufficient to cause a clinically relevant reduction in symptoms of a specific disease indication known to a skilled clinician.

對於活體內使用，某些實施例包括醫藥組合物，其包含如本文所述之抗體或其抗原結合片段及醫藥學上可接受之載劑。為了製備治療或醫藥組合物，將有效量或所需量之一或多種藥劑與熟習此項技術者已知適合於特定藥劑及/或投與模式之任何醫藥載劑或賦形劑混合。醫藥載劑可為液體、半液體或固體。用於非經腸、皮內、眼內、皮下、直接滴注至膀胱中或局部施用之溶液或懸浮液可包括例如無菌稀釋劑（諸如水）、生理食鹽水溶液（例如磷酸鹽緩衝鹽水；PBS）、不揮發性油、聚乙二醇、甘油、丙二醇或其他合成溶劑；抗菌劑（諸如苯甲醇及對羥基苯甲酸甲酯）；抗氧化劑（諸如抗壞血酸及亞硫酸氫鈉）及螯合劑（諸如乙二胺四乙酸（EDTA））；緩衝劑（諸如乙酸鹽、檸檬酸鹽及磷酸鹽）。若經靜脈內投與(例如藉由IV輸注)，則適合載劑包括生理食鹽水或磷酸鹽緩衝生理食鹽水（PBS），及含有增稠劑及增溶劑（諸如葡萄糖、聚乙二醇、聚丙二醇及其混合物）之溶液。For in vivo use, certain embodiments include pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier. To prepare a therapeutic or pharmaceutical composition, an effective amount or a desired amount of one or more agents is mixed with any pharmaceutical carrier or formulation known to those skilled in the art to be suitable for the particular agent and/or mode of administration. The pharmaceutical carrier may be liquid, semi-liquid or solid. Solutions or suspensions for parenteral, intradermal, intraocular, subcutaneous, direct instillation into the bladder, or topical administration may include, for example, a sterile diluent such as water, a physiological saline solution (e.g., phosphate buffered saline; PBS), a non-volatile oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol and methyl paraben; antioxidants such as ascorbic acid and sodium bisulfite and chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates and phosphates. If administered intravenously (e.g., by IV infusion), suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

以純形式或以適當治療或醫藥組合物形式投與本文所述之藥劑可經由投與用於起相似作用之藥劑的任一公認模式來進行。治療或醫藥組合物可藉由將含藥劑之組合物與適當的生理學上可接受之載劑、稀釋劑或賦形劑組合來製備，且可調配成固體、半固體、液體或氣體形式之製劑，諸如錠劑、膠囊、散劑、顆粒、軟膏、溶液、栓劑、注射劑、吸入劑、凝膠、微球體及氣溶膠。另外，其他醫藥活性成分（包括如本文中其他地方所描述之其他小分子）及/或諸如鹽、緩衝劑及穩定劑之適合賦形劑可但無需存在於組合物內。Administration of the agents described herein in pure form or in the form of an appropriate therapeutic or pharmaceutical composition can be carried out by any recognized mode of administration for agents that act similarly. Therapeutic or pharmaceutical compositions can be prepared by combining the composition containing the agent with an appropriate physiologically acceptable carrier, diluent or excipient, and can be formulated into solid, semi-solid, liquid or gaseous preparations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Additionally, other pharmaceutically active ingredients (including other small molecules as described elsewhere herein) and/or suitable excipients such as salts, buffers and stabilizers may but need not be present in the composition.

投與可藉由多種不同途徑達成，包括經口、非經腸、經鼻、靜脈內、眼內、皮內、肌肉內、皮下、滴注至膀胱中或局部。較佳投與模式視待治療或預防之病狀的性質而定。特定實施例包括藉由IV輸注來投與。Administration can be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intraocular, intradermal, intramuscular, subcutaneous, instillation into the bladder or topical. The preferred mode of administration depends on the nature of the condition to be treated or prevented. Specific embodiments include administration by IV infusion.

載劑可包括例如在所採用之劑量及濃度下對暴露於其之細胞或哺乳動物無毒的醫藥學上或生理學上可接受之載劑、賦形劑或穩定劑。通常生理學上可接受之載劑為水性pH緩衝溶液。生理學上可接受之載劑之實例包括：緩衝劑，諸如磷酸鹽、檸檬酸鹽及其他有機酸；抗氧化劑，包括抗壞血酸；低分子量（小於約10個殘基）多肽；蛋白質，諸如血清白蛋白、明膠或免疫球蛋白；親水性聚合物，諸如聚乙烯吡咯啶酮；胺基酸，諸如甘胺酸、麩醯胺酸、天冬醯胺、精胺酸、組胺酸及/或離胺酸；單醣、雙醣及其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合劑，諸如EDTA；糖醇，諸如甘露糖醇或山梨糖醇；成鹽相對離子，諸如鈉；及/或非離子型界面活性劑，諸如聚山梨醇酯20（TWEEN™）、聚乙二醇（PEG）及泊洛沙姆（poloxamer）（PLURONICS™），以及其類似物。Carriers may include, for example, pharmaceutically or physiologically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the doses and concentrations employed. Typically, physiologically acceptable carriers are aqueous pH buffered solutions. Examples of physiologically acceptable carriers include: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, histidine and/or lysine; Sugars, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or non-ionic surfactants, such as polysorbate 20 (TWEEN™), polyethylene glycol (PEG) and poloxamer (PLURONICS™), and their analogs.

在一些實施例中，一或多種藥劑可包覆於例如藉由凝聚技術或藉由界面聚合製備之微膠囊（例如分別為羥甲基纖維素或明膠微膠囊及聚(甲基丙烯酸甲酯)微膠囊）中、膠態藥物遞送系統（例如脂質體、白蛋白微球體、微乳液、奈米粒子及奈米膠囊）中或巨乳液中。此類技術揭示於Remington's Pharmaceutical Sciences, 第16版, Oslo, A.編輯(1980)中。粒子或脂質體可進一步包含其他治療劑或診斷劑。In some embodiments, one or more agents may be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions, for example, prepared by coacervation techniques or by interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. ed. (1980). The particles or liposomes may further contain other therapeutic or diagnostic agents.

治療之精確劑量及持續時間隨所治療之疾病而變化，且可使用已知測試方案憑經驗確定，或藉由測試此項技術中已知之模型系統中之組合物且自其推斷來確定。亦可進行對照臨床試驗。劑量亦可隨待緩解之病狀的嚴重程度而變化。醫藥組合物一般經調配及投與以在使不合期望之副作用降至最低的同時發揮治療學上適用之作用。組合物可一次投與，或可分成多個較小劑量而以各時間間隔投與。對於任何特定個體，特定劑量方案可根據個體需要隨時間調節。The exact dosage and duration of treatment varies with the disease being treated, and can be determined empirically using known test protocols, or by testing the composition in model systems known in the art and extrapolating therefrom. Controlled clinical trials can also be performed. The dosage can also vary with the severity of the condition to be alleviated. Pharmaceutical compositions are generally formulated and administered to exert a therapeutically applicable effect while minimizing undesirable side effects. The composition can be administered at once, or can be divided into multiple smaller doses and administered at various time intervals. For any particular individual, the specific dosage regimen can be adjusted over time according to individual needs.

因此，投與此等及相關治療或醫藥組合物之典型途徑包括但不限於經口、局部、經皮、吸入、非經腸、舌下、經頰、經眼、經直腸、經陰道及鼻內。如本文所用，術語非經腸包括皮下注射、靜脈內、滴注至膀胱中、肌肉內、胸骨內注射或輸注技術。根據本發明之某些實施例之治療或醫藥組合物經調配使其中所含之活性成分在向個體或患者投與組合物時為生物可用的。將向個體或患者投與之組合物可採取一或多個劑量單位之形式，其中例如錠劑可為單個劑量單位，且呈氣溶膠形式之本文所述之藥劑的容器可容納複數個劑量單位。製備此類劑型之實際方法為熟習此項技術者所已知或對於熟習此項技術者而言將為顯而易見的；例如參見Remington: The Science and Practice of Pharmacy, 第20版(Philadelphia College of Pharmacy and Science, 2000)。待投與之組合物將通常含有治療有效量之本文所述之藥劑，以用於治療所關注之疾病或病狀。Thus, typical routes of administration of these and related therapeutic or pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, ocular, rectal, vaginal, and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, instillation into the bladder, intramuscular, intrasternal injection, or infusion techniques. Therapeutic or pharmaceutical compositions according to certain embodiments of the present invention are formulated so that the active ingredients contained therein are bioavailable when the composition is administered to an individual or patient. The composition to be administered to an individual or patient may take the form of one or more dosage units, wherein, for example, a tablet may be a single dosage unit, and a container of the medicament described herein in aerosol form may accommodate a plurality of dosage units. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy , 20th edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will generally contain a therapeutically effective amount of an agent described herein for treating the disease or condition of interest.

治療或醫藥組合物可呈固體或液體形式。在一些實施例中，載劑為微粒，使得組合物例如呈錠劑或散劑形式。載劑可為液體，其中組合物為例如適用於例如吸入投與之口服油、可注射液體或氣溶膠。當意欲經口投與時，醫藥組合物較佳呈固體或液體形式，其中半固體、半液體、懸浮液及凝膠形式包括在本文視為固體或液體之形式內。某些實施例包括無菌可注射溶液。The therapeutic or pharmaceutical composition may be in solid or liquid form. In some embodiments, the carrier is a microparticle, so that the composition is, for example, in the form of a tablet or powder. The carrier may be a liquid, wherein the composition is, for example, an oral oil suitable for administration, such as inhalation, an injectable liquid, or an aerosol. When oral administration is intended, the pharmaceutical composition is preferably in solid or liquid form, wherein semi-solid, semi-liquid, suspension, and gel forms are included in the forms considered as solid or liquid herein. Certain embodiments include sterile injectable solutions.

作為用於經口投與之固體組合物，醫藥組合物可調配為散劑、顆粒、凝膠、壓縮錠劑、丸劑、膠囊、口嚼錠、粉片或其類似物。該固體組合物典型地含有一或多種惰性稀釋劑或可食載劑。另外，可存在以下中之一或多者：黏合劑，諸如羧基甲基纖維素、乙基纖維素、微晶纖維素、黃蓍膠或明膠；賦形劑，諸如澱粉、乳糖或糊精；崩解劑，諸如海藻酸、海藻酸鈉、澱粉羥基乙酸鈉、玉米澱粉及其類似物；潤滑劑，諸如硬脂酸鎂或氫化植物油（Sterotex）；滑動劑，諸如膠態二氧化矽；甜味劑，諸如蔗糖或糖精；調味劑，諸如胡椒薄荷、水楊酸甲酯或柑橘調味劑；及著色劑。當醫藥組合物呈膠囊（例如明膠膠囊）形式時，除以上類型之物質之外，其可含有諸如聚乙二醇或油之液體載劑。As a solid composition for oral administration, the pharmaceutical composition can be formulated as a powder, granules, gel, compressed tablet, pill, capsule, chewable tablet, powder tablet or the like. The solid composition typically contains one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth or gelatin; formulators such as starch, lactose or dextrin; disintegrants such as alginic acid, sodium alginate, starch sodium hydroxyacetate, corn starch and the like; lubricants such as magnesium stearate or hydrogenated vegetable oil (Sterotex); slip agents such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavorings such as peppermint, methyl salicylate or citrus flavoring; and coloring agents. When the pharmaceutical composition is in the form of a capsule (e.g., a gelatin capsule), it may contain, in addition to the above-type substances, a liquid carrier such as polyethylene glycol or oil.

治療或醫藥組合物可呈液體形式，例如酏劑、糖漿、溶液、凝膠、乳液或懸浮液。作為兩個實例，液體可用於經口投與或用於藉由注射遞送。當意欲用於經口投與時，較佳組合物除本發明化合物以外亦含有甜味劑、防腐劑、染料/著色劑及香味增強劑中之一或多者。在意欲藉由注射投與之組合物中，可包括界面活性劑、防腐劑、濕潤劑、分散劑、懸浮劑、緩衝劑、穩定劑及等張劑中之一或多者。The therapeutic or pharmaceutical composition may be in the form of a liquid, such as an elixir, syrup, solution, gel, emulsion or suspension. As two examples, the liquid may be used for oral administration or for delivery by injection. When intended for oral administration, preferred compositions contain one or more of a sweetener, a preservative, a dye/colorant, and a flavor enhancer in addition to the compounds of the invention. In compositions intended for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer, and an isotonic agent may be included.

液體治療或醫藥組合物無論為溶液、懸浮液或其他類似形式，均可包括以下佐劑中之一或多者：無菌稀釋劑，諸如注射用水、生理食鹽水溶液（較佳生理食鹽水）、林格氏溶液（Ringer's solution）、等張氯化鈉、不揮發性油（諸如合成單酸甘油酯或二酸甘油酯，其可充當溶劑或懸浮介質）、聚乙二醇、甘油、丙二醇或其他溶劑；抗菌劑，諸如苯甲醇或對羥基苯甲酸甲酯；抗氧化劑，諸如抗壞血酸或亞硫酸氫鈉；螯合劑，諸如乙二胺四乙酸；緩衝劑，諸如乙酸鹽、檸檬酸鹽或磷酸鹽，及用於調整張力之試劑，諸如氯化鈉或右旋糖。非經腸製劑可封裝於由玻璃或塑膠製成的安瓿、拋棄式注射器或多劑量小瓶中。生理食鹽水為較佳佐劑。可注射的醫藥組合物較佳為無菌的。Liquid therapeutic or pharmaceutical compositions, whether in the form of solutions, suspensions or other similar forms, may include one or more of the following adjuvants: sterile diluents, such as water for injection, physiological saline solution (preferably physiological saline), Ringer's solution (Ringer's solution), isotonic sodium chloride, nonvolatile oils (such as synthetic mono- or diglycerides, which may serve as solvents or suspending media), polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral preparations may be enclosed in ampoules, disposable syringes or multiple-dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

意欲用於非經腸、眼內或經口投與之液體治療或醫藥組合物應含有將獲得合適劑量之量的藥劑。通常，此量為組合物中相關藥劑之至少0.01%。當意欲用於經口投與時，此量可在組合物重量的0.1%與約70%之間變化。某些口服治療或醫藥組合物含有約4%與約75%之間的所關注之藥劑。在某些實施例中，製備治療或醫藥組合物及製劑以使得非經腸劑量單位在稀釋之前含有介於0.01至10重量%之間的所關注的藥劑。Liquid therapeutic or pharmaceutical compositions intended for parenteral, intraocular or oral administration should contain an amount of the agent that will obtain a suitable dosage. Typically, this amount is at least 0.01% of the relevant agent in the composition. When intended for oral administration, this amount may vary between 0.1% and about 70% by weight of the composition. Certain oral therapeutic or pharmaceutical compositions contain between about 4% and about 75% of the agent of interest. In certain embodiments, therapeutic or pharmaceutical compositions and formulations are prepared so that the parenteral dosage unit contains between 0.01 and 10% by weight of the agent of interest before dilution.

治療或醫藥組合物可意欲用於局部投與，在此情況下載劑可適宜地包含溶液、乳液、軟膏或凝膠基質。舉例而言，基質可包含以下中之一或多者：石蠟脂、羊毛蠟、聚乙二醇、蜂蠟、礦物油、稀釋劑（諸如水及醇）及乳化劑及穩定劑。增稠劑可存在於治療或醫藥組合物中以供局部投與。若意欲用於經皮投與，則組合物可包括經皮貼片或離子導入療法裝置。The therapeutic or pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. For example, the base may comprise one or more of: wax, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. A thickener may be present in the therapeutic or pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or ionophoresis device.

治療或醫藥組合物可意欲用於呈例如栓劑形式經直腸投與，其將在直腸中融化且釋放藥物。用於經直腸投與之組合物可含有油性基質作為適合之無刺激性賦形劑。此類基質包括但不限於羊毛蠟、可可脂及聚乙二醇。The therapeutic or pharmaceutical composition may be intended for rectal administration in the form of, for example, a suppository, which will melt in the rectum and release the drug. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, wool wax, cocoa butter and polyethylene glycols.

治療或醫藥組合物可包括各種材料，其改變固體或液體劑量單位之物理形式。舉例而言，組合物可包括圍繞活性成分形成包覆殼層之材料。形成包覆殼層之材料典型地為惰性的，且可選自例如糖、蟲膠及其他腸溶包覆劑。替代地，活性成分可圍封於明膠膠囊中。呈固體或液體形式之治療或醫藥組合物可包括與藥劑結合且藉此幫助遞送化合物之組分。可起此能力作用之適合組分包括單株或多株抗體、一或多種蛋白質或脂質體。Therapeutic or pharmaceutical compositions may include various materials that change the physical form of a solid or liquid dosage unit. For example, a composition may include materials that form a coating shell around an active ingredient. The materials that form the coating shell are typically inert and may be selected from, for example, sugar, wormwood, and other enteric coating agents. Alternatively, the active ingredient may be enclosed in a gelatin capsule. Therapeutic or pharmaceutical compositions in solid or liquid form may include components that are combined with a drug and thereby help deliver the compound. Suitable components that may function in this capacity include single or multiple strains of antibodies, one or more proteins, or liposomes.

治療或醫藥組合物可基本上由可呈氣溶膠形式投與之劑量單位組成。術語氣溶膠用於表示各種系統，範圍為自膠態性質之系統至由加壓封裝組成之系統。遞送可藉由液化或壓縮氣體或藉由分配活性成分的適合之泵系統來進行。氣溶膠可以單相、雙相或三相系統形式遞送以遞送活性成分。氣溶膠之遞送包括必需容器、活化劑、閥門、次容器及其類似物，其在一起可形成套組。一般熟習此項技術者在不進行過度實驗的情況下即可確定較佳氣溶膠。Therapeutic or pharmaceutical compositions may consist essentially of dosage units that can be administered in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from those of a colloidal nature to those consisting of pressurized packaging. Delivery may be by liquefying or compressing a gas or by a suitable pump system that dispenses the active ingredient. Aerosols may be delivered in the form of a single-phase, two-phase or three-phase system to deliver the active ingredient. Delivery of an aerosol includes the necessary container, activator, valve, subcontainer and the like, which together may form a kit. One of ordinary skill in the art can determine the preferred aerosol without undue experimentation.

本文所描述之組合物可用保護藥劑以免自體內快速消除之載劑製備，諸如延時釋放調配物或包衣。此類載劑包括控制釋放調配物，諸如但不限於植入物及微膠囊化遞送系統，及生物可降解、生物相容性聚合物，諸如乙烯乙酸乙烯酯、聚酸酐、聚乙醇酸、聚原酸酯、聚乳酸及一般熟習此項技術者已知之其他載劑。The compositions described herein can be prepared with carriers that protect the agent from rapid elimination from the body, such as delayed release formulations or coatings. Such carriers include controlled release formulations, such as but not limited to implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and other carriers known to those skilled in the art.

醫藥組合物可藉由醫藥技術中熟知之方法製備。舉例而言，意欲藉由注射投與之治療或醫藥組合物可包含鹽、緩衝劑及/或穩定劑中之一或多者，與無菌蒸餾水一起以便形成溶液。可添加界面活性劑以促進形成均質溶液或懸浮液。界面活性劑為與藥劑非共價相互作用，以便促進藥劑溶解或均質懸浮於水性遞送系統中之化合物。Pharmaceutical compositions can be prepared by methods well known in the pharmaceutical art. For example, a therapeutic or pharmaceutical composition intended for administration by injection may include one or more of a salt, a buffer and/or a stabilizer, together with sterile distilled water to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the drug to facilitate dissolution or homogeneous suspension of the drug in an aqueous delivery system.

治療或醫藥組合物可以治療有效量投與，治療有效量將視多種因素而變，該等因素包括所採用之特定化合物之活性；化合物之代謝穩定性及作用時長；個體之年齡、體重、一般健康狀況、性別及膳食；投與模式及時間；排泄速率；藥物組合；特定病症或病狀之嚴重程度；以及經歷療法之個體。在一些情況下，治療有效日劑量（對於70 kg哺乳動物）為約0.001 mg/kg（亦即約0.07 mg）至約100 mg/kg（亦即約7.0 g）；較佳地，治療有效劑量（對於70 kg哺乳動物）為約0.01 mg/kg（亦即約0.7 mg）至約50 mg/kg（亦即約3.5 g）；更佳地，治療有效劑量（對於70 kg哺乳動物）為約1 mg/kg（亦即約70 mg）至約25 mg/kg（亦即約1.75 g）。在一些實施例中，治療有效劑量為每週、每兩週或每月投與一次。在具體實施例中，治療有效劑量每週、每兩週或每月例如以約1-10或1-5 mg/kg或約1、2、3、4、5、6、7、8、9或10 mg/kg之劑量投與一次。The therapeutic or pharmaceutical composition may be administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the specific compound employed; the metabolic stability and duration of action of the compound; the age, weight, general health, sex, and diet of the individual; the mode and timing of administration; the rate of excretion; the drug combination; the severity of the particular disease or condition; and the individual undergoing therapy. In some cases, the therapeutically effective daily dose (for a 70 kg mammal) is about 0.001 mg/kg (ie, about 0.07 mg) to about 100 mg/kg (ie, about 7.0 g); preferably, the therapeutically effective dose (for a 70 kg mammal) is about 0.01 mg/kg (ie, about 0.7 mg) to about 50 mg/kg (ie, about 3.5 g); more preferably, the therapeutically effective dose (for a 70 kg mammal) is about 1 mg/kg (ie, about 70 mg) to about 25 mg/kg (ie, about 1.75 g). In some embodiments, the therapeutically effective dose is administered once a week, every two weeks, or every month. In specific embodiments, a therapeutically effective dose is administered once weekly, biweekly or monthly, for example at a dose of about 1-10 or 1-5 mg/kg, or about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg.

亦包括患者護理套組，其包含（a）如本文所述之與IL-18BP結合之抗體或其抗原結合片段；及視情況存在之（b）至少一種額外治療劑。在某些套組中，（a）及（b）處於分開的治療組合物中。在一些套組中，（a）及（b）處於相同的治療組合物中。Also included are patient care kits comprising (a) an antibody or antigen-binding fragment thereof that binds to IL-18BP as described herein; and optionally (b) at least one additional therapeutic agent. In some kits, (a) and (b) are in separate therapeutic compositions. In some kits, (a) and (b) are in the same therapeutic composition.

本文中之套組亦可包括一或多種適合於所治療之適應症或為其所需或者達成所需診斷應用之額外治療劑或其他組分。本文中之套組亦可包括一或多種注射器或幫助所欲遞送模式所需或所期望之其他組件（例如，支架、可植入儲槽等）。The kits herein may also include one or more additional therapeutic agents or other components suitable for or required for the indication being treated or to achieve the desired diagnostic application. The kits herein may also include one or more syringes or other components required or desired to assist in the desired mode of delivery (e.g., stents, implantable reservoirs, etc.).

在一些實施例中，患者護理套組含有用於組合物及資訊材料的分開容器、隔板或區室。舉例而言，組合物可包含於瓶、小瓶或注射器中，且資訊材料可與容器相聯而包含。在一些實施例中，分開的套組元件包含於單一未分隔容器內。舉例而言，組合物包含於附著有呈標籤形式之資訊材料的瓶、小瓶或注射器中。在一些實施例中，套組包括複數個（例如一組）個別容器，各容器含有抗體及視情況存在之至少一種額外治療劑之一或多個單位劑型（例如本文所述之劑型）。舉例而言，套組包括複數個注射器、安瓿、箔封包或泡殼包裝，其各自含有單一單位劑量之抗體及視情況存在之至少一種額外治療劑。套組之容器可為氣密、防水（例如不可因滲透而改變水分或蒸發）及/或不透光的。In some embodiments, the patient care kit contains separate containers, partitions or compartments for the composition and the informational material. For example, the composition can be contained in a bottle, vial or syringe, and the informational material can be contained in association with the container. In some embodiments, the separate kit elements are contained in a single undivided container. For example, the composition is contained in a bottle, vial or syringe with the informational material in the form of a label attached. In some embodiments, the kit includes a plurality (e.g., a set) of individual containers, each container containing one or more unit dosage forms (e.g., dosage forms described herein) of the antibody and, optionally, at least one additional therapeutic agent. For example, the kit includes a plurality of syringes, ampoules, foil packets or blister packs, each of which contains a single unit dose of an antibody and, if appropriate, at least one additional therapeutic agent. The container of the kit can be airtight, waterproof (e.g., cannot change moisture or evaporate due to penetration) and/or light-proof.

患者護理套組視情況包括適合於投與組合物之裝置，例如注射器、吸入器、滴管（例如滴眼管）、拭子（例如棉簽或木拭子）或任何此類遞送裝置。在一些實施例中，裝置為分配定量劑量之藥劑的可植入裝置。亦包括提供套組之方法，其例如藉由將本文所述之組分組合來提供。表現及純化系統The patient care kit optionally includes a device suitable for administering the composition, such as a syringe, an inhaler, a dropper (e.g., an eye dropper), a swab (e.g., a cotton swab or a wooden swab), or any such delivery device. In some embodiments, the device is an implantable device that dispenses a metered dose of a medicament. Also included are methods of providing a kit, for example, by combining the components described herein.Expression and Purification Systems

某些實施例包括用於表現及純化本文所述之抗IL-18BP抗體或其抗原結合片段的方法及相關組合物。此類重組抗IL-18BP抗體宜使用如例如以下中所述之標準方案製備：Sambrook等人(1989, 同前文獻)，尤其第16章及第17章；Ausubel等人(1994, 同前文獻)，尤其第10章及第16章；及Coligan等人, Current Protocols in Protein Science (John Wiley & Sons, Inc. 1995-1997)，尤其第1章、第5章及第6章。作為一個通用實例，抗IL-18BP抗體可藉由包括以下步驟中之一或多者的程序製備：（a）製備包含聚核苷酸序列之構築體，該聚核苷酸序列編碼抗IL-18BP抗體重鏈及/或輕鏈且可操作地連接於調控元件；（b）將構築體引入宿主細胞中；（c）培養宿主細胞以表現抗IL-18BP抗體；及（d）自宿主細胞分離抗IL-18BP。Certain embodiments include methods and related compositions for expressing and purifying the anti-IL-18BP antibodies or antigen-binding fragments thereof described herein. Such recombinant anti-IL-18BP antibodies are preferably prepared using standard protocols as described, for example, in Sambrook et al. (1989, supra), especially Chapters 16 and 17; Ausubel et al. (1994, supra), especially Chapters 10 and 16; and Coligan et al., Current Protocols in Protein Science (John Wiley & Sons, Inc. 1995-1997), especially Chapters 1, 5, and 6. As a general example, an anti-IL-18BP antibody can be prepared by a process comprising one or more of the following steps: (a) preparing a construct comprising a polynucleotide sequence encoding the anti-IL-18BP antibody heavy chain and/or light chain and operably linked to a regulatory element; (b) introducing the construct into host cells; (c) culturing the host cells to express the anti-IL-18BP antibody; and (d) isolating the anti-IL-18BP from the host cells.

因此，某些實施例包括編碼本文所述之抗IL-18BP抗體或其抗原結合片段的聚核苷酸，包括包含該等聚核苷酸之載體，及包含該等聚核苷酸及/或載體之宿主細胞。為了表現所需多肽，可將編碼抗IL-18BP之核苷酸序列或功能等效物插入至適當表現載體（亦即，含有用於所插入編碼序列之轉錄及轉譯之必要元件的載體）。熟習此項技術者熟知之方法可用於構築含有編碼所關注多肽之序列以及適當轉錄及轉譯控制元件之表現載體。此等方法包括活體外重組DNA技術、合成技術及活體內基因重組。此類技術描述於Sambrook等人, Molecular Cloning, A Laboratory Manual (1989)及Ausubel等人, Current Protocols in Molecular Biology (1989)中。Thus, certain embodiments include polynucleotides encoding the anti-IL-18BP antibodies or antigen-binding fragments thereof described herein, including vectors comprising such polynucleotides, and host cells comprising such polynucleotides and/or vectors. To express the desired polypeptide, the nucleotide sequence encoding the anti-IL-18BP or a functional equivalent may be inserted into an appropriate expression vector (i.e., a vector containing the necessary elements for transcription and translation of the inserted coding sequence). Methods well known to those skilled in the art can be used to construct expression vectors containing sequences encoding the polypeptide of interest and appropriate transcription and translation control elements. Such methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1989).

已知多種表現載體/宿主系統，且可用以含有及表現聚核苷酸序列。此等表現載體/宿主系統包括但不限於微生物，諸如經重組噬菌體、質體或黏質體DNA表現載體轉型之細菌；經酵母表現載體轉型之酵母；經病毒表現載體（例如桿狀病毒）感染之昆蟲細胞系統；經病毒表現載體（例如花椰菜嵌紋病毒，CaMV；菸草嵌紋病毒，TMV）或經細菌表現載體（例如Ti或pBR322質體）轉型之植物細胞系統；或動物細胞系統，包括哺乳動物細胞，且更特定言之人類細胞系統。A variety of expression vector/host systems are known and can be used to contain and express polynucleotide sequences. Such expression vector/host systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., bacilli); plant cell systems transformed with viral expression vectors (e.g., Cauliflower mosaic virus, CaMV; Tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems, including mammalian cells, and more particularly human cell systems.

存在於表現載體中之「控制元件」或「調控序列」為載體之彼等非轉譯區，亦即強化子、啟動子、5'及3'非轉譯區，其與宿主細胞蛋白相互作用以進行轉錄及轉譯。此類元件之強度及特異性可變化。視所利用之載體系統及宿主而定，可使用任何數目之適合的轉錄元件及轉譯元件，包括組成性啟動子及誘導性啟動子。舉例而言，當在細菌系統中選殖時，可使用誘導型啟動子，諸如PBLUESCRIPT噬菌粒（Stratagene，La Jolla, Calif.）或PSPORT1質體（Gibco BRL，Gaithersburg, Md.）之雜合lacZ啟動子及其類似啟動子。在哺乳動物細胞系統中，來自哺乳動物基因或來自哺乳動物病毒之啟動子一般較佳。若需要產生含有編碼多肽之序列之多個複本的細胞株，則基於SV40或EBV之載體宜與適當可選標記一起使用。The "control elements" or "regulatory sequences" present in the expression vector are those non-translated regions of the vector, i.e., enhancers, promoters, 5' and 3' non-translated regions, which interact with host cell proteins for transcription and translation. The strength and specificity of such elements can vary. Depending on the vector system and host utilized, any number of suitable transcriptional elements and translational elements can be used, including constitutive promoters and inducible promoters. For example, when cloning in bacterial systems, inducible promoters can be used, such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or the PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and similar promoters. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If it is desired to generate cell lines containing multiple copies of the sequence encoding the polypeptide, vectors based on SV40 or EBV are preferably used with an appropriate selectable marker.

在細菌系統中，可根據意欲用於所表現之多肽之用途而選擇多種表現載體。舉例而言，當需要大量時，可使用導引容易純化之融合蛋白之高表現量的載體。此類載體包括但不限於多功能大腸桿菌選殖及表現載體，諸如BLUESCRIPT（Stratagene），其中編碼所關注多肽之序列可與β-半乳糖苷酶之胺基端Met及後7個殘基之序列同框接合至載體中，從而產生雜合蛋白質；pIN載體（Van Heeke及Schuster,J. Biol. Chem. 264:5503 5509 (1989)）；及類似載體。pGEX載體（Promega，Madison, Wis.）亦可用於將外源多肽表現為與麩胱甘肽S-轉移酶（GST）之融合蛋白。一般而言，此類融合蛋白可溶且可藉由吸附至麩胱甘肽-瓊脂糖珠粒，接著在游離麩胱甘肽存在下溶離而容易自溶解細胞純化。在此類系統中製成之蛋白質可經設計成包括肝素、凝血酶或因子XA蛋白酶裂解位點，使得可任意自GST部分釋放所關注之經選殖多肽。In bacterial systems, a variety of expression vectors can be selected depending on the intended use of the expressed polypeptide. For example, when large quantities are required, vectors that direct high expression of fusion proteins that are easily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors, such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest can be ligated in frame with the amino-terminal Met and the last 7 residues of β-galactosidase to produce a hybrid protein; pIN vectors (Van Heeke and Schuster,J. Biol. Chem. 264 : 5503 5509 (1989)); and similar vectors. The pGEX vector (Promega, Madison, Wis.) can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor XA protease cleavage sites, allowing for the release of the cloned polypeptide of interest from the GST moiety at will.

某些實施例可採用基於大腸桿菌之表現系統（參見例如，Structural Genomics Consortium等,Nature Methods. 5:135-146, 2008）。此等及相關實施例可部分或全部依賴於非接合依賴型選殖（LIC），以產生適合的表現載體。在具體實施例中，蛋白質表現可由T7 RNA聚合酶（例如pET載體系列）控制。此等及相關實施例可利用表現宿主菌株BL21 (DE3)，即支持T7介導之表現且缺乏改善目標蛋白穩定性之lon及ompT蛋白酶的BL21之λDE3溶源。亦包括攜載編碼很少用於大腸桿菌之tRNA之質體的表現宿主菌株，諸如ROSETTA™ (DE3)及Rosetta 2 (DE3)菌株。細胞溶解及樣品處理亦可使用以商標BENZONASE®核酸酶及BUGBUSTER® 蛋白質萃取試劑出售之試劑改良。對於細胞培養，自誘導培養基可提高許多表現系統之效率，包括高通量表現系統。此類型之培養基（例如OVERNIGHT EXPRESS™自誘導系統）經由不添加人造誘導劑（諸如IPTG）之代謝轉移而逐漸引發蛋白質表現。特定實施例採用六組胺酸標籤（諸如以商標HIS•TAG®融合物出售之該等標籤），之後為固定金屬親和性層析（IMAC）純化，或相關技術。然而，在某些態樣中，臨床級蛋白質可在不使用或不使用親和標籤之情況下自大腸桿菌包涵體分離（參見例如，Shimp等人,Protein Expr Purif. 50:58-67, 2006）。作為另一實例，某些實施例可採用冷震誘導之大腸桿菌高產率產生系統，因為低溫下大腸桿菌中蛋白質之過度表現改善其溶解度及穩定性（參見例如，Qing等人, Nature Biotechnology. 22:877-882, 2004）。Certain embodiments may employ an expression system based on E. coli (see, e.g., Structural Genomics Consortium, et al.,Nature Methods . 5:135-146, 2008). These and related embodiments may rely in part or in whole on independent conjugation dependent cloning (LIC) to generate suitable expression vectors. In specific embodiments, protein expression may be controlled by T7 RNA polymerase (e.g., pET vector series). These and related embodiments may utilize the expression host strain BL21 (DE3), i.e., a λDE3 lysogen of BL21 that supports T7-mediated expression and lacks the lon and ompT proteases that improve the stability of the target protein. Also included are expression host strains that carry plasmids encoding tRNAs that are rarely used in E. coli, such as the ROSETTA™ (DE3) and Rosetta 2 (DE3) strains. Cell lysis and sample handling can also be improved using reagents sold under the trademarks BENZONASE® Nuclease and BUGBUSTER® Protein Extraction Reagent. For cell culture, autoinducing media can improve the efficiency of many expression systems, including high-throughput expression systems. This type of media (e.g., the OVERNIGHT EXPRESS™ Autoinducing System) gradually induces protein expression via metabolic shift without the addition of artificial inducers (such as IPTG). Specific embodiments employ a hexahistidine tag (such as those sold under the trade name HIS•TAG® fusions) followed by immobilized metal affinity chromatography (IMAC) purification, or related techniques. However, in certain aspects, clinical-grade proteins can be isolated from E. coli inclusion bodies without or with an affinity tag (see, e.g., Shimp et al.,Protein Expr Purif . 50:58-67, 2006). As another example, certain embodiments may employ a cold-shock-induced high-yield production system for E. coli, because overexpression of proteins in E. coli at low temperatures improves their solubility and stability (see, e.g., Qing et al., Nature Biotechnology. 22:877-882, 2004).

亦包括高密度細菌醱酵系統。舉例而言，富養羅爾斯通氏菌（Ralstonia eutropha）之高細胞密度培養允許以超過150 g/L之細胞密度產生蛋白質，及以超出10 g/L之效價表現重組蛋白。High-density bacterial fermentation systems are also included. For example, high cell density culture of Ralstonia eutropha allows protein production at cell densities exceeding 150 g/L and expression of recombinant proteins at titers exceeding 10 g/L.

在酵母釀酒酵母（Saccharomyces cerevisiae）中，可使用含有諸如α因子、醇氧化酶及PGH之組成型或誘導型啟動子之多種載體。綜述參見Ausubel等人(同前文獻)及Grant等人, Methods Enzymol. 153:516-544 (1987)。亦包括巴斯德氏畢赤酵母（Pichia pandoris）表現系統（參見例如，Li等人, Nature Biotechnology. 24, 210 - 215, 2006；及Hamilton等人, Science, 301:1244, 2003）。某些實施例包括經工程改造以選擇性地使蛋白質糖基化之酵母系統，尤其包括具有人源化N-糖基化路徑之酵母（參見例如，Hamilton等人, Science. 313:1441-1443, 2006；Wildt等人, Nature Reviews Microbiol. 3:119-28, 2005；及Gerngross等人, Nature-Biotechnology. 22:1409 -1414, 2004；美國專利第7,629,163號、第7,326,681號及第7,029,872號）。僅舉例而言，重組酵母培養物尤其可生長於馮巴赫燒瓶（Fernbach Flask）或15L、50L、100L及200L醱酵器中。In the yeast Saccharomyces cerevisiae, various vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For a general description, see Ausubel et al. (supra) and Grant et al., Methods Enzymol. 153:516-544 (1987). Also included is the Pichia pandoris expression system (see, e.g., Li et al., Nature Biotechnology. 24, 210 - 215, 2006; and Hamilton et al., Science, 301:1244, 2003). Certain embodiments include yeast systems engineered to selectively glycosylate proteins, particularly yeast with humanized N-glycosylation pathways (see, e.g., Hamilton et al., Science. 313:1441-1443, 2006; Wildt et al., Nature Reviews Microbiol. 3:119-28, 2005; and Gerngross et al., Nature-Biotechnology. 22:1409-1414, 2004; U.S. Patent Nos. 7,629,163, 7,326,681, and 7,029,872). Recombinant yeast cultures can be grown, by way of example only, in Fernbach Flasks or 15L, 50L, 100L, and 200L fermentors.

在其中使用植物表現載體之情況下，編碼多肽之序列的表現可由多種啟動子中之任一者驅動。舉例而言，諸如CaMV之35S及19S啟動子的病毒啟動子可單獨或與來自TMV之ω前導序列組合使用（Takamatsu, EMBO J. 6:307-311 (1987)）。可替代地，可使用植物啟動子，諸如RUBISCO或熱休克啟動子之小子單元（Coruzzi等人, EMBO J. 3:1671-1680 (1984)；Broglie等人, Science 224:838-843 (1984)；及Winter等人, Results Probl. Cell Differ. 17:85-105 (1991)）。可藉由直接DNA轉型或病原體介導之轉染將此等構築體引入至植物細胞中。此類技術描述於多個通常可獲得之綜述中（參見例如，Hobbs, McGraw Hill, Yearbook of Science and Technology, 第191-196頁(1992)）。In the case where a plant expression vector is used, expression of the sequence encoding the polypeptide can be driven by any of a variety of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the ω leader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)). Alternatively, plant promoters such as the small subunits of RUBISCO or heat shock promoters can be used (Coruzzi et al., EMBO J. 3:1671-1680 (1984); Broglie et al., Science 224:838-843 (1984); and Winter et al., Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, e.g., Hobbs, McGraw Hill, Yearbook of Science and Technology, pp. 191-196 (1992)).

昆蟲系統亦可用於表現所關注之多肽。舉例而言，在一個此類系統中，苜蓿銀紋夜蛾（Autographa californica）核多角體病毒（AcNPV）用作載體以在草地黏蟲（Spodoptera frugiperda）細胞或粉紋夜蛾（Trichoplusia）細胞中表現外源基因。可將編碼多肽之序列選殖至病毒之非必需區，諸如多角體蛋白基因，且置於多角體蛋白啟動子之控制下。成功插入多肽編碼序列將使得多角體蛋白基因非活性且產生缺乏鞘蛋白之重組病毒。重組病毒接著可用於感染例如可表現所關注多肽之草地黏蟲細胞或粉紋夜蛾細胞（Engelhard等人, Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)）。亦包括桿狀病毒表現系統，包括利用SF9、SF21及Tni細胞之該等表現系統（參見例如，Murphy及Piwnica-Worms, Curr Protoc Protein Sci.第5章:第5.4單元, 2001）。昆蟲系統可提供與哺乳動物系統類似之轉譯後修飾。Insect systems can also be used to express polypeptides of interest. For example, in one such system, the Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or Trichoplusia cells. The polypeptide encoding sequence can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of the polypeptide encoding sequence will render the polyhedrin gene inactive and produce a recombinant virus lacking sheath proteins. The recombinant virus can then be used to infect, for example, Spodoptera frugiperda cells or Trichoplusia spp. cells, which express the polypeptide of interest (Engelhard et al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)). Also included are bacilliform virus expression systems, including those utilizing SF9, SF21, and Tni cells (see, e.g., Murphy and Piwnica-Worms, Curr Protoc Protein Sci. Chapter 5: Unit 5.4, 2001). Insect systems can provide post-translational modifications similar to mammalian systems.

在哺乳動物宿主細胞中，一般可利用多種基於病毒之表現系統。舉例而言，在腺病毒用作表現載體之情況下，編碼所關注多肽之序列可接合至由晚期啟動子及三聯前導序列組成之腺病毒轉錄/轉譯複合物。插入病毒基因體之非必需E1或E3區中可用於獲得能夠在感染宿主細胞中表現多肽的活病毒（Logan及Shenk, Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659 (1984)）。另外，轉錄增強子，諸如勞氏肉瘤病毒（Rous sarcoma virus，RSV）增強子可用於增加哺乳動物宿主細胞中之表現。In mammalian host cells, a variety of viral-based expression systems are generally available. For example, where adenovirus is used as an expression vector, the sequence encoding the polypeptide of interest can be joined to an adenoviral transcription/translation complex consisting of a late promoter and a tripartite leader sequence. Insertion into the non-essential E1 or E3 region of the viral genome can be used to obtain a live virus capable of expressing the polypeptide in infected host cells (Logan and Shenk, Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659 (1984)). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

適用哺乳動物宿主細胞株之實例包括經SV40轉型之猴腎CV1株（COS-7, ATCC CRL 1651）；人胚腎株（293細胞或經次選殖以在懸浮培養物中生長之293細胞，Graham等人, J. Gen Virol. 36:59 (1977)）；幼倉鼠腎細胞（BHK，ATCC CCL 10）；小鼠塞特利氏細胞（mouse sertoli cell）（TM4，Mather, Biol. Reprod. 23:243-251 (1980)）；猴腎細胞（CV1 ATCC CCL 70）；非洲綠猴腎細胞（VERO-76，ATCC CRL-1587）；人類子宮頸癌細胞（HELA，ATCC CCL 2）；犬腎細胞（MDCK，ATCC CCL 34）；水牛鼠肝細胞（BRL 3A，ATCC CRL 1442）；人類肺細胞（W138，ATCC CCL 75）；人類肝細胞（Hep G2，HB 8065）；小鼠乳房腫瘤（MMT 060562，ATCC CCL51）；TR1細胞（Mather等人, Annals N.Y. Acad. Sci. 383:44-68 (1982)）；MRC 5細胞；FS4細胞；及人類肝腫瘤株（Hep G2）。其他適用哺乳動物宿主細胞株包括中國倉鼠卵巢（CHO）細胞，包括DHFR-CHO細胞（Urlaub等人, PNAS USA 77:4216 (1980)）；及骨髓瘤細胞株，諸如NSO及Sp2/0。適於抗體產生之某些哺乳動物宿主細胞株之評述參見例如Yazaki及Wu, Methods in Molecular Biology, 第248卷(B. K.C Lo編, Humana Press, Totowa, N.J., 2003), 第255-268頁。某些較佳哺乳動物細胞表現系統包括基於CHO及HEK293細胞之表現系統。在此項技術中之已知者中，哺乳動物表現系統尤其可利用例如T燒瓶、輥瓶或細胞廠中之附著細胞株；或例如1L及5L旋轉器，5L、14L、40L、100L及200L攪拌槽生物反應器或20/50L及100/200L WAVE生物反應器中之懸浮培養物。Examples of suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 strain (COS-7, ATCC CRL 1651); human embryonic kidney strain (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human liver tumor line (Hep G2). Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNAS USA 77:4216 (1980)); and myeloma cell lines, such as NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268. Certain preferred mammalian cell expression systems include those based on CHO and HEK293 cells. Among those known in the art, mammalian expression systems can particularly utilize attached cell lines in, for example, T-flasks, roller flasks, or cell plants; or suspension cultures in, for example, 1L and 5L rotators, 5L, 14L, 40L, 100L and 200L stirred tank bioreactors, or 20/50L and 100/200L WAVE bioreactors.

亦包括蛋白質之無細胞表現。此等及相關實施例通常利用純化RNA聚合酶、核糖體、tRNA及核糖核苷酸；此等試劑可藉由自細胞或自基於細胞之表現系統提取而產生。Cell-free expression of proteins is also included. These and related embodiments typically utilize purified RNA polymerase, ribosomes, tRNA, and ribonucleotides; these reagents can be produced by extraction from cells or from cell-based expression systems.

特定起始信號亦可用以實現編碼所關注多肽之序列之更有效轉譯。此類信號包括ATG起始密碼子及相鄰序列。在編碼多肽之序列、其起始密碼子及上游序列插入至適當表現載體中之情況下，可能不需要額外的轉錄或轉譯控制信號。然而，在插入僅編碼序列或其一部分之情況下，應提供包括ATG起始密碼子之外源轉譯控制信號。此外，起始密碼子應在正確的閱讀框架中以確保整個插入物之轉譯。外源轉譯元件及起始密碼子可源自各種來源（天然及合成來源）。表現效率可藉由包括適合於使用之特定細胞系統的增強子，諸如文獻中所描述之彼等而增強（Scharf等人, Results Probl. Cell Differ. 20:125-162 (1994)）。Specific start signals can also be used to achieve more efficient translation of sequences encoding polypeptides of interest. Such signals include the ATG start codon and adjacent sequences. When the sequence encoding the polypeptide, its start codon, and upstream sequence are inserted into an appropriate expression vector, additional transcription or translation control signals may not be required. However, when only the coding sequence or a portion thereof is inserted, exogenous translation control signals including the ATG start codon should be provided. In addition, the start codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translation elements and start codons can be derived from a variety of sources (natural and synthetic sources). Expression efficiency may be enhanced by including enhancers appropriate to the particular cell system used, such as those described in the literature (Scharf et al., Results Probl. Cell Differ. 20:125-162 (1994)).

此外，宿主細胞菌株可針對其調節插入序列之表現或以所需方式加工所表現蛋白質之能力來選擇。此類多肽修飾包括但不限於轉譯後修飾，諸如乙醯化、羧化、糖基化、磷酸化、脂質化及醯化。裂解蛋白質「前原」形式之轉譯後加工亦可用於促成正確插入、摺疊及/或功能。除細菌細胞以外，可選擇具有或甚至缺乏針對此類轉譯後活性之特定細胞機構及特徵機制的不同宿主細胞，諸如酵母、CHO、HeLa、MDCK、HEK293及W138，以確保外源蛋白質之正確修飾及加工。In addition, host cell strains can be selected for their ability to modulate the expression of inserted sequences or to process the expressed protein in a desired manner. Such polypeptide modifications include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing of the "prepro" form of the cleaved protein can also be used to facilitate correct insertion, folding, and/or function. In addition to bacterial cells, different host cells such as yeast, CHO, HeLa, MDCK, HEK293, and W138 that have or even lack specific cellular machinery and characteristic mechanisms for such post-translational activities can be selected to ensure the correct modification and processing of foreign proteins.

為長期高產率地產生重組蛋白，穩定表現一般為較佳的。舉例而言，穩定表現所關注聚核苷酸之細胞株可使用表現載體轉型，該等表現載體可在相同或分開的載體上含有病毒複製起點及/或內源性表現元件及可選標記基因。引入載體之後，可使細胞在富集培養基中生長約1-2天，隨後換至選擇性培養基。可選標記之目的在於賦予對選擇之抗性，且其存在允許生長及回收成功表現所引入序列之細胞。可使用適於該細胞類型之組織培養技術增殖穩定轉型細胞之抗性純系。亦可採用諸如藉由短暫轉染或感染之短暫產生。適於短暫產生之例示性哺乳動物表現系統包括基於HEK293及CHO之系統。For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines that stably express the polynucleotide of interest can be transformed using expression vectors that can contain viral replication origins and/or endogenous expression elements and selectable marker genes on the same or separate vectors. After the introduction of the vector, the cells can be grown in an enriched medium for about 1-2 days and then switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows the growth and recovery of cells that successfully express the introduced sequence. Resistant pure lines of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. Transient production, such as by transient transfection or infection, may also be employed. Exemplary mammalian expression systems suitable for transient production include HEK293 and CHO based systems.

任何數目個選擇系統可用於回收經轉型或轉導之細胞株。此等選擇系統包括但不限於單純疱疹病毒胸苷激酶（Wigler等人, Cell 11:223-232 (1977)）及腺嘌呤磷酸核糖轉移酶（Lowy等人, Cell 22:817-823 (1990)）基因，該等基因可分別用於tk-細胞或aprt-細胞。此外，抗代謝物、抗生素或除草劑抗性可用作選擇基礎；舉例而言，賦予甲胺喋呤抗性之dhfr（Wigler等人, PNAS USA. 77:3567-70 (1980)）；賦予胺基糖苷類、新黴素及G-418抗性之npt（Colbere-Garapin等人, J. Mol. Biol. 150:1-14 (1981)）；以及分別賦予氯磺隆及草丁膦乙醯基轉移酶抗性之als或pat（Murry, 同前文獻）。已描述另外的可選基因，例如允許細胞利用吲哚代替色胺酸的trpB，或允許細胞利用組胺醇代替組胺酸的hisD（Hartma及Mulligan, Proc. Natl. Acad. Sci. U.S.A. 85:8047-51 (1988)）。可見標記之使用已得到普及，此類標記諸如綠色螢光蛋白（GFP）及其他螢光蛋白（例如RFP、YFP）、花青素、β-葡糖醛酸酶及其受質GUS以及螢光素酶及其受質螢光素，其不僅廣泛地用於鑑別轉型體，而且用於對由特定載體系統引起之短暫或穩定蛋白表現之量進行定量（參見例如，Rhodes等人, Methods Mol. Biol. 55:121-131 (1995)）。Any number of selection systems can be used to recover transformed or transduced cell lines. Such selection systems include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) genes, which can be used for tk- cells or aprt- cells, respectively. In addition, anti-metabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr confers resistance to methotrexate (Wigler et al., PNAS USA. 77:3567-70 (1980)); npt confers resistance to aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); and als or pat confers resistance to chlorsulfuron and phosphinothricin acetyltransferase, respectively (Murry, supra). Additional alternative genes have been described, such as trpB, which allows cells to utilize indole instead of tryptophan, or hisD, which allows cells to utilize histinol instead of histidine (Hartma and Mulligan, Proc. Natl. Acad. Sci. U.S.A. 85:8047-51 (1988)). The use of visible markers has become popular, such as green fluorescent protein (GFP) and other fluorescent proteins (e.g., RFP, YFP), anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, which are widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression caused by a particular vector system (see, e.g., Rhodes et al., Methods Mol. Biol. 55:121-131 (1995)).

亦包括高通量蛋白質產生系統或微量產生系統。某些態樣可將例如六組胺酸融合標籤（hexa-histidine fusion tag）用於經金屬螯合劑修飾之載片表面或MagneHis Ni粒子上之蛋白質表現及純化（參見例如，Kwon等人, BMC Biotechnol. 9:72, 2009；及Lin等人, Methods Mol Biol. 498:129-41, 2009)）。亦包括高通量無細胞蛋白質表現系統（參見例如，Sitaraman等人, Methods Mol Biol. 498:229-44, 2009）。此等及相關實施例可用於例如產生抗體之微陣列，接著該等微陣列可用於篩選庫以鑑別與所關注IL-18BP多肽相互作用之抗體及抗原結合域。High-throughput protein production systems or micro-production systems are also included. Certain aspects can use, for example, a hexa-histidine fusion tag for protein expression and purification on a slide surface modified with a metal chelator or MagneHis Ni particle (see, for example, Kwon et al., BMC Biotechnol. 9:72, 2009; and Lin et al., Methods Mol Biol. 498:129-41, 2009). High-throughput cell-free protein expression systems are also included (see, for example, Sitaraman et al., Methods Mol Biol. 498:229-44, 2009). These and related embodiments can be used, for example, to generate microarrays of antibodies, which can then be used to screen libraries to identify antibodies and antigen binding domains that interact with an IL-18BP polypeptide of interest.

此項技術中已知用於偵測及量測聚核苷酸編碼產物之表現的多種方案，其使用結合劑或諸如對產物具有特異性之多株或單株抗體之抗體。實例包括酶聯免疫吸附分析（ELISA）、西方免疫墨點、放射免疫分析（RIA）及螢光活化細胞分選（FACS）。此等及其他分析尤其描述於Hampton等人, Serological Methods, a Laboratory Manual (1990)及Maddox等人, J. Exp. Med. 158:1211-1216 (1983)中。A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products are known in the art using binding agents or antibodies such as polyclonal or monoclonal antibodies specific for the product. Examples include enzyme-linked immunosorbent assay (ELISA), Western immunoblot, radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). These and other assays are described, inter alia, in Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216 (1983).

熟習此項技術者已知廣泛多種標記及結合技術，且該等技術可用於多種核酸及胺基酸分析中。用於產生經標記之雜交或PCR探針以偵測與聚核苷酸相關之序列的方式包括寡聚物標記（oligolabeling）、缺口平移、末端標記或使用經標記核苷酸之PCR擴增。可替代地，序列或其任何部分可選殖至用於產生mRNA探針之載體中。此類載體為此項技術中已知，市售，且可用於在活體外藉由添加諸如T7、T3或SP6之適當RNA聚合酶及標記之核苷酸來合成RNA探針。此等程序可使用多種市售套組進行。可使用之適合報導分子或標記包括放射性核素、酶、螢光、化學發光或顯色劑以及受質、輔因子、抑制劑、磁粒及其類似物。A wide variety of labeling and binding techniques are known to those skilled in the art and can be used in a variety of nucleic acid and amino acid analyses. Methods for generating labeled hybridization or PCR probes to detect sequences associated with polynucleotides include oligolabeling, nick translation, end labeling, or PCR amplification using labeled nucleotides. Alternatively, the sequence or any portion thereof can be cloned into a vector for generating mRNA probes. Such vectors are known in the art, commercially available, and can be used to synthesize RNA probes in vitro by adding appropriate RNA polymerases such as T7, T3 or SP6 and labeled nucleotides. These procedures can be performed using a variety of commercially available kits. Suitable reporter molecules or labels that may be used include radionuclides, enzymes, fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.

用所關注之聚核苷酸序列轉型之宿主細胞可在適於自細胞培養物表現及回收蛋白質之條件下培養。某些具體實施例利用不含血清之細胞表現系統。實例包括可在不含血清之培養基中生長之HEK293細胞及CHO細胞（參見例如，Rosser等人, Protein Expr. Purif. 40:237-43, 2005；及美國專利第6,210,922號）。Host cells transformed with the polynucleotide sequence of interest can be cultured under conditions suitable for expression and recovery of the protein from the cell culture. Certain embodiments utilize serum-free cell expression systems. Examples include HEK293 cells and CHO cells that can be grown in serum-free medium (see, e.g., Rosser et al., Protein Expr. Purif. 40:237-43, 2005; and U.S. Patent No. 6,210,922).

由重組細胞產生之抗體或其抗原結合片段可在細胞內分泌或含於細胞內，視所用序列及/或載體而定。如熟習此項技術者將理解，含有聚核苷酸之表現載體可經設計成含有信號序列，該信號序列導引經編碼多肽分泌穿過原核或真核細胞膜。其他重組構築可用於將編碼所關注多肽之序列與編碼將促進可溶蛋白質純化及/或偵測之多肽域的核苷酸序列接合。此類域之實例包括可裂解及不可裂解之親和純化及抗原決定基標籤，諸如抗生物素蛋白、FLAG標籤、聚組胺酸標籤（例如6×His）、cMyc標籤、V5-標籤、麩胱甘肽S-轉移酶（GST）標籤及其他。Antibodies or antigen-binding fragments thereof produced by recombinant cells may be secreted or contained within the cell, depending on the sequence and/or vector used. As will be appreciated by those skilled in the art, expression vectors containing polynucleotides may be designed to contain a signal sequence that directs secretion of the encoded polypeptide across a prokaryotic or eukaryotic cell membrane. Other recombinant constructs may be used to join sequences encoding a polypeptide of interest to nucleotide sequences encoding polypeptide domains that will facilitate purification and/or detection of soluble proteins. Examples of such domains include cleavable and non-cleavable affinity purification and antigenic determinant tags, such as avidin, FLAG tags, polyhistidine tags (e.g., 6×His), cMyc tags, V5-tags, glutathione S-transferase (GST) tags, and others.

由重組細胞產生之蛋白質可根據此項技術中已知之多種技術純化及表徵。進行蛋白質純化及分析蛋白質純度之例示性系統包括快速蛋白質液相層析（FPLC）（例如AKTA及Bio-Rad FPLC系統）、高壓液相層析（HPLC）（例如Beckman及Waters HPLC）。在此項技術中之已知者中，用於純化之例示性化學反應尤其包括離子交換層析（例如Q、S）、尺寸排阻層析、鹽梯度、親和純化（例如Ni、Co、FLAG、麥芽糖、麩胱甘肽、蛋白質A/G）、凝膠過濾、逆相、陶瓷HYPERD®離子交換層析及疏水相互作用管柱（HIC）。亦包括諸如SDS-PAGE（例如考馬斯（Coomassie）、銀染色）、免疫墨點、Bradford及ELISA之分析方法，其通常可在產生或純化過程之任何步驟期間加以利用以量測蛋白質組合物之純度。Proteins produced by recombinant cells can be purified and characterized according to a variety of techniques known in the art. Exemplary systems for protein purification and analysis of protein purity include fast protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems), high pressure liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemical reactions for purification include, among others, ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradient, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A/G), gel filtration, reverse phase, ceramic HYPERD® ion exchange chromatography, and hydrophobic interaction columns (HIC), among those known in the art. Also included are analytical methods such as SDS-PAGE (e.g., Coomassie, silver stain), immunoblot, Bradford, and ELISA, which can generally be utilized during any step of the production or purification process to measure the purity of a protein composition.

亦包括濃縮抗IL-18BP抗體及其抗原結合片段以及包含濃縮之可溶蛋白質之組合物的方法。在某些態樣中，抗IL-18BP抗體之濃縮溶液包含濃度為約5 mg/mL、或約8 mg/mL、或約10 mg/mL、約15 mg/mL或約20 mg/mL或更大之蛋白質。Also included are methods of concentrating anti-IL-18BP antibodies and antigen-binding fragments thereof and compositions comprising the concentrated soluble protein. In certain aspects, the concentrated solution of anti-IL-18BP antibody comprises the protein at a concentration of about 5 mg/mL, or about 8 mg/mL, or about 10 mg/mL, about 15 mg/mL, or about 20 mg/mL or greater.

在一些態樣中，組合物實質上單分散，例如其中當例如藉由尺寸排阻層析、動態光散射及/或分析型超速離心評估時，抗IL-18BP抗體主要（亦即至少約90%或更大）以一種表觀分子量形式存在。In some aspects, the composition is substantially monodisperse, e.g., wherein the anti-IL-18BP antibody exists predominantly (i.e., at least about 90% or greater) in one apparent molecular weight form, e.g., as assessed by size exclusion analysis, dynamic light scattering, and/or analytical ultracentrifugation.

在一些態樣中，組合物之純度（以蛋白質計）為至少約90%，或在一些態樣中，純度為至少約95%，或在一些實施例中，純度為至少約98%。純度可經由如此項技術中已知之任何常規分析方法來測定。In some aspects, the purity of the composition (based on protein) is at least about 90%, or in some aspects, the purity is at least about 95%, or in some embodiments, the purity is at least about 98%. Purity can be determined by any conventional analytical method known in the art.

在一些態樣中，組合物之高分子量聚集體含量小於約10%、小於約5%、小於約3%、小於約1%。高分子量聚集體含量可藉由多種分析技術測定，包括例如藉由尺寸排阻層析、動態光散射及/或分析型超速離心測定。In some aspects, the high molecular weight aggregate content of the composition is less than about 10%, less than about 5%, less than about 3%, less than about 1%. The high molecular weight aggregate content can be determined by a variety of analytical techniques, including, for example, by size exclusion chromatography, dynamic light scattering and/or analytical ultracentrifugation.

本文中涵蓋之濃縮方法之實例包括凍乾，當溶液含有除所關注蛋白質外之少量可溶組分時通常採用凍乾。凍乾常常在HPLC操作之後進行，且可自混合物移除大部分或所有揮發性組分。亦包括超過濾技術，其通常採用一或多種選擇性滲透膜以濃縮蛋白質溶液。該膜允許水及小分子通過且保留蛋白質；在其他技術中，該溶液尤其可藉由機械泵、氣壓或離心相對於該膜加壓。Examples of concentration methods encompassed herein include lyophilization, which is typically employed when the solution contains small amounts of soluble components other than the protein of interest. Lyophilization is often performed after an HPLC run and can remove most or all volatile components from the mixture. Also included are superfiltration techniques, which typically employ one or more selectively permeable membranes to concentrate protein solutions. The membrane allows water and small molecules to pass through and retains the protein; among other techniques, the solution can be pressurized relative to the membrane by, inter alia, a mechanical pump, air pressure, or centrifugation.

在某些實施例中，抗IL-18BP抗體、試劑或相關藥劑具有至少約90%之純度，如根據此項技術中之常規技術所量測。在某些實施例中，抗IL-18BP組合物具有至少約95%之純度。在具體實施例中，諸如治療或醫藥組合物，抗IL-18BP抗體組合物具有至少約97%或98%或99%之純度。在一些實施例中，諸如當用作參考或研究試劑時，抗IL-18BP抗體可具有較小純度，且可具有至少約50%、60%、70%或80%之純度。純度可整體上或相對於選定組分，諸如其他蛋白質，例如以蛋白質計之純度來量測。In certain embodiments, the anti-IL-18BP antibody, reagent, or related agent has a purity of at least about 90%, as measured according to conventional techniques in this art. In certain embodiments, the anti-IL-18BP composition has a purity of at least about 95%. In specific embodiments, such as therapeutic or pharmaceutical compositions, the anti-IL-18BP antibody composition has a purity of at least about 97% or 98% or 99%. In some embodiments, such as when used as a reference or research reagent, the anti-IL-18BP antibody may have a lesser purity and may have a purity of at least about 50%, 60%, 70% or 80%. Purity can be measured overall or relative to selected components, such as other proteins, for example, as a purity measured in terms of protein.

經純化之抗體亦可根據其生物特徵來表徵。結合親和力及結合動力學可根據此項技術中已知之多種技術，諸如Biacore®及利用表面電漿子共振（SPR）之相關技術量測，表面電漿子共振係一種能夠即時偵測未標記相互作用物之光學現象。基於SPR之生物感測器可用於測定活性劑濃度、根據親和力與動力學兩方面進行篩選及表徵。一或多種典型或非典型生物活性之存在情況或水平可根據基於細胞之分析來量測，包括如本文所述之利用所選抗IL-18BP抗體之細胞結合搭配物的分析，該抗體在功能上偶合於讀數（readout）或指示劑，諸如生物活性之螢光或冷光指示劑。Purified antibodies can also be characterized based on their biological characteristics. Binding affinity and binding kinetics can be measured using a variety of techniques known in the art, such as Biacore® and related techniques using surface plasmon resonance (SPR), an optical phenomenon that enables real-time detection of unlabeled interactors. SPR-based biosensors can be used to determine active agent concentrations, screen and characterize based on both affinity and kinetics. The presence or level of one or more typical or atypical biological activities can be measured according to cell-based assays, including assays utilizing cell-binding partners of selected anti-IL-18BP antibodies as described herein, which antibodies are functionally coupled to a readout or indicator, such as a fluorescent or luminescent indicator of the biological activity.

在某些實施例中，如上文所指出，組合物實質上不含內毒素，包括例如約或至少約95%不含內毒素、約或至少約99%不含內毒素或約或至少約99.99%不含內毒素。如本文所描述，可根據此項技術中之常規技術偵測內毒素之存在情況。在具體實施例中，組合物由諸如哺乳動物或人類細胞之真核細胞在實質上不含血清之培養基中製成。在某些實施例中，如本文所指出，組合物具有小於約10 EU/mg抗體、或小於約5 EU/mg抗體、小於約3 EU/mg抗體或小於約1 EU/mg抗體之內毒素含量。In certain embodiments, as noted above, the composition is substantially free of endotoxin, including, for example, about or at least about 95% free of endotoxin, about or at least about 99% free of endotoxin, or about or at least about 99.99% free of endotoxin. As described herein, the presence of endotoxin can be detected according to conventional techniques in the art. In specific embodiments, the composition is made from eukaryotic cells such as mammalian or human cells in a culture medium that is substantially free of serum. In certain embodiments, as noted herein, the composition has an endotoxin content of less than about 10 EU/mg antibody, or less than about 5 EU/mg antibody, less than about 3 EU/mg antibody, or less than about 1 EU/mg antibody.

在某些實施例中，組合物包含小於約10% wt/wt高分子量聚集體、或小於約5% wt/wt高分子量聚集體、或小於約2% wt/wt高分子量聚集體或小於約1% wt/wt高分子量聚集體。In certain embodiments, the composition comprises less than about 10% wt/wt high molecular weight aggregates, or less than about 5% wt/wt high molecular weight aggregates, or less than about 2% wt/wt high molecular weight aggregates, or less than about 1% wt/wt high molecular weight aggregates.

亦包括基於蛋白質之分析型分析及方法，其可用於評估例如蛋白質純度、尺寸、溶解性及聚集程度以及其他特徵。蛋白質純度可以多種方式評估。舉例而言，純度可基於一級結構、高階結構、尺寸、電荷、疏水性及糖基化來評估。用於評估一級結構之方法之實例包括N端及C端定序以及肽定位（參見例如Allen等人, Biologicals. 24:255-275, 1996）。用於評估高階結構之方法之實例包括圓二色性（參見例如Kelly等人, Biochim Biophys Acta. 1751:119-139, 2005）、螢光光譜分析（參見例如Meagher等人, J. Biol. Chem. 273:23283-89, 1998）、FT-IR、醯胺氫-氘交換動力學（amide hydrogen-deuterium exchange kinetics）、差示掃描熱量測定、NMR光譜分析、與構形敏感抗體之免疫反應性。高階結構亦可依據諸如pH值、溫度或所添加鹽之多種參數來評估。用於評估諸如尺寸之蛋白質特徵之方法的實例包括分析型超速離心及尺寸排阻HPLC（SEC-HPLC），且用於量測電荷之示例性方法包括離子交換層析法及等電聚焦。疏水性可例如藉由逆相HPLC及疏水相互作用層析法HPLC評估。糖基化可影響藥物動力學（例如清除率）、構形或穩定性、受體結合及蛋白質功能，且可例如藉由質譜分析及核磁共振（NMR）光譜分析評估。Also included are protein-based analytical assays and methods that can be used to assess, for example, protein purity, size, solubility, and aggregation, among other characteristics. Protein purity can be assessed in a variety of ways. For example, purity can be assessed based on primary structure, higher order structure, size, charge, hydrophobicity, and glycosylation. Examples of methods for assessing primary structure include N-terminal and C-terminal sequencing and peptide localization (see, e.g., Allen et al., Biologicals. 24:255-275, 1996). Examples of methods used to assess higher order structure include circular dichroism (see, e.g., Kelly et al., Biochim Biophys Acta. 1751:119-139, 2005), fluorescence spectroscopy (see, e.g., Meagher et al., J. Biol. Chem. 273:23283-89, 1998), FT-IR, amide hydrogen-deuterium exchange kinetics, differential scanning calorimetry, NMR spectroscopy, and immunoreactivity of conformationally sensitive antibodies. Higher order structure can also be assessed based on a variety of parameters such as pH, temperature, or added salts. Examples of methods for assessing protein characteristics such as size include analytical ultracentrifugation and size exclusion HPLC (SEC-HPLC), and exemplary methods for measuring charge include ion exchange chromatography and isoelectric focusing. Hydrophobicity can be assessed, for example, by reverse phase HPLC and hydrophobic interaction chromatography HPLC. Glycosylation can affect pharmacokinetic (e.g., clearance), conformation or stability, receptor binding, and protein function, and can be assessed, for example, by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

如上文所指出，某些實施例包括使用SEC-HPLC評估諸如純度、尺寸（例如尺寸均質性）或聚集程度之蛋白質特徵及/或純化蛋白質以及其他用途。SEC亦包括凝膠過濾層析法（GFC）及凝膠滲透層析法（GPC），其係指其中溶液中之分子在多孔材料中基於其尺寸或更確切而言基於其流體動力學體積、擴散係數及/或表面特性而分離的層析方法。該方法一般用於分離生物分子，及測定聚合物之分子量及分子量分佈。通常，將生物或蛋白質樣品（諸如根據本文提供及此項技術中已知之蛋白質表現方法產生的蛋白質提取物）負載至所選之具有界定之固定相（多孔材料）、較佳不與樣品中之蛋白質相互作用之相的尺寸排阻管柱中。在某些態樣中，固定相由封裝於玻璃或鋼管柱內之稠密三維基質中之惰性粒子構成。移動相可為純水、水性緩衝液、有機溶劑或其混合物。固定相粒子通常具有僅允許小於某一尺寸之分子進入的小孔及/或通道。因此，大粒子被排除在此等孔及通道外，且其與固定相有限之相互作用導致其在實驗開始時作為「全部排除」峰溶離。剛好放入孔中之較小分子自流動之移動相移除，且其固定於固定相孔中所耗費之時間部分視其滲透至孔中之距離而定。其自移動相流之移除使得其花費更長時間自管柱溶離，且基於其尺寸之差異將粒子分離。給定尺寸排阻管柱具有可分離之分子量範圍。總體而言，固定相將不截留大於上限之分子，小於下限之分子將完全進入固相且作為單一帶溶離，且該範圍內之分子將以由諸如流體動力學體積之其特性界定的不同速率溶離。實際上關於醫藥蛋白質之此等方法的實例參見Bruner等人, Journal of Pharmaceutical and Biomedical Analysis. 15: 1929-1935, 1997。As noted above, certain embodiments include the use of SEC-HPLC to assess protein characteristics such as purity, size (e.g., size homogeneity), or degree of aggregation and/or to purify proteins, among other uses. SEC also includes gel filtration chromatography (GFC) and gel permeation chromatography (GPC), which refer to analytic methods in which molecules in solution are separated in porous materials based on their size, or more precisely, their hydrodynamic volume, diffusion coefficient, and/or surface properties. The method is generally used to separate biomolecules and to determine the molecular weight and molecular weight distribution of polymers. Typically, a biological or protein sample (such as a protein extract produced according to the protein expression methods provided herein and known in the art) is loaded into a selected size exclusion column having a defined stationary phase (porous material), preferably a phase that does not interact with the proteins in the sample. In certain embodiments, the stationary phase is composed of inert particles in a dense three-dimensional matrix encapsulated in a glass or steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent, or a mixture thereof. The stationary phase particles typically have pores and/or channels that only allow molecules smaller than a certain size to enter. Therefore, large particles are excluded from these pores and channels, and their limited interaction with the stationary phase causes them to dissolve as an "all-excluded" peak at the beginning of the experiment. Smaller molecules that fit into the hole are removed from the flowing mobile phase, and the time they spend fixed in the stationary phase hole depends in part on the distance they penetrate into the hole. Their removal from the mobile phase flow causes them to take longer to dissolve from the column, and the particles are separated based on the difference in their size. A given size exclusion column has a range of molecular weights that can be separated. In general, the stationary phase will not retain molecules larger than the upper limit, molecules smaller than the lower limit will completely enter the solid phase and dissolve as a single band, and molecules within this range will dissolve at different rates defined by their properties such as hydrodynamic volume. For examples of these methods for pharmaceutical proteins, see Bruner et al., Journal of Pharmaceutical and Biomedical Analysis. 15: 1929-1935, 1997.

臨床應用之蛋白質純度亦論述於例如Anicetti等人, (Trends in Biotechnology. 7:342-349, 1989)。用於分析蛋白質純度之更新近技術包括但不限於LabChip GXII，其為一種用於快速分析蛋白質及核酸之自動化平台，其提供蛋白質之效價之高通量分析、確定尺寸及純度分析。在某些非限制性實施例中，諸如蛋白質片段及抗體之臨床級蛋白質可藉由在至少兩個正交步驟中利用層析材料之組合以及其他方法來獲得（參見例如Therapeutic Proteins: Methods and Protocols. 第308卷, 編輯Smales及James, Humana Press Inc., 2005）。通常，蛋白質藥劑（例如抗體及抗原結合片段）實質上不含內毒素，如根據此項技術中已知及本文所述之技術所量測。Protein purity for clinical applications is also discussed in, for example, Anicetti et al. (Trends in Biotechnology. 7:342-349, 1989). More recent technologies for analyzing protein purity include, but are not limited to, LabChip GXII, an automated platform for rapid analysis of proteins and nucleic acids that provides high-throughput analysis of protein titers, size determination, and purity analysis. In certain non-limiting embodiments, clinical-grade proteins such as protein fragments and antibodies can be obtained by utilizing a combination of chromatographic materials in at least two orthogonal steps and other methods (see, for example, Therapeutic Proteins: Methods and Protocols. Vol. 308, ed. Smales and James, Humana Press Inc., 2005). Typically, protein agents (e.g., antibodies and antigen-binding fragments) are substantially free of endotoxin as measured according to techniques known in the art and described herein.

亦包括蛋白質溶解度分析。此類分析可用於例如確定重組產生之最佳生長及純化條件，最佳化緩衝液之選擇及最佳化抗體或其抗原結合片段之選擇。溶解度或聚集可根據多種參數來評估，該等參數包括溫度、pH、鹽及其他添加劑存在與否。溶解度篩選分析之實例包括但不限於使用濁度或其他量度作為評估指標量測蛋白質溶解度的基於微量培養盤之方法、用於分析經純化重組蛋白之溶解度的高通量分析（參見例如Stenvall等人, Biochim Biophys Acta. 1752:6-10, 2005）、使用遺傳標記蛋白之結構補充來活體內監測及量測蛋白質摺疊及溶解度的分析（參見例如Wigley等人, Nature Biotechnology. 19:131-136, 2001）及使用掃描電化學顯微法（SECM）電化學篩選重組蛋白在大腸桿菌中之溶解度（參見例如Nagamine等人, Biotechnology and Bioengineering. 96:1008-1013, 2006）等等。可根據此項技術中之常規技術，包括蛋白質溶解度之簡單活體內分析（參見例如Maxwell等人, Protein Sci. 8:1908-11, 1999）鑑別或選擇具有增加之溶解度（或減少之聚集）的抗體。Protein solubility analysis is also included. Such analysis can be used, for example, to determine optimal growth and purification conditions for recombinant production, optimal buffer selection, and optimal antibody or antigen-binding fragment selection. Solubility or aggregation can be assessed based on a variety of parameters, including temperature, pH, and the presence or absence of salt and other additives. Examples of solubility screening assays include, but are not limited to, microplate-based methods for measuring protein solubility using turbidity or other metrics as an assessment metric, high-throughput assays for analyzing the solubility of purified recombinant proteins (see, e.g., Stenvall et al., Biochim Biophys Acta. 1752:6-10, 2005), assays using structural complementation of genetically tagged proteins to monitor and measure protein folding and solubility in vivo (see, e.g., Wigley et al., Nature Biotechnology. 19:131-136, 2001), and electrochemical screening of recombinant proteins for solubility in E. coli using scanning electrochemical microscopy (SECM) (see, e.g., Nagamine et al., Biotechnology and Bioengineering. 96:1008-1013, 2001). 2006), etc. Antibodies with increased solubility (or reduced aggregation) can be identified or selected according to routine techniques in the art, including simple in vivo analysis of protein solubility (see, e.g., Maxwell et al., Protein Sci. 8:1908-11, 1999).

蛋白質溶解度及聚集亦可藉由動態光散射技術量測。聚集係涵蓋若干類型之相互作用或特徵的通用術語，該等相互作用或特徵包括可溶/不溶、共價/非共價、可逆/不可逆及天然/變性相互作用及特徵。對於蛋白質治療劑而言，通常由於如下問題而認為不希望存在聚集體：聚集體可能引起免疫原性反應（例如小聚集體），或可引起關於投與之不良事件（例如微粒）。動態光散射係指可用於測定小粒子在懸浮液或聚合物中（諸如蛋白質在溶液中）之尺寸分佈概況的技術。此技術亦稱光子關聯光譜法（PCS）或準彈性光散射（QELS），使用散射光量測蛋白質粒子之擴散速率。由於分子及粒子在溶液中之布朗運動（Brownian motion），故可觀測散射強度之波動。此運動資料可按照慣例進行加工，以得出樣品之尺寸分佈，其中尺寸由蛋白質粒子之斯托克斯半徑（Stokes radius）或流體動力學半徑給出。流體動力學尺寸視質量及形狀（構形）二者而定。動態散射可偵測極少量之聚集蛋白質（＜0.01重量%）之存在情況，即使在含有大範圍質量之樣品中亦如此。其亦可用於比較不同調配物之穩定性，包括例如依賴於高溫下之改變之即時監測的應用。因此，某些實施例包括使用動態光散射來分析聚集體在含有本發明之抗體之樣品中之溶解度及/或存在情況。Protein solubility and aggregation can also be measured by dynamic light scattering techniques. Aggregation is a general term that covers several types of interactions or characteristics, including soluble/insoluble, covalent/non-covalent, reversible/irreversible, and native/denatured interactions and characteristics. For protein therapeutics, the presence of aggregates is generally considered undesirable due to the following issues: aggregates may cause immunogenic reactions (such as small aggregates) or may cause adverse events associated with administration (such as microparticles). Dynamic light scattering refers to a technique that can be used to determine the size distribution profile of small particles in suspension or aggregates (such as proteins in solution). This technique, also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS), uses scattered light to measure the diffusion rate of protein particles. Due to the Brownian motion of molecules and particles in solution, fluctuations in the scattering intensity can be observed. This motion data can be processed conventionally to obtain the size distribution of the sample, where the size is given by the Stokes radius or the hydrodynamic radius of the protein particle. The hydrodynamic size depends on both mass and shape (configuration). Dynamic scattering can detect the presence of very small amounts of aggregated protein (<0.01% by weight), even in samples containing a wide range of masses. It can also be used to compare the stability of different formulations, including applications such as real-time monitoring that rely on changes at high temperatures. Therefore, certain embodiments include the use of dynamic light scattering to analyze the solubility and/or presence of aggregates in samples containing antibodies of the present invention.

儘管已出於清楚理解之目的藉助於說明及實例相當詳細地描述前述實施例，但鑒於本發明之教示，對一般熟習此項技術者將容易地顯而易見，可在不背離隨附申請專利範圍之精神或範疇的情況下對其進行某些變化及修改。以下實例僅作為說明而非作為限制提供。熟習此項技術者將容易地認識到可改變或修改以產生基本上相似之結果的多種非關鍵性參數。所列舉實施例Although the foregoing embodiments have been described in considerable detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of the present invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. One of ordinary skill in the art will readily recognize a variety of non-critical parameters that may be changed or modified to produce substantially similar results.The examples listed

以下非限制性所列舉實施例作為例示性提供。The following non-limiting examples are provided as illustrations.

實施例I-1. 一種經分離之抗體或其抗原結合片段，其與介白素-18結合蛋白（IL-18BP）結合，其中該至少一種抗體或其抗原結合片段包含： 重鏈可變區（V_H），其包含選自表A1之互補決定區V_HCDR1、V_HCDR2及V_HCDR3序列及其與IL-18BP特異性結合之變異體；以及 輕鏈可變區（V_L），其包含選自表A1之互補決定區V_LCDR1、V_LCDR2及V_LCDR3序列及其與IL-18BP特異性結合之變異體。Embodiment I-1. An isolated antibody or antigen-binding fragment thereof that binds to interleukin-18 binding protein (IL-18BP), wherein the at least one antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (_VH ) comprising complementary determining region_VH CDR1,_VH CDR2 and_VH CDR3 sequences selected fromTableA1 and variants thereof that specifically bind to IL-18BP; and a light chain variable region (_VL ) comprising complementary determining region_VL CDR1,_VL CDR2 and_VL CDR3 sequences selected fromTableA1 and variants thereof that specifically bind to IL-18BP.

實施例I-2. 如實施例I-1之經分離之抗體或其抗原結合片段，其中： 該VHCDR1、VHCDR2及VHCDR3序列分別包含TFX₁X₂X₃X₄X₅H、IX₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅AQKFQG及X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂DY，且該VLCDR1、VLCDR2及VLCDR3序列分別包含X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀WX₃₁A、X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈及QX₃₉X₄₀X₄₁SFPYX₄₂（關於「X」殘基之定義，參見表E11）； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 1-3，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 4-6； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 25-27，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 28-30； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 31-33，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 34-36； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 34-39，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 40-42； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 43-45，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 46-48； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 49-51，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 52-54； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 55-57，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 58-60； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 61-63，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 64-66； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 67-69，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 70-72； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 109-111，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 112-114； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 115-117，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 118-120； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 121-123，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 124-126； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 127-129，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 130-132； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 133-135，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 136-138； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 139-141，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 142-144； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 145-147，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 148-150； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 151-153，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 154-156； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 157-159，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 160-162； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 163-165，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 166-168； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 169-171，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 172-174； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 175-177，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 178-180； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 181-183，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 184-186； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 187-189，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 190-192； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 193-195，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 196-198； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 199-201，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 202-204； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 205-207，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 208-210； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 211-213，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 214-216； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 217-219，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 220-222； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 223-225，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 226-228； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 229-231，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 232-234； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 235-237，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 238-240； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 241-243，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 244-246； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 247-249，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 250-252； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 253-255，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 256-258； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 265-267，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 268-270；或 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 271-273，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 274-276。Embodiment I-2. The isolated antibody or antigen-binding fragment thereof of Embodiment I-1, wherein: the VHCDR1, VHCDR2 and VHCDR3 sequences comprise_TFX1 X₂ X₃ X₄ X₅ H, IX₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ AQKFQG and X₁₆ X₁₇ X₁₈ X₁₉ X₂₀ X₂₁ X₂₂ DY, respectively; and the VLCDR1, VLCDR2 and VLCDR3 sequences comprise X₂₃ X₂₄ X₂₅ X₂₆ X₂₇ X₂₈ X₂₉ X₃₀ WX₃₁ A, X₃₂ X₃₃ X₃₄ X₃₅ X₃₆ X₃₇ X₃₈ and QX₃₉ X₄₀ X₄₁ SFPYX₄₂ (for the definition of "X" residue,seeTable E11 ); the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 1-3, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 4-6, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 25-27, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 28-30, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 31-33, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 34-36, respectively; the_VH CDR1,_VH CDR2 and_VH the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 49-50, and the_VL CDR1,_VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 51-52, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 53-54, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 55-56, respectively; the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 57-58, respectively; the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 59-60, respectively; the_VH CDR1,_VHCDR2 and_VH CDR3 sequences comprise SEQ ID_NOs : 61-62_, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 58-60; said_{VH CDR1, VH CDR2 and VH CDR3 sequences comprise SEQ ID NOs: 61-63, respectively, and said VL} CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 64-66, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 67-69, respectively, and said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 70-72, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 109-111, respectively, and said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 112-114, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 127-129, and said_VL CDR1,_VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 130-132, respectively; said VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 133-135, respectively; said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 136-137, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 137-138, respectively; said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 139-140, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 141-142, respectively; said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 143-144, respectively; the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 136-138, respectively; the_VH CDR1,_VH CDR2 and VH CDR3 sequences comprise SEQ ID NOs: 139-141, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 142-144, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 145-147, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 148-150, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 151-153, respectively, and the_VL CDR1,_{VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 154-156, respectively; the VH CDR1, VH} CDR2 and_VHCDR3 sequences comprise SEQ ID NOs:_157-158 , respectively the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 157-159, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 160-162, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 163-165, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 166-168, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 169-171, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 172-174, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 175-177, respectively, and the_VL the VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 181-183, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 184-186, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 187-189, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 190-192, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 193-195, and_theVL CDR1,_VLCDR2 and_VLCDR3 sequences comprise SEQ ID NOs: said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 196-198; said_{VH CDR1, VH CDR2 and VH CDR3 sequences comprise SEQ ID NOs: 199-201, respectively, and said VL CDR1, VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 202-204, respectively; said VHCDR1,VHCDR2andVH} CDR3 sequences comprise SEQ ID NOs: 205-207, respectively, and said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 208-210, respectively; said_VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 211-213, respectively, and said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 214-216, respectively; said_VH CDR1,_VH CDR2 and_VH the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 229-231, and the_VL CDR1,_VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 232-234, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 235-237, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 236-238, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 237-239, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs_: 240-241, respectively; the_VH CDR1,_VH CDR2 and_VHCDR3 sequences comprise SEQ ID NOs: 242-243, respectively; the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 238-240, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 241-243, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 244-246, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 247-249, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 250-252, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 253-255, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 256-258, respectively; the_VH The VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 265-267, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 268-270, respectively; or the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 271-273, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 274-276, respectively.

實施例I-3. 如實施例I-1或I-2之經分離之抗體或其抗原結合片段，其中該V_H包含與選自表A2之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，視情況其中該V_H在構架區中具有1、2、3、4、5、6、7、8、9或10個改變。Embodiment I-3. An isolated antibody or antigen-binding fragment thereof as described in Embodiment I-1 or I-2, wherein the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected fromTableA2 , wherein the_VH has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations in the framework region.

實施例I-4. 如實施例I-1至I-3中任一項之經分離之抗體或其抗原結合片段，其中該V_L包含與選自表A2之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，視情況其中該V_L在構架區中具有1、2、3、4、5、6、7、8、9或10個改變。Embodiment I-4. An isolated antibody or antigen-binding fragment thereof according to any one of Embodiments I-1 to I-3, wherein the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected fromTableA2 , wherein the_VL has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations in the framework region.

實施例I-5. 如實施例I-1至I-4中任一項之經分離之抗體或其抗原結合片段，其中： 該V_H包含與SEQ ID NO: 277至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 278至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 279至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 280至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 285至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 286至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 287至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 288至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 289至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 290至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 291至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 292至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 293至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 294至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 295至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 296至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 297至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 298至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 299至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 300至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 313至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 314至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 315至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 316至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 317至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 318至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 319至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 320至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 321至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 322至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 323至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 324至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 325至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 326至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 327至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 328至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 329至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 330至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 331至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 332至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 333至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 334至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 335至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 336至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 337至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 338至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 339至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 340至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 341至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 342至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 343至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 344至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 345至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 346至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 347至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 348至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 349至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 350至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 351至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 352至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 353至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 354至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 355至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 356至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 357至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 358至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 359至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 360至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 361至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 362至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 367至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 368至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列；或 該V_H包含與SEQ ID NO: 369至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 370至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列。Embodiment I-5. An isolated antibody or antigen-binding fragment thereof according to any one of Embodiments I-1 to I-4, wherein: the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 277, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 278; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 279, and the VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%_, 98%, 99% or 100% identical to SEQ ID NO: 280; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 285 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 286, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 286; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 287, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 288; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 289, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 290; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 291, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 292; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 293, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 294; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 98%, 99%, 99%, 100% identical to SEQ ID NO: 299; the_VH comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 291, and the VL comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 292; the_VH comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 293, and the_VL comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 294; the_VH comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 295, and the_VL comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 300; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 313, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 314; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 315, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 316; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 317 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 318, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 318; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 319, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 320; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 321, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 322; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 323, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 324; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 325, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 326; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 327 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 328, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 328; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 329, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 330; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 331, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 332; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 333, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 334; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 335, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 336; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 337 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 338, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 338; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 339, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 340; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 341, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 342; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 343, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 344; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 345, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 346; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 347 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 348, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 348; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 349, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 350; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 351, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 352; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 353, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 354; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 355, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 356; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 357 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 358, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 358; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 359, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 360; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 361, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 362; the VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 367, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 368; or the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 369, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 370.

實施例I-6. 如實施例I-1至I-5中任一項之經分離之抗體或其抗原結合片段，其中該抗體與抗原決定基結合，該抗原決定基包含根據UniProt：O95998之編號之I97及V153 （可替代地，如由SEQ ID NO: 1所定義之I95及V151；或如由SEQ ID NO: 2所定義之I67及V123）。Embodiment I-6. An isolated antibody or antigen-binding fragment thereof as described in any one of Embodiments I-1 to I-5, wherein the antibody is bound to an antigenic determinant comprising I97 and V153 numbered according to UniProt: O95998 (alternatively, I95 and V151 as defined by SEQ ID NO: 1; or I67 and V123 as defined by SEQ ID NO: 2).

實施例I-7. 一種經分離之抗體或其抗原結合片段，其中該抗體在抗原決定基處與介白素-18結合蛋白（IL-18BP）結合，該抗原決定基包含根據UniProt：O95998之編號之I97及V153 （可替代地，如由SEQ ID NO: 1所定義之I95及V151；或如由SEQ ID NO: 2所定義之I67及V123）。Embodiment 1-7. An isolated antibody or antigen-binding fragment thereof, wherein the antibody binds to interleukin-18 binding protein (IL-18BP) at an antigenic determinant comprising I97 and V153 numbered according to UniProt: O95998 (alternatively, I95 and V151 as defined by SEQ ID NO: 1; or I67 and V123 as defined by SEQ ID NO: 2).

實施例I-8. 如實施例I-7之經分離之抗體或其抗原結合片段，其中： 該VHCDR1、VHCDR2及VHCDR3序列分別包含TFX₁X₂X₃X₄X₅H、IX₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅AQKFQG及X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂DY，且該VLCDR1、VLCDR2及VLCDR3序列分別包含X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀WX₃₁A、X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈及QX₃₉X₄₀X₄₁SFPYX₄₂（關於「X」殘基之定義，參見表E11）； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 1-3，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 4-6；或 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 265-267，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 268-270。Embodiment I-8. The isolated antibody or antigen-binding fragment thereof of Embodiment I-7, wherein: the VHCDR1, VHCDR2 and VHCDR3 sequences comprise_TFX1 X₂ X₃ X₄ X₅ H, IX₆ X₇ X₈ X 9 X₁₀ X₁₁ X₁₂ X₁₃ X_{14X 15AQKFQG} and X₁₆ X₁₇ X₁₈ X₁₉ X₂₀ X₂₁ X₂₂ DY, respectively, and the VLCDR1, VLCDR2 and VLCDR3 sequences comprise X₂₃ X₂₄ X₂₅ X₂₆ X₂₇ X₂₈ X₂₉ X₃₀ WX₃₁ A, X₃₂ X₃₃ X₃₄ X₃₅ X₃₆ X₃₇ X₃₈ and QX₃₉ X₄₀ X_41SFPYX42 (for the definition of "X" residue,seeTable E11 ); the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 1-3, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 4-6, respectively; or the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 265-267, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 268-270, respectively.

實施例I-9. 如實施例I-8之經分離之抗體或其抗原結合片段，其中： 該V_H包含與SEQ ID NO: 277至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 278至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 279至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 280至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列；或 該V_H包含與SEQ ID NO: 367至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 368至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列。Embodiment I-9. An isolated antibody or antigen-binding fragment thereof as described in Embodiment I-8, wherein: the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 277, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 278; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 279, and the VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 280; or the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100_% identical to SEQ ID NO: The V L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 367, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 368.

實施例I-10. 一種經分離之抗體或其抗原結合片段，其中該抗體與介白素-18結合蛋白（IL-18BP）結合且其中該抗體干擾IL-18與IL-18BP之結合。Embodiment I-10. An isolated antibody or an antigen-binding fragment thereof, wherein the antibody binds to interleukin-18 binding protein (IL-18BP) and wherein the antibody interferes with the binding of IL-18 to IL-18BP.

實施例I-11. 如實施例I-10之經分離之抗體或其抗原結合片段，其中該抗體與IL-18BP之構形抗原決定基結合。Example I-11. The isolated antibody or antigen-binding fragment thereof of Example I-10, wherein the antibody binds to a conformational epitope of IL-18BP.

實施例I-12. 如實施例I-10或I-11之經分離之抗體或其抗原結合片段，其中IL-18BP之該構形抗原決定基包含選自由以下組成之群的兩個或更多個胺基酸殘基：SEQ ID NO: 372之殘基K32、T40、S60、R61、S66、Y69、R91、R93、T96、K102、R131及H132。Example I-12. An isolated antibody or antigen-binding fragment thereof as described in Example I-10 or I-11, wherein the conformational antigenic determinant of IL-18BP comprises two or more amino acid residues selected from the group consisting of residues K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131 and H132 of SEQ ID NO: 372.

實施例I-13. 如實施例I-10至I-12中任一項之經分離之抗體或其抗原結合片段，其中IL-18BP之該構形抗原決定基包含SEQ ID NO: 372之K32、T40、S60、R61、S66、Y69、R91、R93、T96、K102、R131及H132之該等胺基酸殘基。Embodiment I-13. An isolated antibody or antigen-binding fragment thereof as described in any one of Embodiments I-10 to I-12, wherein the conformational antigenic determinant of IL-18BP comprises the amino acid residues K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131 and H132 of SEQ ID NO: 372.

實施例I-14. 如實施例I-10之經分離之抗體或其抗原結合片段，其中該抗體與IL-18BP之線性抗原決定基結合。Example I-14. The isolated antibody or antigen-binding fragment thereof of Example I-10, wherein the antibody binds to a linear antigenic determinant of IL-18BP.

實施例I-15. 如實施例I-10至I-14中任一項之經分離之抗體或其抗原結合片段，其中該抗體與IL-18與成熟形式之IL-18BP之間的結合界面結合。Embodiment I-15. The isolated antibody or antigen-binding fragment thereof of any one of Embodiments I-10 to I-14, wherein the antibody binds to the binding interface between IL-18 and the mature form of IL-18BP.

實施例I-16. 如實施例I-15之經分離之抗體或其抗原結合片段，其中該抗體結合SEQ ID NO: 372之該等胺基酸殘基R61、Y69及R131。Example I-16. The isolated antibody or antigen-binding fragment thereof as described in Example I-15, wherein the antibody binds to the amino acid residues R61, Y69 and R131 of SEQ ID NO: 372.

實施例I-17. 如實施例I-12至I-16中任一項之經分離之抗體或其抗原結合片段，其中： 該V_H包含與SEQ ID NO: 333至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 334至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列。Embodiment I-17. An isolated antibody or antigen-binding fragment thereof as described in any one of Embodiments I-12 to I-16, wherein: the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 333, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 334.

實施例I-18. 如實施例I-1至I-17中任一項之經分離之抗體或其抗原結合片段，其中該抗體與人類IL-18BP及食蟹獼猴IL-18BP結合，但不與小鼠IL-18BP（特異性或實質上）結合。Embodiment I-18. The isolated antibody or antigen-binding fragment thereof of any one of Embodiments I-1 to I-17, wherein the antibody binds to human IL-18BP and cynomolgus macaque IL-18BP, but does not bind to mouse IL-18BP (specifically or substantially).

實施例I-19. 如實施例I-1至I-17中任一項之經分離之抗體或其抗原結合片段，其中該抗體與人類IL-18BP、食蟹獼猴IL-18BP及小鼠IL-18BP結合。Embodiment I-19. The isolated antibody or antigen-binding fragment thereof of any one of Embodiments I-1 to I-17, wherein the antibody binds to human IL-18BP, cynomolgus macaque IL-18BP and mouse IL-18BP.

實施例I-20. 如實施例I-1至I-19中任一項之經分離之抗體或其抗原結合片段，其中該抗體與IL-18與成熟形式之IL-18BP之間的該結合界面結合。Embodiment I-20. The isolated antibody or antigen-binding fragment thereof of any one of Embodiments I-1 to I-19, wherein the antibody binds to the binding interface between IL-18 and the mature form of IL-18BP.

實施例I-21. 如實施例I-1至I-20中任一項之經分離之抗體或其抗原結合片段，其以比IL-18與IL-18BP之間的結合親和力（K_D為約650 pM）更強的結合親和力，視情況以約1 pm至約650 pm，或約或小於約1、5、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200或300、400、500、600或650 pM之結合親和力與IL-18BP結合。Embodiment I-21. An isolated antibody or antigen-binding fragment thereof according to any one of Embodiments I-1 to I-20, which binds to IL-18BP with a stronger binding affinity than the binding affinity between IL-18 and IL-18BP (_KD is about 650 pM), optionally with a binding affinity of about 1 pm to about 650 pm, or about or less than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 300, 400, 500, 600 or 650 pM.

實施例I-22. 如實施例I-1至I-21中任一項之經分離之抗體或其抗原結合片段，其為IL-18BP拮抗劑，其拮抗IL-18BP與IL-18之間的結合活性。Example I-22. The isolated antibody or antigen-binding fragment thereof according to any one of Examples I-1 to I-21, which is an IL-18BP antagonist that antagonizes the binding activity between IL-18BP and IL-18.

實施例I-23. 如實施例I-22之經分離之抗體或其抗原結合片段，其中該抗體阻斷IL-18BP對IL-18之抑制活性，且藉此增加IL-18介導之信號傳導，包括IFN-γ、CXCL10及/或TNFα之誘導。Example I-23. The isolated antibody or antigen-binding fragment thereof of Example I-22, wherein the antibody blocks the inhibitory activity of IL-18BP on IL-18 and thereby increases IL-18-mediated signaling, including the induction of IFN-γ, CXCL10 and/or TNFα.

實施例I-24. 如實施例I-1至I-8中任一項之經分離之抗體或其抗原結合片段，其包含IgA（包括子類IgA1及IgA2）、IgD、IgE、IgG（包括子類IgG1、IgG2、IgG3及IgG4）或IgM Fc域，視情況人類Fc域或其雜合體及/或變異體。Embodiment I-24. An isolated antibody or antigen-binding fragment thereof according to any one of Embodiments I-1 to I-8, comprising an IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3 and IgG4) or IgM Fc domain, optionally a human Fc domain or a hybrid and/or variant thereof.

實施例I-25. 如實施例I-24之經分離之抗體或其抗原結合片段，其包含在人類中具有高效應功能之IgG Fc域，視情況IgG1或IgG3 Fc域。Example I-25. An isolated antibody or antigen-binding fragment thereof according to Example I-24, comprising an IgG Fc domain having high efficacy in humans, optionally an IgG1 or IgG3 Fc domain.

實施例I-26. 如實施例I-24之經分離之抗體或其抗原結合片段，其包含在人類中具有低效應功能之IgG Fc域，視情況IgG2或IgG4 Fc域。Example I-26. An isolated antibody or antigen-binding fragment thereof according to Example I-24, comprising an IgG Fc domain having low efficiency function in humans, optionally an IgG2 or IgG4 Fc domain.

實施例I-27. 如實施例I-1至I-26中任一項之經分離之抗體或其抗原結合片段，其為單株抗體。Embodiment I-27. The isolated antibody or antigen-binding fragment thereof according to any one of Embodiments I-1 to I-26, which is a monoclonal antibody.

實施例I-28. 如實施例I-1至I-27中任一項之經分離之抗體或其抗原結合片段，其為人源化抗體。Embodiment I-28. The isolated antibody or antigen-binding fragment thereof according to any one of Embodiments I-1 to I-27, which is a humanized antibody.

實施例I-29. 如實施例I-1至I-28中任一項之經分離之抗體或其抗原結合片段，其係選自Fv片段、單鏈Fv（scFv）多肽、纖連蛋白（adnectin）、抗運載蛋白（anticalin）、適體、高親和性多聚體（avimer）、駱駝科抗體、經設計之錨蛋白重複蛋白（DARPin）、微型抗體、奈米抗體及單抗體（unibody）。Embodiment I-29. An isolated antibody or antigen-binding fragment thereof according to any one of Embodiments I-1 to I-28, which is selected from Fv fragments, single-chain Fv (scFv) polypeptides, adnectins, anticalins, aptamers, avimers, camel antibodies, designed anchor protein repeat proteins (DARPins), minibodies, nanobodies and unibodies.

實施例I-30. 一種經分離之聚核苷酸，其編碼如實施例I-1至I-29中任一項之經分離之抗IL-18BP抗體或其抗原結合片段；一種包含該經分離之聚核苷酸的表現載體；或一種包含該載體之經分離之宿主細胞。Embodiment I-30. An isolated polynucleotide encoding the isolated anti-IL-18BP antibody or antigen-binding fragment thereof of any one of Embodiments I-1 to I-29; an expression vector comprising the isolated polynucleotide; or an isolated host cell comprising the vector.

實施例I-31. 一種醫藥組合物，其包含如實施例I-1至I-29中任一項之經分離之抗IL-18BP抗體或其抗原結合片段及醫藥學上可接受之載劑。Example I-31. A pharmaceutical composition comprising the isolated anti-IL-18BP antibody or antigen-binding fragment thereof according to any one of Examples I-1 to I-29 and a pharmaceutically acceptable carrier.

實施例I-32. 如實施例I-31之醫藥組合物，其中該組合物具有就該至少一種抗體或抗原結合片段而言以蛋白質計至少約80%、85%、90%、95%、98%或99%之純度且實質上不含聚集體及內毒素。Embodiment I-32. The pharmaceutical composition of Embodiment I-31, wherein the composition has a purity of at least about 80%, 85%, 90%, 95%, 98% or 99% on a protein basis for the at least one antibody or antigen-binding fragment and is substantially free of aggregates and endotoxins.

實施例I-33. 如實施例I-31或I-32之醫藥組合物，其中該組合物為視情況適合於靜脈內、肌肉內、皮下或腹膜內投與之無菌可注射溶液。Embodiment I-33. The pharmaceutical composition of Embodiment I-31 or I-32, wherein the composition is a sterile injectable solution suitable for intravenous, intramuscular, subcutaneous or intraperitoneal administration as appropriate.

實施例I-34. 一種治療有需要之個體之疾病或病狀的方法，其包含向該個體投與如實施例I-31至I-33中任一項之醫藥組合物。Embodiment I-34. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of any one of Embodiments I-31 to I-33.

實施例I-35. 如實施例I-34之方法，其中該疾病或病狀為癌症或腫瘤或增生性疾病或病症，視情況選自以下之增生性疾病或病症：淋巴增生性病症、骨髓增生性病症、增生性腸炎、增生性糖尿病視網膜病變及增生性腎病。Embodiment I-35. The method of embodiment I-34, wherein the disease or condition is cancer or a tumor or a proliferative disease or disorder, optionally selected from the following proliferative diseases or disorders: lymphoproliferative disorders, myeloproliferative disorders, proliferative enteritis, proliferative diabetic retinopathy and proliferative nephropathy.

實施例I-36. 如實施例I-35之方法，其中該癌症或腫瘤表現或過度表現IL-18BP及/或IL-18，或其中該增生性疾病或病症與IL-18BP及/或IL-18之表現增加相關。Embodiment I-36. The method of embodiment I-35, wherein the cancer or tumor expresses or overexpresses IL-18BP and/or IL-18, or wherein the proliferative disease or disorder is associated with increased expression of IL-18BP and/or IL-18.

實施例I-37. 如實施例I-35或I-36之方法，其中該癌症係選自以下中之一或多者：骨癌、前列腺癌、黑色素瘤（例如，轉移性黑色素瘤）、胰臟癌、小細胞肺癌、非小細胞肺癌（NSCLC）、間皮瘤、白血病（例如，淋巴球性白血病、慢性骨髓性白血病、急性骨髓性白血病、復發性急性骨髓性白血病、毛細胞白血病、急性淋巴母細胞性白血病）、淋巴瘤（例如，非何傑金氏淋巴瘤（non-Hodgkin's lymphoma）、何傑金氏淋巴瘤）、肝癌（肝細胞癌）、肉瘤、B細胞惡性病、乳癌、卵巢癌、結腸直腸癌、神經膠質瘤、多形性神經膠質母細胞瘤、腦膜瘤、垂體腺瘤、前庭神經鞘瘤、原發性CNS淋巴瘤、原始神經外胚層腫瘤（神經管胚細胞瘤）、腎癌（例如，腎細胞癌）、膀胱癌、子宮癌、尿道上皮癌、食道癌、腦癌、頭頸癌、子宮頸癌、睪丸癌、甲狀腺癌及胃癌。Embodiment I-37. The method of embodiment I-35 or I-36, wherein the cancer is selected from one or more of the following: bone cancer, prostate cancer, melanoma (e.g., metastatic melanoma), pancreatic cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, hairy cell leukemia, acute lymphoblastic leukemia), lymphoma (e.g., non-Hodgkin's lymphoma lymphoma), Hodgkin's lymphoma), liver cancer (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, neuroglioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (neurotubular cell tumor), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, urothelial carcinoma, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.

實施例I-38. 如實施例I-34至I-37中任一項之方法，其包含投與該醫藥組合物（具有抗IL18BP抗體或其抗原結合片段）與IL-18之組合。Embodiment I-38. The method of any one of Embodiments I-34 to I-37, comprising administering the pharmaceutical composition (having an anti-IL18BP antibody or an antigen-binding fragment thereof) in combination with IL-18.

實施例I-39. 如實施例I-35至I-38中任一項之方法，其包含投與該醫藥組合物（具有該抗IL18BP抗體或其抗原結合片段）與免疫檢查點調節劑之組合，該免疫檢查點調節劑選自抑制性免疫檢查點分子之拮抗劑及刺激性免疫檢查點分子之促效劑。Embodiment I-39. The method of any one of Embodiments I-35 to I-38, comprising administering the pharmaceutical composition (having the anti-IL18BP antibody or antigen-binding fragment thereof) in combination with an immune checkpoint regulator selected from antagonists of inhibitory immune checkpoint molecules and agonists of stimulatory immune checkpoint molecules.

實施例I-40. 如實施例I-39之方法，其中該免疫檢查點調節劑為多肽，視情況抗體或其抗原結合片段，或配位體或小分子。Embodiment I-40. The method of embodiment I-39, wherein the immune checkpoint modulator is a polypeptide, an antibody or an antigen-binding fragment thereof, or a ligand or a small molecule.

實施例I-41. 如實施例I-39或I-40之方法，其中該抑制性免疫檢查點分子係選自以下中之一或多者：程式化死亡-配位體1（PD-L1）、程式化死亡1（PD-1）、程式化死亡-配位體2（PD-L2）、細胞毒性T淋巴球相關蛋白4（CTLA-4）、吲哚胺2,3-雙加氧酶（IDO）、色胺酸2,3-雙加氧酶（TDO）、T細胞免疫球蛋白域及黏蛋白域3（TIM-3）、淋巴球活化基因-3（LAG-3）、T細胞活化之V域Ig抑制因子（VISTA）、B及T淋巴球衰減因子（BTLA）、CD160、疱疹病毒侵入介體（HVEM）以及具有Ig及ITIM域之T細胞免疫受體（TIGIT）。Embodiment I-41. The method of embodiment I-39 or I-40, wherein the inhibitory immune checkpoint molecule is selected from one or more of the following: programmed death-ligand 1 (PD-L1), programmed death 1 (PD-1), programmed death-ligand 2 (PD-L2), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene-3 (LAG-3), V-domain Ig inhibitory factor of T cell activation (VISTA), B and T lymphocyte depletion factor (BTLA), CD160, herpes virus entry mediator (HVEM) and T cell immune receptor with Ig and ITIM domains (TIGIT).

實施例I-42. 如實施例I-41之方法，其中： 該拮抗劑為視情況選自以下中之一或多者的PD-L1及/或PD-L2拮抗劑：與PD-L1及/或PD-L2特異性結合之抗體或抗原結合片段或小分子、阿替利珠單抗（atezolizumab）（MPDL3280A）、阿維魯單抗（avelumab）（MSB0010718C）及度伐利尤單抗（durvalumab）（MEDI4736），視情況其中該癌症係選自以下中之一或多者：結腸直腸癌、黑色素瘤、乳癌、非小細胞肺癌、膀胱癌及腎細胞癌； 該拮抗劑為視情況選自以下中之一或多者的PD-1拮抗劑：與PD-1特異性結合之抗體或抗原結合片段或小分子、納武單抗（nivolumab）、帕博利珠單抗（pembrolizumab）、MK-3475、AMP-224、AMP-514PDR001及皮立珠單抗（pidilizumab），視情況其中該PD-1拮抗劑為納武單抗，且該癌症視情況選自以下中之一或多者：何傑金氏淋巴瘤、黑色素瘤、非小細胞肺癌、肝細胞癌、腎細胞癌及卵巢癌； 該PD-1拮抗劑為帕博利珠單抗，且該癌症視情況選自以下中之一或多者：黑色素瘤、非小細胞肺癌、小細胞肺癌、頭頸癌及尿道上皮癌； 該拮抗劑為視情況選自以下中之一或多者的CTLA-4拮抗劑：與CTLA-4特異性結合之抗體或抗原結合片段或小分子、伊匹單抗（ipilimumab）、曲美木單抗（tremelimumab），視情況其中該癌症係選自以下中之一或多者：黑色素瘤、前列腺癌、肺癌及膀胱癌； 該拮抗劑為視情況選自以下中之一或多者的IDO拮抗劑：與IDO特異性結合之抗體或抗原結合片段或小分子、因多莫得（indoximod）（NLG-8189）、1-甲基-色胺酸（1MT）、β-咔啉（去甲哈爾滿（norharmane）；9H-吡啶并[3,4-b]吲哚）、迷迭香酸及艾帕斯塔（epacadostat），且其中該癌症視情況選自以下中之一或多者：轉移性乳癌及腦癌、視情況多形性神經膠質母細胞瘤、神經膠質瘤、神經膠質肉瘤或惡性腦瘤； 該拮抗劑為視情況選自以下中之一或多者的TDO拮抗劑：與TDO特異性結合之抗體或抗原結合片段或小分子、680C91及LM10； 該拮抗劑為視情況選自與TIM-3特異性結合之抗體或抗原結合片段或小分子中之一或多者的TIM-3拮抗劑； 該拮抗劑為視情況選自以下中之一或多者的LAG-3拮抗劑：與LAG-3特異性結合之抗體或抗原結合片段或小分子,及BMS-986016； 該拮抗劑為視情況選自與VISTA特異性結合之抗體或抗原結合片段或小分子中之一或多者的VISTA拮抗劑； 該拮抗劑為視情況選自與BTLA、CD160及/或HVEM特異性結合之抗體或抗原結合片段或小分子中之一或多者的BTLA、CD160及/或HVEM拮抗劑； 該拮抗劑為視情況選自與TIGIT特異性結合之抗體或抗原結合片段或小分子中之一或多者的TIGIT拮抗劑。Embodiment I-42. The method of Embodiment I-41, wherein: The antagonist is a PD-L1 and/or PD-L2 antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to PD-L1 and/or PD-L2, atezolizumab (MPDL3280A), avelumab (MSB0010718C) and durvalumab (MEDI4736), wherein the cancer is selected from one or more of the following: colorectal cancer, melanoma, breast cancer, non-small cell lung cancer, bladder cancer and renal cell carcinoma; The antagonist is a PD-1 antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to PD-1, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514PDR001 and pidilizumab, wherein the PD-1 antagonist is nivolumab, and the cancer is selected from one or more of the following: Hodgkin's lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma and ovarian cancer;The PD-1 antagonist is pembrolizumab, and the cancer is selected from one or more of the following: melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial carcinoma;The antagonist is a CTLA-4 antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to CTLA-4, ipilimumab, tremelimumab, and the cancer is selected from one or more of the following: melanoma, prostate cancer, lung cancer, and bladder cancer;The antagonist is an IDO antagonist selected from one or more of the following: antibodies or antigen-binding fragments or small molecules that specifically bind to IDO, indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carbolines (norharmane; 9H-pyrido[3,4-b]indole), rosmarinic acid and epacadostat, and the cancer is selected from one or more of the following: metastatic breast cancer and brain cancer, glioblastoma multiforme, neuroglioma, neurosarcoma or malignant brain tumor;The antagonist is a TDO antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to TDO, 680C91 and LM10;The antagonist is a TIM-3 antagonist selected from one or more of the antibodies or antigen-binding fragments or small molecules that specifically bind to TIM-3;The antagonist is a LAG-3 antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to LAG-3, and BMS-986016;The antagonist is a VISTA antagonist selected from one or more of the antibodies or antigen-binding fragments or small molecules that specifically bind to VISTA;The antagonist is a BTLA, CD160 and/or HVEM antagonist selected from one or more of antibodies, antigen-binding fragments or small molecules that specifically bind to BTLA, CD160 and/or HVEM as appropriate;The antagonist is a TIGIT antagonist selected from one or more of antibodies, antigen-binding fragments or small molecules that specifically bind to TIGIT as appropriate.

實施例I-43. 如實施例I-39或I-40之方法，其中該刺激性免疫檢查點分子係選自以下中之一或多者：OX40、CD40、糖皮質激素誘導之TNFR家族相關基因（GITR）、CD137（4-1BB）、CD27、CD28、CD226及疱疹病毒侵入介體（HVEM）。Embodiment I-43. The method of embodiment I-39 or I-40, wherein the stimulatory immune checkpoint molecule is selected from one or more of the following: OX40, CD40, glucocorticoid-induced TNFR family-related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226 and herpes virus entry mediator (HVEM).

實施例I-44. 如實施例I-43之方法，其中： 該促效劑為視情況選自以下中之一或多者的OX40促效劑：與OX40特異性結合之抗體或抗原結合片段或小分子或配位體、OX86、Fc-OX40L及GSK3174998； 該促效劑為視情況選自以下中之一或多者的CD40促效劑：與CD40特異性結合之抗體或抗原結合片段或小分子或配位體、CP-870,893、達西組單抗（dacetuzumab）、Chi Lob 7/4、ADC-1013及rhCD40L，且其中該癌症視情況選自以下中之一或多者：黑色素瘤、胰臟癌、間皮瘤及血液癌，視情況淋巴瘤，諸如非何傑金氏淋巴瘤； 該促效劑為視情況選自以下中之一或多者的GITR促效劑：與GITR特異性結合之抗體或抗原結合片段或小分子或配位體、INCAGN01876、DTA-1及MEDI1873； 該促效劑為視情況選自以下中之一或多者的CD137促效劑：與CD137特異性結合之抗體或抗原結合片段或小分子或配位體、烏托米單抗（utomilumab）及4-1BB配位體； 該促效劑為視情況選自以下中之一或多者的CD27促效劑：與CD27特異性結合之抗體或抗原結合片段或小分子或配位體、瓦利魯單抗（varlilumab）及CDX-1127 (1F5)； 該促效劑為視情況選自以下中之一或多者的CD28促效劑：與CD28特異性結合之抗體或抗原結合片段或小分子或配位體，及TAB08；及/或 該促效劑為視情況選自與HVEM特異性結合之抗體或抗原結合片段或小分子或配位體中之一或多者的HVEM促效劑。Example I-44. The method of Example I-43, wherein: The agonist is an OX40 agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to OX40, OX86, Fc-OX40L and GSK3174998; The agonist is a CD40 agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD40, CP-870,893, dacetuzumab, Chi Lob 7/4, ADC-1013 and rhCD40L, and wherein the cancer is selected from one or more of the following: melanoma, pancreatic cancer, mesothelioma and blood cancer, and lymphoma, such as non-Hodgkin's lymphoma; The agonist is a GITR agonist selected from one or more of the following: an antibody or antigen binding fragment or small molecule or ligand that specifically binds to GITR, INCAGN01876, DTA-1 and MEDI1873; The agonist is a CD137 agonist selected from one or more of the following: an antibody or antigen binding fragment or small molecule or ligand that specifically binds to CD137, utomilumab and 4-1BB ligand; The agonist is a CD27 agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD27, varlilumab, and CDX-1127 (1F5);The agonist is a CD28 agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD28, and TAB08; and/orThe agonist is a HVEM agonist selected from one or more of the antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to HVEM.

實施例I-45. 如實施例I-35至I-44中任一項之方法，其包含投與該醫藥組合物（具有該抗IL18BP抗體或其抗原結合片段）與至少一種化學治療劑之組合。Embodiment I-45. The method of any one of Embodiments I-35 to I-44, comprising administering the pharmaceutical composition (having the anti-IL18BP antibody or antigen-binding fragment thereof) in combination with at least one chemotherapeutic agent.

實施例I-46. 如實施例I-45之方法，其中該至少一種化學治療劑係選自以下中之一或多者：烷基化劑、抗代謝物、細胞毒性抗生素、拓樸異構酶抑制劑（1型或II型）及抗微管劑。Embodiment I-46. The method of embodiment I-45, wherein the at least one chemotherapeutic agent is selected from one or more of the following: an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an anti-microtubule agent.

實施例I-47. 如實施例I-46之方法，其中： 該烷基化劑係選自以下中之一或多者：氮芥（nitrogen mustard）（視情況甲氮芥（mechlorethamine）、環磷醯胺、氮芥（mustine）、美法侖（melphalan）、苯丁酸氮芥（chlorambucil）、異環磷醯胺（ifosfamide）及硫酸布他卡因（busulfan））、亞硝基脲（視情況N-亞硝基-N-甲基脲（MNU）、卡莫司汀（carmustine）（BCNU）、洛莫司汀（lomustine）（CCNU）、司莫司汀（semustine）（MeCCNU）、福莫司汀（fotemustine）及鏈佐黴素（streptozotocin））、四（視情況達卡巴嗪（dacarbazine）、米托唑胺（mitozolomide）及替莫唑胺（temozolomide））、氮丙啶（視情況噻替派（thiotepa）、絲裂黴素（mytomycin）及地吖醌（diaziquone）（AZQ））、順鉑及其衍生物（視情況卡鉑（carboplatin）及奧沙利鉑（oxaliplatin））及非典型烷基化劑（視情況丙卡巴肼（procarbazine）及六甲三聚氰胺）； 該抗代謝物係選自以下中之一或多者：抗葉酸劑（視情況甲胺喋呤（methotrexate）及培美曲塞（pemetrexed））、氟嘧啶（視情況5-氟尿嘧啶及卡培他濱（capecitabine））、去氧核苷類似物（視情況安西他濱（ancitabine）、依諾他濱（enocitabine）、阿糖胞苷（cytarabine）、吉西他濱（gemcitabine）、地西他濱（decitabine）、阿紮胞苷（azacitidine）、氟達拉濱（fludarabine）、奈拉濱（nelarabine）、克拉屈濱（cladribine）、氯法拉濱（clofarabine）、氟達拉濱（fludarabine）及噴司他汀（pentostatin））及硫代嘌呤（視情況硫鳥嘌呤及巰基嘌呤）； 該細胞毒性抗生素係選自以下中之一或多者：蒽環黴素（視情況小紅莓（doxorubicin）、道諾黴素（daunorubicin）、表柔比星（epirubicin）、艾達黴素（idarubicin）、吡柔比星（pirarubicin）、阿克拉黴素（aclarubicin）及米托蒽醌（mitoxantrone））、博來黴素（bleomycin）、絲裂黴素（mitomycin）C、米托蒽醌及放線菌素（actinomycin）； 該拓樸異構酶抑制劑係選自以下中之一或多者：喜樹鹼（camptothecin）、伊立替康（irinotecan）、拓樸替康（topotecan）、依託泊苷（etoposide）、小紅莓、米托蒽醌、替尼泊苷（teniposide）、新生黴素（novobiocin）、麥爾巴隆（merbarone）及阿克拉黴素；及/或 該抗微管劑係選自以下中之一或多者：紫杉烷（視情況太平洋紫杉醇（paclitaxel）及多西他賽（docetaxel））及長春花生物鹼（視情況長春鹼（vinblastine）、長春新鹼（vincristine）、長春地辛（vindesine）、長春瑞濱（vinorelbine））。Embodiment I-47. The method of embodiment I-46, wherein: the alkylating agent is selected from one or more of the following: nitrogen mustard (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide and busulfan), nitrosourea (optionally N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine and streptozotocin), tetracycline (dacarbazine, mitozolomide and temozolomide, as appropriate), aziridines (thiotepa, mytomycin and diaziquone (AZQ), as appropriate), cis-platinum and its derivatives (carboplatin and oxaliplatin, as appropriate) and atypical alkylating agents (procarbazine and hexamethonium, as appropriate); The anti-metabolite is selected from one or more of the following: antifolates (methotrexate and pemetrexed as appropriate), fluoropyrimidines (5-fluorouracil and capecitabine as appropriate), deoxynucleoside analogs (ancitabine, enocitabine, cytarabine, gemcitabine as appropriate), itabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine and pentostatin) and thiopurines (thioguanine and thioguanine as appropriate); The cytotoxic antibiotic is one or more selected from the group consisting of anthracyclines (depending on the situation, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone), bleomycin, mitomycin C, mitoxantrone and actinomycin; The topoisomerase inhibitor is selected from one or more of the following: camptothecin, irinotecan, topotecan, etoposide, cranberry, mitoxantrone, teniposide, novobiocin, merbarone and aclatomycin; and/or the anti-microtubule agent is selected from one or more of the following: taxanes (paclitaxel and docetaxel as appropriate) and vinca alkaloids (vinblastine, vincristine, vindesine, vinorelbine as appropriate).

實施例I-48. 如實施例I-34之方法，其中該疾病或病狀為骨髓發育不良症候群（MDS）。Embodiment I-48. The method of Embodiment I-34, wherein the disease or condition is myelodysplastic syndrome (MDS).

實施例I-49. 如實施例I-34之方法，其中該疾病或病狀為感染性疾病。Embodiment I-49. The method of Embodiment I-34, wherein the disease or condition is an infectious disease.

實施例I-50. 如實施例I-49之方法，其中該感染性疾病係選自病毒、細菌、真菌（視情況酵母）及原蟲感染。Embodiment I-50. The method of Embodiment I-49, wherein the infectious disease is selected from viral, bacterial, fungal (optionally yeast) and protozoan infections.

實施例I-51. 如實施例I-48至I-50中任一項之方法，其包含投與該醫藥組合物（具有該抗IL18BP抗體或其抗原結合片段）與IL-18之組合。Embodiment I-51. The method of any one of Embodiments I-48 to I-50, comprising administering the pharmaceutical composition (having the anti-IL18BP antibody or antigen-binding fragment thereof) in combination with IL-18.

實施例I-52. 一種針對阻斷或抑制IL-18與IL-18BP之間的結合之能力篩選抗IL-18BP抗體或其抗原結合片段之方法，其包含 a) 測定該抗體或其抗原結合片段對以下之結合親和力， i) 單獨的IL-18BP，及 ii) 低IL-18融合蛋白，其中該低IL-18融合蛋白包含經由可撓性連接子（及其間的視情況存在之蛋白酶裂解位點）與IL-18BP融合的IL-18，其中該融合蛋白之IL-18部分結合至該融合蛋白之IL-18BP部分且空間阻斷該融合蛋白之該IL-18BP部分之IL-18結合位點； b) 比較（i）之該結合親和力與（ii）之該結合親和力；及 c) 若（i）之該結合親和力顯著強於（ii）之該結合親和力，則將該抗體或其抗原結合片段鑑別或選擇為能夠阻斷或抑制IL-18與IL-18BP之間的結合。Example I-52. A method for screening an anti-IL-18BP antibody or an antigen-binding fragment thereof for its ability to block or inhibit the binding between IL-18 and IL-18BP, comprising:a) determining the binding affinity of the antibody or antigen-binding fragment thereof to,i) IL-18BP alone, andii) a low IL-18 fusion protein, wherein the low IL-18 fusion protein comprises IL-18 fused to IL-18BP via a flexible linker (and a protease cleavage site therebetween, if appropriate), wherein the IL-18 portion of the fusion protein binds to the IL-18BP portion of the fusion protein and sterically blocks the IL-18 binding site of the IL-18BP portion of the fusion protein;b) comparing the binding affinity of (i) with the binding affinity of (ii); andc) If the binding affinity of (i) is significantly stronger than the binding affinity of (ii), the antibody or antigen-binding fragment thereof is identified or selected as being capable of blocking or inhibiting the binding between IL-18 and IL-18BP.

實施例I-53. 如實施例I-52之方法，其中該IL-18及該IL-18BP為小鼠IL-18及小鼠IL-18BP。Embodiment I-53. The method of Embodiment I-52, wherein the IL-18 and the IL-18BP are mouse IL-18 and mouse IL-18BP.

實施例I-54. 如實施例I-52之方法，其中該IL-18及該IL-18BP為人類IL-18及人類IL-18BP。Embodiment I-54. The method of Embodiment I-52, wherein the IL-18 and the IL-18BP are human IL-18 and human IL-18BP.

實施例I-55. 如實施例I-52至I-54中任一項之方法，其中該低IL-18融合蛋白在N端至C端定向上包含信號肽、IL-18、第一可撓性連接子、蛋白酶裂解位點（視情況TEV蛋白酶裂解位點）、可撓性連接子及IL-18BP。Embodiment I-55. The method of any one of Embodiments I-52 to I-54, wherein the low IL-18 fusion protein comprises a signal peptide, IL-18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker and IL-18BP in an N-terminal to C-terminal orientation.

實施例I-56. 如實施例I-55之方法，其中該低IL-18融合蛋白包含與來自表S1之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的胺基酸序列。Embodiment I-56. The method of Embodiment I-55, wherein the low IL-18 fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the sequence fromTableS1 .

實施例I-57. 一種低IL-18融合蛋白，其在N端至C端定向上包含信號肽、IL-18、第一可撓性連接子、蛋白酶裂解位點（視情況TEV蛋白酶裂解位點）、可撓性連接子及IL-18BP。Example 1-57. A low IL-18 fusion protein comprising a signal peptide, IL-18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker and IL-18BP in the N-terminal to C-terminal orientation.

實施例I-58. 如實施例I-57之低IL-18融合蛋白，其包含與來自表S1之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的胺基酸序列。Example I-58. The low IL-18 fusion protein of Example I-57, comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the sequence fromTableS1 .

實施例I-59. 一種刺激有需要之個體之免疫反應的方法，其包含向該個體投與如實施例31-33中任一項之醫藥組合物。Embodiment I-59. A method of stimulating an immune response in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to any one of Embodiments 31-33.

實施例I-60. 如實施例I-59之方法，其中該免疫反應為IL-18介導之免疫反應。Embodiment I-60. The method of Embodiment I-59, wherein the immune response is an IL-18-mediated immune response.

實施例I-61. 如實施例I-60之方法，其中該IL-18介導之免疫反應包含誘導該有需要之個體中的IFN-γ、CXCL10及/或TNFα。實例實例1產生針對介白素18結合蛋白（IL-18BP）之拮抗性單株抗體Example I-61. The method of Example I-60, wherein the IL-18 mediated immune response comprises inducing IFN-γ, CXCL10 and/or TNFα in the individual in need thereof.ExampleExample1 Producingantagonistic monoclonal antibodiesagainst interleukin18binding protein(IL-18BP)

進行研究以產生強效人類及人源化抗體，其與IL-18/IL-18BP結合且抑制IL-18/IL-18BP結合，且藉此釋放IL-18以刺激免疫活性。鑑別與人類及食蟹獼猴IL-18BP具有交叉反應性之潛在治療候選物。此外，亦產生與抗人類/食蟹獼猴對應物密切相關之強效抗小鼠IL-18BP mAb，以允許在鼠類腫瘤模型系統中研究此等藥劑。材料及方法Studies were conducted to generate potent human and humanized antibodies that bind to IL-18/IL-18BP and inhibit IL-18/IL-18BP binding and thereby release IL-18 to stimulate immune activity. Potential therapeutic candidates with cross-reactivity to human and cynomolgus macaque IL-18BP were identified. In addition, potent anti-mouse IL-18BP mAbs were generated that were closely related to the anti-human/cynomolgus macaque counterparts to allow the study of these agents in murine tumor model systems.Materials and Methods

免疫接種及自單一B細胞分離抗原特異性抗體。針對IL-18BP之單株抗體係藉由以下產生：用IL-18BP免疫接種小鼠，隨後使用Berkeley Lights Beacon儀器分離抗原特異性單一B細胞且自所關注之各細胞選殖編碼抗體之基因。使用來自Alloy Therapeutics之ATX-GK小鼠，其為小鼠免疫球蛋白基因已經人類免疫球蛋白基因置換且因此產生編碼人類抗體序列之抗原特異性B細胞的轉殖基因小鼠品系。用his或Fc標籤將小鼠以交替時程之人類IL-18BP及食蟹獼猴IL-18BP進行免疫接種，同時在各情況下使用另一標籤以人類、食蟹獼猴或小鼠IL-18BP測試效價，以避免標籤特異性抗體之偵測。Immunization andIsolation of Antigen-Specific Antibodies from SingleB Cells. Monoclonal antibodies against IL-18BP were generated by immunizing mice with IL-18BP, followed by isolation of antigen-specific single B cells using a Berkeley Lights Beacon instrument and selection of the antibody-encoding gene from each cell of interest. ATX-GK mice from Alloy Therapeutics were used, which is a transgenic mouse strain in which mouse immunoglobulin genes have been replaced with human immunoglobulin genes and thus generate antigen-specific B cells encoding human antibody sequences. Mice were immunized with alternating schedules of human IL-18BP and cynomolgus IL-18BP using either his or Fc tags, while titers were tested in each case with human, cynomolgus or mouse IL-18BP using an alternative tag to avoid detection of tag-specific antibodies.

在對IL-18BP產生較高效價之後，如所描述使用Berkeley Lights Beacon儀器，用製造商建議之程序（Mullen等人, Antibody Therapeutics. 4(3): 185-196, 2021）將來自合適小鼠之抗原特異性B細胞分離為單細胞。獲得來自抗原特異性B細胞之免疫球蛋白基因序列且將其用於使用既定方法來產生重組抗體。After generating high titers for IL-18BP, antigen-specific B cells from appropriate mice were isolated as single cells using the Berkeley Lights Beacon instrument as described, using the manufacturer's recommended procedures (Mullen et al., Antibody Therapeutics. 4(3): 185-196, 2021). Immunoglobulin gene sequences from antigen-specific B cells were obtained and used to generate recombinant antibodies using established methods.

命名抗體。一些抗體經命名為具有「SA」字首、連續的2數位編號及指示HC恆定域之屬性的單字母字尾：對於人類IgG1為「a」、對於鼠類IgG2a為「d」。因此，SA01a為人類IgG1抗體，而SA51d為小鼠IgG2a抗體。Naming the Antibodies .Some antibodies are named with the prefix "SA", a sequential 2-digit number, and a single-letter suffix indicating the nature of the HC constant domain: "a" for human IgG1, "d" for mouse IgG2a. Thus, SA01a is a human IgG1 antibody, and SA51d is a mouse IgG2a antibody.

變異體及突變按以下次序命名：原始胺基酸，隨後為Kabat位置編號（Kabat 1991），隨後為替代胺基酸。對於胺基酸，使用標準單字母碼。因此，Y32E指示Kabat位置32處之酪胺酸（Y）已經麩胺酸（E）置換。Variants and mutations are named in the following order: the original amino acid, followed by the Kabat position number (Kabat 1991), followed by the substituted amino acid. For the amino acids, the standard single-letter code is used. Thus, Y32E indicates that the tyrosine (Y) at Kabat position 32 has been replaced by glutamine (E).

製備庫/定點突變誘發。為使起始mAb親和力成熟，製備變異體之庫，依次聚焦於HC及LC CDR中之各者。為了實現此目的，各CDR胺基酸依次用至多17個胺基酸取代置換。庫中不包括半胱胺酸及色胺酸以免引入非所需潛在序列傾向（sequence liabilities），篩選中所包括之位置中亦不存在親本胺基酸。為了在各位置產生變異體，將兩組含有簡併密碼子NDT或VHG之突變誘發寡核苷酸（Integrated DNA Technologies（IDT），San Diego）（其中N = A/C/G/T；D = A/G/T，V = A/C/G；H = A/C/T）用於各位置（Kille, 2013；Acevedo-Rocha, 2015），與經設計以准許擴增及選殖的適當5'及3'遠端寡核苷酸（IDT）配對。Preparation of libraries/site-directed mutagenesis . To affinity mature the starting mAb, a library of variants was prepared, focusing sequentially on each of the HC and LC CDRs. To achieve this, each CDR amino acid was replaced sequentially with up to 17 amino acid substitutions. Cysteine and tryptophan were not included in the library to avoid introducing undesirable potential sequence liabilities, and no parental amino acids were present in the positions included in the screen. To generate variants at each position, two sets of mutation-inducing oligonucleotides (Integrated DNA Technologies (IDT), San Diego) containing the degenerate codons NDT or VHG (where N = A/C/G/T; D = A/G/T, V = A/C/G; H = A/C/T) were used for each position (Kille, 2013; Acevedo-Rocha, 2015), paired with appropriate 5' and 3' distal oligonucleotides (IDT) designed to allow amplification and selection.

根據製造商之方案，用高保真DNA聚合酶（Q5，New England Biolabs）進行PCR。將親本質體（重鏈與輕鏈）稀釋至10 ng/µL，且使用1 µL作為各50 µL反應之模板。具有如上文所描述之簡併密碼子的V區基因片段係藉由PCR擴增，且加以純化（按照製造商說明書使用Qiagen PCR純化套組）。基因片段經由重疊延伸PCR（OE-PCR）或Gibson選殖而組裝成重鏈及輕鏈純系。對於OE-PCR，將片段用含有限制性位點（用於重鏈之AgeI-NheI，用於輕鏈之SbfI-MfeI）之相應正向及反向引子進行擴增且經管柱純化（Qiagen PCR純化套組）。使用高保真酶（New England Biolabs）進行限制消化，且使用T4 DNA接合酶（New England Biolabs，目錄號M0202L）將片段接合至用於重鏈及輕鏈之適當載體中。空重鏈載體含有人類IgG1恆定區之大部分，其中經工程改造之NheI位點（藉由改變擺動位置產生）12個胺基酸引入至該恆定區中。輕鏈空載體含有人類κ恆定區之大部分，其中經工程改造之MfeI位點18個胺基酸引入至該恆定區中。使用具有相同空載體之片段實現Gibson選殖（Gibson Assembly^®主混合物套組，New England Biolabs目錄號ES2611L）。將插入物正規化為1 ng/µL且使用總共2 ng插入DNA（每個片段1 ng）。10 µL反應體積由5 µL Gibson主混合物及QS與純化水構成。將反應物在50℃下培育15-60分鐘。PCR was performed with a high-fidelity DNA polymerase (Q5, New England Biolabs) according to the manufacturer's protocol. Parental plasmids (heavy and light chains) were diluted to 10 ng/µL, and 1 µL was used as template for each 50 µL reaction. V region gene fragments with degenerate codons as described above were amplified by PCR and purified (using the Qiagen PCR purification kit according to the manufacturer's instructions). Gene fragments were assembled into heavy and light chain clones by overlap extension PCR (OE-PCR) or Gibson selection. For OE-PCR, the fragments were amplified with corresponding forward and reverse primers containing restriction sites (AgeI-NheI for heavy chain, SbfI-MfeI for light chain) and column purified (Qiagen PCR purification kit). Restriction digestion was performed using high-fidelity enzymes (New England Biolabs), and the fragments were ligated into appropriate vectors for heavy and light chains using T4 DNA ligase (New England Biolabs, catalog number M0202L). The empty heavy chain vector contains a large portion of the human IgG1 constant region, in which an engineered NheI site (generated by changing the swing position) 12 amino acids were introduced into the constant region. The light chain empty vector contains a large portion of the human kappa constant region with an engineered MfeI site 18 amino acids introduced into the constant region. Gibson cloning was performed using fragments with the same empty vector (Gibson Assembly^® Master Mix Kit, New England Biolabs Catalog No. ES2611L). Inserts were normalized to 1 ng/µL and a total of 2 ng of insert DNA was used (1 ng per fragment). A 10 µL reaction volume was made up of 5 µL Gibson Master Mix and QS with purified water. Reactions were incubated at 50°C for 15-60 minutes.

藉由將2-5 µL之接合反應混合物添加至細胞且在冰上培育5分鐘而將來自OE-PCR或Gibson組裝之接合產物轉型成勝任型大腸桿菌（Monserate Biotechnology，San Diego）。將細胞在42℃下熱休克30秒且置於冰上。添加250 µL SOC培養基（BioPioneer，San Diego或Teknova, San Diego）且將試管在37℃、200 rpm下培育1小時。將100 µL培養物塗鋪於抗生素選擇盤（BioPioneer，San Diego或Teknova, San Diego）上，且在37℃下培育隔夜。Ligation products from OE-PCR or Gibson assembly were transformed into competent E. coli (Monserate Biotechnology, San Diego) by adding 2-5 µL of ligation reaction mixture to cells and incubating on ice for 5 minutes. Cells were heat shocked at 42°C for 30 seconds and placed on ice. 250 µL of SOC medium (BioPioneer, San Diego or Teknova, San Diego) was added and the tubes were incubated at 37°C, 200 rpm for 1 hour. 100 µL of culture was spread on antibiotic selection plates (BioPioneer, San Diego or Teknova, San Diego) and incubated at 37°C overnight.

輸送盤以對抗體基因進行群落定序（Genewiz或Eton），其中每培養盤24-48個純系經定序。用SnapGene軟體（GSL Biotech）針對參考序列對序列進行分析，該參考序列為庫之電腦模擬選殖。基於序列比對及由突變引子編碼之胺基酸來挑選純系，且該等純系用於產生個別質體微型製備物（mini-preps）。Plates were sent for colony sequencing (Genewiz or Eton) of the antibody gene, with 24-48 clones sequenced per plate. Sequences were analyzed using SnapGene software (GSL Biotech) against a reference sequence, which was an in silico selection of the library. Clones were selected based on sequence alignment and the amino acids encoded by the mutagenesis primers, and used to generate individual plastid mini-preps.

使用下文描述之方法，藉由在48孔盤中培養之Expi293F細胞中小規模表現庫衍生之質體來實現突變體之篩選。以1:2比率（0.5 ng HC:1 ng LC質體/孔）進行重鏈及輕鏈配對。各盤上包括親本抗體對照之複製物。使細胞在盤中生長3天，其後收集來自各孔之上清液用於篩選。Screening of mutants was achieved by small-scale expression library-derived plasmids in Expi293F cells cultured in 48-well plates using the method described below. Heavy and light chain pairing was performed at a 1:2 ratio (0.5 ng HC:1 ng LC plasmid/well). Replicates of the parental antibody control were included on each plate. Cells were grown in plates for 3 days, after which supernatants from each well were collected for screening.

使用生物膜干涉技術（BLI）完成盤轉染之篩選。最初，在篩選對IL-18BP之結合親和力之前，使用BLI測定各樣品中所存在之抗體的濃度。在Fortébio Octet RED96e儀器上獲取初始表觀結合動力學量測值。將mAb裝載至抗人類恆定域（AHC）生物感測器（FortéBio）於由PBS、0.1% BSA、0.02% Tween 20組成之10×動力學緩衝液中120秒，以達到0.8至1.2 nm之光譜移位值。在20 nM人類、食蟹獼猴或鼠類IL-18BP直系同源物存在下進行締合階段且使其進行120 s；量測解離300 s以確定任何變異體是否展現相比於親本抗體提高之締合速率或解離速率。接著針對完全結合動力學對比各直系同源物，再次測試基於單一篩選濃度呈現明顯改善之結合動力學的候選物，且與如下文所描述之其他突變重組。Screening of plate transfections was done using biofilm interferometry (BLI). Initially, the concentration of antibody present in each sample was determined using BLI prior to screening for binding affinity to IL-18BP. Initial apparent binding kinetic measurements were obtained on a Fortébio Octet RED96e instrument. mAbs were loaded onto anti-human constant domain (AHC) biosensors (FortéBio) in 10× kinetic buffer consisting of PBS, 0.1% BSA, 0.02% Tween 20 for 120 seconds to achieve spectral shift values of 0.8 to 1.2 nm. The association phase was performed in the presence of 20 nM human, cynomolgus monkey or mouse IL-18BP orthologs and allowed to proceed for 120 s; dissociation was measured for 300 s to determine whether any variant exhibited an increased association rate or dissociation rate compared to the parent antibody. Each ortholog was then compared for full binding kinetics, and candidates that exhibited significantly improved binding kinetics based on a single screening concentration were retested and recombined with additional mutations as described below.

重組抗體之表現及純化。使來自Expi293表現系統套組（Thermo Fisher，目錄號A14635）之Expi293F細胞在Expi293F表現培養基（目錄號A1435101）中生長。使細胞生長至3-6×10⁶個細胞/mL之密度且接著使用血球計進行計數。將質體DNA（1.0 μg/1.0 mL培養物）稀釋於Opti-MEM還原血清培養基（RSM）（目錄號31985062）中。自製造商之建議獲取Opti-MEM RSM之值進行轉染。將Expifectamine 293試劑稀釋於Opti-MEM RSM中且在室溫下培育5 min，隨後與經稀釋之質體DNA混合。使此混合物靜置以在室溫下培育10-20分鐘。在培育expifectamine/質體DNA複合物時，將Expi293F細胞稀釋至3×10⁶個細胞/mL之密度且添加至所需體積之愛倫美氏燒瓶（Erlenmeyer flask）（BioPioneer，125 mL燒瓶之DGFPC0125S）中。接著將expifectamine/質體DNA複合物緩慢轉移至具有Expi293F細胞之搖瓶中，且將燒瓶在37℃、8% CO₂、125 rpm下，在具有25 mm迴旋擺度（orbital throw）之振盪培育箱（Infors-HT Multitron）中置放。轉染後18-22 h，將ExpiFectamine 293轉染增強子1（#100013863）及2（#A14350-01）添加至細胞，且將該等細胞放回至振盪培育箱。接著使細胞再培育4天，接著在冷凍離心機中以4000×g快速離心20分鐘且在純化之前以0.22 μm過濾。Expression and purification of recombinant antibodies . Grow Expi293F cells from the Expi293 Expression System Kit (Thermo Fisher, Catalog No. A14635) in Expi293F Expression Medium (Catalog No. A1435101). Grow cells to a density of 3-6×10⁶ cells/mL and then count using a hemacytometer. Dilute plasmid DNA (1.0 μg/1.0 mL culture) in Opti-MEM Reduced Serum Medium (RSM) (Catalog No. 31985062). Obtain values for Opti-MEM RSM from the manufacturer's recommendations for transfection. Expifectamine 293 reagent was diluted in Opti-MEM RSM and incubated at room temperature for 5 min, then mixed with diluted plasmid DNA. This mixture was allowed to stand to incubate at room temperature for 10-20 minutes. While incubating the expifectamine/plasmid DNA complex, Expi293F cells were diluted to a density of 3×10⁶ cells/mL and added to the required volume of Erlenmeyer flask (BioPioneer, DGFPC0125S for 125 mL flask). The expifectamine/plasmid DNA complex was then slowly transferred to the shake flask with Expi293F cells and the flask was placed at 37°C, 8% CO₂ , 125 rpm in a shaking incubator (Infors-HT Multitron) with a 25 mm orbital throw. 18-22 h after transfection, ExpiFectamine 293 Transfection Enhancer 1 (#100013863) and 2 (#A14350-01) were added to the cells and the cells were returned to the shaking incubator. The cells were then incubated for an additional 4 days before rapid centrifugation at 4000×g for 20 minutes in a refrigerated centrifuge and filtered at 0.22 μm before purification.

在AKTA Explorer FPLC系統上使用5 mL HiTrap MabSelect SuRe（蛋白A）管柱純化抗體。首先藉由添加50 mL 0.1 M甘胺酸，pH 3.0（溶離緩衝液），隨後添加50 mL 50 mM甘胺酸、50 mM甘胺酸鹽pH 8（結合/洗滌緩衝液）清除管柱中的任何殘餘結合蛋白。以5 mL/min將抗體（25-400 mL）負載至管柱上，且進一步用25 mL平衡/洗滌緩衝液洗滌，直至UV讀數達到基線。隨後藉由使用0-100%溶離緩衝液之25 mL線性梯度以5 mL/min溶離MAb 2 min。藉由在280 nm下之吸光度監測抗體溶離。收集峰溶離份且彙集於15 mL錐形管中。接著使用PD10管柱（Cytiva目錄號17085101）將材料之緩衝液更換成儲存緩衝液（PBS，pH 7.4），且隨後將其過濾滅菌（GenClone針筒過濾器，目錄號25-244，附接至BD 5 mL [目錄號309646]及20 mL [目錄號302830] BD Luer-Lok^TM注射器）至15 mL錐形管中且用於後續表徵分析。The antibody was purified using a 5 mL HiTrap MabSelect SuRe (Protein A) column on an AKTA Explorer FPLC system. Any residual bound protein in the column was first cleared by adding 50 mL of 0.1 M glycine, pH 3.0 (elution buffer), followed by 50 mL of 50 mM glycine, 50 mM glycine, pH 8 (binding/wash buffer). The antibody (25-400 mL) was loaded onto the column at 5 mL/min and further washed with 25 mL of equilibration/wash buffer until the UV reading reached baseline. The MAb was then eluted at 5 mL/min for 2 min using a 25 mL linear gradient of 0-100% elution buffer. Antibody elution was monitored by absorbance at 280 nm. Peak fractions were collected and pooled in 15 mL conical tubes. The material was then buffer exchanged into storage buffer (PBS, pH 7.4) using a PD10 column (Cytiva Catalog No. 17085101) and subsequently filter-sterilized (GenClone syringe filter, Catalog No. 25-244, attached to BD 5 mL [Catalog No. 309646] and 20 mL [Catalog No. 302830] BD Luer-Lok^™ syringes) into 15 mL conical tubes and used for subsequent characterization analysis.

藉由尺寸排阻HPLC（SEC-HPLC）之分析。SEC-HPLC係在5 μm粒度、7.8 mm I.D.×30 cm TSKgel G3000SW_XL上執行，且在Agilent 1100 HPLC上以1 mL/分鐘之流動速率，使用50 mM磷酸鈉、200 mM精胺酸pH 6.8等度運行。使用二極體陣列偵測器在280 nm下偵測且使用Agilent ChemStation軟體對峰進行積分。用於校準管柱之SEC-HPLC標準物係由牛甲狀腺球蛋白、牛IgG、雞白蛋白、牛核糖核酸酶A及對胺基苯甲酸組成（Sigma Aldrich #69385）。Analysisby size exclusionHPLC(SEC-HPLC ). SEC-HPLC was performed on a 5 μm particle size, 7.8 mm ID×30 cm TSKgel G3000SW_XL and isocratically run on an Agilent 1100 HPLC at a flow rate of 1 mL/min using 50 mM sodium phosphate, 200 mM arginine pH 6.8. Peaks were detected at 280 nm using a diode array detector and integrated using Agilent ChemStation software. The SEC-HPLC standards used to calibrate the column consisted of bovine thyroglobulin, bovine IgG, chicken albumin, bovine ribonuclease A, and p-aminobenzoic acid (Sigma Aldrich #69385).

藉由生物膜干涉技術（BLI）分析結合親和力。在Fortébio（現為Sartorius）Octet RED96e儀器上獲取結合動力學量測值。將mAb裝載至抗人類恆定域（AHC）生物感測器（FortéBio）於由含有0.1% BSA、0.02% Tween 20之PBS組成之10×動力學緩衝液中90-120 s，以達到0.8至1.2 nm之光譜移位值。在2倍稀釋系列之hIL-18BP存在下進行締合且通常使其進行90-120 s；通常量測解離300至1200 s。對於較弱變異體，稀釋系列以100 nM開始，或對於最強效的mAb，則以10 nM開始。使用與適當物種之IL-18BP相同的方法，測定對食蟹獼猴IL-18BP及小鼠IL-18BP之交叉反應性。Binding affinity was analyzedby biofilm interferometry (BLI) . Binding kinetic measurements were obtained on a Fortébio (now Sartorius) Octet RED96e instrument. mAbs were loaded onto anti-human constant domain (AHC) biosensors (FortéBio) in 10× kinetic buffer consisting of PBS containing 0.1% BSA, 0.02% Tween 20 for 90–120 s to achieve spectral shift values of 0.8 to 1.2 nm. Association was performed in the presence of a 2-fold dilution series of hIL-18BP and was typically allowed to proceed for 90–120 s; dissociation was typically measured for 300 to 1200 s. Dilution series started at 100 nM for weaker variants or 10 nM for the most potent mAb. Cross-reactivity to cynomolgus monkey IL-18BP and mouse IL-18BP was determined using the same method as for IL-18BP of the appropriate species.

IL-18報導HEK 293細胞中抗IL-18BP mAb之活性。來自InvivoGen之IL-18報導HEK 293細胞（hkb-hmil18）藉由表現NF-κB/AP-1誘導型分泌胚胎鹼性磷酸酶（SEAP）報導基因而對外部添加之IL-18作出反應。為進行分析，使細胞在具有1×HEK-Blue Selection（Invivogen，hb-sel）之完全DMEM培養基（10% HI FBS，1% PS）中生長。用1×PBS小心地沖洗細胞兩次且在37℃下用1×PBS提昇5 min。對細胞進行計數，以200×g快速離心5 min，且以2.5×10⁵個細胞/mL再懸浮於無選擇之完全DMEM培養基中。接著將細胞以25,000個細胞以100 µL/孔塗鋪於96孔盤（Genesee Scientific，25-109）中。接著將塗鋪細胞置於37℃、5% CO₂下之培育箱中。在不含選擇之DMEM中以90 μg/mL製備測試抗體溶液，且在室溫下將其與人類IL-18BP以120 ng/mL一起培育30 min。接著製備0.6 ng/mL重組人類IL-18儲備液（SinoBiological，10119-HNCE），且在培育之後添加至抗體/IL-18BP複合物中。立即將所得溶液添加至細胞中，得到15 μg/mL測試抗體、20 ng/mL人類IL-18BP及0.1 ng/mL人類IL-18之最終濃度。使細胞在37℃、5% CO₂下靜置培育18-22小時。獲取10 µL/孔之細胞上清液，且將其與90 µL/孔之完全Quanti-Blue溶液（Invivogen）在新96孔盤中混合，且置於37℃下之培育箱中1-3小時，注意到自紫色至藍色之比色變化。接著在620 nm下讀取盤且使用GraphPad Prism分析資料。Activity of anti-IL-18BP mAbinIL-18reporterHEK 293 cells. IL-18 reporter HEK 293 cells from InvivoGen (hkb-hmil18) respond to exogenously added IL-18 by expressing the NF-κB/AP-1-induced secreted embryonic alkaline phosphatase (SEAP) reporter gene. For analysis, cells were grown in complete DMEM medium (10% HI FBS, 1% PS) with 1× HEK-Blue Selection (Invivogen, hb-sel). Cells were carefully rinsed twice with 1× PBS and raised with 1× PBS for 5 min at 37°C. The cells were counted, centrifuged at 200 × g for 5 min, and resuspended in complete DMEM without selection at 2.5 × 10⁵ cells/mL. The cells were then plated at 25,000 cells in 100 µL/well in a 96-well plate (Genesee Scientific, 25-109). The plated cells were then placed in an incubator at 37°C, 5% CO_2. The test antibody solution was prepared at 90 μg/mL in DMEM without selection and incubated with human IL-18BP at 120 ng/mL for 30 min at room temperature. Then prepare 0.6 ng/mL recombinant human IL-18 stock solution (SinoBiological, 10119-HNCE) and add to the antibody/IL-18BP complex after incubation. Immediately add the resulting solution to the cells to give a final concentration of 15 μg/mL test antibody, 20 ng/mL human IL-18BP, and 0.1 ng/mL human IL-18. Incubate the cells statically at 37°C, 5% CO₂ for 18-22 hours. Obtain 10 µL/well of cell supernatant and mix it with 90 µL/well of complete Quanti-Blue solution (Invivogen) in a new 96-well plate and place in an incubator at 37°C for 1-3 hours, noting a colorimetric change from purple to blue. The plate was then read at 620 nm and the data analyzed using GraphPad Prism.

KG-1細胞中抗IL-18BP mAb之IFNγ表現的除抑制作用。以150k個細胞/孔塗鋪人類KG-1細胞（ATCC目錄號CCL-246）。將IL-18BP（最終為50 ng/mL，Sino Biologicals，目錄號10357-H08H）在室溫下用抗體連續稀釋液預阻斷20 min。將IL-18（最終為10 ng/mL，R&D Systems，目錄號9124-IL/CF）添加至此混合物中且在室溫下再培育20 min。將此混合物添加至細胞中且在37℃下培育隔夜。在Nunc MaxiSorp平底盤（目錄號44-2404-21）上使用來自R&D Systems之人類IFN-γ DuoSet ELISA（目錄號DY285B）在細胞培養物上清液中量測分泌之IFNγ。遵循製造商之方案，且用試劑稀釋劑將上清液按1:2稀釋。使用Spectramax iD5盤讀取器在450 nm下量測吸光度。使用GraphPad Prism分析資料。De-inhibition of IFNγ expression byanti-IL-18BP mAbinKG-1 cells. Human KG-1 cells (ATCC catalog number CCL-246) were plated at 150k cells/well. IL-18BP (50 ng/mL final, Sino Biologicals, catalog number 10357-H08H) was pre-blocked with serial dilutions of the antibody for 20 min at room temperature. IL-18 (10 ng/mL final, R&D Systems, catalog number 9124-IL/CF) was added to this mixture and incubated for another 20 min at room temperature. This mixture was added to the cells and incubated overnight at 37°C. Secreted IFNγ was measured in cell culture supernatants using the Human IFN-γ DuoSet ELISA (Cat. No. DY285B) from R&D Systems on Nunc MaxiSorp flat-bottom plates (Cat. No. 44-2404-21). The manufacturer's protocol was followed and supernatants were diluted 1:2 with reagent diluent. Absorbance was measured at 450 nm using a Spectramax iD5 plate reader. Data were analyzed using GraphPad Prism.

人類IL-18活性之PBMC分析。將獲自San Diego血庫之人類周邊血液單核細胞（PBMC）以2×10⁵個細胞/孔接種至96孔平底盤（GenClone，目錄號25-109）中或以1.7×10⁵個細胞/孔接種至圓底盤（GenClone，目錄號25-221）中的含有100 U青黴素、100 µg鏈黴素（Gibco目錄號10378016）及10% FBS（RPMIc）之RPMI+GlutaMAX（Gibco目錄號61870036）中。將測試抗體以50 µL之體積以4×最終濃度添加至孔中。接著以4 ng/mL添加50 µL重組人類IL-12（R&D system，目錄號219-IL-005），隨後以8 ng/mL添加50 µL重組人類IL-18（Sino Biological，目錄號10119-HNCE），分別達到1 ng/mL及2 ng/mL之最終濃度。對於使用食蟹獼猴PBMC（iQ Biosciences，目錄號IQB-MnPB102）之分析，在圓底盤中以1.7×10⁵個細胞/孔接種細胞。以1 ng/mL之最終濃度添加重組食蟹獼猴IL-12（R&D Systems，目錄號10215-CL），且以2 ng/mL之最終濃度添加重組恆河猴（rhesus macaque）IL-18（R&D Systems目錄號2548-RM-025/CF [應注意恆河猴及食蟹獼猴之胺基酸IL-18序列一致]）。所有稀釋均在RPMIc中進行。將細胞在37℃、5% CO₂下培育48 h。在48 h時，自各孔取出50 µL上清液等分試樣，且根據製造商說明書使用DuoSet ELISA套組（R&D Systems，目錄號DY285B，用於人類IFNγ；及目錄號DY961，用於靈長類動物IFNγ）在Nunc MaxiSorp平底盤（Invitrogen，目錄號44-2404-21）上分析IFNγ之存在情況。使用Spectramax iD5盤讀取器在450 nm下量測吸光度，且使用GraphPad Prism軟體分析資料。PBMCAssay forHumanIL-18Activity . Human peripheral blood mononuclear cells (PBMCs) obtained from the San Diego Blood Bank were seeded at 2×10⁵ cells/well in 96-well flat-bottom plates (GenClone, catalog #25-109) or 1.7×10⁵ cells/well in round-bottom plates (GenClone, catalog #25-221) in RPMI+GlutaMAX (Gibco catalog #61870036) containing 100 U penicillin, 100 µg streptomycin (Gibco catalog #10378016), and 10% FBS (RPMIc). Test antibodies were added to the wells in a volume of 50 µL at 4× final concentration. Then 50 µL of recombinant human IL-12 (R&D system, catalog #219-IL-005) at 4 ng/mL was added, followed by 50 µL of recombinant human IL-18 (Sino Biological, catalog #10119-HNCE) at 8 ng/mL, to reach final concentrations of 1 ng/mL and 2 ng/mL, respectively. For assays using cynomolgus macaque PBMCs (iQ Biosciences, catalog #IQB-MnPB102), cells were seeded at 1.7×10⁵ cells/well in round-bottom plates. Recombinant cynomolgus macaque IL-12 (R&D Systems, catalog #10215-CL) was added at a final concentration of 1 ng/mL, and recombinant rhesus macaque IL-18 (R&D Systems catalog #2548-RM-025/CF [note that the amino acid sequence of rhesus macaque and cynomolgus macaque IL-18 is identical]) was added at a final concentration of 2 ng/mL. All dilutions were made in RPMIc. Cells were incubated at 37°C, 5%_CO2 for 48 h. At 48 h, a 50 µL aliquot of supernatant was removed from each well and analyzed for the presence of IFNγ using the DuoSet ELISA kit (R&D Systems, catalog number DY285B for human IFNγ and catalog number DY961 for primate IFNγ) on Nunc MaxiSorp flat-bottom plates (Invitrogen, catalog number 44-2404-21) according to the manufacturer's instructions. Absorbance was measured at 450 nm using a Spectramax iD5 plate reader, and data were analyzed using GraphPad Prism software.

在預複合之hIL-18/hIL-18BP存在下進行反應之實驗中，設計如下。將80 ng/mL之重組人類或食蟹獼猴IL-18與重組人類（SinoBiological，目錄號10357-H08H）或食蟹獼猴IL-18BP（內部產生）分別在400 ng/mL下在室溫下一起培育30 min。將mAb之連續稀釋液以50 µL/孔以4×濃度添加至96孔圓底盤中。將50 µL/孔之IL-18-IL-18BP複合物添加至含有mAb之各孔中且在37℃下培育1 h。1 h之後，將呈4 ng/mL之50 µL/孔重組人類或食蟹獼猴IL-12及呈2×10⁶個細胞/mL之50 µL PBMC添加至各孔中，以使各孔中之最終濃度為20 ng/mL IL-18，100 ng/mL IL-18BP、1 ng/mL IL-12及1×10⁵個PBMC。所有稀釋均在RPMIc中進行。對照孔含有單獨的IL-12+IL-18或單獨的IL-12+IL-18+IL-18BP。將細胞在37℃、5% CO₂下培育48 h，收集上清液且分析IFNγ及CCL2之存在情況。In experiments where the reaction was performed in the presence of pre-complexed hIL-18/hIL-18BP, the design was as follows. Recombinant human or cynomolgus IL-18 at 80 ng/mL was incubated with recombinant human (SinoBiological, Catalog No. 10357-H08H) or cynomolgus IL-18BP (produced in-house) at 400 ng/mL, respectively, for 30 min at room temperature. Serial dilutions of mAb were added to a 96-well round bottom plate at 50 µL/well at 4× concentration. 50 µL/well of IL-18-IL-18BP complex was added to each well containing mAb and incubated at 37°C for 1 h. One hour later, 50 µL/well recombinant human or cynomolgus macaque IL-12 at 4 ng/mL and 50 µL PBMC at 2×10⁶ cells/mL were added to each well so that the final concentrations in each well were 20 ng/mL IL-18, 100 ng/mL IL-18BP, 1 ng/mL IL-12, and 1×10⁵ PBMC. All dilutions were performed in RPMIc. Control wells contained IL-12+IL-18 alone or IL-12+IL-18+IL-18BP alone. Cells were incubated for 48 h at 37°C, 5% CO₂ , and supernatants were collected and analyzed for the presence of IFNγ and CCL2.

小鼠IL-18報導子分析。使IL-18報導HEK 293細胞（Invivogen，hkb-hmil18）在具有1×HEK-Blue Selection（Invivogen，hb-sel）之完全DMEM培養基（10% HI FBS，1% PS）中生長。用1×PBS小心地沖洗細胞兩次且在37℃下用1×PBS提昇5分鐘。對細胞進行計數，以200×g快速離心5分鐘，且以2.5E⁵個細胞/mL再懸浮於無選擇之完全DMEM培養基中。接著將細胞以100 µL/孔塗鋪於96孔盤（Genesee Scientific，25-109）中以得到25k個細胞/孔。接著將塗鋪細胞置於37℃、5% CO₂下之培育箱中。在不含選擇之DMEM中以90 μg/mL製備測試抗體溶液，且在室溫下將其與小鼠IL-18BP以120 ng/mL一起培育30分鐘。接著製備60 ng/mL重組小鼠IL-18儲備液，且在培育之後添加至抗體/IL-18BP複合物中。立即將所得溶液添加至細胞中，得到15 μg/mL測試抗體、20 ng/mL小鼠IL-18BP及10 ng/mL小鼠IL-18之最終濃度。使細胞在37℃、5% CO₂下靜置培育18-22小時。獲取10 µL/孔之細胞上清液且將其與90 µL/孔之完全Quanti-Blue溶液（Invivogen，rep-qbs）在新96孔盤中混合且置於37℃之培育箱中1-3小時，注意到自紫色至藍色之比色變化。接著在620 nm下讀取盤且使用GraphPad Prism分析資料。MouseIL-18reporter assay . IL-18 reporter HEK 293 cells (Invivogen, hkb-hmil18) were grown in complete DMEM (10% HI FBS, 1% PS) with 1× HEK-Blue Selection (Invivogen, hb-sel). Cells were carefully rinsed twice with 1× PBS and raised with 1× PBS at 37°C for 5 minutes. Cells were counted, centrifuged quickly at 200×g for 5 minutes, and resuspended in complete DMEM without selection at 2.5E⁵ cells/mL. Cells were then plated at 100 µL/well in a 96-well plate (Genesee Scientific, 25-109) to give 25k cells/well. The plated cells were then placed in an incubator at 37°C, 5% CO_2. A solution of test antibody was prepared at 90 μg/mL in DMEM without selection and incubated with mouse IL-18BP at 120 ng/mL for 30 minutes at room temperature. A 60 ng/mL stock solution of recombinant mouse IL-18 was then prepared and added to the antibody/IL-18BP complex after the incubation. The resulting solution was immediately added to the cells to give a final concentration of 15 μg/mL test antibody, 20 ng/mL mouse IL-18BP, and 10 ng/mL mouse IL-18. The cells were incubated statically at 37°C, 5% CO₂ for 18-22 hours. 10 µL/well of cell supernatant was obtained and mixed with 90 µL/well of complete Quanti-Blue solution (Invivogen, rep-qbs) in a new 96-well plate and placed in a 37°C incubator for 1-3 hours, and a colorimetric change from purple to blue was noted. The plate was then read at 620 nm and the data analyzed using GraphPad Prism.

小鼠脾細胞分析-鼠類脾細胞分離。將小鼠脾臟添加至10 mm皮氏培養皿（petri dish）中，且每一脾臟添加1 mL無酶解離緩衝液（Gibco，目錄號13151014）。接著用1 mL注射器之背部搗碎脾臟，直至解離。使此溶液通過70 µm細胞濾器，將皮氏培養皿用RPMI 10% FBS 1× P/S洗滌，且使此亦通過細胞濾器。使細胞以300×g快速離心5 min，移除上清液，且根據製造商之方案添加1×RBC溶解緩衝液（Biolegend，目錄號420301）以溶解RBC。將細胞在-80℃下在細胞回收培養基（Gibco,目錄號12648010）中冷凍，且接著次日移動至液氮以用於長期儲存。Mouse spleen cell analysis -Murine spleen cell isolation . Mouse spleens were added to a 10 mm petri dish and 1 mL of non-enzymatic dissociation buffer (Gibco, catalog number 13151014) was added per spleen. The spleen was then mashed with the back of a 1 mL syringe until dissociated. This solution was passed through a 70 µm cell filter, the petri dish was washed with RPMI 10% FBS 1× P/S and this was also passed through the cell filter. Cells were quickly centrifuged at 300×g for 5 min, the supernatant was removed, and 1× RBC lysis buffer (Biolegend, catalog number 420301) was added to lyse the RBCs according to the manufacturer's protocol. Cells were frozen at -80°C in cell recovery medium (Gibco, catalog number 12648010) and then moved to liquid nitrogen the next day for long-term storage.

鼠類脾細胞mIL-18BP抑制分析。將小鼠脾細胞解凍至RPMI 10% HI FBS +Pen-strep中。對細胞進行計數且以110k個細胞/孔接種至96孔u底盤之中心60個孔中。製備抗體之9點連續稀釋液，且向此添加0.1 ng/mL最終濃度之mIL-18（R&D Systems，目錄號9139-IL-010）及10 ng/mL最終濃度之mIL-12（R&D Systems，目錄號419-ML-010）以製備2×溶液。將此溶液添加於鼠類脾細胞頂部上且在37℃下培育隔夜。第二天，按照製造商之方案使用小鼠IFN-γ DuoSet ELISA（R&D Systems，DY485）在Nunc MaxiSorp平底盤（Invitrogen，目錄號44-2404-21）上量測mIFNγ，且在SpectraMax iD5盤式讀取器上量測在450 nm下之吸光度。結果Murine splenocytemIL- 18BPinhibition assay . Thawmouse splenocytes into RPMI 10% HI FBS + Pen-strep. Count cells and plate 110k cells/well into the center 60 wells of a 96-well u-bottom plate. Prepare a 9-point serial dilution of the antibody and to this add 0.1 ng/mL final concentration of mIL-18 (R&D Systems, Catalog No. 9139-IL-010) and 10 ng/mL final concentration of mIL-12 (R&D Systems, Catalog No. 419-ML-010) to make a 2× solution. Add this solution on top of the murine splenocytes and incubate overnight at 37°C. The next day, mIFNγ was measured using the Mouse IFN-γ DuoSet ELISA (R&D Systems, DY485) according to the manufacturer'sprotocol on Nunc MaxiSorp flat-bottom plates (Invitrogen, catalog number 44-2404-21), and the absorbance at 450 nm was measured on a SpectraMax iD5 plate reader.

自免疫接種小鼠分離單株抗體序列。將Alloy ATX-GK小鼠群組用人類及食蟹獼猴IL-18BP免疫接種。藉由ELISA量測對人類、食蟹獼猴及小鼠IL-18BP之效價，且選擇具有高效價之小鼠以使用Berkeley Lights Beacon Optofluidic系統（Mullen, 2021）分離分泌B細胞之抗體。篩選鑑別出分泌單細胞之抗體，其與人類及食蟹獼猴IL-18BP交叉反應且其中一些亦結合小鼠IL-18BP。另外，新穎篩選經設計以鑑別分泌細胞之抗體，該等抗體無法識別其中活性位點已被阻斷之IL-18BP形式，其稱為「低IL-18」。此使得篩選能夠鑑別推定的配位體阻斷抗體。Monoclonal antibody sequences were isolated from immunized mice . Groups of Alloy ATX-GK mice were immunized with human and cynomolgus macaque IL-18BP. Titers to human, cynomolgus macaque, and mouse IL-18BP were measured by ELISA, and mice with high titers were selected for isolation of antibodies to secretory B cells using the Berkeley Lights Beacon Optofluidic system (Mullen, 2021). Screens identified antibodies to secretory monoclonal cells that cross-reacted with human and cynomolgus macaque IL-18BP and some of them also bound mouse IL-18BP. In addition, novel screens were designed to identify antibodies to secretory cells that were unable to recognize a form of IL-18BP in which the active site had been blocked, termed “low IL-18.” This enables screening to identify putative ligand-blocking antibodies.

由於IL-18呈現對結合蛋白之高親和力（KD小於1 nM）（Kim等人, PNAS 97(3): 1190-1195, 2000；Kimura等人, Allergol Int. 57(4):367-76, 2008），因此親和力在該nM親和力範圍內之mAb（諸如通常自抗原特異性B細胞分離之mAb）可能不可能拮抗IL-18/IL-18BP相互作用。因此，為評估新鑑別之mAb候選物之潛在阻斷能力，產生新穎嵌合蛋白「低IL-18」。低IL-18包含繫栓至其對應IL-18BP但藉由可撓性連接肽分離之人類或小鼠IL-18（示意性地展示於圖3A-圖3B中）。低IL-18之序列提供於下表S1中。表S1.低IL-18序列物種序列SEQ ID NO:人類MRAWIFFLLCLAGRALAYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDGGSGGGSGENLYFQSGGGSGGGGTPVSQTTTAATASVRSTKDPCPSQPPVFPAAKQCPALEVTWPEVEVPLNGTLSLSCVACSRFPNFSILYWLGNGSFIEHLPGRLWEGSTSRERGSTGTQLCKALVLEQLTPALHSTNFSCVLVDPEQVVQRHVVLAQLWAGLRATLPPTQEALPSSHSSPQQQGHHHHHH377小鼠MRAWIFFLLCLAGRALANFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQSGGSGGGSGENLYFQSGGGSGGGGTSAPQTTATVLTGSSKDPCSSWSPAVPTKQYPALDVIWPEKEVPLNGTLTLSCTACSRFPYFSILYWLGNGSFIEHLPGRLKEGHTSREHRNTSTWLHRALVLEELSPTLRSTNFSCLFVDPGQVAQYHIILAQLWDGLKTAPSPSQETLSSHSPVSRSAGPGVAHHHHHH378Since IL-18 exhibits a high affinity for the binding protein (KD less than 1 nM) (Kim et al., PNAS 97(3): 1190-1195, 2000; Kimura et al., Allergol Int. 57(4):367-76, 2008), mAbs with affinities in this nM affinity range (such as mAbs typically isolated from antigen-specific B cells) are unlikely to antagonize the IL-18/IL-18BP interaction. Therefore, to evaluate the potential blocking ability of the newly identified mAb candidates, a novel chimeric protein "Low IL-18" was generated. Low IL-18 comprises human or mouse IL-18 tethered to its corresponding IL-18BP but separated by a flexible linker peptide (schematically shown inFigures3A -3B ). The sequence of low IL-18 is provided inTableS1below .TableS1.LowIL-18sequencesSpeciessequenceSEQ ID NO: Human MRAWIFFLLCLAGRALAYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDGGSGGGSGENLYFQS GGGSGGGGTPVSQTTTAATASVRSTKDPCPS QPPVFPAAKQCPALEVTWPEVEVPLNGTLSLSCVACSRFPNFSILYWLGNGSFIEHLPGRLWEGSTSRERGSTGTQLCKALVLEQLTPALHSTNFSCVLVDPEQVVQRHVVLAQLWAGLRATLPPTQEALPSSHSSPQQQGHHHHHH 377 Mouse MRAWIFFLLCLAGRALANFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTNLHQSGGSGGGSGENLYFQS GGGSGGGGTSAPQTTATVLTGSSKDP CSSWSPAVPTKQYPALDVIWPEKEVPLNGTLTLSCTACSRFPYFSILYWLGNGSFIEHLPGRLKEGHTSREHRNTSTWLHRALVLEELSPPTLRSTNFSCLFVDPGQVAQYHIILAQLWDGLKTAPSPSQETLSSHSPVSRSAGPGVAHHHHHH 378

產生此分子之理論基礎為IL-18BP活性位點將被阻斷，此係因為其繫栓配位體將不能解離。因此，識別BP之活性結合位點之mAb將對於識別低IL-18造成空間位阻，而大部分非阻斷抗體將能夠同等地與IL-18BP及低IL-18結合。TEV蛋白酶裂解位點亦包括於該設計中以提供使hIL-18與其結合蛋白分離之能力。The theoretical basis for the generation of this molecule is that the IL-18BP active site will be blocked because its tethered ligand will not be able to dissociate. Therefore, mAbs that recognize the active binding site of BP will be sterically hindered from recognizing low IL-18, while most non-blocking antibodies will be able to bind to IL-18BP and low IL-18 equally. A TEV protease cleavage site was also included in the design to provide the ability to separate hIL-18 from its binding proteins.

基於晶體結構（蛋白質資料庫[PDB]結構3F62）設計低IL-18，其將IL-18之C端接合至IL-18BP之N端，該晶體結構包括與鼠痘病毒IL-18BP複合之人類IL-18。因為包括鼠痘之正痘病毒（Orthopoxviruse）編碼功能性IL-18BP同源物，其與哺乳動物直系同源IL-18BP展現17-34%胺基酸一致性（Calderara, 2001），所以認為3F62晶體結構將在低IL-18構築體之設計中具有指導性。經解析之晶體結構指示IL-18之C端相對接近結合蛋白之N端且因此融合蛋白為可能的。因此，某些實施例包括低IL-18融合蛋白，其在N端至C端定向上包含信號肽、IL-18、第一可撓性連接子、蛋白酶裂解位點（視情況TEV蛋白酶裂解位點）、可撓性連接子及IL-18BP；其中融合蛋白之IL-18部分結合至融合蛋白之IL-18BP部分且空間阻斷融合蛋白之IL-18BP部分之IL-18結合位點。在具體實施例中，低IL-18融合蛋白包含與來自表S1之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的胺基酸序列，由其組成或基本上由其組成。亦包括編碼低IL-18融合蛋白之核酸分子。Low IL-18 was designed based on a crystal structure (Protein Data Bank [PDB] structure 3F62) that joins the C-terminus of IL-18 to the N-terminus of IL-18BP, which includes human IL-18 in complex with mousepox virus IL-18BP. Because orthopoxviruses, including mousepox, encode functional IL-18BP homologs that exhibit 17-34% amino acid identity with the mammalian orthologous IL-18BP (Calderara, 2001), it was thought that the 3F62 crystal structure would be instructive in the design of the low IL-18 construct. The solved crystal structure indicated that the C-terminus of IL-18 was relatively close to the N-terminus of the bound protein and therefore a fusion protein was possible. Thus, certain embodiments include a low IL-18 fusion protein comprising a signal peptide, IL-18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker, and an IL-18BP in an N-terminal to C-terminal orientation; wherein the IL-18 portion of the fusion protein binds to the IL-18BP portion of the fusion protein and sterically blocks the IL-18 binding site of the IL-18BP portion of the fusion protein. In specific embodiments, the low IL-18 fusion protein comprises, consists of, or consists essentially of anaminoacid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a sequence from Table S1. Also included are nucleic acid molecules encoding the low IL-18 fusion protein.

確認再表現之mAb之活性，分析結合動力學、阻斷能力及生物活性。抗體序列係來源於Beacon儀器上之單一B細胞篩選。為確認活性，使用與人類IgG1 κ恆定區融合之經分離之人類可變區序列以重組方式再表現抗體。抗體表現於HEK293細胞中且自細胞培養物上清液進行測試或經純化以供更詳細的分析。Confirm activity of re-expressedmAbs, analyze binding kinetics, blocking capacity, and biological activity . Antibody sequences were derived from single B cell screens on the Beacon instrument. To confirm activity, antibodies were re-expressed recombinantly using isolated human variable region sequences fused to human IgG1 kappa constant regions. Antibodies were expressed in HEK293 cells and tested from cell culture supernatants or purified for more detailed analysis.

初始表徵使用BLI測試與人類、食蟹獼猴及小鼠IL-18BP之結合親和力。另外，測試mAb與低IL-18之結合以鑑別最可能為阻斷抗體之mAb。亦針對mAb之表現量及是否認為序列有問題來對mAb進行評分。有問題之序列之實例為CDR3中之聚酪胺酸序列，或低水平之體細胞超突變，其指示該抗體未經歷顯著活體內成熟。結果在表E1中加以說明。表E1.初始抗IL-18BP mAb之表徵mAbhIL-18BP (KD nM)cyIL-18BP (KD nM)mIL-18BP (KD nM)低IL-18結合表現量序列特徵SA01a2.82.719否+++良好SA08a1214否是+良好SA09a0.41.832否+++差SA10a1.80.4否否+++差SA12a0.80.5否否+良好SA13a1.30.6否是++良好SA14a8.013.0否是+++良好SA16a8.94.1否是+++良好SA17a3.86.7否是++良好ND，未測定；NT，未測試。對於表現，+++，＞200 µg/mL；++，100-200 µg/mL；+，50-100 µg/mL；±，＜50 µg/mL。Initial characterization used BLI to test binding affinity to human, cynomolgus macaque, and mouse IL-18BP. In addition, mAbs were tested for binding to low IL-18 to identify mAbs that were most likely blocking antibodies. mAbs were also scored for their expression and whether the sequence was considered problematic. Examples of problematic sequences are polytyrosine sequences in CDR3, or low levels of somatic hypermutations, which indicate that the antibody has not undergone significant in vivo maturation. The results are illustrated inTableE1 .TableE1.Characterization ofinitial anti-IL-18BP mAbsmAbshIL-18BP (KD nM)cyIL-18BP (KD nM)mIL-18BP (KD nM)LowIL-18bindingPerformanceSequence characteristics SA01a 2.8 2.7 19 no +++ good SA08a 12 14 no yes + good SA09a 0.4 1.8 32 no +++ Difference SA10a 1.8 0.4 no no +++ Difference SA12a 0.8 0.5 no no + good SA13a 1.3 0.6 no yes ++ good SA14a 8.0 13.0 no yes +++ good SA16a 8.9 4.1 no yes +++ good SA17a 3.8 6.7 no yes ++ good ND, not determined; NT, not tested. For performance, +++, >200 µg/mL; ++, 100-200 µg/mL; +, 50-100 µg/mL; ±, <50 µg/mL.

亦在基於細胞之報導子分析中測試所關注抗體之活性。此分析使用經工程改造之細胞株，其中IL-18信號傳導引起鹼性磷酸酶之分泌，此容易量測。藉由添加IL-18BP抑制鹼性磷酸酶之IL-18誘導，且此抑制藉由添加抗體至IL-18BP得到減輕，其限制條件為該等抗體能夠阻斷IL-18BP/IL-18結合。如表E2中所示，若干抗體能夠阻IL-18/IL-18BP相互作用且允許誘導IL-18驅動之反應。在此分析中產生活性之抗體與不能結合低IL-18之抗體一致，從而驗證用於分離所關注抗體之篩選方法。表E2. HEK293 IL-18報導子分析中之抗IL-18BP mAb之活性（EC50以nM展示）抗體N=1N=2SA011.61.17SA02無活性無活性SA05＞100＞100SA06無活性無活性SA08無活性無活性SA090.080.18SA100.340.28SA11＞100＞100SA120.370.38SA13無活性無活性SA14無活性無活性SA16無活性無活性SA17＞100＞100The activity of the antibodies of interest was also tested in a cell-based reporter assay. This assay uses an engineered cell line in which IL-18 signaling causes the secretion of alkaline phosphatases, which is easily measured. IL-18 induction of alkaline phosphatases is inhibited by the addition of IL-18BP, and this inhibition is alleviated by the addition of antibodies to IL-18BP, with the proviso that the antibodies are able to block IL-18BP/IL-18 binding. As shown inTableE2 , several antibodies are able to block IL-18/IL-18BP interaction and allow induction of IL-18 driven reactions. Antibodies that are active in this assay are consistent with antibodies that are unable to bind low IL-18, thereby validating the screening method used to isolate the antibodies of interest.TableE2. Activity of anti-IL-18BP mAbsin HEK293 IL-18 reporter assay(EC50 showninnM)antibodyN=1N=2 SA01 1.6 1.17 SA02 Inactive Inactive SA05 >100 >100 SA06 Inactive Inactive SA08 Inactive Inactive SA09 0.08 0.18 SA10 0.34 0.28 SA11 >100 >100 SA12 0.37 0.38 SA13 Inactive Inactive SA14 Inactive Inactive SA16 Inactive Inactive SA17 >100 >100

隨後測試最有前景的候選物在KG-1細胞中的生物活性。KG-1為藉由產生IFNγ而對IL-18作出反應之人類骨髓衍生之巨噬細胞株。IL-18BP之添加阻斷IL-18誘導IFNγ表現之能力，由此抑制該反應。進一步添加中和抗IL-18BP抗體（其與IL-18BP結合）破壞與IL-18之相互作用，因此釋放IL-18以誘導IFNγ。此亦可稱為除抑制作用。圖4展示初始組的抗體在KG-1細胞中實現IFNγ之除抑制的能力。在mAb候選物之間存在明顯等級次序的效力，其中SA12a ＞ SA09a ＞ SA10a ≥ SA01a。mAb最佳化The most promising candidates were then tested for biological activity in KG-1 cells. KG-1 is a human bone marrow-derived macrophage cell line that responds to IL-18 by producing IFNγ. The addition of IL-18BP blocks the ability of IL-18 to induce IFNγ expression, thereby inhibiting the response. Further addition of a neutralizing anti-IL-18BP antibody (which binds to IL-18BP) disrupts the interaction with IL-18, thereby releasing IL-18 to induce IFNγ. This may also be referred to as de-inhibition.Figure4 shows the ability of the initial set of antibodies to achieve de-inhibition of IFNγ in KG-1 cells. There was a clear rank order of potency between the mAb candidates, with SA12a > SA09a > SA10a ≥ SA01a.mAboptimization

SA01a優先用於最佳化（參見表E3）。SA01a衍生自Alloy ATX-GK小鼠且為人類IgG。表E3mAb人類KDpM ± SEM（N）食蟹獼猴KDpM ± SEM（N）小鼠KDpM ± SEM（N）人類/食蟹獼猴倍數差異SA01a2400 ± 300 (N3)6000 ± 2000 (N3)17600 ± 1800 (N5)2.5SA01a was prioritized for optimization (seeTable E3 ). SA01a was derived from Alloy ATX-GK mice and is a human IgG.TableE3mAbsHumanKDpM ± SEM(N)Cynomolgus macaqueKDpM ± SEM(N)MouseKDpM ± SEM(N)Human/ Crab-eating macaque fold difference SA01a 2400 ± 300 (N3) 6000 ± 2000 (N3) 17600 ± 1800 (N5) 2.5

SA01a之成熟。為使完全人類mAb SA01a親和力成熟，吾人製備以HC及LC CDR為中心之mAb變異體庫。為了實現此目的，CDR胺基酸依次用至多17個胺基酸取代置換-庫中不包括半胱胺酸及色胺酸以免引入非所需潛在序列傾向，篩選中所包括之位置中亦不存在親本胺基酸。在產生後，首先藉由BLI在人類、食蟹獼猴及鼠類IL-18BP之單一濃度下對變異體進行篩選，以確定與親本SA01a相比是否有任何變異體具有經改善之締合速率或解離速率。篩選鑑別出多種單點變異體，其確實實際上改善表觀結合動力學。個別突變可以能夠重組以進一步改善針對目標抗原之結合親和力，但並非在所有情況下皆如此。因此，胺基酸置換以組合方式重組以進一步使抗體成熟。有趣地，改善對鼠類IL-18BP之組合通常對於人類而言並未改善，且反之亦然。因此，實行兩個成熟路徑，一個用於最佳化與小鼠IL-18BP之結合且另一個用於共同最佳化與人類及食蟹獼猴IL-18BP之結合（參見例如圖1，下圖區）。Maturation ofSA01a . To affinity mature the fully human mAb SA01a, we prepared a library of mAb variants centered around the HC and LC CDRs. To achieve this, the CDR amino acids were sequentially replaced with up to 17 amino acid substitutions - cysteine and tryptophan were not included in the library to avoid introducing undesirable potential sequence tendencies, and the parent amino acids were not present in the positions included in the screen. After generation, the variants were first screened by BLI at a single concentration of human, cynomolgus macaque, and murine IL-18BP to determine if any variants had improved on- or off-rates compared to the parent SA01a. The screening identified a variety of single-point variants that did actually improve the apparent binding kinetics. Individual mutations may be able to recombine to further improve binding affinity for the target antigen, but this is not the case in all cases. Therefore, amino acid substitutions are recombined in a combinatorial manner to further mature the antibody. Interestingly, combinations that improve for murine IL-18BP generally do not improve for humans, and vice versa. Therefore, two maturation pathways were implemented, one for optimizing binding to mouse IL-18BP and another for co-optimizing binding to human and cynomolgus macaque IL-18BP (see, e.g.,Figure1 , lower panel).

組合庫之初始篩選鑑別出兩種mAb，其藉由BLI量測對mIL-18BP具有極高親和力，命名為SA51d及SA52d。此等兩種mAb經再表現且針對完全結合動力學進行再測試，分別產生＜50 pM及140之KD值。儘管兩種mAb亦藉由BLI對比hIL-18BP量測為超過標度值（off-scale），但親和力對比食蟹獼猴IL-18BP弱約2個數量級，表明此等實體對於進一步人類臨床前開發而言並非較佳。Initial screening of the library identified two mAbs with very high affinity for mIL-18BP as measured by BLI, designated SA51d and SA52d. These two mAbs were re-expressed and re-tested for full binding kinetics, yielding KD values of <50 pM and 140, respectively. Although both mAbs were also measured off-scale by BLI versus hIL-18BP, the affinity was approximately 2 orders of magnitude weaker than cynomolgus macaque IL-18BP, indicating that these entities were not optimal for further human preclinical development.

該篩選亦鑑別出特異性改善與人類及食蟹獼猴IL-18BP二者之結合的胺基酸取代子集。因此，此等變化以各種組合重組用於進一步分析。一種突變，尤其A99S，極大地改善與食蟹獼猴結合蛋白之結合，但稍微使人類直系同源物之識別表現變差。The screen also identified a subset of amino acid substitutions that specifically improved binding to both human and cynomolgus macaque IL-18BP. Therefore, these changes were recombined in various combinations for further analysis. One mutation in particular, A99S, greatly improved binding to the cynomolgus macaque binding protein, but slightly impaired recognition of the human ortholog.

表E4展示引起與人類及食蟹獼猴IL-18BP之結合改善的變異體。括弧的（N）值指示平均值中包括了多少獨立運行實驗的資料點；±指示S.E.M.；未測試，NT；ND，未測定。在測試mAb僅兩次之情況下，提供兩個值。食蟹獼猴/人類提供兩個物種之間的比率差異。表E4.與人類及食蟹獼猴IL-18BP之結合改善的變異體HC變異LC變異mAb人類KD（pM）食蟹獼猴KD（pM）食蟹獼猴/人類Y33P52aN53I96A99R24L33N92T25SA01a2400 ± 300 (N3)6000 ± 2000 (N3)2.5SA45a900NDI96LN92YSA54a87 ± 29 (N4)59 ± 16 (N4)0.7Y33FN53EI96LA99SN92YSA55a78 ± 22 (N5)127 ± 19 (N5)1.6Y33FP52aVN53EI96LN92YSA56a＜50420＞8.4N53EI96LN92YSA57a88 ± 29 (N4)60 ± 16 (N4)0.7Y33FN53EI96LN92YSA59a44 ± 9 (N9)45 ± 10 (N9)1.5Y33FP52aVN53EI96LA99SN92YSA60a760, 590 (N2)NTNDN92YSA61a280NTNDN53EN92YSA62a170, ＜50 (N2)4802.8P52aVN53EN92YSA63a＜50 (N3)430 (N2)＞8.6Y33FN53EN92YSA64a63 ± 10 (N16)71 ± 13 (N16)1.2Y33FP52aVN53EI96LA99SL33TN92YT25MSA65a94 ± 25 (N4)136 ± 14 (N5)1.4Y33FP52aVN53EI96LA99SN92YSA66a77 ± 9 (N7)94 ± 14 (N7)1.2Y33FP52aVN53EI96LA99SR24DN92YSA73a150, 93 (N2)120, 800 (N2)3.8P52aVN53EI96LA99SR24DL33TN92YSA77a380 ± 150 (N3)373 ± 244 (N3)1N53EI96LA99SR24DL33TN92YSA59a-R24QND140NEY33FP52aVN53EI96LA99SR24QN92YSA59a-S26I140, 570 (N2)140, 120 (N2)0.37Y33FP52aVN53EI96LA99SN92YS26ISA59a-S26R220150, 160 (N2)0.7Y33FP52aVN53EI96LA99SN92YS26RSA59a-G28Q140120, 89 (N2)0.75Y33FP52aVN53EI96LA99SN92YG28QSA55a-R24D1293202.5Y33FP52aVN53EI96LR24DN92YSA55a-T25M199, 110 (N2)3802.5Y33FP52aVN53EI96LN92YT25MSA55a-S26I113, 180 (N2)3302.2Y33FP52aVN53EI96LN92YS26ISA55a-S26R1304703.6Y33FP52aVN53EI96LN92YS26RSA55a-G28Q813003.7Y33FP52aVN53EI96LN92YG28QSA55a-L33T＜90 ± 17 (N5)227 ± 15 (N3)2.5Y33FP52aVN53EI96LL33TN92YTableE4 shows variants that cause improved binding to human and cynomolgus macaque IL-18BP. The bracketed (N) values indicate how many data points from independent runs are included in the mean; ± indicates SEM; not tested, NT; ND, not determined. In cases where the mAb was tested only twice, two values are provided. Cynomolgus macaque/human provides the difference in ratio between the two species.TableE4.Variants with improved binding tohuman and cynomolgus macaqueIL-18BPHCmutationLCvariationmAbsHumanKD(pM)Crab-eating macaqueKD(pM)Crab-eating macaque/humanY33P52aN53I96A99R24L33N92T25SA01a 2400 ± 300 (N3) 6000 ± 2000 (N3) 2.5SA45a 900 ND I96L N92SA54a 87 ± 29 (N4) 59 ± 16 (N4) 0.7 Y33F N53E I96L A99S N92SA55a 78 ± 22 (N5) 127 ± 19 (N5) 1.6 Y33F P52 N53E I96L N92SA56a ＜50 420 ＞8.4 N53E I96L N92SA57a 88 ± 29 (N4) 60 ± 16 (N4) 0.7 Y33F N53E I96L N92SA59a 44 ± 9 (N9) 45 ± 10 (N9) 1.5 Y33F P52 N53E I96L A99S N92SA60a 760, 590 (N2) NT ND N92SA61a 280 NT ND N53E N92SA62a 170, ＜50 (N2) 480 2.8 P52 N53E N92SA63a ＜50 (N3) 430 (N2) ＞8.6 Y33F N53E N92SA64a 63 ± 10 (N16) 71 ± 13 (N16) 1.2 Y33F P52 N53E I96L A99S L33T N92 T25MSA65a 94 ± 25 (N4) 136 ± 14 (N5) 1.4 Y33F P52 N53E I96L A99S N92SA66a 77 ± 9 (N7) 94 ± 14 (N7) 1.2 Y33F P52 N53E I96L A99S R24D N92SA73a 150, 93 (N2) 120, 800 (N2) 3.8 P52 N53E I96L A99S R24D L33T N92SA77a 380 ± 150 (N3) 373 ± 244 (N3) 1 N53E I96L A99S R24D L33T N92SA59a-R24Q ND 140 NE Y33F P52 N53E I96L A99S R24Q N92SA59a-S26I 140, 570 (N2) 140, 120 (N2) 0.37 Y33F P52 N53E I96L A99S N92 S26ISA59a-S26R 220 150, 160 (N2) 0.7 Y33F P52 N53E I96L A99S N92 S26RSA59a-G28Q 140 120, 89 (N2) 0.75 Y33F P52 N53E I96L A99S N92 G28QSA55a-R24D 129 320 2.5 Y33F P52 N53E I96L R24D N92SA55a-T25M 199, 110 (N2) 380 2.5 Y33F P52 N53E I96L N92 T25MSA55a-S26I 113, 180 (N2) 330 2.2 Y33F P52 N53E I96L N92 S26ISA55a-S26R 130 470 3.6 Y33F P52 N53E I96L N92 S26RSA55a-G28Q 81 300 3.7 Y33F P52 N53E I96L N92 G28QSA55a-L33T ＜90 ± 17 (N5) 227 ± 15 (N3) 2.5 Y33F P52 N53E I96L L33T N92

以上資料指示SA59a、SA64a及SA66a含有相同HC但包括一些額外LC可變性且併入所有人類HC（改善變異）加上食蟹獼猴（增強A99S改變），對兩個物種均達成極高結合親和力。The above data indicate that SA59a, SA64a and SA66a contain the same HC but include some additional LC variability and incorporate all human HC (improved variation) plus cynomolgus macaque (enhanced A99S change), achieving very high binding affinity to both species.

對小鼠IL-18BP之高親和力mAb的進一步表徵。使用報導子分析及小鼠脾細胞分析來測試所選高親和力mAb對小鼠IL-18BP之生物活性。報導子分析利用細胞株，其中鼠類IL-18刺激如上文所述之鹼性磷酸酶（SEAP）之釋放。IL-18活性藉由添加之鼠類IL-18BP得到抑制，且此抑制藉由對IL-18BP而言高親和力中和mAb來減輕。如圖5中所示，親和力之改善與此分析中之改善活性相關。SA01a以19.8 nM之IC50展現活性，而成熟mAb SA51d及SA52d產生之IC50值更強效＞100倍-分別為0.14及0.19 nM。Further characterization ofhigh affinitymAbsto mouseIL-18BP . The selected high affinity mAbs were tested for biological activity against mouse IL-18BP using a reporter assay and a mouse spleen cell assay. The reporter assay utilized a cell line in which murine IL-18 stimulates the release of a basic phosphatase (SEAP) as described above. IL-18 activity was inhibited by the addition of murine IL-18BP, and this inhibition was alleviated by a high affinity neutralizing mAb to IL-18BP.As shown in Figure5 , the improvement in affinity correlated with improved activity in this assay. SA01a exhibited activity with an IC50 of 19.8 nM, while the mature mAbs SA51d and SA52d produced IC50 values that were >100-fold more potent - 0.14 and 0.19 nM, respectively.

在額外生物分析中使用小鼠脾細胞來測試抗小鼠IL-18BP mAb。小鼠脾細胞回應於IL-18而釋放IFNγ，此藉由添加IL-18BP來抑制。活性mAb減輕此抑制，從而恢復IFNγ之產生。圖6展示SA01a、SA51d及SA52d在此分析中顯著增加IFNγ產生。在報導子分析及脾細胞分析二者中具有高親和力及高功能性效力之SA51d視為對於活體內評估而言最佳。Mouse spleen cells were used to test anti-mouse IL-18BP mAbs in an additional bioassay. Mouse spleen cells release IFNγ in response to IL-18, which is inhibited by the addition of IL-18BP. Active mAbs alleviate this inhibition, thereby restoring IFNγ production.Figure6 shows that SA01a, SA51d, and SA52d significantly increased IFNγ production in this assay. SA51d, which had high affinity and high functional potency in both the reporter assay and spleen cell assay, was considered optimal for in vivo evaluation.

在MC38腫瘤模型中抗小鼠IL-18BP之活性。在小鼠同基因型腫瘤模型中使用MC38癌細胞株測試高親和力抗小鼠IL-18BP mAb（SA51d）。此處，將1×10⁶個MC38細胞皮下植入至C57BL/6小鼠之後側腹中。腫瘤達到49 - 120 mm³之尺寸後，將小鼠隨機分為5個獨立組（n = 10），其中各組平均腫瘤尺寸範圍介於79 - 81 mm3。每三天（q3d）藉由腹膜內（i.p.）注射施以處理，對於研究時長期間存活之動物，至多7個總劑量。出於多種原因自研究移除動物，包括如研究指南所概述，達到所准許之尺寸限制或觀測到嚴重腫瘤潰瘍。處理包括PBS媒劑對照、mIL-18（0.32 mg/kg）、SA0051d（10 mg/kg）或mIL-18（0.32 mg/kg）+ SA0051d（10 mg/kg）。Activity of anti-mouseIL-18BPin theMC38tumor model . A high affinity anti-mouse IL-18BP mAb (SA51d) was tested in a mouse syngeneic tumor model using the MC38 cancer cell line. Here, 1×10⁶ MC38 cells were implanted subcutaneously into the flank of C57BL/6 mice. After tumors reached a size of 49 - 120 mm³ , mice were randomized into 5 independent groups (n = 10), with mean tumor size in each group ranging from 79 - 81 mm3. Treatments were administered every three days (q3d) by intraperitoneal (ip) injection, up to 7 total doses for animals surviving for the duration of the study. Animals were removed from the study for a variety of reasons, including reaching the permitted size limit or observation of severe tumor ulceration as outlined in the study guidelines. Treatments included PBS vehicle control, mIL-18 (0.32 mg/kg), SA0051d (10 mg/kg), or mIL-18 (0.32 mg/kg) + SA0051d (10 mg/kg).

藉由測徑規量測來一週量測腫瘤兩次且平均化以計算各組之平均腫瘤生長抑制。在各時間點藉由GraphPad Prism軟體（9.4版），使用2因子變異數分析，之後使用龐費洛尼多重比較檢定（Bonferroni's multiple comparison test）進行統計分析。如圖7A-圖7B中所示，mIL-18及SA51d之組合截至第15天展現出顯著MC38腫瘤生長抑制（對比媒劑，p＜0.001，相較於單獨mIL-18，p = 0.0018）。Tumors were measured twice a week by caliper measurement and averaged to calculate the mean tumor growth inhibition for each group. Statistical analysis was performed at each time point by GraphPad Prism software (version 9.4) using a 2-way variance analysis followed by Bonferroni's multiple comparison test.As shown inFigures7A-7B , the combination of mIL-18 and SA51d exhibited significant MC38 tumor growth inhibition by day 15 (p < 0.001 vs. vehicle, p = 0.0018 vs. mIL-18 alone).

對人類及食蟹獼猴IL-18BP之高親和力mAb的進一步表徵。對人類及食蟹獼猴IL-18BP具有高親和力之MAb經進一步表徵以鑑別適用於進一步臨床開發之MAb。此包括評估抗體結合特徵（例如阻斷IL-18BP介導之IL-18信號傳導中和的抗人類IL-18BP；人類IgG1/κ；與食蟹獼猴IL-18BP交叉反應；與人類IL-18BP之KD高親和力，低於IL-18對IL-18BP之親和力，無可偵測非特異性結合或與同源蛋白質之結合）、功能特性（例如阻斷IL-18與IL-18BP之結合；中和IL-18BP，由此拮抗其對IL-18介導之IFNγ誘導的抑制作用）及可開發性。Further Characterization ofHigh AffinitymAbsfor Human and Cynomolgus MascotIL-18BP . MAbs with high affinity for human and cynomolgus macaque IL-18BP were further characterized to identify MAbs suitable for further clinical development. This includes assessment of antibody binding characteristics (e.g., anti-human IL-18BP that blocks IL-18BP-mediated neutralization of IL-18 signaling; human IgG1/κ; cross-reactive with cynomolgus macaque IL-18BP; high affinity with KD for human IL-18BP, lower than the affinity of IL-18 for IL-18BP, no detectable non-specific binding or binding to homologous proteins), functional properties (e.g., blocks IL-18 binding to IL-18BP; neutralizes IL-18BP, thereby antagonizing its inhibitory effect on IL-18-mediated IFNγ induction), and developability.

功能活性。所關注mAb藉由IL-18BP抑制IL-18中和之能力在多種不同生物分析中加以評估：IL-18驅動報導子分析（圖8A-圖8B），自KG-1細胞分泌IFNγ（圖9、表E7）及自人類及獼猴PBMC分泌IFNγ（圖10A-圖10B；表E9）。Functional Activity . The ability of the mAbs of interest to inhibit IL-18 neutralization by IL-18BP was assessed in a variety of different bioassays: IL-18 driven reporter assay (Fig.8A-B) , IFNγ secretion from KG-1 cells (Fig.9 ,TableE7 ), and IFNγ secretion from human and macaque PBMCs (Fig.10A-B;TableE9 ).

如圖8A-圖8B中所示，所測試mAb能夠結合IL-18BP，減輕IL-18之抑制且允許IL-18在報導子分析中信號傳導（引起報導蛋白鹼性磷酸酶之分泌）。效力次序與阻斷mAb之結合親和力相關；亦即，最高親和力mAb在此分析中最有效。As shown inFigures8A-8B , the tested mAbs were able to bind IL-18BP, relieve IL-18 inhibition and allow IL-18 signaling in the reporter assay (causing secretion of the reporter protein alkaline phosphatase). The order of potency correlated with the binding affinity of the blocking mAbs; that is, the highest affinity mAbs were the most potent in this assay.

圖9展示所測試之mAb在KG-1分析中亦展示高效力。表E7提供EC50值。表E7.KG-1細胞中之IFNγ分泌（EC50以nM展示）mAb運行1運行2運行3平均值SA01a384518188SA45a1.84.16.94.3SA59a0.550.470.930.65SA64a0.590.630.560.59SA66a0.730.781.170.89SA73a2.53.64.63.6SA77a2.63.474.3Figure9 shows that the mAbs tested also exhibited high potency in the KG-1 assay.TableE7 provides EC50 values.TableE7.IFNγ SecretioninKG-1 Cells(EC50 showninnM) mAbs Run 1 Run 2 Run 3 average value SA01a 38 45 181 88 SA45a 1.8 4.1 6.9 4.3 SA59a 0.55 0.47 0.93 0.65 SA64a 0.59 0.63 0.56 0.59 SA66a 0.73 0.78 1.17 0.89 SA73a 2.5 3.6 4.6 3.6 SA77a 2.6 3.4 7 4.3

IL-18結合IL-12誘導PBMC中之多個細胞類型（包括NK細胞及T細胞）產生IFNγ。IL-18BP由PBMC內源性產生且由IFNγ上調，從而提供抑制IFNγ反應之負回饋迴路。添加中和抗IL-18BP抗體將結合內源性IL-18BP且防止負回饋迴路，從而引起IFNγ產生之誘導。在健康供體PBMC中活體外測試一組mAb與IL-18競爭結合至IL-18BP且允許IL-18介導之IFNγ誘導的能力。分析中所用IL-18之濃度僅足以在PBMC中提供低水平IFNγ反應，此可能歸因於由內源性IL-18BP之抑制。如圖10A-圖10B中所示，添加mAb移除此抑制，從而引起IFNγ產生之劑量依賴性增加。人類（10A）及食蟹獼猴（10B）PBMC二者均用於測試抗體之活性以及交叉反應性。該等抗體的效力在針對大多數抗體之食蟹獼猴PBMC分析中較低。在此等分析中亦觀測到IFNγ產生與mAb親和力之間的等級次序相關性。表E8提供EC50值。表E8. PBMC中之IFNγ分泌（EC50以nM展示）mAb人類食蟹獼猴SA014.9391SA4510723SA593.423SA643.25.6SA668.417SA735.447SA77＞900227IL-18 binds to IL-12 to induce IFNγ production by multiple cell types in PBMCs, including NK cells and T cells. IL-18BP is endogenously produced by PBMCs and is upregulated by IFNγ, providing a negative feedback loop that inhibits the IFNγ response. Addition of a neutralizing anti-IL-18BP antibody will bind endogenous IL-18BP and prevent the negative feedback loop, leading to induction of IFNγ production. A panel of mAbs was tested in vitro in healthy donor PBMCs for their ability to compete with IL-18 for binding to IL-18BP and allow IL-18-mediated IFNγ induction. The concentration of IL-18 used in the assay was only sufficient to provide a low level of IFNγ response in PBMCs, which may be due to inhibition by endogenous IL-18BP.As shown inFigures10A -10B , addition of mAb removed this inhibition, resulting in a dose-dependent increase in IFNγ production. Both human (10A ) and cynomolgus macaque (10B ) PBMCs were used to test the activity and cross-reactivity of the antibodies. The potency of the antibodies was lower in the cynomolgus macaque PBMC assay for most antibodies. A rank order correlation between IFNγ production and mAb affinity was also observed in these assays.TableE8 provides the EC50 values.TableE8.IFNγ Secretionin PBMCs(EC50 showninnM) mAbs Human Crab-eating macaque SA01 4.9 391 SA45 107 twenty three SA59 3.4 twenty three SA64 3.2 5.6 SA66 8.4 17 SA73 5.4 47 SA77 ＞900 227

在PBMC中進行額外分析，其中IL-18與IL-18BP預複合，隨後添加至PBMC中。此分析測試抗體自預先存在之與IL-18BP之複合物釋放IL-18的能力。如圖11A-圖11B中所示，亦在此分析中觀測到活性，其中IFNγ誘導之效力再次與最高親和力mAb之親和力相關。表E9提供EC50值。表E9.預複合PBMC中之IFNγ分泌（EC50以nM展示）mAb人類食蟹獼猴SA015.47.4SA45849SA590.550.5SA640.690.75SA6610.46抗原決定基定位An additional assay was performed in PBMCs in which IL-18 was pre-complexed with IL-18BP and then added to PBMCs. This assay tests the ability of the antibodies to release IL-18 from a pre-existing complex with IL-18BP.As shown inFigures11A-11B , activity was also observed in this assay, where the potency of IFNγ induction was again correlated with the affinity of the highest affinity mAb.TableE9 provides the EC50 values.TableE9.IFNγ SecretioninPre-complexedPBMCs(EC50 showninnM) mAbs Human Crab-eating macaque SA01 5.4 7.4 SA45 8 49 SA59 0.55 0.5 SA64 0.69 0.75 SA66 1 0.46Epitope location

若干SA01衍生之抗體對IL-18BP直系同源物之差異反應性使得能夠鑑別某些mAb之結合抗原決定基。特定言之，因為SA51d之親和力成熟至超出小鼠及人類IL-18BP之Octet偵測極限（KD＜50 pM），但對比食蟹獼猴IL-18BP（8 nM）基本上不變，所以此提供一種用於研究SA51d及其前身（antecedent）之抗原決定基結合位點的適用工具。IL-18BP直系同源物之同相對齊（lineup）揭示存在四個位置，在該等位置處，人類及小鼠彼此相同但不同於食蟹獼猴（參見圖2）。The differential reactivity of several SA01-derived antibodies to IL-18BP orthologs enabled the identification of the binding epitopes of certain mAbs. Specifically, because the affinity of SA51d was matured beyond the Octet detection limit for mouse and human IL-18BP (KD < 50 pM), but was essentially unchanged compared to cynomolgus macaque IL-18BP (8 nM), this provided a useful tool for studying the epitope binding sites of SA51d and its antecedents. A lineup of IL-18BP orthologs revealed the presence of four positions where human and mouse are identical to each other but different from cynomolgus macaque (seeFigure2 ).

最近描述人類IL-18BP結合至IL-18之共晶體結構（參見Detry等人, J. Biol. Chem. 298(5): 101908, 2022；PDB資料庫條目7AL7）且展示於圖12中。此結構准許對結合蛋白中之4個人類/小鼠對比食蟹獼猴的差異中之各者進行定位。此等變化中之兩者I97M及V153M（如由UniProt：O95998所定義）定位至活性結合位點，從而證實此等變化可潛在地涉及結合抗原決定基，此係因為吾人知曉其作為阻斷抗原決定基。亦鑑別出細胞介素本身有4個人類/小鼠對比食蟹獼猴的差異，其中無一者定位至結合位點。The co-crystal structure of human IL-18BP bound to IL-18 was recently described (see Detry et al., J. Biol. Chem. 298(5): 101908, 2022; PDB entry 7AL7) and is shown inFigure12. This structure allowed the localization of each of the four human/mouse versus cynomolgus macaque differences in the binding protein. Two of these changes, I97M and V153M (as defined by UniProt: O95998), were localized to the active binding site, demonstrating that these changes could potentially be involved in the binding epitope, as it is known to act as a blocking epitope. Four differences in the interleukins themselves were identified between humans/mouse and cynomolgus macaques, none of which were mapped to the binding site.

作為此等發現之結果，產生含有兩個食蟹獼猴置換之人類IL-18BP之型式，亦即hIL-18BP_{-I97M及V153M}。測試此型式針對mAb SA51d（其表明人類對比食蟹獼猴物種的選擇性）及mAb SA58a（其展示出人類/食蟹獼猴結合無差異）之結合動力學。SA58a充當對照以證明hIL-18BP_{-I97M及V153M}經正確摺疊。如圖13中所示，SA51d與人類良好地結合，與食蟹獼猴不良地結合且與hIL-18BP_{-I97M及V153M}完全不結合。相比之下，對照SA58a識別所有三種型式。此指示當人類及鼠類殘基I97及V153經其食蟹獼猴直系同源對應物取代時，SA51d之結合經阻斷。此資料表明，此兩個殘基（I97及V153，如由UniProt：O95998所定義且在圖2中說明）為用於SA51d之人類IL-18BP結合抗原決定基的一部分。As a result of these findings, a version of human IL-18BP containing two cynomolgus macaque substitutions was generated, namely hIL-18BP_{-I97MandV153M} . This version was tested for binding kinetics against mAb SA51d, which demonstrated selectivity for human vs. cynomolgus macaque species, and mAb SA58a, which showed no difference in human/cynomolgus macaque binding. SA58a served as a control to demonstrate that hIL-18BP_{-I97MandV153M} were correctly folded.As shown in Figure13 , SA51d bound well to humans, poorly to cynomolgus macaques, and did not bind at all to hIL-18BP_{-I97MandV153M} . In contrast, the control SA58a recognized all three versions. This indicates that when human and mouse residues I97 and V153 were replaced by their cynomolgus monkey orthologous counterparts, binding of SA51d was blocked. This data suggests that these two residues (I97 and V153, as defined by UniProt: O95998 and illustrated inFigure2 ) are part of the human IL-18BP binding epitope for SA51d.

mAb SA64a在IL-18BP上之抗原決定基藉由CovalX AG開發之交聯/高解析質譜法（Pimenova等人, 2008, J. Mass Spectrometry 43: 185）測定。簡而言之，使人類IL-18BP與SA64a結合且與異雙官能連接子交聯。將所得複合物用5種不同蛋白酶（胰蛋白酶、胰凝乳蛋白酶、ASP-N、彈性蛋白酶及嗜熱菌蛋白酶）消化，且所得肽無論是否交聯均係藉由高解析質譜法進行分析。The antigenic determinant of mAb SA64a on IL-18BP was determined by a cross-linking/HDMS method developed by CovalX AG (Pimenova et al., 2008, J. Mass Spectrometry 43: 185). Briefly, human IL-18BP was conjugated to SA64a and cross-linked with a heterobifunctional linker. The resulting complex was digested with 5 different proteases (trypsin, chymotrypsin, ASP-N, elastin, and thermolysin), and the resulting peptides, whether cross-linked or not, were analyzed by HDMS.

結果證明SA64a識別構形抗原決定基（圖14），包括下文所指示之IL-18BP中的殘基。另外，使用相同技術類似地定位與IL-18相互作用之IL-18BP上的殘基。以下結果證明IL-18BP上涉及IL-18之識別的三個殘基亦為SA64a所識別之殘基，支持此為功能性阻斷抗體之證據。SA64a與在IL-18BP上之以下殘基相互作用：K32、T40、S60、R61、S66、Y69、R91、R93、T96、K102、R131、H132。IL-18BP上與IL-18相互作用之殘基鑑別為：R61、Y69、S75、H79、T116、S119及R131。成熟IL-18BP之序列顯示如下，其中殘基與如所指示突出顯示之SA64a及IL-18相互作用。 1 TPVSQTTTAA TASVRSTKDP CPSQPPVFPA AKQCPALEVTWPEVEVPLNG 51 TLSLSCVACSRFPNFSILYW LGNGSFIEHL PGRLWEGSTSRERGSTGTQL 101 CKALVLEQLT PALHSTNFSC VLVDPEQVVQRHVVLAQLWA GLRATLPPTQ 151 EALPSSHSSP QQQG (SEQ ID NO: 372)The results demonstrate that SA64a recognizes conformational epitopes (Figure14 ), including the residues in IL-18BP indicated below. In addition, the residues on IL-18BP that interact with IL-18 were similarly located using the same technique. The following results demonstrate that the three residues on IL-18BP involved in the recognition of IL-18 are also residues recognized by SA64a, supporting evidence that this is a functional blocking antibody. SA64a interacts with the following residues on IL-18BP: K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131, H132. Residues on IL-18BP that interact with IL-18 were identified as: R61, Y69, S75, H79, T116, S119 and R131. The sequence of mature IL-18BP is shown below with residues interacting with SA64a and IL-18 highlighted as indicated. 1 TPVSQTTTAA TASVRSTKDP CPSQPPVFPA AK QCPALEVT WPEVEVPLNG 51 TLSLSCVACSR FPNFS ILY W LGNGS FIEH L PGRLWEGSTSR ER GST GTQL 101 CK ALVLEQLT PALHST NFS C VLVDPEQVVQR H VVLAQLWA GLRATLPPTQ 151 EALPSSHSSP QQQG (SEQ ID NO: 372)

發現與IL-18相互作用之殘基以粗體且加下劃線來突出顯示。形成SA64a之抗原決定基的殘基以粗體以斜體字型來突出顯示。殘基R61、Y69及R131皆由IL-18及SA64a二者識別且以粗體、斜體及加下劃線展示。庫篩選資料Residues found to interact with IL-18 are highlighted in bold and underlined. Residues that form the antigenic determinant of SA64a are highlighted in bold and italic. Residues R61, Y69 and R131 are recognized by both IL-18 and SA64a and are shown in bold, italic and underlined.Library Screening Data

測試親本SA01a及SA60a抗體之變異體的結合特徵。Variants of the parental SA01a and SA60a antibodies were tested for their binding properties.

重鏈庫篩選。在＞50 nM下量測之Octet產生之動力學值列為「非活性」。考慮對人類及食蟹獼猴二者或小鼠提供≳1.5至2.0倍改善的位置以供進一步分析。對於各物種呈現之值為表觀KD，此係因為此等值係根據以半高通量型式收集之單一結合濃度計算。將樣品與產生其之親本背景相比（字型在親本為SA01a的情況下為正常的，及在親本為SA60a（具有LC-N92Y之SA01a）的情況下為斜體的）。表H1裝載樣品IDKD nM huKD (nM) cyKD (nM) mo裝載樣品IDKD nM huKD (nM) cyKD (nM) moG95V8.33.4非活性A97L6.02.828.5G95H1.02.5非活性A97I6.12.332.3G95S3.84.238.6A97R2.62.728.4G95F非活性非活性非活性A97S5.92.7非活性G95D2.71.7非活性A97K2.22.514.0G95R非活性3.2非活性A97T5.62.6非活性親本3.03.718.4親本2.82.820.7G95N8.91.1非活性A97E2.1非活性35.5G95I非活性2.2非活性A97P0.7非活性非活性G95P6.22.7非活性A97M5.64.0非活性G95T2.83.015.1L100N6.2非活性21.1G95Q非活性0.3非活性L100G1.5非活性非活性G95A6.77.529.8L100D1.9非活性非活性親本2.93.415.0親本3.17.627.0G95L0.5非活性非活性L100H1.1非活性非活性G95K非活性1.5非活性L100R0.1非活性非活性G95Q1.00.4非活性L100Y7.822.542.5I96G0.60.4非活性L100I6.06.925.9I96D非活性非活性非活性L100Q4.211.927.7I96N11.42.0非活性L100E1.9非活性非活性親本3.03.319.8親本2.97.414.5I96V3.64.119.0L100P2.321.7非活性I96H5.34.438.9L100T8.727.2非活性I96R1.82.511.3L100V5.78.644.1I96S5.90.7非活性L100M3.917.520.2I96L1.21.88.8F100aV6.217.044.1I96Y12.21.6非活性F100aY7.0非活性非活性親本2.93.023.4親本2.97.318.2I96Q4.23.923.1F100aR非活性非活性非活性I96K1.52.510.7F100aG非活性非活性非活性I96E6.21.9非活性F100aN6.750.428.7I96P0.81.027.6F100aH1.76.16.3I96M3.43.329.1F100aT9.611.132.2A97Y5.93.9非活性F100aK4.3非活性20.4親本2.83.022.3親本3.06.914.4表H2裝載樣品IDKD nM huKD (nM) cyKD (nM) mo裝載樣品IDKD nM huKD (nM) cyKD (nM) moF100aM1.97.27.5I34P4.99.711.1F100aL2.712.411.5I34Q5.08.617.8F100aE9.7非活性17.2I34T6.510.220.1F100aA4.812.119.9I34M4.77.217.3F100aP6.92.824.0I34V5.78.819.8F100aQ4.5非活性21.0I34K5.07.511.0親本2.95.615.7親本非活性非活性10.1T30L3.310.115.5I34H6.47.723.9T30V3.29.314.5I34D非活性非活性非活性T30H3.56.519.4I34N4.17.718.4T30N2.96.214.5I34V4.67.422.4T30S3.25.718.0I34F6.69.728.1T30R3.525.619.3I34L7.09.619.1親本2.95.915.5親本非活性非活性10.9H35L非活性37.09.8I34C5.38.2非活性H35P35.219.939.4I34Y5.312.024.1H35Q非活性47.6非活性I34S6.910.719.4H35M非活性非活性非活性Y33V10.812.8非活性H35T非活性19.7非活性Y33K非活性非活性非活性H35V16.87.724.3Y33P24.720.5非活性親本非活性非活性10.1H35A非活性27.224.9Y33A33.114.7非活性H35K非活性非活性非活性Y33Q非活性非活性非活性H35S14.112.339.7Y33H14.320.9非活性H35L非活性16.944.2Y33G9.77.4非活性H35D42.919.944.7Y33R非活性11.4非活性H35F非活性非活性8.0Y33N非活性16.0非活性親本非活性非活性9.8親本6.212.020.8H35N29.319.636.1Y33S非活性9.9非活性H35Y非活性非活性非活性Y33L25.919.9非活性H35I44.520.9非活性Y33D15.716.5非活性I34A5.48.316.4Y33F3.06.713.4I34L5.48.719.7Y32K9.38.936.3I34E22.624.822.3Y32P非活性17.3非活性親本30.4非活性9.9親本6.611.421.8表H3裝載樣品IDKD nM huKD (nM) cyKD (nM) mo裝載樣品IDKD nM huKD (nM) cyKD (nM) moY32Q5.611.817.0T30P0.80.715.9Y32T9.916.730.7T30A0.70.727.8Y32E5.412.310.1T30K0.90.819.0Y32A14.611.948.3T30E0.70.621.6Y32V8.312.723.9T30Y0.80.822.9Y32H7.512.236.8T30I0.80.717.6親本6.311.018.7親本0.70.731.6Y32C4.78.39.6I50E非活性非活性非活性Y32N7.313.221.7I50A非活性非活性非活性Y32L6.29.920.1I50L11.85.3非活性Y32F4.99.917.2I50V4.53.0非活性Y32I6.610.125.1I50P9.36.4非活性Y32G17.813.2非活性I51P非活性非活性非活性親本6.210.520.8親本0.70.721.9Y32R6.310.418.7I51A9.63.3非活性Y32D6.415.915.1I51V1.00.9非活性N31M6.711.921.0I51K2.01.536.2N31L5.39.413.9I51Q1.00.825.2N31E5.612.915.7I51T1.41.139.1N31T6.511.922.0I51L0.80.717.0親本5.910.121.1親本0.80.728.1N31Q5.410.415.0I51R非活性7.5非活性N31P10.317.829.7I51H1.91.9非活性N31A6.312.420.0I51Y1.41.038.6N31S5.711.419.4I51V1.00.930.5I51F0.90.727.4N31F7.112.018.1N52P非活性非活性非活性親本5.910.318.3親本0.70.623.7N31H1.01.116.2N52Q1.00.823.0N31Y1.01.218.7N52T1.71.038.7N31L0.70.710.9N52M1.51.028.0N31I0.60.79.3N52A1.10.817.8T30M0.90.824.9N52V5.01.9非活性T30Q0.90.820.1N52S1.20.711.6親本0.70.729.3親本0.70.623.3表H4裝載樣品IDKD nM huKD (nM) cyKD (nM) mo裝載樣品IDKD nM huKD (nM) cyKD (nM) moN52F4.32.943.0G54H1.22.112.9P52aA0.92.124.1G54F1.32.313.9P52aM1.82.533.1G54N4.73.8非活性P52aK5.01.722.7G54R1.32.226.3P52aQ1.41.831.1G54I1.52.018.6P52aL0.81.417.0G54Y1.12.19.1親本0.91.745.1親本0.91.525.7P52aV0.61.18.6N92P非活性非活性非活性P52aD3.74.227.4N92Q9.012.533.6P52aR5.22.920.8N92V9.013.432.1P52aY4.75.4非活性N92L9.09.731.2P52aF5.82.7非活性N92E20.428.9非活性P52aN0.71.314.5N92M14.319.7非活性親本0.91.531.4親本6.47.830.0P52aG1.11.724.7N92A非活性非活性非活性P52aH1.21.548.2N92T10.012.231.6N53Q0.61.113.3N92K2.52.211.1N53T0.50.915.2N92Y13.011.536.8N53P0.60.712.4N92S6.26.4非活性N53K0.51.013.4N92H8.28.6非活性親本0.71.224.5親本6.27.923.7N53M0.71.313.7N92R11.814.123.5N53L0.71.220.4N92I13.27.128.6N53A0.71.213.8N92D15.417.436.7N53E0.40.79.7S93P0.81.3非活性N53H1.01.413.7S93K6.27.820.4N53Y1.01.820.5S93T24.337.7非活性親本0.91.535.0親本5.67.222.5N53R1.01.816.0S93A6.99.615.9N53S0.81.422.2S93Q4.66.216.2N53G0.61.112.2S93E非活性非活性非活性N53F0.81.517.8S93M4.76.314.4N53V0.71.116.2S93I6.68.626.8G54D2.12.9非活性S93R5.86.721.5親本0.91.527.1親本5.46.519.4表H5裝載樣品IDKD nM huKD (nM) cyKD (nM) mo裝載樣品IDKD nM huKD (nM) cyKD (nM) moS93H17.619.2非活性Y59A14.921.934.9S93G13.811.340.5Y59M12.417.429.2S93D30.0非活性非活性Y59L10.814.923.0S92N9.29.933.2Y59K11.516.022.6S92L6.28.023.4Y59E13.117.830.9S92V8.012.334.5Y59Q17.725.942.3親本5.66.824.8親本7.59.312.9G54Q6.99.19.6Y59G14.218.329.2G54A6.08.115.5Y59F9.813.624.0G54T6.78.925.5Y59N17.122.8非活性G54K6.17.314.9Y59R12.017.031.2G54E5.77.010.6Y59I7.18.817.0G54L8.89.720.6Y59S16.820.134.8親本5.36.615.4親本7.49.216.0T57N21.229.9非活性Y59H14.119.927.1T57S20.946.9非活性Y33F3.95.69.3T57D19.125.1非活性N53E3.84.56.3T57H12.916.635.7I96L3.04.66.4S58K9.915.426.3I96K3.86.08.0S58P18.5非活性36.4水非活性非活性非活性親本7.99.514.1親本7.49.713.9S58M4.66.78.0M98D非活性非活性非活性S58V9.712.422.2M98H非活性非活性非活性S58E20.229.5非活性M98I37.534.9非活性S58T9.811.319.1M98N非活性4.3非活性S58A9.210.521.0M98R非活性非活性非活性S58I6.08.810.0M98G非活性非活性非活性親本7.69.416.5親本6.08.729.9S58Y4.75.38.0M98S非活性非活性非活性S58N11.614.729.0M98Y非活性非活性非活性S58D21.017.5非活性M98L5.44.814.0S58G30.147.2非活性M98F13.74.45.8S58F5.05.89.3M98Q3.10.2非活性Y59V10.411.819.4M98P非活性非活性非活性親本7.68.915.6親本5.37.88.7表H6裝載樣品IDKD nM huKD (nM) cyKD (nM) mo裝載樣品IDKD nM huKD (nM) cyKD (nM) moM98T非活性非活性非活性G55D7.97.010.7M98A0.83.5非活性G55S10.07.512.8M98K非活性非活性非活性G55R9.77.010.7M98V42.57.90.6G55F8.67.68.5M98E非活性非活性非活性G55H7.46.39.8A99V非活性非活性非活性S56P5.55.56.9親本6.08.19.3親本4.76.06.1A99Y非活性非活性非活性S56A5.25.96.9A99L非活性非活性非活性S56E5.25.78.1A99H1.414.83.1S56T4.33.06.9A99G13.27.621.3S56L6.85.68.4A99I15.46.2非活性S56V5.04.26.5A99S5.92.719.4S56M6.35.87.9親本6.18.312.0親本4.35.76.3A99R非活性非活性非活性S56K8.27.210.4A99N6.91.215.7S56I8.36.89.8A99D37.86.8非活性S56H7.26.39.4A99Q15.92.0非活性S56R8.77.29.7A99K非活性非活性非活性S56N5.95.87.7A99E非活性非活性非活性S56F6.25.48.0親本5.98.011.4親本4.86.16.6A99P非活性12.8非活性S56D7.26.710.6A99M49.623.3非活性T57M10.79.915.1A99T20.915.649.5T57K7.15.89.4G55A6.68.914.5T57P10.67.58.7G55V11.013.433.1T57E9.89.715.9G55M9.912.045.0T57A10.110.313.0親本5.97.58.8親本4.65.96.8G55E5.75.77.4T57Q8.89.411.8G55T5.63.28.3T57V4.24.55.7G55K9.36.510.1T57L9.36.311.0G55Q6.35.97.5T57G13.09.911.7G55L8.86.411.4T57R9.97.813.6G55Y7.75.97.9T57I4.95.26.5親本5.06.06.4親本5.25.87.0Rechain library screening . Octet-generated kinetic values measured at >50 nM were classified as "inactive". Positions providing ≳1.5 to 2.0-fold improvement over both human and cynomolgus macaque or mouse were considered for further analysis. The values presented for each species are apparent KDs as these are calculated from single binding concentrations collected in a semi-high throughput format. Samples were compared to the parental background from which they were generated (font is normal if the parent is SA01a and italicized if the parent is SA60a (SA01a with LC-N92Y)).TableH1Load sampleIDKDKD (nM) cyKD (nM) moLoad sampleIDKDKD (nM) cyKD (nM) mo G95V 8.3 3.4 Inactive A97L 6.0 2.8 28.5 G95H 1.0 2.5 Inactive A97I 6.1 2.3 32.3 G95S 3.8 4.2 38.6 A97R 2.6 2.7 28.4 G95F Inactive Inactive Inactive A97S 5.9 2.7 Inactive G95D 2.7 1.7 Inactive A97K 2.2 2.5 14.0 G95R Inactive 3.2 Inactive A97T 5.6 2.6 Inactive Parent 3.0 3.7 18.4 Parent 2.8 2.8 20.7 G95N 8.9 1.1 Inactive A97E 2.1 Inactive 35.5 G95I Inactive 2.2 Inactive A97P 0.7 Inactive Inactive G95P 6.2 2.7 Inactive A97M 5.6 4.0 Inactive G95T 2.8 3.0 15.1 L100N 6.2 Inactive 21.1 G95Q Inactive 0.3 Inactive L100G 1.5 Inactive Inactive G95A 6.7 7.5 29.8 L100D 1.9 Inactive Inactive Parent 2.9 3.4 15.0 Parent 3.1 7.6 27.0 G95L 0.5 Inactive Inactive L100H 1.1 Inactive Inactive G95K Inactive 1.5 Inactive L100R 0.1 Inactive Inactive G95Q 1.0 0.4 Inactive L100Y 7.8 22.5 42.5 I96G 0.6 0.4 Inactive L100I 6.0 6.9 25.9 I96D Inactive Inactive Inactive L100Q 4.2 11.9 27.7 I96N 11.4 2.0 Inactive L100E 1.9 Inactive Inactive Parent 3.0 3.3 19.8 Parent 2.9 7.4 14.5 I96V 3.6 4.1 19.0 L100P 2.3 21.7 Inactive I96H 5.3 4.4 38.9 L100T 8.7 27.2 Inactive I96R 1.8 2.5 11.3 L100V 5.7 8.6 44.1 I96S 5.9 0.7 Inactive L100M 3.9 17.5 20.2 I96L 1.2 1.8 8.8 F100V 6.2 17.0 44.1 Y 12.2 1.6 Inactive F100 7.0 Inactive Inactive Parent 2.9 3.0 23.4 Parent 2.9 7.3 18.2 Q6 4.2 3.9 23.1 F100 Inactive Inactive Inactive I96K 1.5 2.5 10.7 F100 Inactive Inactive Inactive I96E 6.2 1.9 Inactive F100a 6.7 50.4 28.7 I96P 0.8 1.0 27.6 F100H 1.7 6.1 6.3 I96M 3.4 3.3 29.1 F100aT 9.6 11.1 32.2 A97 5.9 3.9 Inactive F100 4.3 Inactive 20.4 Parent 2.8 3.0 22.3 Parent 3.0 6.9 14.4TableH2Load sampleIDKDKD (nM) cyKD (nM) moLoad sampleIDKDJKD (nM) cyKD (nM) mo F100a 1.9 7.2 7.5 I34P 4.9 9.7 11.1 F100 2.7 12.4 11.5 Q3Q 5.0 8.6 17.8 F100a 9.7 Inactive 17.2 I34T 6.5 10.2 20.1 F100A 4.8 12.1 19.9 I34M 4.7 7.2 17.3 F100 6.9 2.8 24.0 I34V 5.7 8.8 19.8 QF 4.5 Inactive 21.0 I34K 5.0 7.5 11.0 Parent 2.9 5.6 15.7 Parent Inactive Inactive 10.1 T30L 3.3 10.1 15.5 I34H 6.4 7.7 23.9 T30V 3.2 9.3 14.5 I3D Inactive Inactive Inactive T30H 3.5 6.5 19.4 I34N 4.1 7.7 18.4 T30N 2.9 6.2 14.5 I34V 4.6 7.4 22.4 T30S 3.2 5.7 18.0 I34F 6.6 9.7 28.1 T30R 3.5 25.6 19.3 I34L 7.0 9.6 19.1 Parent 2.9 5.9 15.5 Parent Inactive Inactive 10.9 H35L Inactive 37.0 9.8 I34C 5.3 8.2 Inactive H35P 35.2 19.9 39.4 I3Y 5.3 12.0 24.1 H35Q Inactive 47.6 Inactive I34S 6.9 10.7 19.4 H35M Inactive Inactive Inactive Y33V 10.8 12.8 Inactive H35T Inactive 19.7 Inactive Y33K Inactive Inactive Inactive H35V 16.8 7.7 24.3 Y33P 24.7 20.5 Inactive Parent Inactive Inactive 10.1 H35A Inactive 27.2 24.9 Y33A 33.1 14.7 Inactive H35K Inactive Inactive Inactive Y3 Inactive Inactive Inactive H35S 14.1 12.3 39.7 Y33H 14.3 20.9 Inactive H35L Inactive 16.9 44.2 Y33G 9.7 7.4 Inactive H35D 42.9 19.9 44.7 Y33R Inactive 11.4 Inactive H35F Inactive Inactive 8.0 Y33N Inactive 16.0 Inactive Parent Inactive Inactive 9.8 Parent 6.2 12.0 20.8 H35N 29.3 19.6 36.1 Y33S Inactive 9.9 Inactive H35Y Inactive Inactive Inactive Y33L 25.9 19.9 Inactive H35I 44.5 20.9 Inactive Y3D 15.7 16.5 Inactive I34A 5.4 8.3 16.4 Y33F 3.0 6.7 13.4 I34L 5.4 8.7 19.7 Y32K 9.3 8.9 36.3 I34E 22.6 24.8 22.3 Y32P Inactive 17.3 Inactive Parent 30.4 Inactive 9.9 Parent 6.6 11.4 21.8TableH3Load sampleIDKDKD (nM) cyKD (nM) moLoad sampleIDKDJKD (nM) cyKD (nM) mo Y3 5.6 11.8 17.0 T30P 0.8 0.7 15.9 Y32T 9.9 16.7 30.7 T30A 0.7 0.7 27.8 Y32E 5.4 12.3 10.1 T30K 0.9 0.8 19.0 Y32A 14.6 11.9 48.3 T30E 0.7 0.6 21.6 Y32V 8.3 12.7 23.9 T30Y 0.8 0.8 22.9 Y32H 7.5 12.2 36.8 T30I 0.8 0.7 17.6 Parent 6.3 11.0 18.7 Parent 0.7 0.7 31.6 Y32C 4.7 8.3 9.6 I50E Inactive Inactive Inactive Y32N 7.3 13.2 21.7 I50A Inactive Inactive Inactive Y32L 6.2 9.9 20.1 I50L 11.8 5.3 Inactive Y32F 4.9 9.9 17.2 I50V 4.5 3.0 Inactive Y32I 6.6 10.1 25.1 I50P 9.3 6.4 Inactive Y32G 17.8 13.2 Inactive I51P Inactive Inactive Inactive Parent 6.2 10.5 20.8 Parent 0.7 0.7 21.9 Y32R 6.3 10.4 18.7 I51A 9.6 3.3 Inactive Y32D 6.4 15.9 15.1 I51V 1.0 0.9 Inactive N31M 6.7 11.9 21.0 I51K 2.0 1.5 36.2 N31L 5.3 9.4 13.9 I51Q 1.0 0.8 25.2 N31E 5.6 12.9 15.7 I51T 1.4 1.1 39.1 N31T 6.5 11.9 22.0 I51L 0.8 0.7 17.0 Parent 5.9 10.1 21.1 Parent 0.8 0.7 28.1 Q 5.4 10.4 15.0 I51R Inactive 7.5 Inactive N31P 10.3 17.8 29.7 I51 1.9 1.9 Inactive N31A 6.3 12.4 20.0 I5 1.4 1.0 38.6 N31S 5.7 11.4 19.4 I51V 1.0 0.9 30.5 I51F 0.9 0.7 27.4 N31F 7.1 12.0 18.1 N52P Inactive Inactive Inactive Parent 5.9 10.3 18.3 Parent 0.7 0.6 23.7 N31H 1.0 1.1 16.2 QUR 1.0 0.8 23.0 N31Y 1.0 1.2 18.7 N52T 1.7 1.0 38.7 N31L 0.7 0.7 10.9 N52M 1.5 1.0 28.0 N31I 0.6 0.7 9.3 N52A 1.1 0.8 17.8 T30M 0.9 0.8 24.9 N52V 5.0 1.9 Inactive T30Q 0.9 0.8 20.1 N52S 1.2 0.7 11.6 Parent 0.7 0.7 29.3 Parent 0.7 0.6 23.3TableH4Load sampleIDKDJKD (nM) cyKD (nM) moLoad sampleIDKDKD (nM) cyKD (nM) mo N52F 4.3 2.9 43.0 G54H 1.2 2.1 12.9 P52a 0.9 2.1 24.1 G54F 1.3 2.3 13.9 P52 1.8 2.5 33.1 G54N 4.7 3.8 Inactive P52 5.0 1.7 22.7 G54R 1.3 2.2 26.3 QUR 1.4 1.8 31.1 G54I 1.5 2.0 18.6 P52 0.8 1.4 17.0 G54Y 1.1 2.1 9.1 Parent 0.9 1.7 45.1 Parent 0.9 1.5 25.7 P52 0.6 1.1 8.6 N92P Inactive Inactive Inactive P52 3.7 4.2 27.4 Q9 9.0 12.5 33.6 P52 5.2 2.9 20.8 N92V 9.0 13.4 32.1 P5Y 4.7 5.4 Inactive N92L 9.0 9.7 31.2 P52 5.8 2.7 Inactive N92E 20.4 28.9 Inactive P52 0.7 1.3 14.5 N92M 14.3 19.7 Inactive Parent 0.9 1.5 31.4 Parent 6.4 7.8 30.0 P52 1.1 1.7 24.7 N92A Inactive Inactive Inactive P5H 1.2 1.5 48.2 N92T 10.0 12.2 31.6 Q5Q 0.6 1.1 13.3 N92K 2.5 2.2 11.1 N53T 0.5 0.9 15.2 N92 13.0 11.5 36.8 N53P 0.6 0.7 12.4 N92S 6.2 6.4 Inactive N53K 0.5 1.0 13.4 N92H 8.2 8.6 Inactive Parent 0.7 1.2 24.5 Parent 6.2 7.9 23.7 N53M 0.7 1.3 13.7 N92R 11.8 14.1 23.5 N53L 0.7 1.2 20.4 N92I 13.2 7.1 28.6 N53A 0.7 1.2 13.8 N92D 15.4 17.4 36.7 N53E 0.4 0.7 9.7 S93P 0.8 1.3 Inactive N53H 1.0 1.4 13.7 S93K 6.2 7.8 20.4 N53 1.0 1.8 20.5 S93T 24.3 37.7 Inactive Parent 0.9 1.5 35.0 Parent 5.6 7.2 22.5 N53R 1.0 1.8 16.0 S93A 6.9 9.6 15.9 N53S 0.8 1.4 22.2 S93Q 4.6 6.2 16.2 N53G 0.6 1.1 12.2 S93E Inactive Inactive Inactive N53F 0.8 1.5 17.8 S93M 4.7 6.3 14.4 N53V 0.7 1.1 16.2 S93I 6.6 8.6 26.8 G54D 2.1 2.9 Inactive S93R 5.8 6.7 21.5 Parent 0.9 1.5 27.1 Parent 5.4 6.5 19.4TableH5Load sampleIDKDKD (nM) cyKD (nM) moLoad sampleIDKDKD (nM) cyKD (nM) mo S93H 17.6 19.2 Inactive Y59A 14.9 21.9 34.9 S93G 13.8 11.3 40.5 Y59M 12.4 17.4 29.2 S93D 30.0 Inactive Inactive Y59L 10.8 14.9 23.0 S92N 9.2 9.9 33.2 Y59K 11.5 16.0 22.6 S92L 6.2 8.0 23.4 Y59E 13.1 17.8 30.9 S92V 8.0 12.3 34.5 Y9Q 17.7 25.9 42.3 Parent 5.6 6.8 24.8 Parent 7.5 9.3 12.9 G54Q 6.9 9.1 9.6 Y59G 14.2 18.3 29.2 G54A 6.0 8.1 15.5 Y59F 9.8 13.6 24.0 G54T 6.7 8.9 25.5 Y59N 17.1 22.8 Inactive G54K 6.1 7.3 14.9 Y59R 12.0 17.0 31.2 G54E 5.7 7.0 10.6 Y59I 7.1 8.8 17.0 G54L 8.8 9.7 20.6 Y59S 16.8 20.1 34.8 Parent 5.3 6.6 15.4 Parent 7.4 9.2 16.0 T57N 21.2 29.9 Inactive Y59H 14.1 19.9 27.1 T57S 20.9 46.9 Inactive Y33F 3.9 5.6 9.3 T57D 19.1 25.1 Inactive N53E 3.8 4.5 6.3 T57H 12.9 16.6 35.7 I96L 3.0 4.6 6.4 S58K 9.9 15.4 26.3 I96K 3.8 6.0 8.0 S58P 18.5 Inactive 36.4 water Inactive Inactive Inactive Parent 7.9 9.5 14.1 Parent 7.4 9.7 13.9 S58M 4.6 6.7 8.0 M98D Inactive Inactive Inactive S58V 9.7 12.4 22.2 M98H Inactive Inactive Inactive S58E 20.2 29.5 Inactive M98I 37.5 34.9 Inactive S58T 9.8 11.3 19.1 M98N Inactive 4.3 Inactive S58A 9.2 10.5 21.0 M98R Inactive Inactive Inactive S58I 6.0 8.8 10.0 M98G Inactive Inactive Inactive Parent 7.6 9.4 16.5 Parent 6.0 8.7 29.9 S58 4.7 5.3 8.0 M98S Inactive Inactive Inactive S58N 11.6 14.7 29.0 M98Y Inactive Inactive Inactive S58D 21.0 17.5 Inactive M98L 5.4 4.8 14.0 S58G 30.1 47.2 Inactive M98F 13.7 4.4 5.8 S58F 5.0 5.8 9.3 M98Q 3.1 0.2 Inactive Y59V 10.4 11.8 19.4 M98P Inactive Inactive Inactive Parent 7.6 8.9 15.6 Parent 5.3 7.8 8.7TableH6Load sampleIDKDKD (nM) cyKD (nM) moLoad sampleIDKDKD (nM) cyKD (nM) mo M98T Inactive Inactive Inactive G55D 7.9 7.0 10.7 M98A 0.8 3.5 Inactive G55S 10.0 7.5 12.8 M98K Inactive Inactive Inactive G55R 9.7 7.0 10.7 M98V 42.5 7.9 0.6 G55F 8.6 7.6 8.5 M98E Inactive Inactive Inactive G55H 7.4 6.3 9.8 A99V Inactive Inactive Inactive S56P 5.5 5.5 6.9 Parent 6.0 8.1 9.3 Parent 4.7 6.0 6.1 A99Y Inactive Inactive Inactive S56A 5.2 5.9 6.9 A99L Inactive Inactive Inactive S56E 5.2 5.7 8.1 A99H 1.4 14.8 3.1 S56T 4.3 3.0 6.9 A99G 13.2 7.6 21.3 S56L 6.8 5.6 8.4 A99I 15.4 6.2 Inactive S56V 5.0 4.2 6.5 A99S 5.9 2.7 19.4 S56M 6.3 5.8 7.9 Parent 6.1 8.3 12.0 Parent 4.3 5.7 6.3 A99R Inactive Inactive Inactive S56K 8.2 7.2 10.4 A99N 6.9 1.2 15.7 S56I 8.3 6.8 9.8 A99D 37.8 6.8 Inactive S56H 7.2 6.3 9.4 A99Q 15.9 2.0 Inactive S56R 8.7 7.2 9.7 A99K Inactive Inactive Inactive S56N 5.9 5.8 7.7 A99E Inactive Inactive Inactive S56F 6.2 5.4 8.0 Parent 5.9 8.0 11.4 Parent 4.8 6.1 6.6 A99P Inactive 12.8 Inactive S56D 7.2 6.7 10.6 A99M 49.6 23.3 Inactive T57M 10.7 9.9 15.1 A99T 20.9 15.6 49.5 T57K 7.1 5.8 9.4 G55A 6.6 8.9 14.5 T57P 10.6 7.5 8.7 G55V 11.0 13.4 33.1 T57E 9.8 9.7 15.9 G55M 9.9 12.0 45.0 T57A 10.1 10.3 13.0 Parent 5.9 7.5 8.8 Parent 4.6 5.9 6.8 G55E 5.7 5.7 7.4 T57Q 8.8 9.4 11.8 G55T 5.6 3.2 8.3 T57V 4.2 4.5 5.7 G55K 9.3 6.5 10.1 T57L 9.3 6.3 11.0 G55Q 6.3 5.9 7.5 T57G 13.0 9.9 11.7 G55L 8.8 6.4 11.4 T57R 9.9 7.8 13.6 G55Y 7.7 5.9 7.9 T57I 4.9 5.2 6.5 Parent 5.0 6.0 6.4 Parent 5.2 5.8 7.0

輕鏈庫篩選。在＞50 nM下量測之Octet產生之動力學值列為「非活性」。考慮對人類及食蟹獼猴二者提供≳1.5至2.0倍改善的位置以供進一步分析。對於各物種呈現之值為表觀KD，此係因為此等值係根據以半高通量型式收集之單一結合濃度計算。將樣品與產生其之親本背景相比，亦即，字型在親本為SA01a的情況下為正常的，及在親本為SA60a（具有LC-N92Y之SA01a）的情況下為斜體的。表L1裝載樣品IDKD nM huKD (nM) cyKD (nM) mo裝載樣品IDKD nM huKD (nM) cyKD (nM) moQ89V非活性非活性非活性A91D非活性非活性非活性Q89N20.526.7非活性Q91Y13.710.9非活性Q89I非活性非活性非活性Q91F21.014.126.0Q89F非活性非活性非活性Q91N非活性非活性非活性Q89D28.614.6非活性Q91H5.112.916.5Q89G非活性非活性非活性Q91V12.619.7非活性親本4.18.213.7親本4.37.712.5Q89M非活性非活性非活性Q91I40.1NT非活性Q89L非活性非活性非活性Q91R非活性NT非活性Q89E7.116.8非活性Q91S16.7NT非活性Q89A35.444.3非活性Q91M8.7NT非活性Q89P非活性非活性非活性Q91E1.3NT非活性Q89T非活性非活性非活性Q91P非活性NT非活性親本3.87.813.3親本7.6NT25.0Q90S13.716.9非活性Q91Q19.7NT非活性Q90G37.8非活性非活性Q91T16.7NT非活性Q90F非活性非活性非活性Q91K37.8NT非活性Q90R33.617.1非活性F94G非活性NT非活性Q90Y非活性非活性非活性F94V非活性NT非活性Q90H17.626.8非活性F94D非活性NT非活性親本3.87.415.3親本10.6NT34.9Q90V非活性非活性非活性F94Y33.2NT非活性Q90N4.96.720.1F94S非活性NT非活性Q90A11.614.5非活性F94R非活性NT非活性Q90L23.410.5非活性F94K非活性NT非活性Q90M11.113.2非活性F94M23.5NT非活性Q90T非活性非活性非活性F94L非活性NT非活性親本4.07.410.4親本8.4NT16.0Q90L非活性非活性非活性F94A非活性NT非活性Q90E非活性非活性非活性F94T非活性NT非活性Q90K0.1非活性非活性F94P非活性NT非活性Q90P30.715.1非活性F94E非活性NT非活性A91G16.911.6非活性F94Q21.1NT非活性A91L18.514.9非活性P95S非活性NT非活性親本4.17.412.0親本8.7NT21.3表L2裝載樣品IDKD nM huKD (nM) cyKD (nM) mo裝載樣品IDKD nM huKD (nM) cyF95N非活性NT非活性A51N非活性55.1F95Y非活性NT非活性A51L30.111.7F95D20.1NT34.0A51S9.810.6F95H13.8NT0.1A51H非活性3.2F95F0.0NT非活性A51Y39.813.5F95R非活性NT非活性A51K22.026.8親本7.4NT21.1親本9.810.2F95I非活性NT非活性A51M19.516.4F95V非活性NT非活性A51T14.914.7F95A24.4NT35.0A51Q18.215.9F95T21.8NT非活性A51V非活性4.5F95E非活性NT52.9A51E非活性15.2F95Q非活性NT非活性A51P非活性非活性親本8.0NT15.7親本10.710.7A50R21.7非活性NTS52G12.614.4A50V11.815.2NTS52D22.319.0A50D非活性非活性NTS52F44.223.9A50G0.09.6NTS52H0.07.8A50S25.636.3NTS52I18.316.1A50N12.911.7NTS52V24.821.6親本8.411.5NT親本10.810.3A50I12.711.5NTS52P8.1非活性A50Y11.111.5NTS52T4.68.6A50M非活性35.9NTS52E7.815.5A50Q0.93.7NTS52L8.88.1A50E非活性非活性NTS52M7.812.9A50P非活性38.2NTS52K5.917.1親本8.511.1NT親本4.310.3A50L非活性27.3NTS52A5.012.5A50T21.111.7NTS53H22.441.6A50K非活性50.4NTS53F非活性非活性A51F非活性0.9NTS53Y39.230.8A51R10.912.9NTS53V49.524.8A51D非活性非活性NTS53G16.9非活性親本9.110.8NT親本4.610.3表L3裝載樣品IDKD nM huKD (nM) cy裝載樣品IDKD nM huKD (nM) cyS53L非活性非活性A50I12.711.5S53R非活性39.3A50Y11.111.5S53T8.810.7A50M非活性35.9S53A6.813.0A50Q0.93.7S53P非活性14.0A50E非活性非活性S53M非活性非活性A50P非活性38.2親本5.210.3親本8.511.1S53E非活性非活性A50L非活性27.3L54D8.613.8A50T21.111.7L54R6.711.5A50K非活性50.4L54I6.610.5A51F非活性0.9L54F16.414.6A51R10.912.9L54Y10.814.3A51D非活性非活性親本5.69.9親本9.110.8L54H5.811.6A51N非活性55.1L54G12.814.4A51L30.111.7L54N6.111.6A51S9.810.6L54T7.111.1A51H非活性3.2L54K6.59.7A51Y39.813.5L54P7.716.9A51K22.026.8親本5.410.3親本9.810.2L54A6.710.6A51M19.516.4L54M6.29.9A51T14.914.7L54V7.912.7A51Q18.215.9Q55R6.610.3A51V非活性4.5Q55L4.86.2A51E非活性15.2Q55F4.06.6A51P非活性非活性親本5.69.8親本10.710.7A50R21.7非活性S52G12.614.4A50V11.815.2S52D22.319.0A50D非活性非活性S52F44.223.9A50G0.09.6S52H0.07.8A50S25.636.3S52I18.316.1A50N12.911.7S52V24.821.6親本8.411.5親本10.810.3表L4裝載樣品IDKD nM huKD (nM) cy裝載樣品IDKD nM huKD (nM) cyS52P8.1非活性L54A6.710.6S52T4.68.6L54M6.29.9S52E7.815.5L54V7.912.7S52L8.88.1Q55R6.610.3S52M7.812.9Q55L4.86.2S52K5.917.1Q55F4.06.6親本4.310.3親本5.69.8S52A5.012.5Q55D18.219.7S53H22.441.6Q55V18.223.9S53F非活性非活性Q55Y11.210.0S53Y39.230.8Q55H14.415.6S53V49.524.8Q55S17.413.4S53G16.9非活性Q55A21.925.8親本4.610.3親本16.020.8S53L非活性非活性Q55T22.415.0S53R非活性39.3Q55P22.617.3S53T8.810.7Q55E21.323.0S53A6.813.0S56H11.513.8S53P非活性14.0S56I15.111.3S53M非活性非活性S56Y19.317.9親本5.210.3親本18.817.6S53E非活性非活性S56N14.110.0L54D8.613.8S56R14.812.7L54R6.711.5S56G19.916.2L54I6.610.5S56L13.49.7L54F16.414.6S56F19.014.0L54Y10.814.3S56V18.414.0親本5.69.9親本16.617.9L54H5.811.6S56Q20.016.2L54G12.814.4S56T16.220.3L54N6.111.6S56P15.711.4L54T7.111.1S56E22.222.2L54K6.59.7S56A19.217.6L54P7.716.9S56K18.714.9親本5.410.3親本19.521.1表L5裝載樣品IDKD nM huKD (nM) cy裝載樣品IDKD nM huKD (nM) cyF95M0.2NTT97Q15.7F95L0.2NTT97M12.1Y96R非活性NTT97E26.1Y96V非活性NT0.0100.0Y96I非活性NT0.0100.0Y96G非活性NT0.0100.0親本8.3NT親本7.6Y96F12.2NTR24I0.54.6Y96D非活性NTR24N0.44.9Y96S非活性NTR24F0.94.8Y96T非活性NTR24YY96E非活性NTR24S2.66.2Y96L非活性NTR24H3.36.8親本7.5NTN92Y親本3.67.4Y96P22.0NTR24D0.94.0Y96A非活性NTR24K0.75.1Y96M非活性NTR24A1.44.7Y96Q非活性NTR24E1.64.6Y96K非活性NTR24L2.04.5Y96G17.0NTR24P2.54.3親本8.2NTN92Y親本3.47.1T97Y22.0NTR24V1.45.1T97D非活性NTR24Q0.63.6T97F35.3NTR24T1.95.6T97L30.4NTR24M1.64.9T87V10.6NTT25G3.35.8T97I18.3NTT25F7.38.7親本8.5NTN92Y親本3.46.7T97R13.0NTT25R5.17.6T97F31.8NTT25I1.15.1T97N20.0NTT25D6.89.1T97K10.2NTT25H3.96.8T97P非活性NTT25N3.15.3T97A12.9NTT25S4.06.7親本7.3NTN92Y親本3.35.9表L6裝載樣品IDKD nM huKD (nM) cy裝載樣品IDKD nM huKD (nM) cyT25L1.64.7Q27I2.26.6T25Q1.74.7Q27S2.36.7T25P2.85.5Q27L2.46.4T25E2.23.9Q27P2.16.5T25K2.44.1Q27E4.99.4T25V3.36.2Q27K3.07.6N92Y親本3.66.6N92Y親本3.57.6T25A2.85.4Q27M1.96.3T25M1.43.8Q27T2.16.0S26R1.93.6G28I2.56.5S26F1.83.7G28S2.26.0S26D3.65.9G28Y3.98.3S26N3.55.5G28L3.78.7N92Y親本3.96.6N92Y親本3.37.6S26V1.56.2G28R2.46.5S26H1.35.7G28H2.47.7S26I0.32.8G28F3.36.8S26Y1.44.7G28N3.66.9S26L2.46.4G28P3.57.5S26M3.48.2G28V4.69.6N92Y親本3.07.9N92Y親本3.88.2S26P2.05.5G28Q1.02.4S26E2.36.8G28A1.32.8S26K2.66.8G28E1.93.1S26Q2.16.2G28K1.32.7S26T2.97.8G28T2.13.2Q27Y3.29.5G28M2.23.4N92Y親本3.27.7N92Y親本2.33.7Q27V1.75.2I29D0.21.4Q27H1.65.3I29R2.45.3Q27R1.95.9S30T3.73.0Q27N1.95.1S31F6.57.8Q27D3.77.7S31L6.010.8Q27F3.07.2S31P1.93.4N92Y親本3.36.9N92Y親本2.43.1表L7裝載樣品IDKD nM huKD (nM) cy裝載樣品IDKD nM huKD (nM) cyS31M2.36.8S30A2.85.8L33K2.15.1S30E5.09.2L33T0.92.0S30Q5.18.5N92Y親本1.52.8S30K7.08.3I29N0.63.1S30T6.86.6I29L6.28.4S30M9.414.7親本4.88.6親本5.49.0I29H0.22.5S31H6.48.3I29D2.33.5S31N5.67.8I29F1.02.8S31D26.113.9I29G0.93.4S31L9.25.8I29R1.12.8S31R6.318.6I29S12.07.1S31F17.414.4親本5.68.3親本5.68.6I29Y非活性1.5S31Y9.05.3I29E0.04.4S31G8.08.8I29K2.24.4S31I8.912.5I29V2.94.4S31V3.66.4I29Q1.65.5S31T5.58.3I29P2.98.8S31E7.612.1親本5.78.6親本6.08.0I29T100.00.1S31P16.711.2I29A13.710.7S31Q4.49.8S30F6.08.3S31M13.115.1S30D5.48.3S31K12.98.1S30H5.58.4S31A9.211.6S30N7.88.9W32V非活性非活性親本5.88.5親本6.68.2S30Y3.66.3W32S7.411.8S30I9.57.7W32N15.19.7S30G6.29.2W32L18.2非活性S30L7.17.3W32F9.64.9S30V7.38.3空白21.3非活性S30P26.522.3W32H非活性非活性親本5.29.1親本6.37.5表L8裝載樣品IDKD nM huKD (nM) cy裝載樣品IDKD nM huKD (nM) cyW32I0.1非活性A34H4.35.8W32G非活性1.6A34G6.73.8W32R非活性非活性A34L22.046.2W32D非活性2.2A34I14.814.2W32K非活性0.6A34F0.71.0W32P0.02.1A34Q37.738.5親本13.516.7親本15.318.2W32E0.00.1W32A1.50.1W32M15.99.3W32Q10.54.5W32T1.70.6W32K3.013.0親本12.816.9L33S9.48.6L33H8.19.7L33R0.00.2L33D非活性0.2L33F14.212.6L33N11.813.6親本14.217.2L33I16.413.7L33T7.210.8L33K0.40.0L33Q23.918.5L33A10.313.2L33V15.615.3親本13.418.0L33P0.01.5L33M11.913.6A34R非活性非活性A34Y28.649.6A34N25.219.2A34D16.816.3親本14.115.0Light chain library screening. Octet-generated kinetic values measured at >50 nM were classified as "inactive". Positions that provided ≳1.5 to 2.0-fold improvement over both human and cynomolgus macaque were considered for further analysis. The values presented for each species are apparent KDs because these values are calculated based on single binding concentrations collected in a semi-high throughput format. Samples are compared to the parental background from which they were generated, i.e., fonts are normal if the parent is SA01a and italic if the parent is SA60a (SA01a with LC-N92Y).TableL1Load sampleIDKDJKD (nM) cyKD (nM) moLoad sampleIDKDJKD (nM) cyKD (nM) mo Q89V Inactive Inactive Inactive A91D Inactive Inactive Inactive Q89N 20.5 26.7 Inactive Q91 13.7 10.9 Inactive Q89I Inactive Inactive Inactive Q91F 21.0 14.1 26.0 Q89F Inactive Inactive Inactive Q91N Inactive Inactive Inactive Q89D 28.6 14.6 Inactive Q91H 5.1 12.9 16.5 Q89G Inactive Inactive Inactive Q91V 12.6 19.7 Inactive Parent 4.1 8.2 13.7 Parent 4.3 7.7 12.5 Q89M Inactive Inactive Inactive Q91I 40.1 NT Inactive Q89L Inactive Inactive Inactive Q91R Inactive NT Inactive Q89E 7.1 16.8 Inactive Q91S 16.7 NT Inactive Q89A 35.4 44.3 Inactive Q91M 8.7 NT Inactive Q89P Inactive Inactive Inactive Q91E 1.3 NT Inactive Q89T Inactive Inactive Inactive Q91P Inactive NT Inactive Parent 3.8 7.8 13.3 Parent 7.6 NT 25.0 Q90S 13.7 16.9 Inactive Q91 19.7 NT Inactive Q90G 37.8 Inactive Inactive Q91T 16.7 NT Inactive Q90F Inactive Inactive Inactive Q91K 37.8 NT Inactive Q90R 33.6 17.1 Inactive F94G Inactive NT Inactive Q90 Inactive Inactive Inactive F94V Inactive NT Inactive Q90H 17.6 26.8 Inactive F94D Inactive NT Inactive Parent 3.8 7.4 15.3 Parent 10.6 NT 34.9 Q90V Inactive Inactive Inactive F94Y 33.2 NT Inactive Q90N 4.9 6.7 20.1 F94S Inactive NT Inactive Q90A 11.6 14.5 Inactive F94R Inactive NT Inactive Q90L 23.4 10.5 Inactive F94K Inactive NT Inactive Q90M 11.1 13.2 Inactive F94M 23.5 NT Inactive Q90T Inactive Inactive Inactive F94L Inactive NT Inactive Parent 4.0 7.4 10.4 Parent 8.4 NT 16.0 Q90L Inactive Inactive Inactive F94A Inactive NT Inactive Q90E Inactive Inactive Inactive F94T Inactive NT Inactive Q90K 0.1 Inactive Inactive F94P Inactive NT Inactive Q90P 30.7 15.1 Inactive F94E Inactive NT Inactive A91G 16.9 11.6 Inactive QF9 21.1 NT Inactive A91L 18.5 14.9 Inactive P95S Inactive NT Inactive Parent 4.1 7.4 12.0 Parent 8.7 NT 21.3TableL2Load sampleIDKDKD (nM) cyKD (nM) moLoad sampleIDKDKD (nM) cy F95N Inactive NT Inactive A51N Inactive 55.1 F95Y Inactive NT Inactive A51L 30.1 11.7 F95D 20.1 NT 34.0 A51S 9.8 10.6 F95H 13.8 NT 0.1 A51H Inactive 3.2 F95F 0.0 NT Inactive A51Y 39.8 13.5 F95R Inactive NT Inactive A51K 22.0 26.8 Parent 7.4 NT 21.1 Parent 9.8 10.2 F95I Inactive NT Inactive A51M 19.5 16.4 F95V Inactive NT Inactive A51T 14.9 14.7 F95A 24.4 NT 35.0 A51Q 18.2 15.9 F95T 21.8 NT Inactive A51V Inactive 4.5 F95E Inactive NT 52.9 A51E Inactive 15.2 F95Q Inactive NT Inactive A51P Inactive Inactive Parent 8.0 NT 15.7 Parent 10.7 10.7 A50R 21.7 Inactive NT S52G 12.6 14.4 A50V 11.8 15.2 NT S52D 22.3 19.0 A50D Inactive Inactive NT S52F 44.2 23.9 A50G 0.0 9.6 NT S52H 0.0 7.8 A50S 25.6 36.3 NT S52I 18.3 16.1 A50N 12.9 11.7 NT S52V 24.8 21.6 Parent 8.4 11.5 NT Parent 10.8 10.3 A50I 12.7 11.5 NT S52P 8.1 Inactive A50Y 11.1 11.5 NT S52T 4.6 8.6 A50M Inactive 35.9 NT S52E 7.8 15.5 A50Q 0.9 3.7 NT S52L 8.8 8.1 A50E Inactive Inactive NT S52M 7.8 12.9 A50P Inactive 38.2 NT S52K 5.9 17.1 Parent 8.5 11.1 NT Parent 4.3 10.3 A50L Inactive 27.3 NT S52A 5.0 12.5 A50T 21.1 11.7 NT S53H 22.4 41.6 A50K Inactive 50.4 NT S53F Inactive Inactive A51F Inactive 0.9 NT S53Y 39.2 30.8 A51R 10.9 12.9 NT S53V 49.5 24.8 A51D Inactive Inactive NT S53G 16.9 Inactive Parent 9.1 10.8 NT Parent 4.6 10.3TableL3Load sampleIDKDKD (nM) cyLoad sampleIDKDJKD (nM) cy S53L Inactive Inactive A50I 12.7 11.5 S53R Inactive 39.3 A50Y 11.1 11.5 S53T 8.8 10.7 A50M Inactive 35.9 S53A 6.8 13.0 A50Q 0.9 3.7 S53P Inactive 14.0 A50E Inactive Inactive S53M Inactive Inactive A50P Inactive 38.2 Parent 5.2 10.3 Parent 8.5 11.1 S53E Inactive Inactive A50L Inactive 27.3 L54D 8.6 13.8 A50T 21.1 11.7 L54R 6.7 11.5 A50K Inactive 50.4 L54I 6.6 10.5 A51F Inactive 0.9 L54F 16.4 14.6 A51R 10.9 12.9 L54 10.8 14.3 A51D Inactive Inactive Parent 5.6 9.9 Parent 9.1 10.8 L54H 5.8 11.6 A51N Inactive 55.1 L54G 12.8 14.4 A51L 30.1 11.7 L54N 6.1 11.6 A51S 9.8 10.6 L54T 7.1 11.1 A51H Inactive 3.2 L54K 6.5 9.7 A51Y 39.8 13.5 L54P 7.7 16.9 A51K 22.0 26.8 Parent 5.4 10.3 Parent 9.8 10.2 L54A 6.7 10.6 A51M 19.5 16.4 L54M 6.2 9.9 A51T 14.9 14.7 L54V 7.9 12.7 A51Q 18.2 15.9 Q55R 6.6 10.3 A51V Inactive 4.5 Q55L 4.8 6.2 A51E Inactive 15.2 Q55F 4.0 6.6 A51P Inactive Inactive Parent 5.6 9.8 Parent 10.7 10.7 A50R 21.7 Inactive S52G 12.6 14.4 A50V 11.8 15.2 S52D 22.3 19.0 A50D Inactive Inactive S52F 44.2 23.9 A50G 0.0 9.6 S52H 0.0 7.8 A50S 25.6 36.3 S52I 18.3 16.1 A50N 12.9 11.7 S52V 24.8 21.6 Parent 8.4 11.5 Parent 10.8 10.3TableL4Load sampleIDKDKD (nM) cyLoad sampleIDKDKD (nM) cy S52P 8.1 Inactive L54A 6.7 10.6 S52T 4.6 8.6 L54M 6.2 9.9 S52E 7.8 15.5 L54V 7.9 12.7 S52L 8.8 8.1 Q55R 6.6 10.3 S52M 7.8 12.9 Q55L 4.8 6.2 S52K 5.9 17.1 Q55F 4.0 6.6 Parent 4.3 10.3 Parent 5.6 9.8 S52A 5.0 12.5 Q55D 18.2 19.7 S53H 22.4 41.6 Q55V 18.2 23.9 S53F Inactive Inactive Q5Y 11.2 10.0 S53Y 39.2 30.8 Q55H 14.4 15.6 S53V 49.5 24.8 Q55S 17.4 13.4 S53G 16.9 Inactive Q55A 21.9 25.8 Parent 4.6 10.3 Parent 16.0 20.8 S53L Inactive Inactive Q55T 22.4 15.0 S53R Inactive 39.3 Q55P 22.6 17.3 S53T 8.8 10.7 Q55E 21.3 23.0 S53A 6.8 13.0 S56H 11.5 13.8 S53P Inactive 14.0 S56I 15.1 11.3 S53M Inactive Inactive S56 19.3 17.9 Parent 5.2 10.3 Parent 18.8 17.6 S53E Inactive Inactive S56N 14.1 10.0 L54D 8.6 13.8 S56R 14.8 12.7 L54R 6.7 11.5 S56G 19.9 16.2 L54I 6.6 10.5 S56L 13.4 9.7 L54F 16.4 14.6 S56F 19.0 14.0 L54 10.8 14.3 S56V 18.4 14.0 Parent 5.6 9.9 Parent 16.6 17.9 L54H 5.8 11.6 S56Q 20.0 16.2 L54G 12.8 14.4 S56T 16.2 20.3 L54N 6.1 11.6 S56P 15.7 11.4 L54T 7.1 11.1 S56E 22.2 22.2 L54K 6.5 9.7 S56A 19.2 17.6 L54P 7.7 16.9 S56K 18.7 14.9 Parent 5.4 10.3 Parent 19.5 21.1TableL5Load sampleIDKDKD (nM) cyLoad sampleIDKDKD (nM) cy F95M 0.2 NT T97Q 15.7 F95L 0.2 NT T97M 12.1 Y96R Inactive NT T97E 26.1 Y96V Inactive NT 0.0 100.0 Y96I Inactive NT 0.0 100.0 Y96G Inactive NT 0.0 100.0 Parent 8.3 NT Parent 7.6 Y96F 12.2 NTR24I0.54.6 Y96D Inactive NTR24N0.44.9 Y96S Inactive NTR24F0.94.8 Y96T Inactive NTR24 Y96E Inactive NTR24S2.66.2 Y96L Inactive NTR24H3.36.8 Parent 7.5 NTN92Yparent3.67.4 Y96P 22.0 NTR24D0.94.0 Y96A Inactive NTR24K0.75.1 Y96M Inactive NTR24A1.44.7 Y96 Inactive NTR24E1.64.6 Y96K Inactive NTR24L2.04.5 Y96G 17.0 NTR24P2.54.3 Parent 8.2 NTN92Yparent3.47.1 T97Y 22.0 NTR24V1.45.1 T97D Inactive NTR24Q0.63.6 T97F 35.3 NTR24T1.95.6 T97L 30.4 NTR24M1.64.9 T87V 10.6 NTT25G3.35.8 T97I 18.3 NTT25F7.38.7 Parent 8.5 NTN92Yparent3.46.7 T97R 13.0 NTT25R5.17.6 T97F 31.8 NTT25I1.15.1 T97N 20.0 NTT25D6.89.1 T97K 10.2 NTT25H3.96.8 T97P Inactive NTT25N3.15.3 T97A 12.9 NTT25S4.06.7 Parent 7.3 NTN92Yparent3.35.9TableL6Load sampleIDKDKD (nM) cyLoad sampleIDKDKD (nM) cyT25L1.64.7Q27I2.26.6T25Q1.74.7Q27S2.36.7T25P2.85.5Q27L2.46.4T25E2.23.9Q27P2.16.5T25K2.44.1Q27E4.99.4T25V3.36.2Q27K3.07.6N92Yparent3.66.6N92Yparent3.57.6T25A2.85.4Q27M1.96.3T25M1.43.8Q27T2.16.0S26R1.93.6G28I2.56.5S26F1.83.7G28S2.26.0S26D3.65.9G28Y3.98.3S26N3.55.5G28L3.78.7N92Yparent3.96.6N92Yparent3.37.6S26V1.56.2G28R2.46.5S26H1.35.7G28H2.47.7S26I0.32.8G28F3.36.8S261.44.7G28N3.66.9S26L2.46.4G28P3.57.5S26M3.48.2G28V4.69.6N92Yparent3.07.9N92Yparent3.88.2S26P2.05.5G28Q1.02.4S26E2.36.8G28A1.32.8S26K2.66.8G28E1.93.1S26Q2.16.2G28K1.32.7S26T2.97.8G28T2.13.2Q7Y3.29.5G28M2.23.4N92Yparent3.27.7N92Yparent2.33.7Q27V1.75.2I29D0.21.4Q7H1.65.3I29R2.45.3Q27R1.95.9S30T3.73.0Q27N1.95.1S31F6.57.8Q27D3.77.7S31L6.010.8Q27F3.07.2S31P1.93.4N92Yparent3.36.9N92Yparent2.43.1TableL7Load sampleIDKDKD (nM) cyLoad sampleIDKDKD (nM) cyS31M2.36.8 S30A 2.8 5.8L33K2.15.1 S30E 5.0 9.2L33T0.92.0 S30Q 5.1 8.5N92Yparent1.52.8 S30K 7.0 8.3 I29N 0.6 3.1 S30T 6.8 6.6 I29L 6.2 8.4 S30M 9.4 14.7 Parent 4.8 8.6 Parent 5.4 9.0 I29 0.2 2.5 S31H 6.4 8.3 I29D 2.3 3.5 S31N 5.6 7.8 I29F 1.0 2.8 S31D 26.1 13.9 I29G 0.9 3.4 S31L 9.2 5.8 I29R 1.1 2.8 S31R 6.3 18.6 I29S 12.0 7.1 S31F 17.4 14.4 Parent 5.6 8.3 Parent 5.6 8.6 I29 Inactive 1.5 S31Y 9.0 5.3 I29E 0.0 4.4 S31G 8.0 8.8 I29K 2.2 4.4 S31I 8.9 12.5 I29V 2.9 4.4 S31V 3.6 6.4 Q9Q 1.6 5.5 S31T 5.5 8.3 I29P 2.9 8.8 S31E 7.6 12.1 Parent 5.7 8.6 Parent 6.0 8.0 I29T 100.0 0.1 S31P 16.7 11.2 I29A 13.7 10.7 S31Q 4.4 9.8 S30F 6.0 8.3 S31M 13.1 15.1 S30D 5.4 8.3 S31K 12.9 8.1 S30H 5.5 8.4 S31A 9.2 11.6 S30N 7.8 8.9 W32V Inactive Inactive Parent 5.8 8.5 Parent 6.6 8.2 S30Y 3.6 6.3 W32S 7.4 11.8 S30I 9.5 7.7 W32N 15.1 9.7 S30G 6.2 9.2 W32L 18.2 Inactive S30L 7.1 7.3 W32F 9.6 4.9 S30V 7.3 8.3 blank 21.3 Inactive S30P 26.5 22.3 W32H Inactive Inactive Parent 5.2 9.1 Parent 6.3 7.5TableL8Load sampleIDKDKD (nM) cyLoad sampleIDKDKD (nM) cy W32I 0.1 Inactive A34H 4.3 5.8 W32G Inactive 1.6 A34G 6.7 3.8 W32R Inactive Inactive A34L 22.0 46.2 W32D Inactive 2.2 A34I 14.8 14.2 W32K Inactive 0.6 A34F 0.7 1.0 W32P 0.0 2.1 A34Q 37.7 38.5 Parent 13.5 16.7 Parent 15.3 18.2 W32E 0.0 0.1 W32A 1.5 0.1 W32M 15.9 9.3 Wlq 10.5 4.5 W32T 1.7 0.6 W32K 3.0 13.0 Parent 12.8 16.9 L33S 9.4 8.6 L33H 8.1 9.7 L33R 0.0 0.2 L33D Inactive 0.2 L33F 14.2 12.6 L33N 11.8 13.6 Parent 14.2 17.2 L33I 16.4 13.7 L33T 7.2 10.8 L33K 0.4 0.0 L3Q 23.9 18.5 L33A 10.3 13.2 L33V 15.6 15.3 Parent 13.4 18.0 L33P 0.0 1.5 L33M 11.9 13.6 A34R Inactive Inactive A34Y 28.6 49.6 A34N 25.2 19.2 A34D 16.8 16.3 Parent 14.1 15.0

自前述組合庫篩選資料鑑別來自SA01a抗體之CDR共同序列（參見圖15）。共同序列結合變異體概述於表E10及表E11中，其展示該等變異體在CDR內之相對位置及保留或改善結合之各胺基酸變異體。表E10：來自變異體測試之抗體CDR共同序列鏈序列V_HCDR1TFX₁X₂X₃X₄X₅HV_HCDR2IX₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅AQKFQGV_HCDR3X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂DYV_LCDR1X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀WX₃₁AV_LCDR2X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈V_LCDR3QX₃₉X₄₀X₄₁SFPYX₄₂表E11：對於CDR共同序列之胺基酸定義X₁T, A, E, H, I, K, L, M, N, P, Q, R, S, V, YX₂₂F, HX₂N, A, D, E, L, M, Q, R, S, TX₂₃R, A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, VX₃Y, A, D, E, F, I, L, N, Q, R, TX₂₄T, A, E, G, I, L, M, N, P, Q, S, VX₄Y, FX₂₅S, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, YX₅I, D, H, N, S, V, YX₂₆Q, D, E, F, H, I, K, L, M, N, P, R, S, T, V, YX₆I, L, QX₂₇G, A, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, YX₇N, A, SX₂₈I, L, VX₈P, A, L, N, VX₂₉S, A, D, E, F, G, H, K, N, Q, T, YX₉N, A, E, F, G, H, K, L, M, P, Q, R, S, T, V, YX₃₀S, A, E, G, H, M, N, P, Q, R, T, V,X₁₀G, A, E, F, I, K, Q, R, T, YX₃₁L, A, H, I, K, M, N, S, T, VX₁₁G, E, H, Q, T, YX₃₂A, G, YX₁₂S, A, E, F, H, I, N, T, VX₃₃A, SX₁₃T, I, VX₃₄S, A, G, H, TX₁₄S, A, F, I, M, Y, YX₃₅S, TX₁₅Y, F, I, L,X₃₆L, A, H, I, K, M, N, R, T, VX₁₆G, TX₃₇Q, D, E, F, L, P, R, S, T, V, YX₁₇I, K, L, R, SX₃₈S, A, E, F, G, H, I, K, L, N, P, Q, R, T, V, YX₁₈A, I, K, L, M, R, TX₃₉Q, NX₁₉M, LX₄₀A, H, MX₂₀A, D, G, H, N, SX₄₁N, YX₂₁L, IX₄₂T, K, V實例2MC38同基因型腫瘤模型The consensus sequences of the CDRs from the SA01a antibody were identified from the aforementioned library screening data (seeFigure15 ). The consensus sequence binding variants are summarized inTablesE10 andE11 , which show the relative positions of the variants within the CDRs and the individual amino acid variantsthat retain or improve binding.TableE10 : AntibodyCDRconsensus sequencesfrom variant testing Chain sequence_VH CDR1 TFX₁ X₂ X₃ X₄ X₅ H_VH CDR2 IX₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ AQKFQG_VH CDR3 X₁₆ X₁₇ X₁₈ X₁₉ X₂₀ X₂₁ X₂₂ DY V_L CDR1 X₂₃ X₂₄ X₂₅ X₂₆ X₂₇ X₂₈ X₂₉ X₃₀ WX₃₁ A V_L CDR2 X₃₂ X₃₃ X₃₄ X₃₅ X₃₆ X₃₇ X₃₈ V_L CDR3 QX₃₉ X₄₀ X₄₁ SFPYX₄₂TableE11: Amino acid definitions forCDRconsensus sequences X₁ T, A, E, H, I, K, L, M, N, P, Q, R, S, V, Y X₂₂ F, H X₂ N, A, D, E, L, M, Q, R, S, T X₂₃ R, A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V X₃ Y, A, D, E, F, I, L, N, Q, R, T X₂₄ T, A, E, G, I, L, M, N, P, Q, S, V X₄ Y, F X₂₅ S, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, Y X₅ I, D, H, N, S, V, Y X₂₆ Q, D, E, F, H, I, K, L, M, N, P, R, S, T, V, Y X₆ I, L, Q X₂₇ G, A, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, Y X₇ N, A, S X₂₈ I, L, V X₈ P, A, L, N, V X₂₉ S, A, D, E, F, G, H, K, N, Q, T, Y X₉ N, A, E, F, G, H, K, L, M, P, Q, R, S, T, V, Y X₃₀ S, A, E, G, H, M, N, P, Q, R, T, V, X₁₀ G, A, E, F, I, K, Q, R, T, Y X₃₁ L, A, H, I, K, M, N, S, T, V X₁₁ G, E, H, Q, T, Y X₃₂ A, G, Y X₁₂ S, A, E, F, H, I, N, T, V X₃₃ A, S X₁₃ T, I, V X₃₄ S, A, G, H, T X₁₄ S, A, F, I, M, Y, Y X₃₅ S, T X₁₅ Y, F, I, L, X₃₆ L, A, H, I, K, M, N, R, T, V X₁₆ G, T X₃₇ Q, D, E, F, L, P, R, S, T, V, Y X₁₇ I, K, L, R, S X₃₈ S, A, E, F, G, H, I, K, L, N, P, Q, R, T, V, Y X₁₈ A, I, K, L, M, R, T X₃₉ Q, N X₁₉ M, L X₄₀ A, H, M X₂₀ A, D, G, H, N, S_X41 N, Y X₂₁ L, I X₄₂ T, K, VExample2MC38syngeneic tumor model

進行實驗以在MC38同基因型腫瘤模型中測試單獨或與抗PD-1抗體及IL-18組合的抗IL18BP抗體之活性。此處，將5×10⁵個MC38細胞植入至C57BL/6小鼠之後側腹中。腫瘤達到32 - 44 mm³之尺寸後，將小鼠隨機分為6個獨立組（n = 10），其中各組平均腫瘤尺寸為36 mm³。每三天（q3d）藉由腹膜內（i.p.）注射施以處理，對於研究時長期間存活之動物，至多7個總劑量。出於多種原因自研究移除動物，包括如研究指南所概述，達到所准許之尺寸限制或觀測到瀕死狀態。處理包括PBS媒劑對照、抗mPD-1（5 mg/kg）、抗mPD-1（5 mg/kg）+ SA0051d（10 mg/kg）、抗mPD-1（5 mg/kg）+ mIL-18（0.32 mg/kg）、mIL18（0.32 mg/kg）+ SA0051d（10 mg/kg）或抗mPD-1（5 mg/kg）+ mIL-18（0.32 mg/kg）+ SA0051d（10 mg/kg）。藉由測徑規量測來一週量測腫瘤兩次且平均化以計算各組之平均腫瘤生長抑制。Experiments were conducted to test the activity of anti-IL18BP antibodies alone or in combination with anti-PD-1 antibodies and IL-18 in the MC38 syngeneic tumor model. Here, 5×10⁵ MC38 cells were implanted into the flank of C57BL/6 mice. After tumors reached a size of 32 - 44 mm³ , mice were randomized into 6 independent groups (n = 10), with an average tumor size of 36 mm³ in each group. Treatments were administered every three days (q3d) by intraperitoneal (ip) injection, up to 7 total doses for animals that survived the duration of the study. Animals were removed from the study for a variety of reasons, including reaching the permitted size limit or observation of moribund conditions, as outlined in the study guidelines. Treatments included PBS vehicle control, anti-mPD-1 (5 mg/kg), anti-mPD-1 (5 mg/kg) + SA0051d (10 mg/kg), anti-mPD-1 (5 mg/kg) + mIL-18 (0.32 mg/kg), mIL18 (0.32 mg/kg) + SA0051d (10 mg/kg), or anti-mPD-1 (5 mg/kg) + mIL-18 (0.32 mg/kg) + SA0051d (10 mg/kg). Tumors were measured twice a week by caliper measurement and averaged to calculate the mean tumor growth inhibition for each group.

如圖16中所示，相較於單獨的抗小鼠PD-1或抗小鼠PD-1 + mIL-18，結果展示利用小鼠PD-1 +抗小鼠IL18BP（SA51d）+ mIL-18之功效改善。As shown in Figure16 , the results showed improved efficacy using mouse PD-1 + anti-mouse IL18BP (SA51d) + mIL-18 compared to anti-mouse PD-1 or anti-mouse PD-1 + mIL-18 alone.

為了進一步理解抗IL18BP抗體在MC38模型中之作用，收集研究結束時採集之腫瘤且藉由RNA及蛋白質分析來分析免疫相關分子之水平。用抗IL18BP處理之動物展現出腫瘤部位處之免疫反應增強的證據。在用抗IL18BP處理之動物中促炎性細胞介素IL-18及干擾素-γ之水平增加（圖17）。藉由RT-qPCR進行之分析證實，在用抗IL18BP及IL-18處理後，NCR1（NK細胞活性之標記）之表現及顆粒酶B（活化免疫細胞之標記）之表現均有所增加。與抗PD-1療法組合之情況下觀測到進一步增加，支持經處理動物中之免疫反應增強（圖18）。To further understand the role of the anti-IL18BP antibody in the MC38 model, tumors harvested at the end of the study were collected and analyzed for levels of immune-related molecules by RNA and protein analysis. Animals treated with anti-IL18BP showed evidence of enhanced immune response at the tumor site. Levels of the proinflammatory interleukins IL-18 and interferon-γ were increased in animals treated with anti-IL18BP (Figure17 ). Analysis by RT-qPCR confirmed that the expression of NCR1 (a marker of NK cell activity) and granzyme B (a marker of activated immune cells) were increased after treatment with anti-IL18BP and IL-18. Further increases were observed in combination with anti-PD-1 therapy, supporting the enhanced immune response in the treated animals (Figure18 ).

為測試經處理動物中反應之持久性及持久免疫性之存在，將對處理展示完全反應之所有動物（n = 12）靜置24天且接著用皮下植入至對側後側腹中之1×10⁶個MC38細胞進行再攻擊。藉由測徑規量測來每週評估腫瘤生長兩次，持續總共24天。如圖19A-圖19B中所示，在再植入腫瘤細胞時，對療法具有完全反應之12隻動物中的11隻對腫瘤再生具有抗性。同時向對照動物植入來自相同批次之MC38腫瘤細胞。腫瘤在所有對照動物中生長。此等結果展示對療法有反應之小鼠對再攻擊免疫，表明其已對腫瘤產生持久免疫性。實例3基於抗人類IL-18BP下游細胞之活性To test the persistence of the response in treated animals and the presence of persistent immunity, all animals (n = 12) that showed a complete response to the treatment were left stationary for 24 days and then re-challenged with 1×10⁶ MC38 cells implanted subcutaneously into the contralateral posterior abdomen. Tumor growth was assessed twice a week by caliper measurement for a total of 24 days.As shown inFigures19A-19B , 11 of the 12 animals that had a complete response to the treatment were resistant to tumor regeneration when tumor cells were re-implanted. MC38 tumor cells from the same batch were implanted into control animals at the same time. Tumors grew in all control animals. These results show that mice that responded to therapy were immune to re-challenge, indicating that they had developed long-lasting immunity to the tumor.Example3Anti-humanIL-18BPdownstream cell activity

測試抗人類IL-18BP允許IL-18誘導自人類PBMC產生額外細胞介素的能力。如實例1中所述分離且測試人類PBMC。對於多種趨化介素之同時分析，使用LEGENDplex人類必需免疫反應集合多重分析（BioLegend目錄號740930）。將樣品在分析緩衝液中以1:50稀釋且根據製造商說明書進行量測，不同之處在於在各次培育期間，將所有樣品及試劑之體積減半且藉由在方案之各步驟處移液以及在450 rpm下振盪來混合樣品。緊接著將樣品在NovoCyte流式細胞儀（Agilent Technologies）上運行且藉由LEGENDplex軟體（BioLegend）分析。測試用媒劑對照、IL-12（1 ng/mL）、IL-18（2 ng/mL）、IL-12 + IL-18或IL-12 + IL-18 +抗人類IL-18BP mAb之處理72 h。添加抗IL-18BP mAb引起PBMC產生之CXCL10及CCL2增加（圖20）。實例4額外小鼠腫瘤模型Anti-human IL-18BP was tested for its ability to allow IL-18 to induce the production of additional interleukins from human PBMCs. Human PBMCs were isolated and tested as described in Example 1. For simultaneous analysis of multiple interleukins, the LEGENDplex Human Essential Immune Response Panel Multiplex Assay (BioLegend Catalog No. 740930) was used. Samples were diluted 1:50 in assay buffer and measured according to the manufacturer's instructions, except that during each incubation, the volume of all samples and reagents was halved and the samples were mixed by pipetting at each step of the protocol and shaking at 450 rpm. Samples were then run on a NovoCyte flow cytometer (Agilent Technologies) and analyzed by LEGENDplex software (BioLegend). Treatments with vehicle control, IL-12 (1 ng/mL), IL-18 (2 ng/mL), IL-12 + IL-18, or IL-12 + IL-18 + anti-human IL-18BP mAb were tested for 72 h. Addition of anti-IL-18BP mAb resulted in increased production of CXCL10 and CCL2 by PBMCs (FIG.20 ).Example4Additional Mouse Tumor Model

進行實驗以測試抗IL-18BP抗體在額外動物模型中之活性。在EMT6同基因型腫瘤模型中，並行及組合地測試SA0051d與抗PD-1抗體。Experiments were performed to test the activity of anti-IL-18BP antibodies in additional animal models. SA0051d was tested in parallel and in combination with anti-PD-1 antibodies in the EMT6 syngeneic tumor model.

將EMT6細胞皮下植入至Balb/c小鼠中且使其形成腫瘤。將1×10⁶個EMT6細胞植入至小鼠之乳腺脂肪墊中。腫瘤達到20-80 mm³之尺寸後，將小鼠區組隨機分為4個獨立組（n = 8）。藉由腹膜內（i.p.）注射施以處理。如研究指南所概述，在達到所准許之腫瘤尺寸限制時或在觀測到腫瘤潰瘍時，自研究移除動物。處理包括PBS媒劑對照、抗PD-1（5 mg/kg，q2w×3）、SA0051d（10 mg/kg q2w）或抗PD-1（5 mg/kg q2w×3）+ SA51d（10 mg/kg q2w）。每日檢測動物，且藉由測徑規量測一週量測腫瘤兩次。如圖21B-圖21E中關於個別動物所示，隨時間推移量測腫瘤尺寸。使用GraphPad Prism軟體進行存活率分析以產生卡普蘭-邁爾（Kaplan-Meier）存活率曲線（圖21A）。EMT6 cells were implanted subcutaneously into Balb/c mice and allowed to form tumors. 1×10⁶ EMT6 cells were implanted into the mammary fat pad of mice. After the tumors reached a size of 20-80 mm³ , the mice were randomly divided into 4 independent groups (n = 8). Treatment was administered by intraperitoneal (ip) injection. As outlined in the research guidelines, animals were removed from the study when the permitted tumor size limit was reached or when tumor ulcers were observed. Treatments included PBS vehicle control, anti-PD-1 (5 mg/kg, q2w×3), SA0051d (10 mg/kg q2w), or anti-PD-1 (5 mg/kg q2w×3) + SA51d (10 mg/kg q2w). Animals were examined daily, and tumors were measured twice a week by caliper measurement. Tumor size was measured over timeas shown in Figures21B-21Efor individual animals. Survival analysis was performed using GraphPad Prism software to generate Kaplan-Meier survival curves (Figure21A ).

結果證實，相比於對照，用SA0051d處理會提高動物存活率（p＜0.0001），然而用單獨的抗PD-1處理並未提高。用SA0051d與抗PD-1之組合處理進一步提高存活率且在一些動物中引起腫瘤消退。此實例證實抗IL-18BP具有單一藥劑抗腫瘤活性，其與用抗PD-1之處理相容。Results demonstrated that treatment with SA0051d increased animal survival compared to controls (p<0.0001), whereas treatment with anti-PD-1 alone did not. Combination treatment with SA0051d and anti-PD-1 further increased survival and caused tumor regression in some animals. This example demonstrates that anti-IL-18BP has single-agent anti-tumor activity that is compatible with treatment with anti-PD-1.

在額外實驗中，亦在E0771小鼠同基因型腫瘤模型中測試抗IL-18BP抗體之活性。將1×10⁶個E0771細胞植入至C57BL/6小鼠之乳腺脂肪墊中。腫瘤達到50-100 mm³之尺寸後，將小鼠隨機分為4個獨立組（n = 8）。藉由腹膜內（i.p.）注射施以處理。處理包括PBS媒劑對照、抗mPD-1（5 mg/kg）、SA0051d（10 mg/kg）或抗mPD-1（5 mg/kg）+ SA0051d（10 mg/kg）。所有動物均以q3d腹膜內給藥。藉由測徑規量測來一週量測腫瘤兩次且平均化以計算各組之平均腫瘤生長抑制。在各時間點藉由GraphPad Prism軟體（9.4版），使用2因子變異數分析，之後使用杜凱氏多重比較檢定（Tukey's multiple comparison test）進行統計分析。計算各組之平均腫瘤尺寸且將其繪製成圖，如圖22A中所示。與媒劑對照組（p = 0.162）或抗mPD-1處理組（p = 0.0004）相比，SA0051d顯著地抑制腫瘤生長。與抗mPD-1處理組（p = 0.0034）相比，SA0051d +抗mPD-1之組合亦顯著抑制腫瘤生長。個別動物中之腫瘤尺寸顯示於圖22B-圖22E中。In an additional experiment, the activity of anti-IL-18BP antibodies was also tested in the E0771 mouse syngeneic tumor model. 1×10⁶ E0771 cells were implanted into the mammary fat pad of C57BL/6 mice. After tumors reached a size of 50-100 mm³ , mice were randomized into 4 independent groups (n = 8). Treatments were administered by intraperitoneal (ip) injection. Treatments included PBS vehicle control, anti-mPD-1 (5 mg/kg), SA0051d (10 mg/kg), or anti-mPD-1 (5 mg/kg) + SA0051d (10 mg/kg). All animals were dosed ip q3d. Tumors were measured twice a week by caliper measurement and averaged to calculate the average tumor growth inhibition for each group. Statistical analysis was performed at each time point by GraphPad Prism software (version 9.4) using a 2-way variance analysis followed by Tukey's multiple comparison test. The mean tumor size for each group was calculated and plottedas shown inFIG22A . SA0051d significantly inhibited tumor growth compared to the vehicle control group (p = 0.162) or the anti-mPD-1 treated group (p = 0.0004). The combination of SA0051d + anti-mPD-1 also significantly inhibited tumor growth compared to the anti-mPD-1 treated group (p = 0.0034). The tumor size in individual animals is showninFIG22B-FIG22E .

來自此研究之結果進一步證實，使用抗IL-18BP抗體SA0051d之單一藥劑處理具有抗腫瘤活性。亦證實與抗PD1處理之組合的抗腫瘤活性。Results from this study further demonstrate that single agent treatment with the anti-IL-18BP antibody SA0051d has anti-tumor activity. Anti-tumor activity in combination with anti-PD1 treatment was also demonstrated.

圖1展示本發明之抗體的譜系學。FIG1 shows the phylogeny ofthe antibodies of the present invention.

圖2展示食蟹獼猴（SEQ ID NO: 373）、人類（SEQ ID NO: 371）及小鼠（SEQ ID NO: 375）IL-18BP直系同源物之比對。信號肽（不存在於成熟肽中）為加下劃線的。用向下箭頭（↓）標記之四個區域指示人類及小鼠中一致但在食蟹獼猴IL-18BP中不同的胺基酸。Figure2 shows an alignment of cynomolgus macaque (SEQ ID NO: 373), human (SEQ ID NO: 371), and mouse (SEQ ID NO: 375) IL-18BP orthologs. The signal peptide (not present in the mature peptide) is underlined. The four regions marked with downward arrows (↓) indicate amino acids that are consistent in human and mouse but different in cynomolgus macaque IL-18BP.

圖3A-圖3B展示低IL-18之設計。圖3A展示低IL-18表現卡匣之示意圖。自N'端至C'端之基因中之標誌包括：黏骨素信號肽、人類IL-18編碼區、間雜有菸草蝕刻病毒（TEV）蛋白酶裂解位點之可撓性gly-ser連接子、人類IL-18BP編碼序列及6x HIS標籤。圖3B展示人類IL-18與鼠痘病毒（Ectromelia virus）IL-18BP（如所指示）複合之晶體結構衍生模型的視圖。IL-18BP N端及IL-18 C端用方框及箭頭指示。Figures3A-3B show the design of low IL-18.Figure3A shows a schematic diagram of the low IL-18 expression cassette. The landmarks in the gene from N' to C' terminus include: mycosin signal peptide, human IL-18 coding region, flexible gly-ser linker interspersed with tobacco etch virus (TEV) protease cleavage site, human IL-18BP coding sequence and 6x HIS tag.Figure3B shows a view of the crystal structure derived model of human IL-18 in complex with Ectromelia virus IL-18BP (as indicated). The IL-18BP N-terminus and IL-18 C-terminus are indicated by boxes and arrows.

圖4展示在人類KG-1細胞中由抗IL-18BP mAb對IFNγ表現的除抑制作用。mAb1191（R&D Systems）為市售的小鼠抗人類IL-18BP中和抗體，其用作此實驗中之陽性對照（目錄號mab1191）。Figure4 shows the de-inhibition of IFNγ expression by anti-IL-18BP mAb in human KG-1 cells. mAb1191 (R&D Systems) is a commercially available mouse anti-human IL-18BP neutralizing antibody that was used as a positive control in this experiment (Catalog No. mab1191).

圖5展示在鼠類IL-18報導子分析中測試之抗mIL-18BP mAb。Figure5 shows anti-mIL-18BP mAbs tested in the murine IL-18 reporter assay.

圖6展示在小鼠脾細胞分析中測試之抗mIL-18BP mAb。不存在明確的上限平穩段；因此，IC50值不可確定。Figure6 shows anti-mIL-18BP mAbs tested in the mouse spleen cell assay. There is no clear upper plateau; therefore, an IC50 value could not be determined.

圖7A-圖7B展示在已建立的MC38結腸腫瘤中mIL-18與SA51d（SA0051d）之組合的抗腫瘤活性。圖7A展示皮下植入至C57BL/6小鼠中且使其形成腫瘤之MC38細胞的腫瘤生長。接著將50隻小鼠隨機分為5組（n = 10）且用PBS媒劑對照、mIL-18、SA51d，或mIL-18 + SA51d之組合處理。每3天向動物給藥至多7次總劑量，如由箭頭所指示。計算各組之平均腫瘤大小且圖示，且與對照相比，mIL-18 + SA0051d之組合能夠顯著抑制生長。圖7B展示與媒劑對照相比，截至第15天，mIL-18 + SA51d之組合能夠顯著抑制生長（p＜0.0001）。Figures7A-7Bshow the anti-tumor activity of the combination of mIL-18 and SA51d (SA0051d) in established MC38 colon tumors.Figure7A shows tumor growth of MC38 cells implanted subcutaneously into C57BL/6 mice and allowed to form tumors. Then 50 mice were randomly divided into 5 groups (n = 10) and treated with PBS vehicle control, mIL-18, SA51d, or a combination of mIL-18 + SA51d. Animals were dosed up to 7 total doses every 3 days, as indicated by arrows. The average tumor size of each group was calculated and shown, and the combination of mIL-18 + SA0051d was able to significantly inhibit growth compared to the control.FIG.7B shows that the combination of mIL-18 + SA51d was able to significantly inhibit growth compared to vehicle control by day 15 (p < 0.0001).

圖8A-圖8B展示mAb在IL-18介導之HEK293報導子分析中之評估。圖8A展示mAb與IL-18BP之預培育；圖8B展示IL-18與IL-18BP之預培育。Figures8A-8B show the evaluation of mAbs in IL-18- mediated HEK293 reporter assay.Figure8A shows the pre-incubation of mAbs with IL-18BP;Figure8B shows the pre-incubation of IL-18 with IL-18BP.

圖9展示mAb對KG-1細胞中IFNγ分泌之影響。單獨的複合物指示在不存在IL-18的情況下添加IL-18BP及mAb。Figure9 shows the effect of mAb on IFNγ secretion in KG-1 cells. Individual complexes indicate the addition of IL-18BP and mAb in the absence of IL-18.

圖10A-圖10B展示高親和力mAb誘導人類（10A）及食蟹獼猴（10B）PBMC中之IFNγ反應。IFNγ產生回應於mAb對IL-18BP之抑制的劑量反應。將2×10⁵個PBMC/孔與IL-12以1 ng/mL及IL-18以2 ng/mL在增加濃度之各mAb存在下一起培育。在48 h時獲取上清液等分試樣且藉由ELISA分析IFNγ。Figures10A-10Bshow that high affinity mAbs induce IFNγ responses in human (10A ) and cynomolgus macaque (10B ) PBMCs. IFNγ is produced in a dose-responsive manner in response to mAb inhibition of IL-18BP. 2×10⁵ PBMCs/well were incubated with IL-12 at 1 ng/mL and IL-18 at 2 ng/mL in the presence of increasing concentrations of each mAb. Supernatant aliquots were harvested at 48 h and analyzed for IFNγ by ELISA.

圖11A-圖11B展示藉由高親和力mAb自預複合IL-18/IL-18BP誘導PBMC中之IFNγ反應。Figures11A-11Bshow the induction of IFNγ responses in PBMCs by high affinity mAb self-complexed with IL-18/IL-18BP.

圖12展示IL-18/IL-18BP複合物之共晶體結構模型。分別以較暗字體（V75M、I97M、R113Q、V153M）及較淺字體（V47I、T99A、K115R、F170Y）展示由人類及小鼠共有但未由食蟹獼猴共有的四個差異各自在IL-18BP及IL-18中之位置。應注意I98M及V153M鄰近且存在於結合界面處，而V75M及R113Q不接近活性位點。Figure12 shows the co-crystal structure model of the IL-18/IL-18BP complex. The positions of the four differences shared by humans and mice but not by cynomolgus macaques in IL-18BP and IL-18 are shown in darker font (V75M, I97M, R113Q, V153M) and lighter font (V47I, T99A, K115R, F170Y), respectively. It should be noted that I98M and V153M are adjacent and present at the binding interface, while V75M and R113Q are not close to the active site.

圖13展示mAb與具有兩個直系同源食蟹獼猴胺基酸置換之人類IL-18BP的結合。所量測之動力學的品質在相關表中對於不結合概述為「-」，且對於不同程度之結合，概述為1至4 +。Figure13 shows the binding of mAbs to human IL-18BP with two orthologous cynomolgus monkey amino acid substitutions. The quality of the measured kinetics is summarized in the associated table as "-" for no binding and 1 to 4+ for varying degrees of binding.

圖14展示如藉由XL-MS質譜分析測定之SA64a之構形抗原決定基。IL-18BP之部分序列展示為具有與指示為抗原決定基之SA64a交聯的殘基。Figure14 shows the conformational epitopes of SA64a as determined by XL-MS mass spectrometry. A partial sequence of IL-18BP is shown with residues cross-linked to SA64a indicated as epitopes.

圖15展示來自組合篩選之SA01a抗體之胺基酸變異體，其保留或改善與IL-18BP之結合（x =對於hu、cy及mo之良好結合；hc =對於hu及cy但不對於mo之良好結合；hm =對於hu及mo但不對於cy之良好結合；cm =對於cy及mo但不對於hu之良好結合；m =對於mo但不對於hu或cy之良好結合）。Figure15 shows amino acid variants of the SA01a antibody from the combinatorial screening that retain or improve binding to IL-18BP (x = good binding to hu, cy, and mo; hc = good binding to hu and cy but not mo; hm = good binding to hu and mo but not cy; cm = good binding to cy and mo but not hu; m = good binding to mo but not hu or cy).

圖16展示抗小鼠IL-18BP抗體在MC38同基因型腫瘤模型中之功效的蛛網圖。結果證實相比於單獨的抗小鼠PD-1或抗小鼠PD-1 + mIL-18，使用抗小鼠PD-1 +抗小鼠IL-18BP（SA51d，亦為SA0051d）+ mIL-18之顯著改善之功效。Figure16 shows a spider graph of the efficacy of anti-mouse IL-18BP antibodies in the MC38 syngeneic tumor model. The results demonstrate significantly improved efficacy using anti-mouse PD-1 + anti-mouse IL-18BP (SA51d, also SA0051d) + mIL-18 compared to anti-mouse PD-1 or anti-mouse PD-1 + mIL-18 alone.

圖17展示相比於經媒劑處理之動物，經抗IL-18BP處理之小鼠的MC38腫瘤中之IL-18（左）及IFNγ（右）水平。在功效研究之終點收集腫瘤且藉由ELISA評估促炎性細胞介素。Figure17 shows IL-18 (left) and IFNγ (right) levels in MC38 tumors of mice treated with anti-IL-18BP compared to vehicle-treated animals. Tumors were harvested at the end of the efficacy study and pro-inflammatory interleukins were assessed by ELISA.

圖18展示在功效研究之終點收集之MC38腫瘤中的相對NK細胞標記（左）及顆粒酶B（右）表現，如藉由qPCR所評估。NK細胞數目及活性之標記回應於抗IL-18BP而增加且在抗PD-1組合療法下進一步增加。Figure18 shows relative NK cell markers (left) and granzyme B (right) expression in MC38 tumors collected at the endpoint of the efficacy study, as assessed by qPCR. Markers of NK cell number and activity increased in response to anti-IL-18BP and were further increased under anti-PD-1 combination therapy.

圖19A-圖19B展示抗小鼠IL-18BP抗體在MC38同基因型腫瘤再攻擊模型中之功效。結果表明在再攻擊後12隻動物中有11隻出現持久反應。Figures19A-19Bshow the efficacy of anti-mouse IL-18BP antibodies in the MC38 syngeneic tumor rechallenge model. The results show that 11 of 12 animals had durable responses after rechallenge.

圖20展示回應於高親和力mAb之在人類PBMC中之CXCL10（左）及CCL2（右）產生。在添加IL-12+ IL-18後分泌出CXCL10及CCL2，且在添加mAb情況下得到增強，如藉由ELISA所評估。Figure20 shows CXCL10 (left) and CCL2 (right) production in human PBMCs in response to high affinity mAbs. CXCL10 and CCL2 were secreted after the addition of IL-12 + IL-18 and were enhanced with the addition of mAbs as assessed by ELISA.

圖21A-圖21E展示抗小鼠IL-18BP抗體SA0051d在EMT6小鼠同基因型腫瘤模型中之功效。圖21A展示經抗IL-18BP抗體SA0051d處理之攜帶EMT6腫瘤之小鼠的存活得到顯著增強，且此在與抗小鼠PD-1抗體組合的情況下得到進一步改善。圖21B-圖21E展示監測各動物中之腫瘤生長的蛛網圖。用SA0051d處理減少腫瘤生長，且與抗PD-1組合之情況下，在一些動物中觀測到腫瘤消退。Figure21A-Figure21E shows the efficacy of anti-mouse IL-18BP antibody SA0051d in the EMT6 mouse syngeneic tumor model. Figure 21A shows that the survival of mice carrying EMT6 tumors treated with anti-IL-18BP antibody SA0051d is significantly enhanced, and this is further improved in combination with anti-mouse PD-1 antibodies. Figure 21B-Figure 21E shows spider graphs monitoring tumor growth in each animal. Treatment with SA0051d reduces tumor growth, and tumor regression is observed in some animals in combination with anti-PD-1.

圖22A-圖22E展示抗小鼠IL-18BP抗體SA0051d在E0771小鼠同基因型腫瘤模型中之功效。圖22A展示各組中之平均腫瘤生長，且圖22B-圖22E展示監測各動物中之腫瘤生長的蛛網圖。用SA0051d處理顯著地減少腫瘤生長，用SA0051d與抗小鼠PD-1抗體之組合處理亦如此。Figures22A-22Eshow the efficacy of the anti-mouse IL-18BP antibody SA0051d in the E0771 mouse syngeneic tumor model. Figure 22A shows the average tumor growth in each group, and Figures 22B-22E show spider graphs monitoring tumor growth in each animal. Treatment with SA0051d significantly reduced tumor growth, as did treatment with a combination of SA0051d and an anti-mouse PD-1 antibody.

TW202430560A_113100655_SEQL.xmlTW202430560A_113100655_SEQL.xml

Claims (61)

Translated fromChinese

一種經分離之抗體或其抗原結合片段，其與介白素-18結合蛋白（IL-18BP）結合，其中該至少一種抗體或其抗原結合片段包含： 重鏈可變區（V_H），其包含選自表A1之互補決定區V_HCDR1、V_HCDR2及V_HCDR3序列及其與IL-18BP特異性結合之變異體；以及 輕鏈可變區（V_L），其包含選自表A1之互補決定區V_LCDR1、V_LCDR2及V_LCDR3序列及其與IL-18BP特異性結合之變異體。An isolated antibody or antigen-binding fragment thereof, which binds to interleukin-18 binding protein (IL-18BP), wherein the at least one antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (_VH ) comprising complementary determining region_VH CDR1,_VH CDR2 and_VH CDR3 sequences selected fromTableA1 and variants thereof that specifically bind to IL-18BP; and a light chain variable region (_VL ) comprising complementary determining region_VL CDR1,_VL CDR2 and_VL CDR3 sequences selected fromTableA1 and variants thereof that specifically bind to IL-18BP.如請求項1之經分離之抗體或其抗原結合片段，其中： 該VHCDR1、VHCDR2及VHCDR3序列分別包含TFX₁X₂X₃X₄X₅H、IX₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅AQKFQG及X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂DY，且該VLCDR1、VLCDR2及VLCDR3序列分別包含X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀WX₃₁A、X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈及QX₃₉X₄₀X₄₁SFPYX₄₂（關於「X」殘基之定義，參見表E11）； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 1-3，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 4-6； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 25-27，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 28-30； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 31-33，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 34-36； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 34-39，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 40-42； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 43-45，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 46-48； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 49-51，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 52-54； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 55-57，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 58-60； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 61-63，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 64-66； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 67-69，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 70-72； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 109-111，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 112-114； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 115-117，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 118-120； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 121-123，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 124-126； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 127-129，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 130-132； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 133-135，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 136-138； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 139-141，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 142-144； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 145-147，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 148-150； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 151-153，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 154-156； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 157-159，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 160-162； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 163-165，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 166-168； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 169-171，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 172-174； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 175-177，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 178-180； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 181-183，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 184-186； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 187-189，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 190-192； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 193-195，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 196-198； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 199-201，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 202-204； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 205-207，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 208-210； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 211-213，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 214-216； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 217-219，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 220-222； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 223-225，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 226-228； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 229-231，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 232-234； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 235-237，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 238-240； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 241-243，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 244-246； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 247-249，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 250-252； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 253-255，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 256-258； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 265-267，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 268-270；或 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 271-273，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 274-276。The isolated antibody or antigen-binding fragment thereof of claim 1, wherein: the VHCDR1, VHCDR2 and VHCDR3 sequences comprise_TFX1 X₂ X₃ X₄ X₅ H, IX₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ AQKFQG and X₁₆ X₁₇ X₁₈ X₁₉ X₂₀ X₂₁ X₂₂ DY, respectively; and the VLCDR1, VLCDR2 and VLCDR3 sequences comprise X₂₃ X₂₄ X₂₅ X 26 X₂₇ X₂₈ X_{29X 30WX31} A, X₃₂ X₃₃ X₃₄ X₃₅ X₃₆ X₃₇ X₃₈ and QX₃₉ X₄₀ X₄₁ SFPYX₄₂ (For the definition of "X" residue,see TableE11 ); the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 1-3, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 4-6, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 25-27, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 28-30, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 31-33, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 34-36, respectively; the_VH CDR1,_VH CDR2 and_VH the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 49-50, and the_VL CDR1,_VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 51-52, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 53-54, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 55-56, respectively; the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 57-58, respectively; the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 59-60, respectively; the_VH CDR1,_VHCDR2 and_VH CDR3 sequences comprise SEQ ID_NOs : 61-62_, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 58-60; said_{VH CDR1, VH CDR2 and VH CDR3 sequences comprise SEQ ID NOs: 61-63, respectively, and said VL} CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 64-66, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 67-69, respectively, and said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 70-72, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 109-111, respectively, and said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 112-114, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 127-129, and said_VL CDR1,_VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 130-132, respectively; said VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 133-135, respectively; said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 136-137, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 137-138, respectively; said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 139-140, respectively; said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 141-142, respectively; said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 143-144, respectively; the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 136-138, respectively; the_VH CDR1,_VH CDR2 and VH CDR3 sequences comprise SEQ ID NOs: 139-141, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 142-144, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 145-147, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 148-150, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 151-153, respectively, and the_VL CDR1,_{VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 154-156, respectively; the VH CDR1, VH} CDR2 and_VHCDR3 sequences comprise SEQ ID NOs:_157-158 , respectively the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 157-159, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 160-162, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 163-165, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 166-168, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 169-171, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 172-174, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 175-177, respectively, and the_VL the VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 181-183, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 184-186, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 187-189, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 190-192, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 193-195, and_theVL CDR1,_VLCDR2 and_VLCDR3 sequences comprise SEQ ID NOs: said_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 196-198; said_{VH CDR1, VH CDR2 and VH CDR3 sequences comprise SEQ ID NOs: 199-201, respectively, and said VL CDR1, VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 202-204, respectively; said VHCDR1,VHCDR2andVH} CDR3 sequences comprise SEQ ID NOs: 205-207, respectively, and said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 208-210, respectively; said_VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 211-213, respectively, and said_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 214-216, respectively; said_VH CDR1,_VH CDR2 and_VH the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 229-231, and the_VL CDR1,_VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 232-234, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 235-237, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 236-238, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 237-239, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs_: 240-241, respectively; the_VH CDR1,_VH CDR2 and_VHCDR3 sequences comprise SEQ ID NOs: 242-243, respectively; the VH CDR1, VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 238-240, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 241-243, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 244-246, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 247-249, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 250-252, respectively; the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 253-255, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 256-258, respectively; the_VH The VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 265-267, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 268-270, respectively; or the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 271-273, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 274-276, respectively.如請求項1或2之經分離之抗體或其抗原結合片段，其中該V_H包含與選自表A2之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，視情況其中該V_H在構架區中具有1、2、3、4、5、6、7、8、9或10個改變。An isolated antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected fromTableA2 , optionally wherein the_VH has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations in the framework region.如請求項1至3中任一項之經分離之抗體或其抗原結合片段，其中該V_L包含與選自表A2之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，視情況其中該V_L在構架區中具有1、2、3、4、5、6、7、8、9或10個改變。The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected fromTableA2 , optionally wherein the_VL has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations in the framework region.如請求項1至4中任一項之經分離之抗體或其抗原結合片段，其中： 該V_H包含與SEQ ID NO: 277至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 278至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 279至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 280至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 285至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 286至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 287至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 288至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 289至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 290至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 291至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 292至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 293至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 294至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 295至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 296至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 297至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 298至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 299至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 300至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 313至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 314至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 315至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 316至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 317至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 318至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 319至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 320至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 321至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 322至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 323至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 324至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 325至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 326至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 327至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 328至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 329至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 330至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 331至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 332至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 333至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 334至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 335至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 336至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 337至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 338至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 339至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 340至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 341至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 342至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 343至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 344至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 345至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 346至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 347至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 348至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 349至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 350至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 351至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 352至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 353至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 354至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 355至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 356至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 357至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 358至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 359至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 360至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 361至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 362至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 367至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 368至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列；或 該V_H包含與SEQ ID NO: 369至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 370至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列。The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein: the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 277, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 278; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 279, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 280; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 285 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 286, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 286; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 287, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 288; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 289, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 290; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 291, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 292; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 293, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 294; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 98%, 99%, 99%, 100% identical to SEQ ID NO: 299; the_VH comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 291, and the VL comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 292; the_VH comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 293, and the_VL comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 294; the_VH comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 295, and the_VL comprises a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 300; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 313, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 314; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 315, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 316; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 317 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 318, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 318; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 319, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 320; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 321, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 322; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 323, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 324; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 325, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 326; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 327 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 328, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 328; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 329, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 330; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 331, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 332; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 333, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 334; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 335, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 336; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 337 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 338, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 338; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 339, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 340; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 341, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 342; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 343, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 344; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 345, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 346; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 347 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 348, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 348; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 349, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 350; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 351, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 352; The VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 353, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 354; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 355, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 356; The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 357 comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 358, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 358; the V_H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 359, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 360; the V H comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 361, and_{the V Lcomprises} a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: The_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 362; the VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 367, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 368; or the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 369, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 370.如請求項1至5中任一項之經分離之抗體或其抗原結合片段，其中該抗體與抗原決定基結合，該抗原決定基包含根據UniProt：O95998之編號之I97及V153 （可替代地，如由SEQ ID NO: 1所定義之I95及V151；或如由SEQ ID NO: 2所定義之I67及V123）。An isolated antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5, wherein the antibody is bound to an antigenic determinant comprising I97 and V153 numbered according to UniProt: O95998 (alternatively, I95 and V151 as defined by SEQ ID NO: 1; or I67 and V123 as defined by SEQ ID NO: 2).一種經分離之抗體或其抗原結合片段，其中該抗體在抗原決定基處與介白素-18結合蛋白（IL-18BP）結合，該抗原決定基包含根據UniProt：O95998之編號之I97及V153 （可替代地，如由SEQ ID NO: 1所定義之I95及V151；或如由SEQ ID NO: 2所定義之I67及V123）。An isolated antibody or antigen-binding fragment thereof, wherein the antibody binds to interleukin-18 binding protein (IL-18BP) at an antigenic determinant comprising I97 and V153 numbered according to UniProt: O95998 (alternatively, I95 and V151 as defined by SEQ ID NO: 1; or I67 and V123 as defined by SEQ ID NO: 2).如請求項7之經分離之抗體或其抗原結合片段，其中： 該VHCDR1、VHCDR2及VHCDR3序列分別包含TFX₁X₂X₃X₄X₅H、IX₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅AQKFQG及X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂DY，且該VLCDR1、VLCDR2及VLCDR3序列分別包含X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀WX₃₁A、X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈及QX₃₉X₄₀X₄₁SFPYX₄₂（關於「X」殘基之定義，參見表E11）； 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 1-3，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 4-6；或 該V_HCDR1、V_HCDR2及V_HCDR3序列分別包含SEQ ID NO: 265-267，且該V_LCDR1、V_LCDR2及V_LCDR3序列分別包含SEQ ID NO: 268-270。The isolated antibody or antigen-binding fragment thereof of claim 7, wherein: the VHCDR1, VHCDR2 and VHCDR3 sequences comprise_TFX1 X₂ X₃ X₄ X₅ H, IX₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ AQKFQG and X₁₆ X₁₇ X₁₈ X₁₉ X₂₀ X₂₁ X₂₂ DY, respectively; and the VLCDR1, VLCDR2 and VLCDR3 sequences comprise X₂₃ X₂₄ X₂₅ X 26 X₂₇ X₂₈ X_{29X 30WX31} A, X₃₂ X₃₃ X₃₄ X₃₅ X₃₆ X₃₇ X₃₈ and QX₃₉ X₄₀ X₄₁ SFPYX₄₂ (For the definition of "X" residue,see TableE11 ); the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 1-3, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 4-6, respectively; or the_VH CDR1,_VH CDR2 and_VH CDR3 sequences comprise SEQ ID NOs: 265-267, respectively, and the_VL CDR1,_VL CDR2 and_VL CDR3 sequences comprise SEQ ID NOs: 268-270, respectively.如請求項8之經分離之抗體或其抗原結合片段，其中： 該V_H包含與SEQ ID NO: 277至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 278至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列； 該V_H包含與SEQ ID NO: 279至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 280至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列；或 該V_H包含與SEQ ID NO: 367至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 368至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列。The isolated antibody or antigen-binding fragment thereof of claim 8, wherein: the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 277, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 278; the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 279, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 280; or the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 367, and the V_L comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 368.一種經分離之抗體或其抗原結合片段，其中該抗體與介白素-18結合蛋白（IL-18BP）結合且其中該抗體干擾IL-18與IL-18BP之結合。An isolated antibody or antigen-binding fragment thereof, wherein the antibody binds to interleukin-18 binding protein (IL-18BP) and wherein the antibody interferes with the binding of IL-18 to IL-18BP.如請求項10之經分離之抗體或其抗原結合片段，其中該抗體與IL-18BP之構形抗原決定基結合。The isolated antibody or antigen-binding fragment thereof of claim 10, wherein the antibody binds to a conformational antigenic determinant of IL-18BP.如請求項10或11之經分離之抗體或其抗原結合片段，其中IL-18BP之該構形抗原決定基包含選自由以下組成之群的兩個或更多個胺基酸殘基：SEQ ID NO: 372之K32、T40、S60、R61、S66、Y69、R91、R93、T96、K102、R131及H132。The isolated antibody or antigen-binding fragment thereof of claim 10 or 11, wherein the conformational antigenic determinant of IL-18BP comprises two or more amino acid residues selected from the group consisting of: K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131 and H132 of SEQ ID NO: 372.如請求項10至12中任一項之經分離之抗體或其抗原結合片段，其中IL-18BP之該構形抗原決定基包含SEQ ID NO: 372之K32、T40、S60、R61、S66、Y69、R91、R93、T96、K102、R131及H132之該等胺基酸殘基。An isolated antibody or antigen-binding fragment thereof as claimed in any one of claims 10 to 12, wherein the conformational antigenic determinant of IL-18BP comprises the amino acid residues K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131 and H132 of SEQ ID NO: 372.如請求項10之經分離之抗體或其抗原結合片段，其中該抗體與IL-18BP之線性抗原決定基結合。The isolated antibody or antigen-binding fragment thereof of claim 10, wherein the antibody binds to a linear antigenic determinant of IL-18BP.如請求項10至14中任一項之經分離之抗體或其抗原結合片段，其中該抗體與IL-18與成熟形式之IL-18BP之間的結合界面結合。An isolated antibody or antigen-binding fragment thereof as claimed in any one of claims 10 to 14, wherein the antibody binds to the binding interface between IL-18 and the mature form of IL-18BP.如請求項15之經分離之抗體或其抗原結合片段，其中該抗體結合SEQ ID NO: 372之該等胺基酸殘基R61、Y69及R131。An isolated antibody or antigen-binding fragment thereof as claimed in claim 15, wherein the antibody binds to the amino acid residues R61, Y69 and R131 of SEQ ID NO: 372.如請求項12至16中任一項之經分離之抗體或其抗原結合片段，其中： 該V_H包含與SEQ ID NO: 333至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列，且該V_L包含與SEQ ID NO: 334至少80%、85%、90%、95%、97%、98%、99%或100%一致的序列。An isolated antibody or antigen-binding fragment thereof as claimed in any one of claims 12 to 16, wherein: the_VH comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 333, and the_VL comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 334.如請求項1至17中任一項之經分離之抗體或其抗原結合片段，其中該抗體與人類IL-18BP及食蟹獼猴IL-18BP結合，但不與小鼠IL-18BP（特異性或實質上）結合。An isolated antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 17, wherein the antibody binds to human IL-18BP and cynomolgus macaque IL-18BP but does not bind to mouse IL-18BP (specifically or substantially).如請求項1至17中任一項之經分離之抗體或其抗原結合片段，其中該抗體與人類IL-18BP、食蟹獼猴IL-18BP及小鼠IL-18BP結合。An isolated antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 17, wherein the antibody binds to human IL-18BP, cynomolgus macaque IL-18BP and mouse IL-18BP.如請求項1至19中任一項之經分離之抗體或其抗原結合片段，其中該抗體與IL-18與成熟形式之IL-18BP之間的該結合界面結合。The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 19, wherein the antibody binds to the binding interface between IL-18 and the mature form of IL-18BP.如請求項1至20中任一項之經分離之抗體或其抗原結合片段，其以比IL-18與IL-18BP之間的結合親和力（K_D為約650 pM）更強的結合親和力，視情況以約1 pm至約650 pm，或約或小於約1、5、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200或300、400、500、600或650 pM之結合親和力與IL-18BP結合。An isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 20, which binds to IL-18BP with a stronger binding affinity than the binding affinity between IL-18 and IL-18BP (_KD is about 650 pM), optionally with a binding affinity of about 1 pm to about 650 pm, or about or less than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 300, 400, 500, 600 or 650 pM.如請求項1至21中任一項之經分離之抗體或其抗原結合片段，其為IL-18BP拮抗劑，其拮抗IL-18BP與IL-18之間的結合活性。The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 21, which is an IL-18BP antagonist that antagonizes the binding activity between IL-18BP and IL-18.如請求項22之經分離之抗體或其抗原結合片段，其中該抗體阻斷IL-18BP對IL-18之抑制活性，且藉此增加IL-18介導之信號傳導，包括IFN-γ、CXCL10及/或TNFα之誘導。An isolated antibody or antigen-binding fragment thereof as claimed in claim 22, wherein the antibody blocks the inhibitory activity of IL-18BP on IL-18 and thereby increases IL-18-mediated signaling, including the induction of IFN-γ, CXCL10 and/or TNFα.如請求項1至8中任一項之經分離之抗體或其抗原結合片段，其包含IgA（包括子類IgA1及IgA2）、IgD、IgE、IgG（包括子類IgG1、IgG2、IgG3及IgG4）或IgM Fc域，視情況人類Fc域或其雜合體及/或變異體。An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising an IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3 and IgG4) or IgM Fc domain, optionally a human Fc domain or a hybrid and/or variant thereof.如請求項24之經分離之抗體或其抗原結合片段，其包含在人類中具有高效應功能之IgG Fc域，視情況IgG1或IgG3 Fc域。An isolated antibody or antigen-binding fragment thereof as claimed in claim 24, comprising an IgG Fc domain having high affinity for human, optionally an IgG1 or IgG3 Fc domain.如請求項24之經分離之抗體或其抗原結合片段，其包含在人類中具有低效應功能之IgG Fc域，視情況IgG2或IgG4 Fc域。An isolated antibody or antigen-binding fragment thereof as claimed in claim 24, comprising an IgG Fc domain having low efficacy function in humans, optionally an IgG2 or IgG4 Fc domain.如請求項1至26中任一項之經分離之抗體或其抗原結合片段，其為單株抗體。The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 26, which is a monoclonal antibody.如請求項1至27中任一項之經分離之抗體或其抗原結合片段，其為人源化抗體。The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 27, which is a humanized antibody.如請求項1至28中任一項之經分離之抗體或其抗原結合片段，其係選自Fv片段、單鏈Fv（scFv）多肽、纖連蛋白（adnectin）、抗運載蛋白（anticalin）、適體、高親和性多聚體（avimer）、駱駝科抗體、經設計之錨蛋白重複蛋白（DARPin）、微型抗體、奈米抗體及單抗體（unibody）。An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 28, which is selected from Fv fragments, single-chain Fv (scFv) polypeptides, adnectins, anticalins, aptamers, avimers, camelid antibodies, designed anchor protein repeat proteins (DARPins), minibodies, nanobodies and unibodies.一種經分離之聚核苷酸，其編碼如請求項1至29中任一項之經分離之抗IL-18BP抗體或其抗原結合片段；一種包含該經分離之聚核苷酸的表現載體；或一種包含該載體之經分離之宿主細胞。An isolated polynucleotide encoding the isolated anti-IL-18BP antibody or antigen-binding fragment thereof of any one of claims 1 to 29; an expression vector comprising the isolated polynucleotide; or an isolated host cell comprising the vector.一種醫藥組合物，其包含如請求項1至29中任一項之經分離之抗IL-18BP抗體或其抗原結合片段及醫藥學上可接受之載劑。A pharmaceutical composition comprising the isolated anti-IL-18BP antibody or antigen-binding fragment thereof according to any one of claims 1 to 29 and a pharmaceutically acceptable carrier.如請求項31之醫藥組合物，其中該組合物具有就該至少一種抗體或抗原結合片段而言以蛋白質計至少約80%、85%、90%、95%、98%或99%之純度且實質上不含聚集體及內毒素。The pharmaceutical composition of claim 31, wherein the composition has a purity of at least about 80%, 85%, 90%, 95%, 98% or 99% on a protein basis with respect to the at least one antibody or antigen-binding fragment and is substantially free of aggregates and endotoxins.如請求項31或32之醫藥組合物，其中該組合物為視情況適合於靜脈內、肌肉內、皮下或腹膜內投與之無菌可注射溶液。The pharmaceutical composition of claim 31 or 32, wherein the composition is a sterile injectable solution suitable for intravenous, intramuscular, subcutaneous or intraperitoneal administration as appropriate.一種治療有需要之個體之疾病或病狀的方法，其包含向該個體投與如請求項31至33中任一項之醫藥組合物。A method for treating a disease or condition in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of any one of claims 31 to 33.如請求項34之方法，其中該疾病或病狀為癌症或腫瘤或增生性疾病或病症，視情況選自以下之增生性疾病或病症：淋巴增生性病症、骨髓增生性病症、增生性腸炎、增生性糖尿病視網膜病變及增生性腎病。The method of claim 34, wherein the disease or condition is cancer or a tumor or a proliferative disease or disorder, optionally selected from the group consisting of a lymphoproliferative disorder, a myeloproliferative disorder, a proliferative enteritis, a proliferative diabetic retinopathy, and a proliferative nephropathy.如請求項35之方法，其中該癌症或腫瘤表現或過度表現IL-18BP及/或IL-18，或其中該增生性疾病或病症與IL-18BP及/或IL-18之表現增加相關。The method of claim 35, wherein the cancer or tumor expresses or overexpresses IL-18BP and/or IL-18, or wherein the proliferative disease or disorder is associated with increased expression of IL-18BP and/or IL-18.如請求項35或36之方法，其中該癌症係選自以下中之一或多者：骨癌、前列腺癌、黑色素瘤（例如，轉移性黑色素瘤）、胰臟癌、小細胞肺癌、非小細胞肺癌（NSCLC）、間皮瘤、白血病（例如，淋巴球性白血病、慢性骨髓性白血病、急性骨髓性白血病、復發性急性骨髓性白血病、毛細胞白血病、急性淋巴母細胞性白血病）、淋巴瘤（例如，非何傑金氏淋巴瘤（non-Hodgkin's lymphoma）、何傑金氏淋巴瘤）、肝癌（肝細胞癌）、肉瘤、B細胞惡性病、乳癌、卵巢癌、結腸直腸癌、神經膠質瘤、多形性神經膠質母細胞瘤、腦膜瘤、垂體腺瘤、前庭神經鞘瘤、原發性CNS淋巴瘤、原始神經外胚層腫瘤（神經管胚細胞瘤）、腎癌（例如，腎細胞癌）、膀胱癌、子宮癌、尿道上皮癌、食道癌、腦癌、頭頸癌、子宮頸癌、睪丸癌、甲狀腺癌及胃癌。The method of claim 35 or 36, wherein the cancer is selected from one or more of the following: bone cancer, prostate cancer, melanoma (e.g., metastatic melanoma), pancreatic cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, hairy cell leukemia, acute lymphoblastic leukemia), lymphoma (e.g., non-Hodgkin's lymphoma lymphoma), Hodgkin's lymphoma), liver cancer (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, neuroglioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (neurotubular cell tumor), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, urothelial carcinoma, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.如請求項34至37中任一項之方法，其包含投與該醫藥組合物（具有抗IL18BP抗體或其抗原結合片段）與IL-18之組合。The method of any one of claims 34 to 37, comprising administering the pharmaceutical composition (having an anti-IL18BP antibody or an antigen-binding fragment thereof) in combination with IL-18.如請求項35至38中任一項之方法，其包含投與該醫藥組合物（具有該抗IL18BP抗體或其抗原結合片段）與免疫檢查點調節劑之組合，該免疫檢查點調節劑選自抑制性免疫檢查點分子之拮抗劑及刺激性免疫檢查點分子之促效劑。The method of any one of claims 35 to 38, comprising administering the pharmaceutical composition (having the anti-IL18BP antibody or antigen-binding fragment thereof) in combination with an immune checkpoint modulator selected from antagonists of inhibitory immune checkpoint molecules and agonists of stimulatory immune checkpoint molecules.如請求項39之方法，其中該免疫檢查點調節劑為多肽，視情況抗體或其抗原結合片段，或配位體或小分子。The method of claim 39, wherein the immune checkpoint modulator is a polypeptide, an antibody or an antigen-binding fragment thereof, or a ligand or a small molecule.如請求項39或40之方法，其中該抑制性免疫檢查點分子係選自以下中之一或多者：程式化死亡-配位體1（PD-L1）、程式化死亡1（PD-1）、程式化死亡-配位體2（PD-L2）、細胞毒性T淋巴球相關蛋白4（CTLA-4）、吲哚胺2,3-雙加氧酶（IDO）、色胺酸2,3-雙加氧酶（TDO）、T細胞免疫球蛋白域及黏蛋白域3（TIM-3）、淋巴球活化基因-3（LAG-3）、T細胞活化之V域Ig抑制因子（VISTA）、B及T淋巴球衰減因子（BTLA）、CD160、疱疹病毒侵入介體（HVEM）以及具有Ig及ITIM域之T細胞免疫受體（TIGIT）。The method of claim 39 or 40, wherein the inhibitory immune checkpoint molecule is selected from one or more of the following: programmed death-ligand 1 (PD-L1), programmed death 1 (PD-1), programmed death-ligand 2 (PD-L2), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene-3 (LAG-3), V-domain Ig inhibitory factor of T cell activation (VISTA), B and T lymphocyte depletion factor (BTLA), CD160, herpes virus entry mediator (HVEM) and T cell immune receptor with Ig and ITIM domains (TIGIT).如請求項41之方法，其中： 該拮抗劑為視情況選自以下中之一或多者的PD-L1及/或PD-L2拮抗劑：與PD-L1及/或PD-L2特異性結合之抗體或抗原結合片段或小分子、阿替利珠單抗（atezolizumab）（MPDL3280A）、阿維魯單抗（avelumab）（MSB0010718C）及度伐利尤單抗（durvalumab）（MEDI4736），視情況其中該癌症係選自以下中之一或多者：結腸直腸癌、黑色素瘤、乳癌、非小細胞肺癌、膀胱癌及腎細胞癌； 該拮抗劑為視情況選自以下中之一或多者的PD-1拮抗劑：與PD-1特異性結合之抗體或抗原結合片段或小分子、納武單抗（nivolumab）、帕博利珠單抗（pembrolizumab）、MK-3475、AMP-224、AMP-514PDR001及皮立珠單抗（pidilizumab），視情況其中該PD-1拮抗劑為納武單抗，且該癌症視情況選自以下中之一或多者：何傑金氏淋巴瘤、黑色素瘤、非小細胞肺癌、肝細胞癌、腎細胞癌及卵巢癌； 該PD-1拮抗劑為帕博利珠單抗，且該癌症視情況選自以下中之一或多者：黑色素瘤、非小細胞肺癌、小細胞肺癌、頭頸癌及尿道上皮癌； 該拮抗劑為視情況選自以下中之一或多者的CTLA-4拮抗劑：與CTLA-4特異性結合之抗體或抗原結合片段或小分子、伊匹單抗（ipilimumab）、曲美木單抗（tremelimumab），視情況其中該癌症係選自以下中之一或多者：黑色素瘤、前列腺癌、肺癌及膀胱癌； 該拮抗劑為視情況選自以下中之一或多者的IDO拮抗劑：與IDO特異性結合之抗體或抗原結合片段或小分子、因多莫得（indoximod）（NLG-8189）、1-甲基-色胺酸（1MT）、β-咔啉（去甲哈爾滿（norharmane）；9H-吡啶并[3,4-b]吲哚）、迷迭香酸及艾帕斯塔（epacadostat），且其中該癌症視情況選自以下中之一或多者：轉移性乳癌及腦癌、視情況多形性神經膠質母細胞瘤、神經膠質瘤、神經膠質肉瘤或惡性腦瘤； 該拮抗劑為視情況選自以下中之一或多者的TDO拮抗劑：與TDO特異性結合之抗體或抗原結合片段或小分子、680C91及LM10； 該拮抗劑為視情況選自與TIM-3特異性結合之抗體或抗原結合片段或小分子中之一或多者的TIM-3拮抗劑； 該拮抗劑為視情況選自以下中之一或多者的LAG-3拮抗劑：與LAG-3特異性結合之抗體或抗原結合片段或小分子,及BMS-986016； 該拮抗劑為視情況選自與VISTA特異性結合之抗體或抗原結合片段或小分子中之一或多者的VISTA拮抗劑； 該拮抗劑為視情況選自與BTLA、CD160及/或HVEM特異性結合之抗體或抗原結合片段或小分子中之一或多者的BTLA、CD160及/或HVEM拮抗劑； 該拮抗劑為視情況選自與TIGIT特異性結合之抗體或抗原結合片段或小分子中之一或多者的TIGIT拮抗劑。The method of claim 41, wherein: The antagonist is a PD-L1 and/or PD-L2 antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to PD-L1 and/or PD-L2, atezolizumab (MPDL3280A), avelumab (MSB0010718C) and durvalumab (MEDI4736), wherein the cancer is selected from one or more of the following: colorectal cancer, melanoma, breast cancer, non-small cell lung cancer, bladder cancer and renal cell carcinoma; The antagonist is a PD-1 antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to PD-1, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514PDR001 and pidilizumab, wherein the PD-1 antagonist is nivolumab, and the cancer is selected from one or more of the following: Hodgkin's lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma and ovarian cancer;The PD-1 antagonist is pembrolizumab, and the cancer is selected from one or more of the following: melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial carcinoma;The antagonist is a CTLA-4 antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to CTLA-4, ipilimumab, tremelimumab, and the cancer is selected from one or more of the following: melanoma, prostate cancer, lung cancer, and bladder cancer;The antagonist is an IDO antagonist selected from one or more of the following: antibodies or antigen-binding fragments or small molecules that specifically bind to IDO, indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carbolines (norharmane; 9H-pyrido[3,4-b]indole), rosmarinic acid and epacadostat, and the cancer is selected from one or more of the following: metastatic breast cancer and brain cancer, glioblastoma multiforme, neuroglioma, neurosarcoma or malignant brain tumor;The antagonist is a TDO antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to TDO, 680C91 and LM10;The antagonist is a TIM-3 antagonist selected from one or more of the antibodies or antigen-binding fragments or small molecules that specifically bind to TIM-3;The antagonist is a LAG-3 antagonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to LAG-3, and BMS-986016;The antagonist is a VISTA antagonist selected from one or more of the antibodies or antigen-binding fragments or small molecules that specifically bind to VISTA;The antagonist is a BTLA, CD160 and/or HVEM antagonist selected from one or more of antibodies, antigen-binding fragments or small molecules that specifically bind to BTLA, CD160 and/or HVEM as appropriate;The antagonist is a TIGIT antagonist selected from one or more of antibodies, antigen-binding fragments or small molecules that specifically bind to TIGIT as appropriate.如請求項39或40之方法，其中該刺激性免疫檢查點分子係選自以下中之一或多者：OX40、CD40、糖皮質激素誘導之TNFR家族相關基因（GITR）、CD137（4-1BB）、CD27、CD28、CD226及疱疹病毒侵入介體（HVEM）。The method of claim 39 or 40, wherein the stimulatory immune checkpoint molecule is selected from one or more of the following: OX40, CD40, glucocorticoid-induced TNFR family-related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226 and herpes virus entry mediator (HVEM).如請求項43之方法，其中： 該促效劑為視情況選自以下中之一或多者的OX40促效劑：與OX40特異性結合之抗體或抗原結合片段或小分子或配位體、OX86、Fc-OX40L及GSK3174998； 該促效劑為視情況選自以下中之一或多者的CD40促效劑：與CD40特異性結合之抗體或抗原結合片段或小分子或配位體、CP-870,893、達西組單抗（dacetuzumab）、Chi Lob 7/4、ADC-1013及rhCD40L，且其中該癌症視情況選自以下中之一或多者：黑色素瘤、胰臟癌、間皮瘤及血液癌，視情況淋巴瘤，諸如非何傑金氏淋巴瘤； 該促效劑為視情況選自以下中之一或多者的GITR促效劑：與GITR特異性結合之抗體或抗原結合片段或小分子或配位體、INCAGN01876、DTA-1及MEDI1873； 該促效劑為視情況選自以下中之一或多者的CD137促效劑：與CD137特異性結合之抗體或抗原結合片段或小分子或配位體、烏托米單抗（utomilumab）及4-1BB配位體； 該促效劑為視情況選自以下中之一或多者的CD27促效劑：與CD27特異性結合之抗體或抗原結合片段或小分子或配位體、瓦利魯單抗（varlilumab）及CDX-1127 (1F5)； 該促效劑為視情況選自以下中之一或多者的CD28促效劑：與CD28特異性結合之抗體或抗原結合片段或小分子或配位體，及TAB08；及/或 該促效劑為視情況選自與HVEM特異性結合之抗體或抗原結合片段或小分子或配位體中之一或多者的HVEM促效劑。The method of claim 43, wherein: the agonist is an OX40 agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to OX40, OX86, Fc-OX40L, and GSK3174998; the agonist is a CD40 agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD40, CP-870,893, dacetuzumab, Chi Lob 7/4, ADC-1013 and rhCD40L, and wherein the cancer is selected from one or more of the following: melanoma, pancreatic cancer, mesothelioma and blood cancer, and lymphoma, such as non-Hodgkin's lymphoma; The agonist is a GITR agonist selected from one or more of the following: an antibody or antigen binding fragment or small molecule or ligand that specifically binds to GITR, INCAGN01876, DTA-1 and MEDI1873; The agonist is a CD137 agonist selected from one or more of the following: an antibody or antigen binding fragment or small molecule or ligand that specifically binds to CD137, utomilumab and 4-1BB ligand; The agonist is a CD27 agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD27, varlilumab, and CDX-1127 (1F5);The agonist is a CD28 agonist selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD28, and TAB08; and/orThe agonist is a HVEM agonist selected from one or more of the antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to HVEM.如請求項35至44中任一項之方法，其包含投與該醫藥組合物（具有該抗IL18BP抗體或其抗原結合片段）與至少一種化學治療劑之組合。The method of any one of claims 35 to 44, comprising administering the pharmaceutical composition (having the anti-IL18BP antibody or antigen-binding fragment thereof) in combination with at least one chemotherapeutic agent.如請求項45之方法，其中該至少一種化學治療劑係選自以下中之一或多者：烷基化劑、抗代謝物、細胞毒性抗生素、拓樸異構酶抑制劑（1型或II型）及抗微管劑。The method of claim 45, wherein the at least one chemotherapeutic agent is selected from one or more of the following: an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an anti-microtubule agent.如請求項46之方法，其中： 該烷基化劑係選自以下中之一或多者：氮芥（nitrogen mustard）（視情況甲氮芥（mechlorethamine）、環磷醯胺、氮芥（mustine）、美法侖（melphalan）、苯丁酸氮芥（chlorambucil）、異環磷醯胺（ifosfamide）及硫酸布他卡因（busulfan））、亞硝基脲（視情況N-亞硝基-N-甲基脲（MNU）、卡莫司汀（carmustine）（BCNU）、洛莫司汀（lomustine）（CCNU）、司莫司汀（semustine）（MeCCNU）、福莫司汀（fotemustine）及鏈佐黴素（streptozotocin））、四（視情況達卡巴嗪（dacarbazine）、米托唑胺（mitozolomide）及替莫唑胺（temozolomide））、氮丙啶（視情況噻替派（thiotepa）、絲裂黴素（mytomycin）及地吖醌（diaziquone）（AZQ））、順鉑及其衍生物（視情況卡鉑（carboplatin）及奧沙利鉑（oxaliplatin））及非典型烷基化劑（視情況丙卡巴肼（procarbazine）及六甲三聚氰胺）； 該抗代謝物係選自以下中之一或多者：抗葉酸劑（視情況甲胺喋呤（methotrexate）及培美曲塞（pemetrexed））、氟嘧啶（視情況5-氟尿嘧啶及卡培他濱（capecitabine））、去氧核苷類似物（視情況安西他濱（ancitabine）、依諾他濱（enocitabine）、阿糖胞苷（cytarabine）、吉西他濱（gemcitabine）、地西他濱（decitabine）、阿紮胞苷（azacitidine）、氟達拉濱（fludarabine）、奈拉濱（nelarabine）、克拉屈濱（cladribine）、氯法拉濱（clofarabine）、氟達拉濱（fludarabine）及噴司他汀（pentostatin））及硫代嘌呤（視情況硫鳥嘌呤及巰基嘌呤）； 該細胞毒性抗生素係選自以下中之一或多者：蒽環黴素（視情況小紅莓（doxorubicin）、道諾黴素（daunorubicin）、表柔比星（epirubicin）、艾達黴素（idarubicin）、吡柔比星（pirarubicin）、阿克拉黴素（aclarubicin）及米托蒽醌（mitoxantrone））、博來黴素（bleomycin）、絲裂黴素（mitomycin）C、米托蒽醌及放線菌素（actinomycin）； 該拓樸異構酶抑制劑係選自以下中之一或多者：喜樹鹼（camptothecin）、伊立替康（irinotecan）、拓樸替康（topotecan）、依託泊苷（etoposide）、小紅莓、米托蒽醌、替尼泊苷（teniposide）、新生黴素（novobiocin）、麥爾巴隆（merbarone）及阿克拉黴素；及/或 該抗微管劑係選自以下中之一或多者：紫杉烷（視情況太平洋紫杉醇（paclitaxel）及多西他賽（docetaxel））及長春花生物鹼（視情況長春鹼（vinblastine）、長春新鹼（vincristine）、長春地辛（vindesine）、長春瑞濱（vinorelbine））。The method of claim 46, wherein: the alkylating agent is selected from one or more of the following: nitrogen mustard (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosourea (optionally N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetracycline (dacarbazine, mitozolomide and temozolomide, as appropriate), aziridines (thiotepa, mytomycin and diaziquone (AZQ), as appropriate), cis-platinum and its derivatives (carboplatin and oxaliplatin, as appropriate) and atypical alkylating agents (procarbazine and hexamethonium, as appropriate); The anti-metabolite is selected from one or more of the following: antifolates (methotrexate and pemetrexed as appropriate), fluoropyrimidines (5-fluorouracil and capecitabine as appropriate), deoxynucleoside analogs (ancitabine, enocitabine, cytarabine, gemcitabine as appropriate), itabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine and pentostatin) and thiopurines (thioguanine and thioguanine as appropriate); The cytotoxic antibiotic is one or more selected from the group consisting of anthracyclines (depending on the situation, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone), bleomycin, mitomycin C, mitoxantrone and actinomycin; The topoisomerase inhibitor is selected from one or more of the following: camptothecin, irinotecan, topotecan, etoposide, cranberry, mitoxantrone, teniposide, novobiocin, merbarone and aclatomycin; and/or the anti-microtubule agent is selected from one or more of the following: taxanes (paclitaxel and docetaxel as appropriate) and vinca alkaloids (vinblastine, vincristine, vindesine, vinorelbine as appropriate).如請求項34之方法，其中該疾病或病狀為骨髓發育不良症候群（MDS）。The method of claim 34, wherein the disease or condition is myelodysplastic syndrome (MDS).如請求項34之方法，其中該疾病或病狀為感染性疾病。The method of claim 34, wherein the disease or condition is an infectious disease.如請求項49之方法，其中該感染性疾病係選自病毒、細菌、真菌（視情況酵母）及原蟲感染。The method of claim 49, wherein the infectious disease is selected from viral, bacterial, fungal (optionally yeast) and protozoan infections.如請求項48至50中任一項之方法，其包含投與該醫藥組合物（具有該抗IL18BP抗體或其抗原結合片段）與IL-18之組合。The method of any one of claims 48 to 50, comprising administering the pharmaceutical composition (having the anti-IL18BP antibody or antigen-binding fragment thereof) in combination with IL-18.一種針對阻斷或抑制IL-18與IL-18BP之間的結合之能力篩選抗IL-18BP抗體或其抗原結合片段之方法，其包含 a.   測定該抗體或其抗原結合片段對以下之結合親和力， i.    單獨的IL-18BP，及 ii.   低IL-18融合蛋白，其中該低IL-18融合蛋白包含經由可撓性連接子（及其間的視情況存在之蛋白酶裂解位點）與IL-18BP融合的IL-18，其中該融合蛋白之IL-18部分結合至該融合蛋白之IL-18BP部分且空間阻斷該融合蛋白之該IL-18BP部分之IL-18結合位點； b.   比較（i）之該結合親和力與（ii）之該結合親和力；及 c.   若（i）之該結合親和力顯著強於（ii）之該結合親和力，則將該抗體或其抗原結合片段鑑別或選擇為能夠阻斷或抑制IL-18與IL-18BP之間的結合。A method for screening an anti-IL-18BP antibody or an antigen-binding fragment thereof for the ability to block or inhibit binding between IL-18 and IL-18BP, comprising: a.   Determining the binding affinity of the antibody or antigen-binding fragment thereof to, i.   IL-18BP alone, and ii.   A low IL-18 fusion protein, wherein the low IL-18 fusion protein comprises IL-18 fused to IL-18BP via a flexible linker (and optionally a protease cleavage site therebetween), wherein the IL-18 portion of the fusion protein binds to the IL-18BP portion of the fusion protein and sterically blocks the IL-18 binding site of the IL-18BP portion of the fusion protein; b.   Comparing the binding affinity of (i) with the binding affinity of (ii); and c.  If the binding affinity of (i) is significantly stronger than the binding affinity of (ii), the antibody or antigen-binding fragment thereof is identified or selected as being capable of blocking or inhibiting the binding between IL-18 and IL-18BP.如請求項52之方法，其中該IL-18及該IL-18BP為小鼠IL-18及小鼠IL-18BP。The method of claim 52, wherein the IL-18 and the IL-18BP are mouse IL-18 and mouse IL-18BP.如請求項52之方法，其中該IL-18及該IL-18BP為人類IL-18及人類IL-18BP。The method of claim 52, wherein the IL-18 and the IL-18BP are human IL-18 and human IL-18BP.如請求項52至54中任一項之方法，其中該低IL-18融合蛋白在N端至C端定向上包含信號肽、IL-18、第一可撓性連接子、蛋白酶裂解位點（視情況TEV蛋白酶裂解位點）、可撓性連接子及IL-18BP。A method as in any one of claims 52 to 54, wherein the low IL-18 fusion protein comprises a signal peptide, IL-18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker and IL-18BP in an N-terminal to C-terminal orientation.如請求項55之方法，其中該低IL-18融合蛋白包含與來自表S1之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的胺基酸序列。The method of claim 55, wherein the low IL-18 fusion protein comprises an amino acid sequencethatis at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a sequence from Table S1.一種低IL-18融合蛋白，其在N端至C端定向上包含信號肽、IL-18、第一可撓性連接子、蛋白酶裂解位點（視情況TEV蛋白酶裂解位點）、可撓性連接子及IL-18BP。A low IL-18 fusion protein comprises a signal peptide, IL-18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker and IL-18BP in an N-terminal to C-terminal orientation.如請求項57之低IL-18融合蛋白，其包含與來自表S1之序列至少80%、85%、90%、95%、97%、98%、99%或100%一致的胺基酸序列。The low IL-18 fusion protein of claim 57, comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the sequence fromTableS1 .一種刺激有需要之個體之免疫反應的方法，其包含向該個體投與如請求項31至33中任一項之醫藥組合物。A method for stimulating an immune response in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of any one of claims 31 to 33.如請求項59之方法，其中該免疫反應為IL-18介導之免疫反應。The method of claim 59, wherein the immune response is an IL-18 mediated immune response.如請求項60之方法，其中該IL-18介導之免疫反應包含誘導該有需要之個體中的IFN-γ、CXCL10及/或TNFα。The method of claim 60, wherein the IL-18 mediated immune response comprises inducing IFN-γ, CXCL10 and/or TNFα in the individual in need thereof.