CN116102655A

Movatterモバイル変換

Info

Publication number: CN116102655A
Application number: CN202210868109.1A
Authority: CN
Inventors: 康平
Original assignee: Nanjing Jisheng Aoma Biomedical Co ltd
Current assignee: Nanjing Jisheng Aoma Biomedical Co ltd
Priority date: 2021-08-19
Filing date: 2022-07-22
Publication date: 2023-05-12

Abstract

The present application relates to an antibody targeting PD-L1/PD-1, and uses thereof, the antibody comprising a first polypeptide chain comprising a first targeting moiety, a second polypeptide chain comprising a second targeting moiety, the first targeting moiety and the second targeting moiety each independently binding an immune cell associated antigen, the first polypeptide chain being linked to the second polypeptide chain by a disulfide bond; the antibodies of the present application may also bear a cytokine, or the antibodies of the present application may bind to a Tumor Associated Antigen (TAA); the application also provides immunoconjugates comprising said antibodies, methods of making said antibodies and uses thereof.

Description

Translated fromChinese

靶向PD-L1/PD-1的抗体及其应用Antibodies targeting PD-L1/PD-1 and their applications

技术领域technical field

本申请涉及生物医药领域，具体的涉及一种靶向PD-L1/PD-1的抗体及其应用。This application relates to the field of biomedicine, in particular to an antibody targeting PD-L1/PD-1 and its application.

背景技术Background technique

程序性死亡受体配体1(PD-L1)，也称为分化簇274(CD274)或B7同源蛋白1(B7-H1)，是一个40KDa的I型跨膜蛋白，一般在活化的T细胞、B细胞、单核细胞、树突状细胞、巨噬细胞及许多非造血细胞上表达。Programmed death receptor ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homologous protein 1 (B7-H1), is a 40KDa type I transmembrane protein, generally in activated T Cells, B cells, monocytes, dendritic cells, macrophages and many non-hematopoietic cells.

PD-L1能与程序性死亡受体1(PD-1)和B7-1(CD80)结合。其中PD-L1/PD-1信号通路是免疫反应中非常重要的共抑制信号途径，负调节T细胞免疫应答，抑制T细胞活性，减弱细胞因子的分泌。研究发现PD-L1能在许多肿瘤组织中表达，包括胃癌、肺癌、乳腺癌、胰腺癌、卵巢癌、结肠癌、肥大细胞性肿瘤以及恶性黑色素瘤等，以及在浸润肿瘤微环境的骨髓细胞中表达，保护肿瘤细胞逃避免疫攻击。PD-L1 can bind to programmed death receptor 1 (PD-1) and B7-1 (CD80). Among them, the PD-L1/PD-1 signaling pathway is a very important co-inhibitory signaling pathway in the immune response, which negatively regulates the immune response of T cells, inhibits the activity of T cells, and reduces the secretion of cytokines. Studies have found that PD-L1 can be expressed in many tumor tissues, including gastric cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, colon cancer, mast cell tumors, and malignant melanoma, as well as in bone marrow cells infiltrating the tumor microenvironment Expression, protect tumor cells from immune attack.

目前，抗PD-L1抗体和抗PD-1抗体能使很多癌症患者获益，但仍有部分患者对单药治疗不做应答。能够结合PD-1蛋白和PD-L1蛋白的多特异性抗体有望增强抗肿瘤活性。因此，亟需开发具备阻断PD-L1/PD-1信号通路能力的PD1/PD-L1双特异性抗体。At present, anti-PD-L1 antibody and anti-PD-1 antibody can benefit many cancer patients, but some patients still do not respond to monotherapy. Multispecific antibodies that can bind PD-1 protein and PD-L1 protein are expected to enhance anti-tumor activity. Therefore, there is an urgent need to develop PD1/PD-L1 bispecific antibodies capable of blocking the PD-L1/PD-1 signaling pathway.

发明内容Contents of the invention

本申请提供了一种分离的抗原结合蛋白，其具有下述性质的一种或多种：1)既能结合PD-1蛋白又能结合PD-L1蛋白；2)第一多肽链和第二多肽链自然配对不需要非自然的工程化配对，克服了一般多特异性抗体生产过程中的错配问题，降低成本；3)相比一般多特异性抗体，覆盖抗原的面积大30％；和4)能够高效杀伤肿瘤细胞。The application provides an isolated antigen-binding protein, which has one or more of the following properties: 1) can bind both PD-1 protein and PD-L1 protein; 2) the first polypeptide chain and the second polypeptide chain The natural pairing of the two polypeptide chains does not require unnatural engineered pairing, which overcomes the mismatch problem in the production process of general multispecific antibodies and reduces costs; 3) Compared with general multispecific antibodies, the area covering the antigen is 30% larger and 4) capable of efficiently killing tumor cells.

在某些实施方式中，所述抗原结合蛋白包含第一多肽链、第二多肽链，所述第一多肽链包括第一靶向部分，所述第二多肽链包含第二靶向部分，所述第一靶向部分和所述第二靶向部分各自独立地结合免疫细胞相关抗原，所述第一多肽链与所述第二多肽链通过二硫键连接。In certain embodiments, the antigen binding protein comprises a first polypeptide chain, a second polypeptide chain, the first polypeptide chain comprises a first targeting moiety, and the second polypeptide chain comprises a second target targeting moiety, the first targeting moiety and the second targeting moiety independently bind to immune cell-associated antigens, and the first polypeptide chain is connected to the second polypeptide chain through a disulfide bond.

在某些实施方式中，所述免疫细胞包括T细胞、巨噬细胞、树突状细胞和/或NK细胞。In certain embodiments, the immune cells include T cells, macrophages, dendritic cells and/or NK cells.

在某些实施方式中，所述免疫细胞相关抗原包括PD-1、PD-L1、CD3、CD16a和/或CD28。In certain embodiments, the immune cell-associated antigens include PD-1, PD-L1, CD3, CD16a and/or CD28.

在某些实施方式中，所述第一靶向部分和第二靶向部分结合相同的免疫细胞相关抗原。In certain embodiments, the first targeting moiety and the second targeting moiety bind the same immune cell-associated antigen.

在某些实施方式中，所述第一靶向部分和第二靶向部分均结合PD-1蛋白或PD-L1蛋白。In certain embodiments, both the first targeting moiety and the second targeting moiety bind to the PD-1 protein or the PD-L1 protein.

在某些实施方式中，所述第一靶向部分特异性结合PD-1蛋白，且所述第二靶向部分特异性结合PD-L1蛋白。In certain embodiments, the first targeting moiety specifically binds the PD-1 protein, and the second targeting moiety specifically binds the PD-L1 protein.

在某些实施方式中，所述第一靶向部分特异性结合PD-L1蛋白，所述第二靶向部分特异性结合PD-1蛋白。In certain embodiments, the first targeting moiety specifically binds to the PD-L1 protein, and the second targeting moiety specifically binds to the PD-1 protein.

在某些实施方式中，所述第一靶向部分特异性结合PD-1蛋白，且所述第二靶向部分特异性结合CD28或CD16a蛋白。In certain embodiments, the first targeting moiety specifically binds the PD-1 protein, and the second targeting moiety specifically binds the CD28 or CD16a protein.

在某些实施方式中，所述第一靶向部分特异性结合CD28或CD16a蛋白，且所述第二靶向部分特异性结合PD-1蛋白。In certain embodiments, the first targeting moiety specifically binds CD28 or CD16a protein, and the second targeting moiety specifically binds PD-1 protein.

在某些实施方式中，所述第一多肽链还包含第三靶向部分。In certain embodiments, said first polypeptide chain further comprises a third targeting moiety.

在某些实施方式中，所述第一多肽链中第三靶向部分与所述第一靶向部分的C端直接或间接地连接。In certain embodiments, the third targeting moiety in the first polypeptide chain is directly or indirectly linked to the C-terminus of the first targeting moiety.

在某些实施方式中，所述第一多肽链中第三靶向部分与所述第一靶向部分的C端通过连接子连接。In some embodiments, the third targeting moiety in the first polypeptide chain is connected to the C-terminus of the first targeting moiety through a linker.

在某些实施方式中，所述第三靶向部分靶向免疫细胞相关抗原。In certain embodiments, the third targeting moiety targets an immune cell-associated antigen.

在某些实施方式中，所述第三靶向部分靶向CD3或CD16a蛋白。In certain embodiments, the third targeting moiety targets CD3 or CD16a protein.

在某些实施方式中，所述第三靶向部分靶向肿瘤发生相关抗原。In certain embodiments, the third targeting moiety targets a tumorigenesis-associated antigen.

在某些实施方式中，所述第三靶向部分靶向EGFR和/或cMet。In certain embodiments, the third targeting moiety targets EGFR and/or cMet.

在某些实施方式中，所述第三靶向部分靶向肿瘤相关抗原。In certain embodiments, the third targeting moiety targets a tumor-associated antigen.

在某些实施方式中，所述肿瘤相关抗原包括PD-L1、HER2、CD47、CD19和/或CD20。In certain embodiments, the tumor-associated antigens include PD-L1, HER2, CD47, CD19 and/or CD20.

在某些实施方式中，所述第二多肽链还包含第四靶向部分。In certain embodiments, said second polypeptide chain further comprises a fourth targeting moiety.

在某些实施方式中，所述第二多肽链中第四靶向部分与所述第二靶向部分的C端直接或间接地连接。In certain embodiments, the fourth targeting moiety in the second polypeptide chain is directly or indirectly linked to the C-terminus of the second targeting moiety.

在某些实施方式中，所述第二多肽链中第四靶向部分与所述第二靶向部分的C端通过连接子连接。In certain embodiments, the fourth targeting moiety in the second polypeptide chain is connected to the C-terminus of the second targeting moiety through a linker.

在某些实施方式中，所述第四靶向部分靶向免疫细胞相关抗原。In certain embodiments, the fourth targeting moiety targets an immune cell-associated antigen.

在某些实施方式中，所述第四靶向部分靶向肿瘤发生相关抗原。In certain embodiments, the fourth targeting moiety targets a tumorigenesis-associated antigen.

在某些实施方式中，所述第四靶向部分靶向EGFR和/或cMet。In certain embodiments, the fourth targeting moiety targets EGFR and/or cMet.

在某些实施方式中，所述第四靶向部分靶向肿瘤相关抗原。In certain embodiments, the fourth targeting moiety targets a tumor-associated antigen.

在某些实施方式中，所述第一多肽链包含细胞因子。In certain embodiments, the first polypeptide chain comprises a cytokine.

在某些实施方式中，所述细胞因子包括IL-2、IFNα、IFNβ和/或IL-15。In certain embodiments, the cytokines include IL-2, IFNα, IFNβ, and/or IL-15.

在某些实施方式中，所述第二多肽链包含细胞因子。In certain embodiments, the second polypeptide chain comprises a cytokine.

在某些实施方式中，所述第一靶向部分靶向PD-L1，所述第二靶向部分靶向PD-L1，所述第四靶向部分靶向PD-1、CD3或CD16a。In certain embodiments, the first targeting moiety targets PD-L1, the second targeting moiety targets PD-L1, and the fourth targeting moiety targets PD-1, CD3 or CD16a.

在某些实施方式中，所述第一靶向部分靶向PD-1，所述第二靶向部分靶向PD-1，所述第四靶向部分靶向PD-L1、CD3或CD16a。In certain embodiments, the first targeting moiety targets PD-1, the second targeting moiety targets PD-1, and the fourth targeting moiety targets PD-L1, CD3 or CD16a.

在某些实施方式中，所述第一靶向部分靶向PD-L1，所述第二靶向部分靶向PD-1，所述第三靶向部分靶向CD3或CD16a。In certain embodiments, the first targeting moiety targets PD-L1, the second targeting moiety targets PD-1, and the third targeting moiety targets CD3 or CD16a.

在某些实施方式中，所述第一靶向部分靶向PD-1，所述第二靶向部分靶向PD-L1，所述第三靶向部分靶向CD3或CD16a。In certain embodiments, the first targeting moiety targets PD-1, the second targeting moiety targets PD-L1, and the third targeting moiety targets CD3 or CD16a.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向CD28或CD16a，所述第二靶向部分靶向PD-1，所述第四靶向部分靶向PD-L1、HER2、CD47、CD19和/或CD20。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets CD28 or CD16a, the second targeting moiety targets PD-1, and the fourth targeting moiety targets PD-L1, HER2, CD47, CD19 and/or CD20.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向PD-1，所述第二靶向部分靶向CD28或CD16a，所述第四靶向部分靶向PD-L1、HER2、CD47、CD19和/或CD20。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets PD-1, the second targeting moiety targets CD28 or CD16a, and the fourth targeting moiety targets PD-L1, HER2, CD47, CD19 and/or CD20.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向CD28或CD16a，所述第二靶向部分靶向PD-1，所述第三靶向部分靶向EGFR或cMET，所述第四靶向部分靶向PD-L1、HER2、CD47、CD19和/或CD20。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets CD28 or CD16a, the second targeting moiety targets PD-1, and the third targeting moiety targets EGFR or cMET, the fourth targeting moiety targets PD-L1, HER2, CD47, CD19 and/or CD20.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向PD-1，所述第二靶向部分靶向CD28或CD16a，所述第三靶向部分靶向PD-L1、HER2、CD47、CD19和/或CD20，所述第四靶向部分靶向EGFR或cMET。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets PD-1, the second targeting moiety targets CD28 or CD16a, and the third targeting moiety targets PD-L1, HER2, CD47, CD19 and/or CD20, the fourth targeting moiety targets EGFR or cMET.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向PD-1，所述第二靶向部分靶向PD-1，且所述第二多肽链包含IL-2或IL-15。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets PD-1, the second targeting moiety targets PD-1, and the second polypeptide chain comprises IL-2 or IL-15.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向PD-L1，所述第二靶向部分靶向PD-L1，且所述第二多肽链包含IL-2或IL-15。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets PD-L1, the second targeting moiety targets PD-L1, and the second polypeptide chain comprises IL-2 or IL-15.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向PD-1，所述第二靶向部分靶向PD-L1，所述第一多肽链包含IL-2或IL-15，第四靶向部分包含CD3或CD16a。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets PD-1, the second targeting moiety targets PD-L1, and the first polypeptide chain comprises IL -2 or IL-15, the fourth targeting moiety comprises CD3 or CD16a.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向PD-L1，所述第二靶向部分靶向PD-1，所述第一多肽链包含IL-2或IL-15，第四靶向部分包含CD3或CD16a。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets PD-L1, the second targeting moiety targets PD-1, and the first polypeptide chain comprises IL -2 or IL-15, the fourth targeting moiety comprises CD3 or CD16a.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向PD-L1，所述第二靶向部分靶向PD-1，所述第一多肽链包含IL-2或IL-15。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets PD-L1, the second targeting moiety targets PD-1, and the first polypeptide chain comprises IL -2 or IL-15.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向PD-1，所述第二靶向部分靶向PD-L1，所述第一多肽链包含IL-2或IL-15。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets PD-1, the second targeting moiety targets PD-L1, and the first polypeptide chain comprises IL -2 or IL-15.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向PD-1，所述第二靶向部分靶向PD-1，所述第一多肽链包含IL-2或IL-15，且第四靶向部分靶向PD-L1。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets PD-1, the second targeting moiety targets PD-1, and the first polypeptide chain comprises IL -2 or IL-15, and the fourth targeting moiety targets PD-L1.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向PD-L1，所述第二靶向部分靶向PD-L1，所述第一多肽链包含IL-2或IL-15，且第四靶向部分靶向PD-1。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets PD-L1, the second targeting moiety targets PD-L1, and the first polypeptide chain comprises IL -2 or IL-15, and the fourth targeting moiety targets PD-1.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向PD-1，所述第二靶向部分靶向PD-L1，第三靶向部分靶向CD3或CD16a，且所述第二多肽链包含IL-2或IL-15。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets PD-1, the second targeting moiety targets PD-L1, and the third targeting moiety targets CD3 or CD16a, and the second polypeptide chain comprises IL-2 or IL-15.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向PD-L1，所述第二靶向部分靶向PD-1，第三靶向部分靶向CD3或CD16a，且所述第二多肽链包含IL-2或IL-15。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets PD-L1, the second targeting moiety targets PD-1, and the third targeting moiety targets CD3 or CD16a, and the second polypeptide chain comprises IL-2 or IL-15.

在某些实施方式中，所述第一靶向部分包含HCDR3，所述HCDR3包含SEQ ID NO:51所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:51.

在某些实施方式中，所述第一靶向部分包含HCDR3，所述HCDR3包含SEQ ID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO:9 and SEQ ID NO:10.

在某些实施方式中，所述第一靶向部分包含HCDR2，所述HCDR2包含SEQ ID NO:6所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:6.

在某些实施方式中，所述第一靶向部分包含HCDR1，所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:3.

在某些实施方式中，所述第一靶向部分包含SEQ ID NO:52所示的重链可变区VH的HCDR1、HCDR2和HCDR3。In certain embodiments, the first targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in SEQ ID NO:52.

在某些实施方式中，所述第一靶向部分包含SEQ ID NO:29至SEQ ID NO:31中任一项所示的重链可变区VH的HCDR1、HCDR2和HCDR3。In certain embodiments, the first targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in any one of SEQ ID NO:29 to SEQ ID NO:31.

在某些实施方式中，所述第一靶向部分包括重链可变区VH，所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:51所示的氨基酸序列；所述HCDR2包含SEQID NO:6所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。In some embodiments, the first targeting moiety comprises a heavy chain variable region VH, the VH comprises the HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:51; The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.

在某些实施方式中，所述第一靶向部分包括重链可变区VH，所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列；所述HCDR2包含SEQ ID NO:6所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。In certain embodiments, said first targeting moiety comprises a heavy chain variable region VH, said VH comprising said HCDR1, HCDR2 and HCDR3, said HCDR3 comprising SEQ ID NO:9 and SEQ ID NO:10 The amino acid sequence shown in any one; The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6; And the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.

在某些实施方式中，所述第一靶向部分中的HCDR1、HCDR2和HCDR3包含选自下述任意一组的氨基酸序列：In certain embodiments, HCDR1, HCDR2 and HCDR3 in the first targeting moiety comprise an amino acid sequence selected from any of the following groups:

a)HCDR1：SEQ ID NO:3，HCDR2：SEQ ID NO:6和HCDR3：SEQ ID NO:9；和a) HCDR1: SEQ ID NO:3, HCDR2: SEQ ID NO:6 and HCDR3: SEQ ID NO:9; and

b)HCDR1：SEQ ID NO:3，HCDR2：SEQ ID NO:6和HCDR3：SEQ ID NO:10。b) HCDR1: SEQ ID NO:3, HCDR2: SEQ ID NO:6 and HCDR3: SEQ ID NO:10.

在某些实施方式中，所述第一靶向部分包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ ID NO:47所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises SEQ ID NO : the amino acid sequence shown in 47.

在某些实施方式中，所述H-FR1包含SEQ ID NO:1和SEQ ID NO:2中任一项所示的氨基酸序列。In some embodiments, the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:1 and SEQ ID NO:2.

在某些实施方式中，所述第一靶向部分包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:48所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO:48 amino acid sequence.

在某些实施方式中，所述H-FR2包含SEQ ID NO:4和SEQ ID NO:5中任一项所示的氨基酸序列。In some embodiments, the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:4 and SEQ ID NO:5.

在某些实施方式中，所述第一靶向部分包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:49所示的氨基酸序列。In some embodiments, the first targeting moiety comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO:49 amino acid sequence.

在某些实施方式中，所述第一靶向部分中的所述H-FR3包含SEQ ID NO:7和SEQ IDNO:8中任一项所示的氨基酸序列。In certain embodiments, the H-FR3 in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:7 and SEQ ID NO:8.

在某些实施方式中，所述第一靶向部分包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO:50所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO : The amino acid sequence shown in 50.

在某些实施方式中，所述第一靶向部分中的所述H-FR4包含SEQ ID NO:11和SEQID NO:12中任一项所示的氨基酸序列。In certain embodiments, the H-FR4 in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO: 11 and SEQ ID NO: 12.

在某些实施方式中，所述第一靶向部分包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:47所示的氨基酸序列；所述H-FR2包含SEQ ID NO:48所示的氨基酸序列；所述H-FR3包含SEQ ID NO:49所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:50所示的氨基酸序列。In some embodiments, the first targeting moiety comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:47; the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:48; said H-FR3 comprises the amino acid sequence shown in SEQ ID NO:49; and said H-FR4 comprises the amino acid sequence shown in SEQ ID NO:50.

在某些实施方式中，所述第一靶向部分包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:1和SEQ ID NO:2中任一项所示的氨基酸序列；所述H-FR2包含SEQ IDNO:4和SEQ ID NO:5中任一项所示的氨基酸序列；所述H-FR3包含SEQ ID NO:7和SEQ IDNO:8中任一项所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:11和SEQ ID NO:12中任一项所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises any of SEQ ID NO:1 and SEQ ID NO:2 The amino acid sequence shown in one item; the H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO:4 and SEQ ID NO:5; the H-FR3 includes SEQ ID NO:7 and SEQ ID NO:8 The amino acid sequence shown in any one; And the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:11 and SEQ ID NO:12.

在某些实施方式中，所述第一靶向部分中的所述H-FR1、H-FR2、H-FR3和H-FR4包含选自下述任意一组的氨基酸序列：In certain embodiments, said H-FR1, H-FR2, H-FR3 and H-FR4 in said first targeting moiety comprise an amino acid sequence selected from any of the following groups:

a)H-FR1：SEQ ID NO:1，H-FR2：SEQ ID NO:4，H-FR3：SEQ ID NO:7和H-FR4：SEQ IDNO:11；和a) H-FR1: SEQ ID NO:1, H-FR2: SEQ ID NO:4, H-FR3: SEQ ID NO:7 and H-FR4: SEQ ID NO:11; and

b)H-FR1：SEQ ID NO:2，H-FR2：SEQ ID NO:5，H-FR3：SEQ ID NO:8和H-FR4：SEQ IDNO:12。b) H-FR1: SEQ ID NO:2, H-FR2: SEQ ID NO:5, H-FR3: SEQ ID NO:8 and H-FR4: SEQ ID NO:12.

在某些实施方式中，所述第一靶向部分包含重链可变区VH，所述VH包含SEQ IDNO:52所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:52.

在某些实施方式中，所述第一靶向部分包含重链可变区VH，所述VH包含SEQ IDNO:29至SEQ ID NO:31中任一项所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises a heavy chain variable region VH comprising the amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31.

在某些实施方式中，所述第一靶向部分包含LCDR3，所述LCDR3包含SEQ ID NO:57所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises LCDR3 comprising the amino acid sequence shown in SEQ ID NO:57.

在某些实施方式中，所述第一靶向部分包含LCDR3，所述LCDR3包含SEQ ID NO:21至SEQ ID NO:26中任一项所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises LCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26.

在某些实施方式中，所述第一靶向部分包含LCDR2，所述LCDR2包含SEQ ID NO:18所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 18.

在某些实施方式中，所述第一靶向部分包含LCDR1，所述LCDR1包含SEQ ID NO:15所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO:15.

在某些实施方式中，所述第一靶向部分包含SEQ ID NO:58所示的轻链可变区VL的LCDR1、LCDR2和LCDR3。In certain embodiments, the first targeting moiety comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL shown in SEQ ID NO:58.

在某些实施方式中，所述第一靶向部分包含SEQ ID NO:32至SEQ ID NO:38中任一项所示的轻链可变区VL的LCDR1、LCDR2和LCDR3。In certain embodiments, the first targeting moiety comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL shown in any one of SEQ ID NO:32 to SEQ ID NO:38.

在某些实施方式中，所述第一靶向部分包含轻链可变区VL，所述VL包含所述LCDR1、LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:57所示的氨基酸序列；所述LCDR2包含SEQID NO:18所示的氨基酸序列；以及所述LCDR1包含SEQ ID NO:15所示的氨基酸序列。In some embodiments, the first targeting moiety comprises a light chain variable region VL, the VL comprises the LCDR1, LCDR2 and LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:57; The LCDR2 comprises the amino acid sequence shown in SEQ ID NO:18; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:15.

在某些实施方式中，所述第一靶向部分包含轻链可变区VL，所述VL包含所述LCDR1、LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:21至SEQ ID NO:26中任一项所示的氨基酸序列；所述LCDR2包含SEQ ID NO:18所示的氨基酸序列；以及所述LCDR1包含SEQ ID NO:15所示的氨基酸序列。In certain embodiments, said first targeting moiety comprises a light chain variable region VL comprising said LCDR1, LCDR2 and LCDR3, said LCDR3 comprising SEQ ID NO:21 to SEQ ID NO:26 The amino acid sequence shown in any one; The LCDR2 includes the amino acid sequence shown in SEQ ID NO: 18; and the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 15.

在某些实施方式中，所述第一靶向部分中所述LCDR1、LCDR2和LCDR3包含选自下述任意一组的氨基酸序列：In certain embodiments, the LCDR1, LCDR2 and LCDR3 in the first targeting moiety comprise an amino acid sequence selected from any of the following groups:

a)LCDR1：SEQ ID NO:15，LCDR2：SEQ ID NO:18和LCDR3：SEQ ID NO:21；a) LCDR1: SEQ ID NO:15, LCDR2: SEQ ID NO:18 and LCDR3: SEQ ID NO:21;

b)LCDR1：SEQ ID NO:15，LCDR2：SEQ ID NO:18和LCDR3：SEQ ID NO:22；b) LCDR1: SEQ ID NO:15, LCDR2: SEQ ID NO:18 and LCDR3: SEQ ID NO:22;

c)LCDR1：SEQ ID NO:15，LCDR2：SEQ ID NO:18和LCDR3：SEQ ID NO:23；c) LCDR1: SEQ ID NO: 15, LCDR2: SEQ ID NO: 18 and LCDR3: SEQ ID NO: 23;

d)LCDR1：SEQ ID NO:15，LCDR2：SEQ ID NO:18和LCDR3：SEQ ID NO:24；d) LCDR1: SEQ ID NO:15, LCDR2: SEQ ID NO:18 and LCDR3: SEQ ID NO:24;

e)LCDR1：SEQ ID NO:15，LCDR2：SEQ ID NO:18和LCDR3：SEQ ID NO:25；和e) LCDR1: SEQ ID NO: 15, LCDR2: SEQ ID NO: 18 and LCDR3: SEQ ID NO: 25; and

f)LCDR1：SEQ ID NO:15，LCDR2：SEQ ID NO:18和LCDR3：SEQ ID NO:26。f) LCDR1: SEQ ID NO:15, LCDR2: SEQ ID NO:18 and LCDR3: SEQ ID NO:26.

在某些实施方式中，所述第一靶向部分包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接地相连，且所述L-FR1包含SEQ ID NO:53所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises L-FR1, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 comprises SEQ ID NO : the amino acid sequence shown in 53.

在某些实施方式中，所述第一靶向部分中的所述L-FR1包含SEQ ID NO:13和SEQID NO:14中任一项所示的氨基酸序列。In certain embodiments, the L-FR1 in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO: 13 and SEQ ID NO: 14.

在某些实施方式中，所述第一靶向部分包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:54所示的氨基酸序列。In some embodiments, the first targeting moiety comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises SEQ ID NO:54 amino acid sequence.

在某些实施方式中，所述第一靶向部分中的所述L-FR2包含SEQ ID NO:16和SEQID NO:17中任一项所示的氨基酸序列。In certain embodiments, the L-FR2 in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO: 16 and SEQ ID NO: 17.

在某些实施方式中，所述第一靶向部分包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:55所示的氨基酸序列。In some embodiments, the first targeting moiety comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises SEQ ID NO:55 amino acid sequence.

在某些实施方式中，所述第一靶向部分中的所述L-FR3包含SEQ ID NO:19和SEQID NO:20中任一项所示的氨基酸序列。In certain embodiments, the L-FR3 in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:19 and SEQ ID NO:20.

在某些实施方式中，所述第一靶向部分包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端直接或间接地相连，且所述L-FR4包含SEQ ID NO:56所示的氨基酸序列。In some embodiments, the first targeting moiety comprises L-FR4, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 comprises SEQ ID NO : the amino acid sequence shown in 56.

在某些实施方式中，所述第一靶向部分中的所述L-FR4包含SEQ ID NO:27和SEQID NO:28中任一项所示的氨基酸序列。In certain embodiments, the L-FR4 in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:27 and SEQ ID NO:28.

在某些实施方式中，所述第一靶向部分包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:53所示的氨基酸序列；所述L-FR2包含SEQ ID NO:54所示的氨基酸序列；所述L-FR3包含SEQ ID NO:55所示的氨基酸序列；以及所述L-FR4包含SEQ ID NO:56所示的氨基酸序列。In some embodiments, the first targeting moiety comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:53; the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:54; said L-FR3 comprises the amino acid sequence shown in SEQ ID NO:55; and said L-FR4 comprises the amino acid sequence shown in SEQ ID NO:56.

在某些实施方式中，所述第一靶向部分包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:13和SEQ ID NO:14中任一项所示的氨基酸序列；所述L-FR2包含SEQ IDNO:16和SEQ ID NO:17中任一项所示的氨基酸序列；所述L-FR3包含SEQ ID NO:19和SEQ IDNO:20中任一项所示的氨基酸序列；以及所述L-FR4包含SEQ ID NO:27和SEQ ID NO:28中任一项所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1 comprises any of SEQ ID NO:13 and SEQ ID NO:14 The amino acid sequence shown in one item; the L-FR2 includes the amino acid sequence shown in any one of SEQ ID NO:16 and SEQ ID NO:17; the L-FR3 includes SEQ ID NO:19 and SEQ ID NO:20 The amino acid sequence shown in any one; And the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:27 and SEQ ID NO:28.

在某些实施方式中，所述第一靶向部分中的所述L-FR1、L-FR2、L-FR3和L-FR4包含选自下述任意一组的氨基酸序列：In certain embodiments, said L-FR1, L-FR2, L-FR3, and L-FR4 in said first targeting moiety comprise an amino acid sequence selected from any of the following groups:

a)L-FR1：SEQ ID NO:13，L-FR2：SEQ ID NO:16，L-FR3：SEQ ID NO:19和L-FR4：SEQID NO:27；和a) L-FR1: SEQ ID NO: 13, L-FR2: SEQ ID NO: 16, L-FR3: SEQ ID NO: 19 and L-FR4: SEQ ID NO: 27; and

b)L-FR1：SEQ ID NO:14，L-FR2：SEQ ID NO:17，L-FR3：SEQ ID NO:20和L-FR4：SEQID NO:28。b) L-FR1: SEQ ID NO:14, L-FR2: SEQ ID NO:17, L-FR3: SEQ ID NO:20 and L-FR4: SEQ ID NO:28.

在某些实施方式中，所述第一靶向部分包括VL，所述VL包含SEQ ID NO:58中任一项所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises a VL comprising the amino acid sequence shown in any one of SEQ ID NO:58.

在某些实施方式中，所述第一靶向部分中的所述VL包含SEQ ID NO:32至SEQ IDNO:38中任一项所示的氨基酸序列。In certain embodiments, said VL in said first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38.

在某些实施方式中，所述第一靶向部分包含HCDR3，所述HCDR3包含SEQ ID NO:76所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:76.

在某些实施方式中，所述第一靶向部分包含HCDR2，所述HCDR2包含SEQ ID NO:70所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:70.

在某些实施方式中，所述第一靶向部分包含HCDR1，所述HCDR1包含SEQ ID NO:61所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:61.

在某些实施方式中，所述第一靶向部分包含SEQ ID NO:122所示的重链可变区VH的HCDR1、HCDR2和HCDR3。In certain embodiments, the first targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in SEQ ID NO: 122.

在某些实施方式中，所述第一靶向部分包含SEQ ID NO:95至SEQ ID NO:104中任一项所示的重链可变区VH的HCDR1、HCDR2和HCDR3。In certain embodiments, the first targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in any one of SEQ ID NO:95 to SEQ ID NO:104.

在某些实施方式中，所述第一靶向部分包括重链可变区VH，所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:76所示的氨基酸序列；所述HCDR2包含SEQID NO:70所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:61所示的氨基酸序列。In some embodiments, the first targeting moiety comprises a heavy chain variable region VH, the VH comprises the HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:76; The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:61.

在某些实施方式中，所述第一靶向部分包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ ID NO:115所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises SEQ ID NO : the amino acid sequence shown in 115.

在某些实施方式中，所述第一靶向部分中的所述H-FR1包含SEQ ID NO:59至SEQID NO:60中任一项所示的氨基酸序列。In certain embodiments, the H-FR1 in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:59 to SEQ ID NO:60.

在某些实施方式中，所述第一靶向部分包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:116所示的氨基酸序列。In some embodiments, the first targeting moiety comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO: 116 amino acid sequence.

在某些实施方式中，所述第一靶向部分中的所述H-FR2包含SEQ ID NO:62至SEQID NO:69中任一项所示的氨基酸序列。In certain embodiments, the H-FR2 in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:62 to SEQ ID NO:69.

在某些实施方式中，所述第一靶向部分包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:117所示的氨基酸序列。In some embodiments, the first targeting moiety comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO: 117 amino acid sequence.

在某些实施方式中，所述第一靶向部分中的所述H-FR3包含SEQ ID NO:71至SEQID NO:75中任一项所示的氨基酸序列。In certain embodiments, the H-FR3 in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:71 to SEQ ID NO:75.

在某些实施方式中，所述第一靶向部分包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO:118所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO : the amino acid sequence shown in 118.

在某些实施方式中，所述第一靶向部分中的所述H-FR4包含SEQ ID NO:77至SEQID NO:78中任一项所示的氨基酸序列。In certain embodiments, the H-FR4 in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:77 to SEQ ID NO:78.

在某些实施方式中，所述第一靶向部分包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:115所示的氨基酸序列；所述H-FR2包含SEQ ID NO:116所示的氨基酸序列；所述H-FR3包含SEQ ID NO:117所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:118所示的氨基酸序列。In some embodiments, the first targeting moiety comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 115; the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:116; the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:117; and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:118.

在某些实施方式中，所述第一靶向部分包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:59至SEQ ID NO:60中任一项所示的氨基酸序列；所述H-FR2包含SEQ IDNO:62至SEQ ID NO:69中任一项所示的氨基酸序列；所述H-FR3包含SEQ ID NO:71至SEQ IDNO:75中任一项所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:77至SEQ ID NO:78中任一项所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises any of SEQ ID NO:59 to SEQ ID NO:60 The amino acid sequence shown in one item; the H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO:62 to SEQ ID NO:69; the H-FR3 includes SEQ ID NO:71 to SEQ ID NO:75 The amino acid sequence shown in any one; And the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:77 to SEQ ID NO:78.

a)H-FR1：SEQ ID NO:59，H-FR2：SEQ ID NO:62，H-FR3：SEQ ID NO:71和H-FR4：SEQID NO:77；a) H-FR1: SEQ ID NO:59, H-FR2: SEQ ID NO:62, H-FR3: SEQ ID NO:71 and H-FR4: SEQ ID NO:77;

b)H-FR1：SEQ ID NO:60，H-FR2：SEQ ID NO:63，H-FR3：SEQ ID NO:72和H-FR4：SEQID NO:78；b) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:63, H-FR3: SEQ ID NO:72 and H-FR4: SEQ ID NO:78;

c)H-FR1：SEQ ID NO:60，H-FR2：SEQ ID NO:64，H-FR3：SEQ ID NO:73和H-FR4：SEQID NO:78；c) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:64, H-FR3: SEQ ID NO:73 and H-FR4: SEQ ID NO:78;

d)H-FR1：SEQ ID NO:60，H-FR2：SEQ ID NO:65，H-FR3：SEQ ID NO:74和H-FR4：SEQID NO:78；d) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:65, H-FR3: SEQ ID NO:74 and H-FR4: SEQ ID NO:78;

e)H-FR1：SEQ ID NO:60，H-FR2：SEQ ID NO:66，H-FR3：SEQ ID NO:75和H-FR4：SEQID NO:78；e) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:66, H-FR3: SEQ ID NO:75 and H-FR4: SEQ ID NO:78;

f)H-FR1：SEQ ID NO:60，H-FR2：SEQ ID NO:67，H-FR3：SEQ ID NO:72和H-FR4：SEQID NO:78；f) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:67, H-FR3: SEQ ID NO:72 and H-FR4: SEQ ID NO:78;

g)H-FR1：SEQ ID NO:60，H-FR2：SEQ ID NO:65，H-FR3：SEQ ID NO:72和H-FR4：SEQID NO:78；g) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:65, H-FR3: SEQ ID NO:72 and H-FR4: SEQ ID NO:78;

h)H-FR1：SEQ ID NO:60，H-FR2：SEQ ID NO:64，H-FR3：SEQ ID NO:74和H-FR4：SEQID NO:78；h) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:64, H-FR3: SEQ ID NO:74 and H-FR4: SEQ ID NO:78;

i)H-FR1：SEQ ID NO:60，H-FR2：SEQ ID NO:68，H-FR3：SEQ ID NO:72和H-FR4：SEQID NO:78；和i) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:68, H-FR3: SEQ ID NO:72 and H-FR4: SEQ ID NO:78; and

j)H-FR1：SEQ ID NO:60，H-FR2：SEQ ID NO:69，H-FR3：SEQ ID NO:73和H-FR4：SEQID NO:78。j) H-FR1: SEQ ID NO:60, H-FR2: SEQ ID NO:69, H-FR3: SEQ ID NO:73 and H-FR4: SEQ ID NO:78.

在某些实施方式中，所述第一靶向部分包含重链可变区VH，所述VH包含SEQ IDNO:122所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 122.

在某些实施方式中，所述第一靶向部分中的VH包含SEQ ID NO:95至SEQ ID NO:104中任一项所示的氨基酸序列。In certain embodiments, the VH in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:95 to SEQ ID NO:104.

在某些实施方式中，所述第一靶向部分包含LCDR3，所述LCDR3包含SEQ ID NO:94所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises LCDR3 comprising the amino acid sequence shown in SEQ ID NO:94.

在某些实施方式中，所述第一靶向部分包含LCDR2，所述LCDR2包含SEQ ID NO:87所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises LCDR2 comprising the amino acid sequence shown in SEQ ID NO:87.

在某些实施方式中，所述第一靶向部分包含LCDR1，所述LCDR1包含SEQ ID NO:82所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO:82.

在某些实施方式中，所述第一靶向部分包含SEQ ID NO:123中任一项所示的轻链可变区VL的LCDR1、LCDR2和LCDR3。In certain embodiments, the first targeting moiety comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL shown in any one of SEQ ID NO: 123.

在某些实施方式中，所述第一靶向部分包含SEQ ID NO:105至SEQ ID NO:111中任一项所示的轻链可变区VL的LCDR1、LCDR2和LCDR3。In certain embodiments, the first targeting moiety comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL shown in any one of SEQ ID NO: 105 to SEQ ID NO: 111.

在某些实施方式中，所述第一靶向部分包含轻链可变区VL，所述VL包含所述LCDR1、LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:94所示的氨基酸序列；所述LCDR2包含SEQID NO:87所示的氨基酸序列；以及所述LCDR1包含SEQ ID NO:82所示的氨基酸序列。In some embodiments, the first targeting moiety comprises a light chain variable region VL, the VL comprises the LCDR1, LCDR2 and LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:94; The LCDR2 comprises the amino acid sequence shown in SEQ ID NO:87; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:82.

在某些实施方式中，所述第一靶向部分包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接地相连，且所述L-FR1包含SEQ ID NO:119所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises L-FR1, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 comprises SEQ ID NO : the amino acid sequence shown in 119.

在某些实施方式中，所述L-FR1包含SEQ ID NO:79至SEQ ID NO:81中任一项所示的氨基酸序列。In certain embodiments, the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:79 to SEQ ID NO:81.

在某些实施方式中，所述第一靶向部分包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:120所示的氨基酸序列。In some embodiments, the first targeting moiety comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises SEQ ID NO: 120 amino acid sequence.

在某些实施方式中，所述第一靶向部分中的所述L-FR2包含SEQ ID NO:83至SEQID NO:86中任一项所示的氨基酸序列。In certain embodiments, the L-FR2 in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:83 to SEQ ID NO:86.

在某些实施方式中，所述第一靶向部分包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:121所示的氨基酸序列。In some embodiments, the first targeting moiety comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises SEQ ID NO: 121 amino acid sequence.

在某些实施方式中，所述第一靶向部分中的所述L-FR3包含SEQ ID NO:88至SEQID NO:93中任一项所示的氨基酸序列。In certain embodiments, the L-FR3 in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:88 to SEQ ID NO:93.

在某些实施方式中，所述第一靶向部分中的所述L-FR4包含SEQ ID NO:27至SEQID NO:28中任一项所示的氨基酸序列。In certain embodiments, the L-FR4 in the first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:27 to SEQ ID NO:28.

在某些实施方式中，所述第一靶向部分包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:119所示的氨基酸序列；所述L-FR2包含SEQ ID NO:120所示的氨基酸序列；所述L-FR3包含SEQ ID NO:121所示的氨基酸序列；以及所述L-FR4包含SEQ ID NO:56所示的氨基酸序列。In some embodiments, the first targeting moiety comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 119; the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:120; said L-FR3 comprises the amino acid sequence shown in SEQ ID NO:121; and said L-FR4 comprises the amino acid sequence shown in SEQ ID NO:56.

在某些实施方式中，所述第一靶向部分包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:79至SEQ ID NO:81中任一项所示的氨基酸序列；所述L-FR2包含SEQ IDNO:83至SEQ ID NO:86中任一项所示的氨基酸序列；所述L-FR3包含SEQ ID NO:88至SEQ IDNO:93中任一项所示的氨基酸序列；以及所述L-FR4包含SEQ ID NO:27至SEQ ID NO:28中任一项所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1 comprises any of SEQ ID NO:79 to SEQ ID NO:81 The amino acid sequence shown in one item; the L-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 83 to SEQ ID NO: 86; the L-FR3 includes SEQ ID NO: 88 to SEQ ID NO: 93 The amino acid sequence shown in any one; And the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:27 to SEQ ID NO:28.

a)L-FR1：SEQ ID NO:79，L-FR2：SEQ ID NO:83，L-FR3：SEQ ID NO:88和L-FR4：SEQID NO:27；a) L-FR1: SEQ ID NO:79, L-FR2: SEQ ID NO:83, L-FR3: SEQ ID NO:88 and L-FR4: SEQ ID NO:27;

b)L-FR1：SEQ ID NO:80，L-FR2：SEQ ID NO:84，L-FR3：SEQ ID NO:89和L-FR4：SEQID NO:28；b) L-FR1: SEQ ID NO:80, L-FR2: SEQ ID NO:84, L-FR3: SEQ ID NO:89 and L-FR4: SEQ ID NO:28;

c)L-FR1：SEQ ID NO:81，L-FR2：SEQ ID NO:85，L-FR3：SEQ ID NO:90和L-FR4：SEQID NO:28；c) L-FR1: SEQ ID NO:81, L-FR2: SEQ ID NO:85, L-FR3: SEQ ID NO:90 and L-FR4: SEQ ID NO:28;

d)L-FR1：SEQ ID NO:81，L-FR2：SEQ ID NO:85，L-FR3：SEQ ID NO:91和L-FR4：SEQID NO:28；d) L-FR1: SEQ ID NO:81, L-FR2: SEQ ID NO:85, L-FR3: SEQ ID NO:91 and L-FR4: SEQ ID NO:28;

e)L-FR1：SEQ ID NO:80，L-FR2：SEQ ID NO:85，L-FR3：SEQ ID NO:90和L-FR4：SEQID NO:28；e) L-FR1: SEQ ID NO:80, L-FR2: SEQ ID NO:85, L-FR3: SEQ ID NO:90 and L-FR4: SEQ ID NO:28;

f)L-FR1：SEQ ID NO:80，L-FR2：SEQ ID NO:85，L-FR3：SEQ ID NO:92和L-FR4：SEQID NO:28；和f) L-FR1: SEQ ID NO:80, L-FR2: SEQ ID NO:85, L-FR3: SEQ ID NO:92 and L-FR4: SEQ ID NO:28; and

g)L-FR1：SEQ ID NO:80，L-FR2：SEQ ID NO:86，L-FR3：SEQ ID NO:93和L-FR4：SEQID NO:28。g) L-FR1: SEQ ID NO:80, L-FR2: SEQ ID NO:86, L-FR3: SEQ ID NO:93 and L-FR4: SEQ ID NO:28.

在某些实施方式中，所述第一靶向部分包括VL，所述VL包含SEQ ID NO:123所示的氨基酸序列。In certain embodiments, the first targeting moiety comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 123.

在某些实施方式中，所述第一靶向部分中的所述VL包含SEQ ID NO:105至SEQ IDNO:111中任一项所示的氨基酸序列。In certain embodiments, said VL in said first targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO: 105 to SEQ ID NO: 111.

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向CD28，所述第一靶向部分包含VH，所述VH包含SEQ ID NO：176所示的氨基酸序列。In certain embodiments, the first targeting moiety in the isolated antigen binding protein targets CD28, the first targeting moiety comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 176 .

在某些实施方式中，所述分离的抗原结合蛋白中所述第一靶向部分靶向CD16a，所述第一靶向部分包含VH，所述VH包含SEQ ID NO：168所示的氨基酸序列。In some embodiments, the first targeting moiety in the isolated antigen binding protein targets CD16a, the first targeting moiety comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 168 .

在某些实施方式中，所述第二靶向部分包含HCDR3，所述HCDR3包含SEQ ID NO:51所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:51.

在某些实施方式中，所述第二靶向部分包含HCDR3，所述HCDR3包含SEQ ID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO:9 and SEQ ID NO:10.

在某些实施方式中，所述第二靶向部分包含HCDR2，所述HCDR2包含SEQ ID NO:6所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:6.

在某些实施方式中，所述第二靶向部分包含HCDR1，所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:3.

在某些实施方式中，所述第二靶向部分包含SEQ ID NO:52所示的重链可变区VH的HCDR1、HCDR2和HCDR3。In certain embodiments, the second targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in SEQ ID NO:52.

在某些实施方式中，所述第二靶向部分包含SEQ ID NO:29至SEQ ID NO:31中任一项所示的重链可变区VH的HCDR1、HCDR2和HCDR3。In certain embodiments, the second targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in any one of SEQ ID NO:29 to SEQ ID NO:31.

在某些实施方式中，所述第二靶向部分包括重链可变区VH，所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:51所示的氨基酸序列；所述HCDR2包含SEQID NO:6所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。In some embodiments, the second targeting moiety comprises a heavy chain variable region VH, the VH comprises the HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:51; The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.

在某些实施方式中，所述第二靶向部分包括重链可变区VH，所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列；所述HCDR2包含SEQ ID NO:6所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises a heavy chain variable region VH, the VH comprising the HCDR1, HCDR2 and HCDR3, the HCDR3 comprising SEQ ID NO: 9 and SEQ ID NO: 10 The amino acid sequence shown in any one; The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6; And the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.

在某些实施方式中，所述第二靶向部分中的所述HCDR1、HCDR2和HCDR3包含选自下述任意一组的氨基酸序列：In certain embodiments, said HCDR1, HCDR2 and HCDR3 in said second targeting moiety comprise an amino acid sequence selected from any of the following groups:

在某些实施方式中，所述第二靶向部分包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ ID NO:47所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises SEQ ID NO : the amino acid sequence shown in 47.

在某些实施方式中，所述第二靶向部分中的所述H-FR1包含SEQ ID NO:1和SEQ IDNO:2中任一项所示的氨基酸序列。In certain embodiments, the H-FR1 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:1 and SEQ ID NO:2.

在某些实施方式中，所述第二靶向部分包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:48所示的氨基酸序列。In some embodiments, the second targeting moiety comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO:48 amino acid sequence.

在某些实施方式中，所述第二靶向部分中的所述H-FR2包含SEQ ID NO:4和SEQ IDNO:5中任一项所示的氨基酸序列。In certain embodiments, the H-FR2 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:4 and SEQ ID NO:5.

在某些实施方式中，所述第二靶向部分包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:49所示的氨基酸序列。In some embodiments, the second targeting moiety comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO:49 amino acid sequence.

在某些实施方式中，所述第二靶向部分中的所述H-FR3包含SEQ ID NO:7和SEQ IDNO:8中任一项所示的氨基酸序列。In certain embodiments, the H-FR3 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:7 and SEQ ID NO:8.

在某些实施方式中，所述第二靶向部分包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO:50所示的氨基酸序列。In some embodiments, the second targeting moiety comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO : The amino acid sequence shown in 50.

在某些实施方式中，所述第二靶向部分中的所述H-FR4包含SEQ ID NO:11和SEQID NO:12中任一项所示的氨基酸序列。In certain embodiments, the H-FR4 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO: 11 and SEQ ID NO: 12.

在某些实施方式中，所述第二靶向部分包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:47所示的氨基酸序列；所述H-FR2包含SEQ ID NO:48所示的氨基酸序列；所述H-FR3包含SEQ ID NO:49所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:50所示的氨基酸序列。In some embodiments, the second targeting moiety comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:47; the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:48; said H-FR3 comprises the amino acid sequence shown in SEQ ID NO:49; and said H-FR4 comprises the amino acid sequence shown in SEQ ID NO:50.

在某些实施方式中，所述第二靶向部分包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:1和SEQ ID NO:2中任一项所示的氨基酸序列；所述H-FR2包含SEQ IDNO:4和SEQ ID NO:5中任一项所示的氨基酸序列；所述H-FR3包含SEQ ID NO:7和SEQ IDNO:8中任一项所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:11和SEQ ID NO:12中任一项所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises any of SEQ ID NO:1 and SEQ ID NO:2 The amino acid sequence shown in one item; the H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO:4 and SEQ ID NO:5; the H-FR3 includes SEQ ID NO:7 and SEQ ID NO:8 The amino acid sequence shown in any one; And the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:11 and SEQ ID NO:12.

在某些实施方式中，所述第二靶向部分中的所述H-FR1、H-FR2、H-FR3和H-FR4包含选自下述任意一组的氨基酸序列：In certain embodiments, said H-FR1, H-FR2, H-FR3 and H-FR4 in said second targeting moiety comprise an amino acid sequence selected from any of the following groups:

在某些实施方式中，所述第二靶向部分包含重链可变区VH，所述VH包含SEQ IDNO:52所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:52.

在某些实施方式中，所述第二靶向部分包含重链可变区VH，所述VH包含SEQ IDNO:29至SEQ ID NO:31中任一项所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises a heavy chain variable region VH comprising the amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31.

在某些实施方式中，所述第二靶向部分包含LCDR3，所述LCDR3包含SEQ ID NO:57所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises LCDR3 comprising the amino acid sequence shown in SEQ ID NO:57.

在某些实施方式中，所述第二靶向部分包含LCDR3，所述LCDR3包含SEQ ID NO:21至SEQ ID NO:26中任一项所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises LCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26.

在某些实施方式中，所述第二靶向部分包含LCDR2，所述LCDR2包含SEQ ID NO:18所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 18.

在某些实施方式中，所述第二靶向部分包含LCDR1，所述LCDR1包含SEQ ID NO:15所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO:15.

在某些实施方式中，所述第二靶向部分包含SEQ ID NO:58所示的轻链可变区VL的LCDR1、LCDR2和LCDR3。In certain embodiments, the second targeting moiety comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL shown in SEQ ID NO:58.

在某些实施方式中，所述第二靶向部分包含SEQ ID NO:32至SEQ ID NO:38中任一项所示的轻链可变区VL的LCDR1、LCDR2和LCDR3。In certain embodiments, the second targeting moiety comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL shown in any one of SEQ ID NO:32 to SEQ ID NO:38.

在某些实施方式中，所述第二靶向部分包含轻链可变区VL，所述VL包含所述LCDR1、LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:57所示的氨基酸序列；所述LCDR2包含SEQID NO:18所示的氨基酸序列；以及所述LCDR1包含SEQ ID NO:15所示的氨基酸序列。In some embodiments, the second targeting moiety comprises a light chain variable region VL, the VL comprises the LCDR1, LCDR2 and LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:57; The LCDR2 comprises the amino acid sequence shown in SEQ ID NO:18; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:15.

在某些实施方式中，所述第二靶向部分包含轻链可变区VL，所述VL包含所述LCDR1、LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:21至SEQ ID NO:26中任一项所示的氨基酸序列；所述LCDR2包含SEQ ID NO:18所示的氨基酸序列；以及所述LCDR1包含SEQ ID NO:15所示的氨基酸序列。In certain embodiments, said second targeting moiety comprises a light chain variable region VL comprising said LCDR1, LCDR2 and LCDR3, said LCDR3 comprising SEQ ID NO:21 to SEQ ID NO:26 The amino acid sequence shown in any one; The LCDR2 includes the amino acid sequence shown in SEQ ID NO: 18; and the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 15.

在某些实施方式中，所述第二靶向部分中的所述LCDR1、LCDR2和LCDR3包含选自下述任意一组的氨基酸序列：In certain embodiments, said LCDR1, LCDR2, and LCDR3 in said second targeting moiety comprise an amino acid sequence selected from any of the following groups:

在某些实施方式中，所述第二靶向部分包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接地相连，且所述L-FR1包含SEQ ID NO:53所示的氨基酸序列。In some embodiments, the second targeting moiety comprises L-FR1, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 comprises SEQ ID NO : the amino acid sequence shown in 53.

在某些实施方式中，所述第二靶向部分中的所述L-FR1包含SEQ ID NO:13和SEQID NO:14中任一项所示的氨基酸序列。In certain embodiments, the L-FR1 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:13 and SEQ ID NO:14.

在某些实施方式中，所述第二靶向部分包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:54所示的氨基酸序列。In some embodiments, the second targeting moiety comprises L-FR2, and the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises SEQ ID NO:54 amino acid sequence.

在某些实施方式中，所述第二靶向部分中的所述L-FR2包含SEQ ID NO:16和SEQID NO:17中任一项所示的氨基酸序列。In certain embodiments, the L-FR2 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO: 16 and SEQ ID NO: 17.

在某些实施方式中，所述第二靶向部分包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:55所示的氨基酸序列。In some embodiments, the second targeting moiety comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises SEQ ID NO:55 amino acid sequence.

在某些实施方式中，所述第二靶向部分中的所述L-FR3包含SEQ ID NO:19和SEQID NO:20中任一项所示的氨基酸序列。In certain embodiments, the L-FR3 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:19 and SEQ ID NO:20.

在某些实施方式中，所述第二靶向部分包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端直接或间接地相连，且所述L-FR4包含SEQ ID NO:56所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises L-FR4, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 comprises SEQ ID NO : the amino acid sequence shown in 56.

在某些实施方式中，所述第二靶向部分中的所述L-FR4包含SEQ ID NO:27和SEQID NO:28中任一项所示的氨基酸序列。In certain embodiments, the L-FR4 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:27 and SEQ ID NO:28.

在某些实施方式中，所述第二靶向部分包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:53所示的氨基酸序列；所述L-FR2包含SEQ ID NO:54所示的氨基酸序列；所述L-FR3包含SEQ ID NO:55所示的氨基酸序列；以及所述L-FR4包含SEQ ID NO:56所示的氨基酸序列。In some embodiments, the second targeting moiety comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:53; the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:54; said L-FR3 comprises the amino acid sequence shown in SEQ ID NO:55; and said L-FR4 comprises the amino acid sequence shown in SEQ ID NO:56.

在某些实施方式中，所述第二靶向部分包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:13和SEQ ID NO:14中任一项所示的氨基酸序列；所述L-FR2包含SEQ IDNO:16和SEQ ID NO:17中任一项所示的氨基酸序列；所述L-FR3包含SEQ ID NO:19和SEQ IDNO:20中任一项所示的氨基酸序列；以及所述L-FR4包含SEQ ID NO:27和SEQ ID NO:28中任一项所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1 comprises any of SEQ ID NO:13 and SEQ ID NO:14 The amino acid sequence shown in one item; the L-FR2 includes the amino acid sequence shown in any one of SEQ ID NO:16 and SEQ ID NO:17; the L-FR3 includes SEQ ID NO:19 and SEQ ID NO:20 The amino acid sequence shown in any one; And the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:27 and SEQ ID NO:28.

在某些实施方式中，所述第二靶向部分中的所述L-FR1、L-FR2、L-FR3和L-FR4包含选自下述任意一组的氨基酸序列：In certain embodiments, said L-FR1, L-FR2, L-FR3, and L-FR4 in said second targeting moiety comprise an amino acid sequence selected from any of the following groups:

在某些实施方式中，所述第二靶向部分包括VL，所述VL包含SEQ ID NO:58中任一项所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises a VL comprising the amino acid sequence shown in any one of SEQ ID NO:58.

在某些实施方式中，所述第二靶向部分中的所述VL包含SEQ ID NO:32至SEQ IDNO:38中任一项所示的氨基酸序列。In certain embodiments, said VL in said second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38.

在某些实施方式中，所述第二靶向部分包含HCDR3，所述HCDR3包含SEQ ID NO:76所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:76.

在某些实施方式中，所述第二靶向部分包含HCDR2，所述HCDR2包含SEQ ID NO:70所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:70.

在某些实施方式中，所述第二靶向部分包含HCDR1，所述HCDR1包含SEQ ID NO:61所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:61.

在某些实施方式中，所述第二靶向部分包含SEQ ID NO:122所示的重链可变区VH的HCDR1、HCDR2和HCDR3。In certain embodiments, the second targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in SEQ ID NO: 122.

在某些实施方式中，所述第二靶向部分包含SEQ ID NO:95至SEQ ID NO:104中任一项所示的重链可变区VH的HCDR1、HCDR2和HCDR3。In certain embodiments, the second targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in any one of SEQ ID NO:95 to SEQ ID NO:104.

在某些实施方式中，所述第二靶向部分包括重链可变区VH，所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:76所示的氨基酸序列；所述HCDR2包含SEQID NO:70所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:61所示的氨基酸序列。In some embodiments, the second targeting moiety comprises a heavy chain variable region VH, the VH comprises the HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:76; The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:61.

在某些实施方式中，所述第二靶向部分包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ ID NO:115所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises SEQ ID NO : the amino acid sequence shown in 115.

在某些实施方式中，所述第二靶向部分中的所述H-FR1包含SEQ ID NO:59至SEQID NO:60中任一项所示的氨基酸序列。In certain embodiments, the H-FR1 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:59 to SEQ ID NO:60.

在某些实施方式中，所述第二靶向部分包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:116所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO: 116 amino acid sequence.

在某些实施方式中，所述第二靶向部分中的所述H-FR2包含SEQ ID NO:62至SEQID NO:69中任一项所示的氨基酸序列。In certain embodiments, the H-FR2 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:62 to SEQ ID NO:69.

在某些实施方式中，所述第二靶向部分包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:117所示的氨基酸序列。In some embodiments, the second targeting moiety comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO: 117 amino acid sequence.

在某些实施方式中，所述第二靶向部分中的所述H-FR3包含SEQ ID NO:71至SEQID NO:75中任一项所示的氨基酸序列。In certain embodiments, the H-FR3 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:71 to SEQ ID NO:75.

在某些实施方式中，所述第二靶向部分包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO:118所示的氨基酸序列。In some embodiments, the second targeting moiety comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO : the amino acid sequence shown in 118.

在某些实施方式中，所述第二靶向部分中的所述H-FR4包含SEQ ID NO:77至SEQID NO:78中任一项所示的氨基酸序列。In certain embodiments, the H-FR4 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:77 to SEQ ID NO:78.

在某些实施方式中，所述第二靶向部分包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:115所示的氨基酸序列；所述H-FR2包含SEQ ID NO:116所示的氨基酸序列；所述H-FR3包含SEQ ID NO:117所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:118所示的氨基酸序列。In some embodiments, the second targeting moiety comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 115; the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:116; the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:117; and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:118.

在某些实施方式中，所述第二靶向部分包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:59至SEQ ID NO:60中任一项所示的氨基酸序列；所述H-FR2包含SEQ IDNO:62至SEQ ID NO:69中任一项所示的氨基酸序列；所述H-FR3包含SEQ ID NO:71至SEQ IDNO:75中任一项所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:77至SEQ ID NO:78中任一项所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises any of SEQ ID NO:59 to SEQ ID NO:60 The amino acid sequence shown in one item; the H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO:62 to SEQ ID NO:69; the H-FR3 includes SEQ ID NO:71 to SEQ ID NO:75 The amino acid sequence shown in any one; And the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:77 to SEQ ID NO:78.

在某些实施方式中，所述第二靶向部分包含重链可变区VH，所述VH包含SEQ IDNO:122所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 122.

在某些实施方式中，所述VH包含SEQ ID NO:95至SEQ ID NO:104中任一项所示的氨基酸序列。In certain embodiments, the VH comprises the amino acid sequence shown in any one of SEQ ID NO:95 to SEQ ID NO:104.

在某些实施方式中，所述第二靶向部分包含LCDR3，所述LCDR3包含SEQ ID NO:94所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises LCDR3 comprising the amino acid sequence shown in SEQ ID NO:94.

在某些实施方式中，所述第二靶向部分包含LCDR2，所述LCDR2包含SEQ ID NO:87所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises LCDR2 comprising the amino acid sequence shown in SEQ ID NO:87.

在某些实施方式中，所述第二靶向部分包含LCDR1，所述LCDR1包含SEQ ID NO:82所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO:82.

在某些实施方式中，所述第二靶向部分包含SEQ ID NO:123中任一项所示的轻链可变区VL的LCDR1、LCDR2和LCDR3。In certain embodiments, the second targeting moiety comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL shown in any one of SEQ ID NO:123.

在某些实施方式中，所述第二靶向部分包含SEQ ID NO:105至SEQ ID NO:111中任一项所示的轻链可变区VL的LCDR1、LCDR2和LCDR3。In certain embodiments, the second targeting moiety comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL shown in any one of SEQ ID NO: 105 to SEQ ID NO: 111.

在某些实施方式中，所述第二靶向部分包含轻链可变区VL，所述VL包含所述LCDR1、LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:94所示的氨基酸序列；所述LCDR2包含SEQID NO:87所示的氨基酸序列；以及所述LCDR1包含SEQ ID NO:82所示的氨基酸序列。In some embodiments, the second targeting moiety comprises a light chain variable region VL, the VL comprises the LCDR1, LCDR2 and LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:94; The LCDR2 comprises the amino acid sequence shown in SEQ ID NO:87; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:82.

在某些实施方式中，所述第二靶向部分包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接地相连，且所述L-FR1包含SEQ ID NO:119所示的氨基酸序列。In some embodiments, the second targeting moiety comprises L-FR1, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 comprises SEQ ID NO : the amino acid sequence shown in 119.

在某些实施方式中，所述第二靶向部分中的所述L-FR1包含SEQ ID NO:79至SEQID NO:81中任一项所示的氨基酸序列。In certain embodiments, the L-FR1 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:79 to SEQ ID NO:81.

在某些实施方式中，所述第二靶向部分包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:120所示的氨基酸序列。In some embodiments, the second targeting moiety comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises SEQ ID NO: 120 amino acid sequence.

在某些实施方式中，所述第二靶向部分中的所述L-FR2包含SEQ ID NO:83至SEQID NO:86中任一项所示的氨基酸序列。In certain embodiments, the L-FR2 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:83 to SEQ ID NO:86.

在某些实施方式中，所述第二靶向部分包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:121所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises SEQ ID NO: 121 amino acid sequence.

在某些实施方式中，所述第二靶向部分中的所述L-FR3包含SEQ ID NO:88至SEQID NO:93中任一项所示的氨基酸序列。In certain embodiments, the L-FR3 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:88 to SEQ ID NO:93.

在某些实施方式中，所述第二靶向部分中的所述L-FR4包含SEQ ID NO:27至SEQID NO:28中任一项所示的氨基酸序列。In certain embodiments, the L-FR4 in the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO:27 to SEQ ID NO:28.

在某些实施方式中，所述第二靶向部分包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:119所示的氨基酸序列；所述L-FR2包含SEQ ID NO:120所示的氨基酸序列；所述L-FR3包含SEQ ID NO:121所示的氨基酸序列；以及所述L-FR4包含SEQ ID NO:56所示的氨基酸序列。In some embodiments, the second targeting moiety comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 119; the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:120; said L-FR3 comprises the amino acid sequence shown in SEQ ID NO:121; and said L-FR4 comprises the amino acid sequence shown in SEQ ID NO:56.

在某些实施方式中，所述第二靶向部分包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:79至SEQ ID NO:81中任一项所示的氨基酸序列；所述L-FR2包含SEQ IDNO:83至SEQ ID NO:86中任一项所示的氨基酸序列；所述L-FR3包含SEQ ID NO:88至SEQ IDNO:93中任一项所示的氨基酸序列；以及所述L-FR4包含SEQ ID NO:27至SEQ ID NO:28中任一项所示的氨基酸序列。In certain embodiments, said second targeting moiety comprises L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR1 comprising any of SEQ ID NO:79 to SEQ ID NO:81 The amino acid sequence shown in one item; the L-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 83 to SEQ ID NO: 86; the L-FR3 includes SEQ ID NO: 88 to SEQ ID NO: 93 The amino acid sequence shown in any one; And the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:27 to SEQ ID NO:28.

在某些实施方式中，所述第二靶向部分中所述L-FR1、L-FR2、L-FR3和L-FR4包含选自下述任意一组的氨基酸序列：In certain embodiments, the L-FR1, L-FR2, L-FR3 and L-FR4 in the second targeting moiety comprise an amino acid sequence selected from any of the following groups:

在某些实施方式中，所述第二靶向部分包括VL，所述VL包含SEQ ID NO:123所示的氨基酸序列。In certain embodiments, the second targeting moiety comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 123.

在某些实施方式中，所述第二靶向部分的VL包含SEQ ID NO:105至SEQ ID NO:111中任一项所示的氨基酸序列。In certain embodiments, the VL of the second targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO: 105 to SEQ ID NO: 111.

在某些实施方式中，所述分离的抗原结合蛋白中所述第二靶向部分靶向CD28，所述第二靶向部分包含VH，所述VH包含SEQ ID NO：176所示的氨基酸序列。In certain embodiments, the second targeting moiety in the isolated antigen binding protein targets CD28, the second targeting moiety comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 176 .

在某些实施方式中，所述分离的抗原结合蛋白中所述第二靶向部分靶向CD16a，所述第二靶向部分包含VH，所述VH包含SEQ ID NO：168所示的氨基酸序列。In certain embodiments, the second targeting moiety in the isolated antigen binding protein targets CD16a, the second targeting moiety comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 168 .

在某些实施方式中，所述分离的抗原结合蛋白包括第一靶向部分和第二靶向部分，所述第一靶向部分包含SEQ ID NO:29至SEQ ID NO:31、SEQ ID NO:32至SEQ ID NO:38、SEQ ID NO:95至SEQ ID NO:104和SEQ ID NO:105至SEQ ID NO:111中任一项所示的氨基酸序列；所述第二靶向部分包含SEQ ID NO:29至SEQ ID NO:31、SEQ ID NO:32至SEQ ID NO:38、SEQ ID NO:95至SEQ ID NO:104和SEQ ID NO:105至SEQ ID NO:111中任一项所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein comprises a first targeting moiety and a second targeting moiety, the first targeting moiety comprising SEQ ID NO: 29 to SEQ ID NO: 31, SEQ ID NO :32 to the amino acid sequence shown in any one of SEQ ID NO:38, SEQ ID NO:95 to SEQ ID NO:104, and SEQ ID NO:105 to SEQ ID NO:111; the second targeting moiety comprises Any of SEQ ID NO: 29 to SEQ ID NO: 31, SEQ ID NO: 32 to SEQ ID NO: 38, SEQ ID NO: 95 to SEQ ID NO: 104, and SEQ ID NO: 105 to SEQ ID NO: 111 The amino acid sequence shown in item.

在某些实施方式中，所述第一靶向部分和第二靶向部分包含选自下述任意一组的氨基酸序列：In certain embodiments, the first targeting moiety and the second targeting moiety comprise an amino acid sequence selected from any of the following groups:

a)第一靶向部分：SEQ ID NO:97和第二靶向部分：SEQ ID NO:31；a) a first targeting moiety: SEQ ID NO:97 and a second targeting moiety: SEQ ID NO:31;

b)第一靶向部分：SEQ ID NO:98和第二靶向部分：SEQ ID NO:31；b) a first targeting moiety: SEQ ID NO:98 and a second targeting moiety: SEQ ID NO:31;

c)第一靶向部分：SEQ ID NO:100和第二靶向部分：SEQ ID NO:31；c) a first targeting moiety: SEQ ID NO: 100 and a second targeting moiety: SEQ ID NO: 31;

d)第一靶向部分：SEQ ID NO:102和第二靶向部分：SEQ ID NO:31；d) a first targeting moiety: SEQ ID NO: 102 and a second targeting moiety: SEQ ID NO: 31;

e)第一靶向部分：SEQ ID NO:103和第二靶向部分：SEQ ID NO:31；e) a first targeting moiety: SEQ ID NO: 103 and a second targeting moiety: SEQ ID NO: 31;

f)第一靶向部分：SEQ ID NO:31和第二靶向部分：SEQ ID NO:97；f) a first targeting moiety: SEQ ID NO:31 and a second targeting moiety: SEQ ID NO:97;

g)第一靶向部分：SEQ ID NO:31和第二靶向部分：SEQ ID NO:98；g) a first targeting moiety: SEQ ID NO:31 and a second targeting moiety: SEQ ID NO:98;

h)第一靶向部分：SEQ ID NO:31和第二靶向部分：SEQ ID NO:100；h) a first targeting moiety: SEQ ID NO:31 and a second targeting moiety: SEQ ID NO:100;

i)第一靶向部分：SEQ ID NO:31和第二靶向部分：SEQ ID NO:102；i) a first targeting moiety: SEQ ID NO:31 and a second targeting moiety: SEQ ID NO:102;

j)第一靶向部分：SEQ ID NO:31和第二靶向部分：SEQ ID NO:103；j) a first targeting moiety: SEQ ID NO:31 and a second targeting moiety: SEQ ID NO:103;

k)第一靶向部分：SEQ ID NO:97和第二靶向部分：SEQ ID NO:36；k) a first targeting moiety: SEQ ID NO:97 and a second targeting moiety: SEQ ID NO:36;

l)第一靶向部分：SEQ ID NO:98和第二靶向部分：SEQ ID NO:36；l) a first targeting moiety: SEQ ID NO:98 and a second targeting moiety: SEQ ID NO:36;

m)第一靶向部分：SEQ ID NO:100和第二靶向部分：SEQ ID NO:36；m) a first targeting moiety: SEQ ID NO: 100 and a second targeting moiety: SEQ ID NO: 36;

n)第一靶向部分：SEQ ID NO:102和第二靶向部分：SEQ ID NO:36；n) a first targeting moiety: SEQ ID NO: 102 and a second targeting moiety: SEQ ID NO: 36;

o)第一靶向部分：SEQ ID NO:103和第二靶向部分：SEQ ID NO:36；o) a first targeting moiety: SEQ ID NO: 103 and a second targeting moiety: SEQ ID NO: 36;

p)第一靶向部分：SEQ ID NO:36和第二靶向部分：SEQ ID NO:97；p) a first targeting moiety: SEQ ID NO:36 and a second targeting moiety: SEQ ID NO:97;

q)第一靶向部分：SEQ ID NO:36和第二靶向部分：SEQ ID NO:98；q) a first targeting moiety: SEQ ID NO:36 and a second targeting moiety: SEQ ID NO:98;

r)第一靶向部分：SEQ ID NO:36和第二靶向部分：SEQ ID NO:100；r) a first targeting moiety: SEQ ID NO:36 and a second targeting moiety: SEQ ID NO:100;

s)第一靶向部分：SEQ ID NO:36和第二靶向部分：SEQ ID NO:102；s) a first targeting moiety: SEQ ID NO:36 and a second targeting moiety: SEQ ID NO:102;

t)第一靶向部分：SEQ ID NO:36和第二靶向部分：SEQ ID NO:103；t) a first targeting moiety: SEQ ID NO:36 and a second targeting moiety: SEQ ID NO:103;

u)第一靶向部分：SEQ ID NO:31和第二靶向部分：SEQ ID NO:176；u) a first targeting moiety: SEQ ID NO:31 and a second targeting moiety: SEQ ID NO:176;

v)第一靶向部分：SEQ ID NO:31和第二靶向部分：SEQ ID NO:168；v) a first targeting moiety: SEQ ID NO:31 and a second targeting moiety: SEQ ID NO:168;

w)第一靶向部分：SEQ ID NO:176和第二靶向部分：SEQ ID NO:31；w) a first targeting moiety: SEQ ID NO: 176 and a second targeting moiety: SEQ ID NO: 31;

x)第一靶向部分：SEQ ID NO:168和第二靶向部分：SEQ ID NO:31。x) First targeting moiety: SEQ ID NO: 168 and second targeting moiety: SEQ ID NO: 31.

在某些实施方式中，所述第二多肽链包含重链恒定区，且所述重链恒定区包括源自IgG的恒定区或源自IgY的恒定区。In certain embodiments, the second polypeptide chain comprises a heavy chain constant region, and the heavy chain constant region comprises an IgG-derived constant region or an IgY-derived constant region.

在某些实施方式中，所述第二多肽链中所述重链恒定区包括源自IgG的恒定区。In certain embodiments, said heavy chain constant region in said second polypeptide chain comprises an IgG-derived constant region.

在某些实施方式中，所述第二多肽链中所述重链恒定区包括源自IgG1、IgG2、IgG3或IgG4的恒定区。In certain embodiments, said heavy chain constant region in said second polypeptide chain comprises a constant region derived from IgGl, IgG2, IgG3 or IgG4.

在某些实施方式中，所述第二多肽链中所述重链恒定区包括源自IgG1的Fc区。In certain embodiments, said heavy chain constant region in said second polypeptide chain comprises an IgG1 derived Fc region.

在某些实施方式中，所述第二多肽链中所述重链恒定区中的Fc区包含N297A突变，残基根据Kabat系统进行编号。In certain embodiments, the Fc region of said heavy chain constant region in said second polypeptide chain comprises a N297A mutation, and residues are numbered according to the Kabat system.

在某些实施方式中，所述重链恒定区中的Fc区包含M252Y、S254T和T256E突变，残基根据Kabat系统进行编号。In certain embodiments, the Fc region in the heavy chain constant region comprises M252Y, S254T and T256E mutations, and the residues are numbered according to the Kabat system.

在某些实施方式中，所述第二多肽链中所述重链恒定区包含SEQ ID NO:112至SEQID NO:113所示的氨基酸序列。In certain embodiments, said heavy chain constant region in said second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 112 to SEQ ID NO: 113.

在某些实施方式中，所述第一多肽链包含轻链恒定区，且所述轻链恒定区包括源自Igκ的恒定区或源自Igλ的恒定区。In certain embodiments, the first polypeptide chain comprises a light chain constant region, and the light chain constant region comprises a constant region derived from Igκ or a constant region derived from Igλ.

在某些实施方式中，所述轻链恒定区包括源自人Igκ的恒定区。In certain embodiments, the light chain constant region comprises a constant region derived from human Igκ.

在某些实施方式中，所述轻链恒定区包含SEQ ID NO:114所示的氨基酸序列。In certain embodiments, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 114.

在某些实施方式中，所述分离的抗原结合蛋白中所述第三靶向部分包含VH，所述VH包含SEQ ID NO：168、170、172、174、176和178中任一项所示的氨基酸序列。In certain embodiments, the third targeting moiety in the isolated antigen binding protein comprises a VH comprising any one of SEQ ID NOs: 168, 170, 172, 174, 176 and 178 amino acid sequence.

在某些实施方式中，所述分离的抗原结合蛋白中所述第三靶向部分包含VL，所述VL包含SEQ ID NO：169、171、173、175、177和179中任一项所示的氨基酸序列。In certain embodiments, the third targeting moiety in the isolated antigen binding protein comprises a VL comprising any one of SEQ ID NOs: 169, 171, 173, 175, 177 and 179 amino acid sequence.

在某些实施方式中，所述分离的抗原结合蛋白中所述第三靶向部分包括抗体或抗原结合片段。In certain embodiments, said third targeting moiety in said isolated antigen binding protein comprises an antibody or an antigen binding fragment.

在某些实施方式中，所述分离的抗原结合蛋白中所述抗原结合片段选自下组：Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和dAb。In certain embodiments, the antigen-binding fragment of the isolated antigen-binding protein is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di- scFv, VHH and dAb.

在某些实施方式中，所述分离的抗原结合蛋白中所述第三靶向部分为scFv。In certain embodiments, said third targeting moiety in said isolated antigen binding protein is a scFv.

在某些实施方式中，所述分离的抗原结合蛋白中所述第三靶向部分中VH的C端与所述VL的N端直接或间接地连接。In certain embodiments, the C-terminus of the VH in the third targeting moiety of the isolated antigen binding protein is directly or indirectly linked to the N-terminus of the VL.

在某些实施方式中，所述分离的抗原结合蛋白中所述第三靶向部分中VL的C端与所述VH的N端直接或间接地连接。In certain embodiments, the C-terminus of the VL in the third targeting moiety of the isolated antigen binding protein is directly or indirectly linked to the N-terminus of the VH.

在某些实施方式中，所述分离的抗原结合蛋白中所述第四靶向部分包含VH，所述VH包含SEQ ID NO：29-31、95-104、168、170、172、174、176和178中任一项所示的氨基酸序列。In certain embodiments, said fourth targeting moiety in said isolated antigen binding protein comprises a VH comprising SEQ ID NO: 29-31, 95-104, 168, 170, 172, 174, 176 and the amino acid sequence shown in any one of 178.

在某些实施方式中，所述分离的抗原结合蛋白中所述第四靶向部分包含VL，所述VL包含SEQ ID NO：32-38、105-111、169、171、173、175、177和179中任一项所示的氨基酸序列。In certain embodiments, said fourth targeting moiety in said isolated antigen binding protein comprises a VL comprising SEQ ID NO: 32-38, 105-111, 169, 171, 173, 175, 177 and the amino acid sequence shown in any one of 179.

在某些实施方式中，所述分离的抗原结合蛋白中所述第四靶向部分包括抗体或抗原结合片段。In certain embodiments, said fourth targeting moiety in said isolated antigen binding protein comprises an antibody or an antigen binding fragment.

在某些实施方式中，所述分离的抗原结合蛋白中所述第四靶向部分为scFv。In certain embodiments, said fourth targeting moiety in said isolated antigen binding protein is a scFv.

在某些实施方式中，所述分离的抗原结合蛋白中所述第四靶向部分中VH的C端与所述VL的N端直接或间接地连接。In certain embodiments, the C-terminus of the VH of the fourth targeting moiety in the isolated antigen binding protein is directly or indirectly linked to the N-terminus of the VL.

在某些实施方式中，所述分离的抗原结合蛋白中所述第四靶向部分中VL的C端与所述VH的N端直接或间接地连接。In certain embodiments, the C-terminus of the VL of the fourth targeting moiety in the isolated antigen binding protein is directly or indirectly linked to the N-terminus of the VH.

在某些实施方式中，所述抗原结合蛋白包含两条第一多肽链和两条第二多肽链，所述两条第一多肽链之间通过二硫键连接，所述两条第一多肽链和两条第二多肽链形成IgG样的抗体。In certain embodiments, the antigen-binding protein comprises two first polypeptide chains and two second polypeptide chains, the two first polypeptide chains are connected by a disulfide bond, and the two The first polypeptide chain and the two second polypeptide chains form an IgG-like antibody.

在某些实施方式中，所述抗原结合蛋白包括单克隆抗体、单链抗体、嵌合抗体、人源化抗体、多特异性抗体、双特异性抗体和/或全人源抗体。In certain embodiments, the antigen binding protein comprises a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, a multispecific antibody, a bispecific antibody and/or a fully human antibody.

另一方面，本申请提供了一种或多种多肽，其包含所述分离的抗原结合蛋白。In another aspect, the application provides one or more polypeptides comprising said isolated antigen binding protein.

另一方面，本申请提供了一种或多种免疫缀合物，其包含所述分离的抗原结合蛋白或所述的多肽。In another aspect, the application provides one or more immunoconjugates comprising said isolated antigen binding protein or said polypeptide.

另一方面，本申请提供了一种或多种分离的核酸分子，其编码所述分离的抗原结合蛋白，或者所述的多肽。In another aspect, the present application provides one or more isolated nucleic acid molecules encoding said isolated antigen binding protein, or said polypeptide.

另一方面，本申请提供了一种或多种载体，其包含所述分离的核酸分子。In another aspect, the application provides one or more vectors comprising said isolated nucleic acid molecule.

另一方面，本申请提供了一种或多种细胞，其包含所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述分离的核酸分子和/或所述的载体。In another aspect, the application provides one or more cells comprising said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule and/or said carrier.

另一方面，本申请提供了一种制备所述分离的抗原结合蛋白或所述的多肽的方法，所述方法包括再使得所述分离的抗原结合蛋白或所述的多肽表达的条件下，培养所述的细胞。In another aspect, the present application provides a method for preparing the isolated antigen-binding protein or the polypeptide, the method comprising culturing the cells.

另一方面，本申请提供了一种或多种药物组合物，其包含所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述分离的核酸分子，所述的载体，所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。In another aspect, the present application provides one or more pharmaceutical compositions comprising said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said carrier, the cells, and/or pharmaceutically acceptable adjuvants and/or excipients.

另一方面，本申请提供了一种抑制PD-1与PD-L1相互作用的方法，其包括向有需要的受试者施用有效量的所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述分离的核酸分子，所述的载体，和/或所述的细胞。In another aspect, the present application provides a method for inhibiting the interaction between PD-1 and PD-L1, which comprises administering to a subject in need an effective amount of the isolated antigen-binding protein, the polypeptide, the The immunoconjugate, the isolated nucleic acid molecule, the carrier, and/or the cell.

另一方面，本申请提供了所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述分离的核酸分子，所述的载体，所述的细胞和/或所述的药物组合物在制备预防和/或治疗疾病或病症的药物中的用途。In another aspect, the present application provides the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell and/or the Use of the pharmaceutical composition in the preparation of medicines for preventing and/or treating diseases or conditions.

在某些实施方式中，所述疾病或病症包括肿瘤。In certain embodiments, the disease or condition comprises a tumor.

在某些实施方式中，所述肿瘤包括实体瘤。In certain embodiments, the tumor comprises a solid tumor.

在某些实施方式中，所述肿瘤包括非实体瘤。In certain embodiments, the tumor comprises a non-solid tumor.

在某些实施方式中，所述肿瘤包括乳腺癌、肺癌、胃癌、肠癌、肾癌、黑色素瘤、非小细胞肺癌、结肠癌、膀胱癌、卵巢癌、胰腺癌和/或肝癌。In certain embodiments, the tumor comprises breast cancer, lung cancer, gastric cancer, bowel cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and/or liver cancer.

另一方面，本申请提供了所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述分离的核酸分子，所述的载体，所述的细胞和/或所述的药物组合物，其用于预防、缓解和/或治疗疾病或病症。In another aspect, the present application provides the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell and/or the A pharmaceutical composition for preventing, alleviating and/or treating diseases or conditions.

另一方面，本申请提供了一种预防和/或治疗疾病或病症的方法，其包括向有需要的受试者施用有效量的所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述分离的核酸分子，所述的载体，和/或所述的细胞。In another aspect, the present application provides a method for preventing and/or treating a disease or disorder, comprising administering to a subject in need an effective amount of the isolated antigen-binding protein, the polypeptide, the Immunoconjugate, said isolated nucleic acid molecule, said carrier, and/or said cell.

本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

附图说明Description of drawings

本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:

图1A-1B显示的是本申请所述示例性的抗原结合蛋白的结构示意图。Figures 1A-1B show schematic structures of exemplary antigen-binding proteins described herein.

图2显示的是本申请所述示例性的双特异性抗体与PD-1高表达、PDL1高表达或野生型的HEK293细胞的结合结果。Figure 2 shows the binding results of the exemplary bispecific antibody described in this application to HEK293 cells with high expression of PD-1, high expression of PDL1 or wild type.

图3显示的是本申请所述示例性的双特异性抗体的ELISA实验结果。Figure 3 shows the results of the ELISA experiment of the exemplary bispecific antibody described in this application.

具体实施方式Detailed ways

以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

术语定义Definition of Terms

在本申请中，术语“分离的”通常指从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。In the present application, the term "isolated" generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of. The term "isolated" does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.

在本申请中，术语“抗原结合蛋白”通常是指一种能够特异性识别和/或中和特定抗原的多肽分子。例如，在本申请中，术语“抗原结合蛋白”可包括“抗体”或“抗原结合片段”。例如，所述抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链组成的免疫球蛋白，并且可包括任何包含其抗原结合部分的分子。术语“抗体”可包括单克隆抗体、抗体片段或抗体衍生物，包括但不限于鼠源抗体、人抗体(全人源抗体)、人源化抗体、嵌合抗体、双特异性抗体、多特异性抗体、单链抗体(例如，scFv)，以及与抗原结合的抗体片段(例如，Fab、Fab’，VHH和(Fab)2片段)。术语“抗体”还可包括抗体的所有重组体形式，例如在原核细胞中表达的抗体、未糖基化的抗体以及本文所述的任何与抗原结合的抗体片段及其衍生物。每条重链可由重链可变区(VH)和重链恒定区构成。每条轻链可由轻链可变区(VL)和轻链恒定区构成。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为构架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原(例如，人PD-L1)相互作用的结合结构域。抗体的恒定区可介导该免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的多种细胞(例如，效应细胞)和经典补体系统的第一成分(Clq)。所述CDRs的确切边界已根据不同系统不同地限定。由Kabat(Kabat等人，Sequences of Proteins of Immunological Interest(NationalInstitutes of Health，Bethesda，Md.(1987)和(1991))描述的系统，不仅提供了可应用于抗原结合片段的任何可变区的明确残基编号系统，还提供了限定CDRs的精确残基边界。这些CDRs可以被称为Kabat CDRs。Chothia和同事(Chothia和Lesk，J.Mol.Biol.196：901-917(1987)以及Chothia等人，Nature 342：877-883(1989))发现尽管在氨基酸序列水平上具有大的多样性，但是Kabat CDRs内的某些亚部分采取几乎相同的肽主链构象。这些亚部分命名为L1、L2和L3或H1、H2和H3，其中“L”和“H”分别指轻链和重链区域。这些区域可以被称为Chothia CDRs，所述Chothia CDRs具有与Kabat CDRs重叠的边界。与Kabat CDRs重叠的限定CDRs的其他边界已由Padlan(FASEB J.9：133-139(1995))和MacCallum(J Mol Biol262(5)：732-45(1996))描述。另外，其他的CDR边界定义可能不严格地遵循上述系统之一，但仍将与Kabat CDRs重叠，尽管按照特定残基或残基组或甚至整个CDRs并不显著影响抗原结合的预测或实验发现，它们可以缩短或加长。在本申请中，所述CDR可通过使用的是Kabat编号系统定义。In this application, the term "antigen-binding protein" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen. For example, in this application, the term "antigen-binding protein" may include "antibody" or "antigen-binding fragment". For example, the antibody may comprise an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and may include any molecule comprising an antigen-binding portion thereof. The term "antibody" may include monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to murine antibodies, human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, bispecific antibodies, multispecific Antibodies, single-chain antibodies (eg, scFv), and antibody fragments (eg, Fab, Fab', VHH, and (Fab)2 fragments) that bind to an antigen. The term "antibody" may also include all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, aglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein. Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain can be composed of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL may consist of three CDR and four FR regions, which may be arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigen (eg, human PD-L1). The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The exact boundaries of the CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear definition of any variable region that can be applied to an antigen-binding fragment The residue numbering system also provides the precise residue boundaries that define the CDRs. These CDRs can be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, J.Mol.Biol.196:901-917 (1987) and Chothia et al. People, Nature 342:877-883 (1989)) found that although there was a large diversity on the amino acid sequence level, some sub-parts in Kabat CDRs took almost the same peptide backbone conformation. These sub-parts were named L1, L2 and L3 or H1, H2 and H3, where "L" and "H" refer to the light and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, which have overlapping boundaries with Kabat CDRs. With Kabat Other boundaries defining CDRs where CDRs overlap have been described by Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). In addition, other CDR boundary definitions may not strictly follow one of the above systems, but will still overlap with Kabat CDRs, although they can be shortened or lengthened according to predictions or experimental findings that specific residues or groups of residues or even entire CDRs do not significantly affect antigen binding. In this application, the CDRs can be defined by using the Kabat numbering system.

在本申请中，术语“抗原结合片段”通常是指抗体中发挥特异性结合抗原功能的一个或多个片段。抗体的抗原结合功能可通过抗体的全长片段来实现。抗体的抗原结合功能也可通过以下来实现：包括Fv、ScFv、dsFv、Fab、Fab’或F(ab’)2的片段的重链，或者，包括Fv、scFv、dsFv、Fab、Fab’或F(ab’)2的片段的轻链。(1)Fab片段，通常为由VL、VH、CL和CH结构域组成的一价片段；(2)F(ab’)2片段，包含通过铰链区处的二硫键连接的两个Fab片段的二价片段；(3)由VH和CH结构域组成的Fd片段；(4)由抗体单臂的VL和VH结构域组成的Fv片段；(5)由VH结构域组成的dAb片段(Ward等，(1989)Nature341：544-546)；(6)分离的互补决定区(CDR)和(7)可任选地通过接头连接的两个或以上分离的CDR的组合。例如，还可包括由VL和VH配对形成的一价单链分子Fv(scFv)(参见Bird等(1988)Science 242：423-426；以及Huston等(1988)Proc.Natl.Acad.Sci.85：5879-5883)。例如，还可包括缺失抗体轻链而只有重链可变区的一类抗体VHH(例如，可参见康晓圳等，生物工程学报,2018,34(12):1974-1984)。所述“抗原结合部分”还可包括免疫球蛋白融合蛋白，所述融合蛋白包含选自以下的结合结构域：(1)与免疫球蛋白铰链区多肽融合的结合结构域多肽；(2)与铰链区融合的免疫球蛋白重链CH2恒定区；和(3)与CH2恒定区融合的免疫球蛋白重链CH3恒定区。In this application, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that specifically bind to an antigen. The antigen-binding function of antibodies can be realized by full-length fragments of antibodies. The antigen-binding function of an antibody can also be achieved by comprising a heavy chain of a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab'), or by comprising a Fv, scFv, dsFv, Fab, Fab' or The light chain of a fragment of F(ab')2. (1) Fab fragment, usually a monovalent fragment consisting of VL, VH, CL and CH domains; (2) F(ab')2 fragment, comprising two Fab fragments linked by a disulfide bond at the hinge region (3) Fd fragment composed of VH and CH domains; (4) Fv fragment composed of VL and VH domains of antibody single arm; (5) dAb fragment composed of VH domains (Ward et al., (1989) Nature 341 :544-546); (6) an isolated complementarity determining region (CDR) and (7) a combination of two or more isolated CDRs optionally linked by a linker. For example, the monovalent single-chain molecule Fv (scFv) formed by the pairing of VL and VH (see Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl.Acad.Sci.85 :5879-5883). For example, a type of antibody VHH that lacks the light chain of the antibody and only has the variable region of the heavy chain can also be included (for example, see Kang Xiaozhen et al., Acta Biological Engineering, 2018, 34(12): 1974-1984). The "antigen binding portion" may also include an immunoglobulin fusion protein comprising a binding domain selected from: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.

在本申请中，术语“单克隆抗体”通常是指一群基本同源的抗体，即包含该群的各个抗体除了可能的以微量存在的天然发生的突变之外是相同的。单克隆抗体是高度特异性的，直接针对单个抗原性位点。例如，所述单克隆抗体可以通过杂交瘤技术制备或者通过使用重组DNA方法在细菌、真核动物或植物细胞中产生。单克隆抗体也可以得自噬菌体抗体文库，使用例如Clackson etal.,Nature,352:624-628(1991)和Marks et al.,Mol.Biol.，222:581-597(1991)所述的技术进行。In this application, the term "monoclonal antibody" generally refers to a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. For example, the monoclonal antibodies can be produced by hybridoma technology or produced in bacterial, eukaryotic or plant cells by using recombinant DNA methods. Monoclonal antibodies can also be obtained from phage antibody libraries using techniques such as those described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991) conduct.

在本申请中，术语“嵌合抗体”通常是指这样的抗体，其中每个重链或轻链氨基酸序列的一部分与来自特定物种的抗体中相应氨基酸序列同源，或者属于特定的类别，而该链的其余区段则与另一物种中的相应序列同源。例如，轻链和重链的可变区均来自一个动物物种(如小鼠、大鼠等)的抗体的可变区，而恒定部分则与来自另一物种(如人)的抗体序列同源。例如，为获得嵌合抗体，可利用非人源的B细胞或杂交瘤细胞产生可变区，而与其组合的恒定区则来自人。所述可变区具有易于制备的优点，并且其特异性不受与其组合的恒定区的来源的影响。同时，由于嵌合抗体的恒定区可来源于人类，因此嵌合在注射时抗体引发免疫应答的可能性会低于使用恒定区为非人来源的抗体。In this application, the term "chimeric antibody" generally refers to an antibody in which a part of each heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific class, and The remainder of the chain is then homologous to the corresponding sequence in another species. For example, the variable regions of both the light and heavy chains are derived from the variable regions of antibodies from one animal species (e.g., mouse, rat, etc.), while the constant portions are homologous to antibody sequences from another species (e.g., human) . For example, to obtain chimeric antibodies, B cells or hybridoma cells of non-human origin can be used to produce variable regions combined with constant regions of human origin. The variable region has the advantage of being easy to prepare and its specificity is not affected by the source of the constant region it is combined with. At the same time, since the constant region of the chimeric antibody can be derived from humans, the possibility of the chimeric antibody triggering an immune response when injected is lower than that of an antibody whose constant region is of non-human origin.

在本申请中，术语“人源化抗体”通常是指一种嵌合抗体，其含有较少的来自非人免疫球蛋白的序列，从而降低异种抗体引入到人类中时的免疫原性，同时保持抗体的完全抗原结合亲和力和特异性。例如，可以使用CDR移植(Jones et al.,Nature 321:522(1986))及其变体；包括“重塑”(reshaping)，(Verhoeyen,et al.,1988Science 239:1534-1536；Riechmann,et al.,1988Nature 332:323-337；Tempest,et al.,Bio/Technol 19919:266-271)，“高度加成”(hyperchimerization)，(Queen,et al.,1989Proc Natl AcadSci USA 86:10029-10033；Co,et al.,1991Proc Natl Acad Sci USA 88:2869-2873；Co,et al.,1992J Immunol 148:1149-1154)和“贴面”(veneering)，(Mark,et al.,“Derivation of therapeutically active humanized and veneered anti-CD18antibodies.”In:Metcalf B W,Dalton B J,eds.Cellular adhesion:moleculardefinition to therapeutic potential.New York:Plenum Press,1994:291-312)、表面重建(美国专利US5639641)等技术手段，对非人源的结合域进行人源化。如果其他区域，例如铰链区和恒定区结构域也源自非人来源，则这些区域也可以被人源化。In this application, the term "humanized antibody" generally refers to a chimeric antibody that contains less sequence from a non-human immunoglobulin, thereby reducing the immunogenicity of a heterologous antibody when introduced into a human, while simultaneously Preserves the full antigen-binding affinity and specificity of the antibody. For example, CDR grafting (Jones et al., Nature 321:522 (1986)) and variants thereof; including "reshaping", (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988Nature 332:323-337; Tempest, et al., Bio/Technol 19919:266-271), "high addition" (hyperchimerization), (Queen, et al., 1989Proc Natl AcadSci USA 86:10029 -10033; Co, et al., 1991Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992J Immunol 148:1149-1154) and "veneering", (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies." In: Metcalf B W, Dalton B J, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312), surface reconstruction (United States Patent US5639641) and other technical means to humanize the non-human binding domain. Other regions, such as the hinge and constant region domains, can also be humanized if they are also derived from non-human sources.

在本申请中，术语“鼠源抗体”通常是指可变区框架和CDR区得自小鼠种系免疫球蛋白序列的抗体。此外，如果抗体包含恒定区，其也得自小鼠种系免疫球蛋白序列。本申请的鼠源抗体可以包含不由小鼠种系免疫球蛋白序列编码的氨基酸残基，例如可以包括通过体外随机突变或点突变或通过体内体细胞突变而导入的突变。In this application, the term "murine antibody" generally refers to an antibody whose variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, these also are derived from mouse germline immunoglobulin sequences. The murine antibody of the present application may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, may include mutations introduced by random or point mutations in vitro or by somatic mutations in vivo.

在本申请中，术语“双特异性抗体”通常是指含有两种特异性抗原结合位点的抗体或抗原结合片段。在本申请中，双特异性抗体可以是同二聚体或者异二聚体。例如，在本申请中，所述双特异性抗体可以结合PD-1蛋白和PD-L1蛋白，能够有效抑制PD-L1和PD-1活化，进而治疗PD1/PD-L1相关的疾病(例如，肿瘤)。In this application, the term "bispecific antibody" generally refers to an antibody or antigen-binding fragment that contains two specific antigen-binding sites. In the present application, bispecific antibodies can be homodimers or heterodimers. For example, in this application, the bispecific antibody can bind to PD-1 protein and PD-L1 protein, can effectively inhibit the activation of PD-L1 and PD-1, and then treat PD1/PD-L1 related diseases (for example, tumor).

在本申请中，术语“PD-L1”通常是指程序性死亡配体1蛋白、其功能变体和/或其功能活性片段。PD-L1也称为分化簇274(CD274)或B7同源物1(B7-H1)，并且是由(人类中)CD274基因编码的蛋白。PD-L1结合其受体，例如程序性死亡1(PD-1)，所述PD-1在活化的T细胞、B细胞和巨噬细胞中表达(Ishida et al.,1992EMBO J,11:3887-3395；Okazaki etal.,Autoimmune dilated cardiomyopathy in PD-1receptor-deficient mice.Science,2001；291:319-22)。PD-L1和PD-1的络合通过抑制T细胞增殖和产生细胞因子IL-2和IFN-γ发挥免疫抑制作用(Freeman et al.,Engagement of PD-1immunoinhibitory receptorby a novel B7 family member leads to negative regulation of lymphocyteactivation,J.Exp.Med.2000,192:1027-1034；Carter et al.,PD-1:PD-L inhibitorypathway affects both CD4(+)and CD8(+)T cells and is overcome by IL-2.Eur.J.Immunol.2002,32:634–643)。术语“PD-L1”涵盖任何脊椎动物来源的任何天然PD-L1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人)和啮齿类(例如，小鼠和大鼠)。所述术语涵盖“全长”、未加工的PD-L1以及由细胞中的加工所产生的任何形式的PD-L1。PD-L1可作为跨膜蛋白或作为可溶性蛋白存在。所述术语还涵盖天然存在的PD-L1的变体，例如剪接变体或等位基因变体。示例性的全长人PD-L1蛋白的氨基酸序列可在UniProt登录号Q9NZQ7下找到。例如，所述“功能活性片段”可以包括保留至少一种天然存在的蛋白质的内源功能(例如，与本申请所述的抗原结合蛋白结合)的片段。例如，所述“功能活性片段”可以包括与本申请的抗原结合蛋白结合的结构域。In this application, the term "PD-L1" generally refers to the programmed death-ligand 1 protein, its functional variants and/or its functionally active fragments. PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1 ), and is a protein encoded by the CD274 gene (in humans). PD-L1 binds its receptors, such as programmed death 1 (PD-1), which is expressed in activated T cells, B cells and macrophages (Ishida et al., 1992 EMBO J, 11:3887 -3395; Okazaki et al., Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science, 2001; 291:319-22). The complexation of PD-L1 and PD-1 exerts an immunosuppressive effect by inhibiting T cell proliferation and producing cytokines IL-2 and IFN-γ (Freeman et al., Engagement of PD-1immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation, J.Exp.Med.2000,192:1027-1034; Carter et al.,PD-1:PD-L inhibitory pathway affects both CD4(+)and CD8(+)T cells and is overcome by IL- 2. Eur. J. Immunol. 2002, 32:634–643). The term "PD-L1" encompasses any native PD-L1 of any vertebrate origin, including mammals, such as primates (eg, humans) and rodents (eg, mice and rats). The term encompasses "full length", unprocessed PD-L1 as well as any form of PD-L1 that results from processing in the cell. PD-L1 can exist as a transmembrane protein or as a soluble protein. The term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants. The amino acid sequence of an exemplary full-length human PD-L1 protein can be found under UniProt accession number Q9NZQ7. For example, such "functionally active fragments" may include fragments that retain at least one endogenous function of a naturally occurring protein (eg, binding to an antigen binding protein described herein). For example, the "functionally active fragment" may include a domain that binds to the antigen-binding protein of the present application.

在本申请中，术语“PD-1蛋白”，“PD-1”或“PD-1抗原”可以互换使用，并且包括PD-1的任何功能活性片段、变体和同源物，其由细胞天然表达或在用PD-1基因转染的细胞上表达。在本申请中，PD-1可以为人PD-1，其在UniProt/Swiss-Prot中的登录号为Q15116。例如，PD-1可以为人PD-1的功能活性片段。例如，所述“功能活性片段”可以包括保留至少一种天然存在的蛋白质的内源功能(例如，与本申请所述的抗原结合蛋白结合)的片段。例如，所述“功能活性片段”可以包括与本申请的抗原结合蛋白结合的结构域。In this application, the terms "PD-1 protein", "PD-1" or "PD-1 antigen" are used interchangeably and include any functionally active fragments, variants and homologues of PD-1, which are derived from Cells express it naturally or on cells transfected with the PD-1 gene. In this application, PD-1 may be human PD-1, whose accession number in UniProt/Swiss-Prot is Q15116. For example, PD-1 can be a functionally active fragment of human PD-1. For example, such "functionally active fragments" may include fragments that retain at least one endogenous function of a naturally occurring protein (eg, binding to an antigen binding protein described herein). For example, the "functionally active fragment" may include a domain that binds to the antigen-binding protein of the present application.

除了本文提到的特定蛋白质和核苷酸之外，本申请还可包括其功能活性片段、衍生物、类似物、同源物及其片段。In addition to the specific proteins and nucleotides mentioned herein, the present application may also include functionally active fragments, derivatives, analogs, homologues and fragments thereof.

术语“功能活性片段”指与天然存在序列具有基本上同一的氨基酸序列或由基本上同一的核苷酸序列编码并能够具有天然存在序列的一种或多种活性的多肽。在本申请的上下文中，任何给定序列的功能活性片段是指其中残基的特定序列(无论是氨基酸或核苷酸残基)已经经过修饰而使得所述多肽或多核苷酸基本上保留至少一种内源功能的序列。可以通过天然存在的蛋白质和/或多核苷酸中存在的至少一个氨基酸残基和/或核苷酸残基的添加、缺失、取代、修饰、替换和/或变异来获得编码功能活性片段的序列，只要保持原来的功能活性即可。The term "functionally active fragment" refers to a polypeptide having substantially the same amino acid sequence or encoded by a substantially identical nucleotide sequence as the naturally occurring sequence and capable of possessing one or more activities of the naturally occurring sequence. In the context of this application, a functionally active fragment of any given sequence is one in which a specific sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least A sequence of endogenous function. Sequences encoding functionally active fragments can be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in naturally occurring proteins and/or polynucleotides , as long as the original functional activity is maintained.

在本申请中，术语“衍生物”通常是指对本申请的多肽或多核苷酸的一个(或多个)氨基酸残基(或核苷酸)的任何取代、变异、修饰、替换、缺失和/或添加，只要所得的多肽或多核苷酸基本上保留其至少一种内源功能。In this application, the term "derivative" generally refers to any substitution, variation, modification, substitution, deletion and/or Or addition, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.

在本申请中，术语“类似物”通常对多肽或多核苷酸而言，包括多肽或多核苷酸的任何模拟物，即拥有该模拟物模拟的多肽或多核苷酸的至少一种内源功能的化学化合物。In this application, the term "analogue" generally refers to polypeptides or polynucleotides, including any mimetic of polypeptides or polynucleotides, that is, having at least one endogenous function of the polypeptide or polynucleotide simulated by the mimetic of chemical compounds.

通常，可以进行氨基酸取代，例如至少1个(例如，1、2、3、4、5、6、7、8、9、10或20个以上)氨基酸取代，只要经修饰的序列基本上保持需要的活性或能力。氨基酸取代可包括使用非天然存在的类似物。Typically, amino acid substitutions, e.g., at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions can be made so long as the modified sequence remains substantially as desired. activity or ability. Amino acid substitutions may involve the use of non-naturally occurring analogs.

在本申请中，术语“同源物”通常是指与天然存在序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于序列“同一性”。同源序列可以包括可以与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中的任一项具有百分比同一性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同一性的序列。为了确定序列同一性，可进行序列比对，其可通过本领域技术人员了解的各种方式进行，例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。In the present application, the term "homologue" generally refers to an amino acid sequence or nucleotide sequence having a certain homology to a naturally occurring sequence. The term "homology" may be equated with sequence "identity". Homologous sequences may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence . Typically, a homologue will comprise the same active site, etc., as the subject amino acid sequence. Homology can be considered in terms of similarity (ie, amino acid residues having similar chemical properties/functions), or can be expressed in terms of sequence identity. In this application, a sequence having a percentage identity of any one of the SEQ ID NOs of the amino acid sequence or nucleotide sequence mentioned means having said percentage identity over the entire length of the mentioned SEQ ID NO the sequence of. To determine sequence identity, sequence alignment can be performed by various means known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software and the like. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment across the full-length sequences being compared.

用于本申请的蛋白质或多肽也可以具有氨基酸残基的缺失、插入或取代，所述氨基酸残基产生沉默的变化并导致功能上等同的蛋白质。可以根据残基的极性、电荷、溶解性、疏水性、亲水性和/或两性性质的相似性进行有意的氨基酸取代，只要保留内源性功能即可。例如，带负电荷的氨基酸包括天冬氨酸和谷氨酸；带正电荷的氨基酸包括赖氨酸和精氨酸；并且含具有相似亲水性值的不带电极性头基的氨基酸包括天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸和酪氨酸。The proteins or polypeptides used in this application may also have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues, so long as endogenous function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar headgroups with similar hydrophilicity values include aspartic acid. Paragine, Glutamine, Serine, Threonine and Tyrosine.

在本申请中，术语“肿瘤”通常是指在各种致瘤因子作用下，局部组织细胞增生所形成的赘生物。例如，所述肿瘤可包括实体瘤。例如，所述肿瘤可包括非实体瘤。例如，所述肿瘤可包括与PD-L1的表达相关的肿瘤。术语“与PD-L1的表达相关的肿瘤”通常是指PD-L1表达改变导致疾病进展或逃避免疫监视而形成的肿瘤。例如，所述“与PD-L1的表达相关的肿瘤”可以是PD-L1表达量上调导致疾病进展或逃避免疫监视而形成的肿瘤。所述与PD-L1的蛋白表达相关的肿瘤可以是PD-L1阳性的肿瘤。在PD-L1阳性的肿瘤中，与正常细胞相比，肿瘤细胞表面或肿瘤微环境中的PD-L1的蛋白表达量高约1％，5％，10％，15％，20％，25％，30％，35％，40％，50％，60％，70％，80％或更高。In this application, the term "tumor" generally refers to a neoplasm formed by the proliferation of local tissue cells under the action of various tumorigenic factors. For example, the tumor can include a solid tumor. For example, the tumor can include a non-solid tumor. For example, the tumor can include a tumor associated with expression of PD-L1. The term "tumor associated with the expression of PD-L1" generally refers to a tumor formed due to changes in the expression of PD-L1 leading to disease progression or escape from immune surveillance. For example, the "tumor associated with the expression of PD-L1" may be a tumor formed by the upregulation of PD-L1 expression leading to disease progression or escape from immune surveillance. The tumors associated with the protein expression of PD-L1 may be PD-L1 positive tumors. In PD-L1-positive tumors, compared with normal cells, the protein expression of PD-L1 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25% higher , 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher.

在本申请中，术语“实体瘤”通常是指可通过临床检测(例如，X线射片，CT扫描，B超或触诊)到的有形肿块。例如，所述实体瘤可包括乳腺癌、肺癌、胃癌、肠癌、肾癌、黑色素瘤、非小细胞肺癌、结肠癌、膀胱癌、卵巢癌、胰腺癌和/或肝癌。In this application, the term "solid tumor" generally refers to a tangible mass that can be detected clinically (eg, X-ray film, CT scan, B-ultrasound or palpation). For example, the solid tumor may include breast cancer, lung cancer, gastric cancer, bowel cancer, renal cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and/or liver cancer.

在本申请中，术语“非实体瘤”通常是指X线射片、CT扫描、B超以及触诊无法看到或扪及到的肿瘤。例如，所述非实体瘤可以包括白血病。例如，所述非实体瘤可以包括淋巴瘤。例如，所述非实体瘤可包括多发性骨髓瘤。In this application, the term "non-solid tumor" generally refers to a tumor that cannot be seen or touched by X-ray films, CT scans, B-ultrasound and palpation. For example, the non-solid tumor can include leukemia. For example, the non-solid tumor can include lymphoma. For example, the non-solid tumor can include multiple myeloma.

在本申请中，术语“肿瘤相关抗原”通常是指在肿瘤细胞或正常细胞上存在的抗原分子。在本申请中，所述肿瘤相关抗原可微量存在于正常细胞，而在肿瘤细胞中高度表达。例如，所述肿瘤相关抗原可包括但不限于胚胎性蛋白、糖蛋白抗原和/或鳞状细胞抗原。例如，所述肿瘤相关抗原可以包括CD19、CD20、CD47、HER2、EGFR、CD22和/或PD-L1。In this application, the term "tumor-associated antigen" generally refers to an antigenic molecule present on tumor cells or normal cells. In the present application, the tumor-associated antigen may be present in normal cells in a small amount, but highly expressed in tumor cells. For example, the tumor-associated antigens may include, but are not limited to, embryonic proteins, glycoprotein antigens, and/or squamous cell antigens. For example, the tumor-associated antigens may include CD19, CD20, CD47, HER2, EGFR, CD22 and/or PD-L1.

在本申请中，术语“免疫细胞相关抗原”通常是指免疫细胞上存在的抗原分子。例如，所述免疫细胞相关抗原可以包括与免疫抑制相关的抗原(例如，PD-1抗原)。例如，所述免疫细胞相关抗原可以包括T细胞或NK细胞抗原(例如，CD3，CD16a等)。In this application, the term "immune cell-associated antigen" generally refers to antigenic molecules present on immune cells. For example, the immune cell-associated antigens may include antigens associated with immunosuppression (eg, PD-1 antigen). For example, the immune cell-associated antigens may include T cell or NK cell antigens (eg, CD3, CD16a, etc.).

在本申请中，术语“细胞因子”通常是指由免疫细胞和某些非免疫细胞(例如，内皮细胞、表皮细胞、纤维母细胞等)经刺激而合成、分泌的一类具有广泛生物学活性的小分子蛋白质。例如，所述细胞因子可以通过结合相应受体调节细胞生长、分化和效应，调控免疫应答。例如，所述细胞因子可包括但不限于白细胞介素(例如，IL-2、IL-7、IL-15等)、干扰素(例如，IFNα、IFNβ)、肿瘤坏死因子超家族、集落刺激因子、趋化因子、生长因子等。例如，所述细胞因子可包括白细胞介素的变体(例如，IL-2变体R38A，I80F，R81D，L85V，I86V，I92F，其可与IL-2βγ受体结合而和IL-2α受体结合力较弱)。例如，所述细胞因子可包括干扰素变体(例如，IFNα变体R144A突变体，其与IFNAR2受体的结合显著减弱，可减少INFα的毒副作用)。In this application, the term "cytokines" generally refers to a class of cytokines that are synthesized and secreted by immune cells and certain non-immune cells (such as endothelial cells, epidermal cells, fibroblasts, etc.) after stimulation, and have a wide range of biological activities. of small molecular proteins. For example, the cytokines can modulate the immune response by binding to the corresponding receptors to regulate cell growth, differentiation and effects. For example, the cytokines may include, but are not limited to, interleukins (e.g., IL-2, IL-7, IL-15, etc.), interferons (e.g., IFNα, IFNβ), tumor necrosis factor superfamily, colony-stimulating factor , chemokines, growth factors, etc. For example, the cytokines can include variants of interleukins (e.g., IL-2 variants R38A, I80F, R81D, L85V, I86V, I92F, which bind to the IL-2βγ receptor and bind to the IL-2α receptor weak bonding). For example, the cytokines may include interferon variants (eg, IFNα variant R144A mutant, which has significantly reduced binding to the IFNAR2 receptor, which reduces the toxic side effects of IFNα).

在本申请中，术语“免疫缀合物”通常是指所述其他治疗剂与所述分离的抗原结合蛋白缀合(例如，通过连接分子共价相连)而形成的缀合物，该缀合物可以通过所述分离的抗原结合蛋白与靶细胞上的抗原的特异性结合，将所述其他治疗剂递送至靶细胞(例如，肿瘤细胞)。此外，所述抗原也可以由所述靶细胞分泌，并位于所述靶细胞外的间隙。In this application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating (for example, covalently linking through a linker molecule) the other therapeutic agent to the isolated antigen-binding protein, the conjugate The other therapeutic agent can be delivered to a target cell (eg, a tumor cell) by specific binding of the isolated antigen binding protein to an antigen on the target cell. In addition, the antigen may also be secreted by the target cell and located in the space outside the target cell.

在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。In this application, the term "subject" generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.

在本申请中，术语“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。In this application, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length isolated from their natural environment or artificially synthesized.

在本申请中，术语“载体”通常是指一种核酸分子，该种核酸分子能够转运与其连接的另一核酸。所述载体可将插入的核酸分子转移到细胞中和/或细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体可以是当引入合适的细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的细胞，所述载体可以产生期望的表达产物。在本申请中，所述载体可包含慢病毒载体。In this application, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The vectors can transfer inserted nucleic acid molecules into and/or between cells. The vectors may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and vectors mainly used for expression of transcription and/or translation of DNA or RNA. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell. Typically, the vector produces the desired expression product by culturing appropriate cells containing the vector. In the present application, the vector may comprise a lentiviral vector.

在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的多肽或本申请所述的抗原结合蛋白的个体细胞，细胞系或细胞培养物。所述细胞可以包括单个细胞的子代。由于天然的，意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的多肽或抗原结合蛋白即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在一些实施方式中，所述细胞可以是免疫细胞。例如，所述免疫细胞可以选自下组：T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。In this application, the term "cell" generally refers to an individual cell that can or has contained a plasmid or vector comprising a nucleic acid molecule described herein, or is capable of expressing a polypeptide described herein or an antigen binding protein described herein , cell line or cell culture. The cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cells, but it is sufficient to be able to express the polypeptide or antigen-binding protein described in this application. The cells can be obtained by transfecting cells in vitro with the vectors described in this application. The cells can be prokaryotic cells (such as Escherichia coli) or eukaryotic cells (such as yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells). In some embodiments, the cells can be immune cells. For example, the immune cells may be selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

在本申请中，术语“治疗”通常是指：(i)预防可能易患疾病、病症和/或病状、但尚未诊断出患病的患者出现该疾病、病症或病状；(ii)抑制该疾病、病症或病状，亦即遏制其发展；以及(iii)缓解该疾病、病症或病状，亦即使得该疾病、病症和/或病状和/或与该疾病、病症和/或病状相关联的症状消退。As used herein, the term "treating" generally refers to: (i) preventing a disease, disorder, and/or condition from occurring in a patient who may be predisposed to it but has not been diagnosed with it; (ii) inhibiting the disease , disorder or condition, i.e. arresting its development; and (iii) relieving the disease, disorder or condition, i.e. making the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition subside.

在本申请中，术语“多肽”、“肽”、“蛋白”和“蛋白质”可互换地使用，通常是指具有任何长度的氨基酸的聚合物。该聚合物可以是直链或支链的，它可以包含修饰的氨基酸，并且可以被非氨基酸中断。这些术语还涵盖已经被修饰的氨基酸聚合物。这些修饰可以包含：二硫键形成、糖基化、脂化(lipidation)、乙酰化、磷酸化、或任何其他操纵(如与标记组分结合)。术语“氨基酸”包括天然的和/或非天然的或者合成的氨基酸，包括甘氨酸以及D和L旋光异构体、以及氨基酸类似物和肽模拟物。In this application, the terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and generally refer to a polymer of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as binding to a labeling component). The term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine and D and L optical isomers, as well as amino acid analogs and peptidomimetics.

在本申请中，术语“多核苷酸”、“核苷酸”、“核苷酸序列”、“核酸”和“寡核苷酸”可互换地使用，通常是指具有任何长度的核苷酸的聚合形式，如脱氧核糖核苷酸或核糖核苷酸、或其类似物。多核苷酸可具有任何三维结构，并且可以执行已知或未知的任何功能。以下是多核苷酸的非限制性实例：基因或基因片段的编码区或非编码区、根据连接分析定义的多个座位(一个座位)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、短干扰RNA(siRNA)、短发夹RNA(shRNA)、micro-RNA(miRNA)、核酶、cDNA、重组多核苷酸、分支多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针、和引物。多核苷酸可以包含一个或多个经修饰的核苷酸，如甲基化的核苷酸和核苷酸类似物。如果存在，可以在聚合物组装之前或之后进行核苷酸结构的修饰。核苷酸的序列可以被非核苷酸组分中断。多核苷酸可以在聚合后，如通过与标记的组分缀合来进一步修饰。In this application, the terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably and generally refer to nucleosides of any length A polymeric form of an acid, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, multiple loci (one locus) defined by junctional analysis, exons, introns, messenger RNA (mRNA), Transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence Isolated DNA of any sequence, isolated RNA, nucleic acid probes, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be performed before or after polymer assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.

在本申请中，术语“K_D”(同样地，“K_D”或“K_D”)通常指“亲和常数”或“平衡解离常数”，并指在滴定测量中在平衡时、或者通过将解离速率常数(kd)除以结合速率常数(ka)所获得的值。使用结合速率常数(ka)、解离速率常数(kd)和平衡解离常数(K_D)表示结合蛋白(例如本申请所述的分离的抗原结合蛋白)对抗原(例如，PD-L1蛋白、PD-1蛋白)的结合亲和力。确定结合和解离速率常数的方法为本领域熟知。使用基于荧光的技术提供了高灵敏度以及在生理缓冲液中在平衡时检查样品的能力。例如，可以通过Biacore(生物分子相互作用分析)测定(例如，可以从BIAcoreInternationalAB，aGEHealthcarecompany，Uppsala，瑞典获得的仪器)所述K_D值，也可以使用其他实验途径和仪器例如Octet检测。另外，也可以使用可以从SapidyneInstruments(Boise，Idaho)获得的KinExA(动态排阻测定(KineticExclusionAssay))测定所述K_D值，或者使用表面等离子共振仪(SPR)测定所述K_D值。例如，也可以通过胺偶联试剂盒测定所述K_D值。In this application, the term "K_D " (likewise, "K_D " or "K_D ") generally refers to "affinity constant" or "equilibrium dissociation constant" and refers to a titration measurement at equilibrium, or Value obtained by dividing the dissociation rate constant (kd) by the association rate constant (ka). The association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (K_D ) are used to express the binding protein (such as the isolated antigen binding protein described herein) to the antigen (such as PD-L1 protein, binding affinity of PD-1 protein). Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples at equilibrium in physiological buffers. For example, the_KD value can be determined by Biacore (Biomolecular Interaction Analysis) (for example, an instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden), and other experimental approaches and instruments such as Octet can also be used. In addition,_the_KD value can also be determined using KinExA (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, Idaho), or using a surface plasmon resonance (SPR) instrument. For example, the_KD value can also be determined by an amine coupling kit.

在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。In this application, the term "and/or" should be understood as meaning any one of the optional items or both of the optional items.

在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。在某些情形中，“包含”也涵盖仅包括指定的组分的情况。例如，包含也表示为也表示“由……组成”的含义。In this application, the term "comprising" generally means including specifically specified features, but not excluding other elements. In some cases, "comprising" also covers including only the specified components. For example, comprising is also meant to also mean "consisting of".

在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。In this application, the term "about" generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。In this application, the term "comprises" generally refers to the meanings comprising, encompassing, comprising or encompassing. In some cases, it also means "for" and "consisting of".

发明详述Detailed description of the invention

本申请所述分离的抗原结合蛋白The isolated antigen-binding protein described in the application

一方面，本申请提供一种分离的抗原结合蛋白，其可包含第一多肽链、第二多肽链，所述第一多肽链包括第一靶向部分，所述第二多肽链包含第二靶向部分，所述第一靶向部分和所述第二靶向部分各自独立地结合PD-1蛋白或PD-L1蛋白，所述第一靶向部分和第二靶向部分结合的蛋白不同，所述第一多肽链与所述第二多肽链通过二硫键连接。In one aspect, the application provides an isolated antigen-binding protein, which may comprise a first polypeptide chain, a second polypeptide chain, the first polypeptide chain includes a first targeting moiety, and the second polypeptide chain Comprising a second targeting moiety, the first targeting moiety and the second targeting moiety independently bind PD-1 protein or PD-L1 protein, and the first targeting moiety and the second targeting moiety bind Different from the protein, the first polypeptide chain is connected to the second polypeptide chain through a disulfide bond.

例如，所述抗原结合蛋白可包含如图1A-1B所示的示例性结构。For example, the antigen binding protein can comprise the exemplary structures shown in Figures 1A-1B.

双特异性抗体bispecific antibody

在本申请中，抗体的CDR又称互补决定区，是可变区的一部分。该区域的氨基酸残基可以与抗原或抗原表位接触。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、AbM、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。本领域技术人员可以根据抗体的序列和结构，用不同的编码系统确定出CDR区。使用不同的编码系统，CDR区可能存在差别。在本申请中，所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在某些实施方式中，本申请所述分离的抗原结合蛋白通过Kabat编码系统定义。In this application, the CDR of an antibody is also called complementarity determining region, which is a part of the variable region. The amino acid residues in this region may make contacts with the antigen or antigenic epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, comprehensive consideration of Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems. In this application, the CDR covers the CDR sequence divided according to any CDR division method; also covers its variants, the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids . For example 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or or insertions; homologues thereof, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences having about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology. In certain embodiments, the isolated antigen binding proteins described herein are defined by the Kabat coding system.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分可特异性结合PD-L1蛋白，所述双特异性抗体第二多肽链中的第二靶向部分可特异性结合PD-1蛋白。In the present application, the first targeting moiety in the first polypeptide chain of the bispecific antibody can specifically bind PD-L1 protein, and the second targeting moiety in the second polypeptide chain of the bispecific antibody Can specifically bind PD-1 protein.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR3可包含SEQID NO:76所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR3序列可根据Kabat编码系统定义。In the present application, the HCDR3 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:76. For example, the HCDR3 sequence of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR2可包含SEQID NO:70所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR2序列可根据Kabat编码系统定义。In the present application, the HCDR2 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:70. For example, the HCDR2 sequence of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR1可包含SEQID NO:61所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR1序列可根据Kabat编码系统定义。In the present application, the HCDR1 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:61. For example, the HCDR1 sequence of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

例如，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR1可包含SEQ IDNO:61所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:70所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:76所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分可包括抗体13H6D3、hu13H6D3、5BM、AH00228、AH00229、AH00230、AH00231、AH00232、AH00233、CBM或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。For example, the HCDR1 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:61; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:70; And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:76. For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM or have the same HCDR3 (e.g., have the same HCDR1-3) antigen-binding fragments.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分可包含重链可变区，所述重链可变区可包含SEQ ID NO:122所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分包含VH，所述VH与SEQ ID NO:122所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₅，X₇，X₁₀，X₁₁，X₁₂，X₁₆，X₂₀，X₃₇，X₃₈，X₄₀，X₄₆，X₄₈，X₆₇，X₆₈，X₆₉，X₇₅，X₈₁，X₈₆，X₉₀，X₁₁₅和X₁₁₆。In the present application, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:122 . For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody comprises VH. Compared with the sequence shown in SEQ ID NO: 122, the VH has one or more amino acids selected from the group consisting of Amino acid substitutions (eg, conservative amino acid substitutions, etc.): X₅ , X₇ , X₁₀ , X₁₁ , X₁₂ , X₁₆ , X 20 , X₃₇ , X₃₈ ,_{X 40}_, X₄₆ , X₄₈ , X₆₇ , X₆₈ , X₆₉ , X₇₅ , X₈₁ , X₈₆ , X₉₀ , X₁₁₅ and X₁₁₆ .

QVQLX₅QX₇GAX₁₀X₁₁X₁₂KPGX₁₆SVKX₂₀SCKASGYTFTSNWMHWX₃₇X₃₈QX₄₀PGQGLX₄₆WX₄₈GMIHPNSAIKYNEKFKSRX₆₇X₆₈X₆₉TADKSX₇₅STAYMX₈₁LSSLX₈₆SEDX₉₀AVYYCARSYYGSSPYFFDYWGQGTX₁₁₅X₁₁₆TVSS(SEQ ID NO：122)，其中，X₅可以是Q或V，X₇可以是P或S，X₁₀可以是D或E，X₁₁可以是L或V，X₁₂可以是K或V，X₁₆可以是A或S，X₂₀可以是L或V，X₃₇可以是D，M或V，X₃₈可以是K或R，X₄₀可以是A或R，X₄₆可以是D或E，X₄₈可以是I或M，X₆₇可以是A或V，X₆₈可以是S或T，X₆₉可以是I或L，X₇₅可以是S或T，X₈₁可以是E或Q，X₈₆可以是R或T，X₉₀可以是S或T，X₁₁₅可以是M或T，X₁₁₆可以是L或V。QVQLX₅ QX₇ GAX₁₀ X₁₁ X₁₂ KPGX₁₆ SVKX₂₀ SCKASGYTFTSNWMHWX₃₇ X₃₈ QX₄₀ PGQGLX₄₆ WX₄₈ GMIHPNSAIKYNEKFKSRX₆₇ X₆₈ X₆₉ TADKSX₇₅ STAYMX₈₁ LSSLX₈₆ S EDX₉₀ AVYYCARSYYGSSPYFFDYWGQGTX₁₁₅ X₁₁₆ TVSS (SEQ ID NO: 122) , where X₅ can be Q or V, X₇ can be P or S, X₁₀ can be D or E, X₁₁ can be L or V, X₁₂ can be K or V, X₁₆ can be A or S , X₂₀ can be L or V, X₃₇ can be D, M or V, X₃₈ can be K or R, X₄₀ can be A or R, X₄₆ can be D or E, X₄₈ can be I or M , X₆₇ can be A or V, X₆₈ can be S or T, X₆₉ can be I or L, X₇₅ can be S or T, X₈₁ can be E or Q, X₈₆ can be R or T, X₉₀ can be S or T, X₁₁₅ can be M or T, and X₁₁₆ can be L or V.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的重链可变区可包含SEQ ID NO:95至SEQ ID NO:104中任一项所示的氨基酸序列。In the present application, the heavy chain variable region of the first targeting portion in the first polypeptide chain of the bispecific antibody may comprise amino acids shown in any one of SEQ ID NO:95 to SEQ ID NO:104 sequence.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分可以包含轻链可变区VL中的至少一个CDR，所述VL可以包含SEQ ID NO:123所示的氨基酸序列。In the present application, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise at least one CDR in the VL of the light chain variable region, and the VL may comprise the CDR shown in SEQ ID NO: 123. amino acid sequence.

例如，所述双特异性抗体第一多肽链中的第一靶向部分中的VL可以包含SEQ IDNO:105至SEQ ID NO:111中任一项所示的氨基酸序列。在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR可以通过任何形式划分，只要VL与SEQ ID NO:105至SEQID NO:111中任一项所示的氨基酸序列相同，以任何形式划分得到的LCDR都可落入本申请的保护范围内。For example, the VL in the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 105 to SEQ ID NO: 111. In this application, the LCDR of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be divided in any form, as long as VL is shown in any one of SEQ ID NO: 105 to SEQ ID NO: 111 The amino acid sequence is the same, and the LCDR obtained by dividing in any form can fall within the protection scope of the present application.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分可包括轻链可变区VL，所述VL可包含LCDR1、LCDR2和LCDR3中的至少一个，至少两个或至少三个。In the present application, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise a light chain variable region VL, and the VL may comprise at least one, at least two, of LCDR1, LCDR2 and LCDR3 or at least three.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR3可包含SEQID NO:94所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR3可根据Kabat编号系统定义。In the present application, the LCDR3 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:94. For example, the LCDR3 of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR2可包含SEQID NO:87所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR2可根据Kabat编号系统定义。In the present application, LCDR2 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:87. For example, LCDR2 of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:82所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR1可根据Kabat编号系统定义。In the present application, LCDR1 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:82. For example, LCDR1 of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

例如，本申请所述双特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:82所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:87所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:94所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分可包括抗体13H6D3、hu13H6D3、5BM、AH00228、AH00229、AH00230、AH00231、AH00232、AH00233、CBM或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:82; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:87 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:94. For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM or have the same LCDR3 (e.g., Antigen-binding fragments having the same LCDR1-3) as it.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分可包含轻链可变区VL，所述VL可包含SEQ ID NO:123所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的VL与SEQ ID NO:123所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₇，X₈，X₁₅，X₂₂，X₄₁，X₄₂，X₄₃，X₄₄，X₄₆，X₆₀，X₇₁，X₇₂，X₇₇，X₇₉，X₈₀，X₈₃，X₈₇和X₁₀₄。In the present application, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:123. For example, compared with the sequence shown in SEQ ID NO: 123, the VL of the first targeting moiety in the first polypeptide chain of the bispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (e.g., conservative amino acid substitutions, etc.):_X7 ,_X8 ,_X15 ,_X22 ,_X41 ,_X42 ,_X43 , X44_,_X46 ,_X60 ,_X71 ,_X72 ,_X77 ,_X79 , X₈₀ , X₈₃ , X₈₇ and X₁₀₄ .

DIQMTQX₇X₈SSLSASX₁₅GDRVTIX₂₂CRASQDISKYLNWYQQKPX₄₁X₄₂X₄₃X₄₄KX₄₆LIY YTSRLHSGVPX₆₀RFSGSGSGTDX₇₁X₇₂LTISX₇₇LX₇₉X₈₀EDX₈₃ATYX₈₇CQQGDTLPWTFGGGT KX₁₀₄EIK(SEQ ID NO：123)，其中，X₇可以是S或T，X₈可以是P或T，X₁₅可以是L或V，X₂₂可以是S或T，X₄₁可以是D或G，X₄₂可以是G或K，X₄₃可以是A或T，X₄₄可以是P或V，X₄₆可以是F或L，X₆₀可以是A或S，X₇₁可以是F或Y，X₇₂可以是F或T，X₇₇可以是N或S，X₇₉可以是E或Q，X₈₀可以是P或Q，X₈₃可以是F或V，X₈₇可以是F或Y，X₁₀₄可以是L或V。DIQMTQX₇ X₈ SSLSASX₁₅ GDRVTIX₂₂ CRASQDISKYLNWYQQKPX₄₁ X₄₂ X₄₃ X₄₄ KX₄₆ LIY YTSRLHSGVPX₆₀ RFSGSGSGTDX₇₁ X₇₂ LTISX₇₇ LX₇₉ X₈₀ EDX₈₃ ATYX₈₇ CQQ GDTLPWTFGGGT KX₁₀₄ EIK (SEQ ID NO: 123), wherein X₇ can be S or T, X₈ can be P or T, X₁₅ can be L or V, X₂₂ can be S or T, X₄₁ can be D or G, X₄₂ can be G or K, X₄₃ can is A or T, X₄₄ can be P or V, X₄₆ can be F or L, X₆₀ can be A or S, X₇₁ can be F or Y, X₇₂ can be F or T, X₇₇ can be N Or S, X₇₉ can be E or Q, X₈₀ can be P or Q, X₈₃ can be F or V, X₈₇ can be F or Y, X₁₀₄ can be L or V.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的轻链可变区可包含SEQ ID NO:105至SEQ ID NO:111中任一项所示的氨基酸序列。In the present application, the light chain variable region of the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise amino acids shown in any one of SEQ ID NO: 105 to SEQ ID NO: 111 sequence.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分可包含轻链恒定区，所述轻链恒定区可包括源自Igκ的恒定区或源自Igλ的恒定区。In the present application, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise a light chain constant region, which may include a constant region derived from Igκ or a constant region derived from Igλ. district.

例如，所述轻链恒定区可包括源自Igκ的恒定区。For example, the light chain constant region may comprise a constant region derived from Igκ.

例如，所述双特异性抗体第一多肽链中的第一靶向部分的轻链恒定区包含SEQ IDNO:114中任一项所示的氨基酸序列。For example, the light chain constant region of the first targeting moiety in the first polypeptide chain of the bispecific antibody comprises the amino acid sequence shown in any one of SEQ ID NO:114.

例如，所述第一多肽链可包含SEQ ID NO:134至140、SEQ ID NO:144至153中任一项所示的氨基酸序列。For example, the first polypeptide chain may comprise the amino acid sequence shown in any one of SEQ ID NO: 134 to 140, SEQ ID NO: 144 to 153.

例如，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:51所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR3序列可根据Kabat编码系统定义。For example, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:51. For example, the HCDR3 sequence of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

例如，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR3与SEQ ID NO：51所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₁。For example, compared with the sequence shown in SEQ ID NO: 51, the HCDR3 of the second targeting part in the second polypeptide chain of the bispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (eg, conservative amino acid substitutions, etc.): X₁ .

X₁HYGTSPFVY(SEQ ID NO：51)，其中，其中，X₁可以是D或E。X₁ HYGTSPFVY (SEQ ID NO: 51), wherein, wherein, X₁ can be D or E.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR3可包含SEQID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR3序列可根据Kabat编码系统定义。In the present application, the HCDR3 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:9 and SEQ ID NO:10. For example, the HCDR3 sequence of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR2可包含SEQID NO:6所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR2序列可根据Kabat编码系统定义。In the present application, the HCDR2 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:6. For example, the HCDR2 sequence of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR1可包含SEQID NO:3所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR1序列可根据Kabat编码系统定义。In the present application, the HCDR1 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3. For example, the HCDR1 sequence of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

例如，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR1可包含SEQ IDNO:3所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:9所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4F1、hu19D4-25、19D4-25-1A3、19D4-25-1B3、19D4-25-1C2、19D4-25-1E4、19D4-25-2E10或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。For example, the HCDR1 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9. For example, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise antibodies 19D4F1, hu19D4-25, 19D4-25-1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25 - 1E4, 19D4-25-2E10 or an antigen-binding fragment thereof having the same HCDR3 (eg, having the same HCDR1-3).

例如，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR1可包含SEQ IDNO:3所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:10所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4-25-3C11、19D4-25-1C2-3C11或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。For example, the HCDR1 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10. For example, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise antibody 19D4-25-3C11, 19D4-25-1C2-3C11 or have the same HCDR3 (eg, have the same HCDR1- 3) The antigen-binding fragment.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分可包含重链可变区，所述重链可变区可包SEQ ID NO:52所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分包含VH，所述VH与SEQ ID NO:52所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₁，X₅，X₁₃，X₁₆，X₁₇，X₂₀，X₃₇，X₄₈，X₆₇，X₆₈，X₇₃，X₇₆，X₇₉，X₈₂，X₈₃，X₈₅，X₈₆，X₈₇，X₈₈，X₉₂，X₉₈和X₁₁₈。In the present application, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:52 . For example, the second targeting moiety in the second polypeptide chain of the bispecific antibody comprises VH, and compared with the sequence shown in SEQ ID NO:52, the VH has one or more amino acids selected from the group consisting of Amino acid substitutions (eg, conservative amino acid substitutions, etc.): X₁ , X₅ , X₁₃ , X₁₆ , X₁₇ , X 20 , X₃₇ , X₄₈ , X₆₇ ,_{X 68}_, X₇₃ , X₇₆ , X₇₉ , X₈₂ , X₈₃ , X₈₅ , X₈₆ , X₈₇ , X₈₈ , X₉₂ , X₉₈ and X₁₁₈ .

X₁VQLX₅ESGPGLVX₁₃PSX₁₆X₁₇LSX₂₀TCTVSGFSLTSYAISWX₃₇RQPPGKGLEWX₄₈GVI WTGGGTNYNSALKSRX₆₇X₆₈ISKDX₇₃SKX₇₆QVX₇₉LKX₈₂X₈₃SX₈₅X₈₆X₈₇X₈₈DTAX₉₂YYCARX₉₈HYGTSPFVYWGQGTLVTVSX₁₁₈(SEQ ID NO：52)，其中，X₁可以是E或Q，X₅可以是K或Q，X₁₃可以是A或K，X₁₆可以是E或Q，X₁₇可以是S或T，X₂₀可以是I或L，X₃₇可以是I或V，X₄₈可以是I或L，X₆₇可以是L或V，X₆₈可以是S或T，X₇₃可以是N或T，X₇₆可以是N或S，X₇₉可以是F或S，X₈₂可以是L或M，X₈₃可以是N或S，X₈₅可以是L或V，X₈₆可以是Q或T，X₈₇可以是A或T，X₈₈可以是A或E，X₉₂可以是S或V，X₉₈可以是D或E，X₁₁₈可以是A或S。X₁ VQLX₅ ESGPGLVX₁₃ PSX₁₆ X₁₇ LSX₂₀ TCTVSGFSLTSYAISWX₃₇ RQPPGKGLEWX₄₈ GVI WTGGGTNYNSALKSRX₆₇ X₆₈ ISKDX₇₃ SKX₇₆ QVX₇₉ LKX₈₂ X₈₃ SX₈₅ X₈₆ X₈₇ X₈₈ DTAX₉₂ YYCARX₉₈ HYGTSPFVYWGQGTLVTVSX₁₁₈ (SEQ ID NO: 52), wherein, X₁ can be E or Q, X₅ can be K or Q, X₁₃ can be A or K, X₁₆ can be E or Q, X₁₇ can be S or T, X₂₀ can be I or L, X₃₇ can be I or V, X₄₈ can be I or L, X₆₇ can be L or V, X₆₈ can be S or T, X₇₃ can be N or T, X₇₆ can be N or S , X₇₉ can be F or S, X₈₂ can be L or M, X₈₃ can be N or S, X₈₅ can be L or V, X₈₆ can be Q or T, X₈₇ can be A or T, X₈₈ can be A or E, X₉₂ can be S or V, X₉₈ can be D or E, X₁₁₈ can be A or S.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的重链可变区可包含SEQ ID NO:29至SEQ ID NO:31中任一项所示的氨基酸序列。In the present application, the heavy chain variable region of the second targeting portion in the second polypeptide chain of the bispecific antibody may comprise amino acids shown in any one of SEQ ID NO:29 to SEQ ID NO:31 sequence.

例如，所述第二多肽链包含SEQ ID NO:39和SEQ ID NO:40中任一项所示的氨基酸序列。For example, the second polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NO:39 and SEQ ID NO:40.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分可以包含抗体轻链可变区VL中的至少一个CDR，所述VL可以包含SEQ ID NO:58所示的氨基酸序列。In the present application, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise at least one CDR in the VL of the light chain variable region of the antibody, and the VL may comprise SEQ ID NO:58 amino acid sequence.

例如，所述VL可以包含SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列。在本申请中，所述分离的双特异性抗体第二多肽链中的第二靶向部分的LCDR可以通过任何形式划分，只要VL与SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列相同，以任何形式划分得到的LCDR都可落入本申请的保护范围内。For example, the VL may comprise the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38. In the present application, the LCDR of the second targeting moiety in the second polypeptide chain of the isolated bispecific antibody can be divided in any form, as long as VL is related to any one of SEQ ID NO:32 to SEQ ID NO:38 The amino acid sequences shown in the items are the same, and the LCDR obtained by dividing in any form can fall within the protection scope of the present application.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分可包括轻链可变区VL，所述VL可包含LCDR1、LCDR2和LCDR3中的至少一个，至少两个或至少三个。In the present application, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise a light chain variable region VL, and the VL may comprise at least one, at least two, of LCDR1, LCDR2 and LCDR3 or at least three.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR3可包含SEQID NO:57所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR3可根据Kabat编号系统定义。In the present application, the LCDR3 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:57. For example, the LCDR3 of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

例如，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR3与SEQ ID NO:57所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₁，X₄，X₅，X₇和X₉。For example, compared with the sequence shown in SEQ ID NO: 57, the LCDR3 of the second targeting part in the second polypeptide chain of the bispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (eg, conservative amino acid substitutions, etc.): X₁ , X₄ , X₅ , X₇ and X₉ .

X₁QSX₄X₅VX₇WX₉(SEQ ID NO：57)，其中，X₁可以是Q或S，X₄可以是K，L或S，X₅可以是E，H，K或R，X₇可以是N或P，X₉可以是S或T。X₁ QSX₄ X₅ VX₇ WX₉ (SEQ ID NO: 57), wherein, X₁ can be Q or S, X₄ can be K, L or S, X₅ can be E, H, K or R, X₇ can be N or P, and X₉ can be S or T.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR3可包含SEQID NO:21至SEQ ID NO:26中任一项所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR3可根据Kabat编号系统定义。In the present application, the LCDR3 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26. For example, the LCDR3 of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR2可包含SEQID NO:18所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR2可根据Kabat编号系统定义。In the present application, the LCDR2 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:18. For example, LCDR2 of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR1可根据Kabat编号系统定义。In the present application, the LCDR1 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:15. For example, LCDR1 of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

例如，本申请所述双特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:21所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4F1、hu19D4-25、19D4-25-3C11或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:21. For example, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise antibody 19D4F1, hu19D4-25, 19D4-25-3C11 or an antibody having the same LCDR3 (e.g., having the same LCDR1-3) Antigen-binding fragments.

例如，本申请所述双特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:22所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4-25-1A3或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:22. For example, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise antibody 19D4-25-1A3 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

例如，本申请所述双特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:23所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4-25-1B3或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:23. For example, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise antibody 19D4-25-1B3 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

例如，本申请所述双特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:24所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4-25-1C2、19D4-25-1C2-3C11或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:24. For example, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise antibody 19D4-25-1C2, 19D4-25-1C2-3C11 or have the same LCDR3 (e.g., have the same LCDR1-3 ) antigen-binding fragment.

例如，本申请所述双特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:25所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4-25-1E4或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:25. For example, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise antibody 19D4-25-1E4 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

例如，本申请所述双特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:26所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4-25-2E10或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:26. For example, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise antibody 19D4-25-2E10 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分可包含轻链可变区VL，所述VL可包含SEQ ID NO:58所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的VL与SEQ ID NO:58所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₇，X₉，X₁₂，X₁₄，X₁₅，X₁₈，X₂₀，X₄₁，X₄₉，X₆₄，X₇₆，X₇₈，X₈₀，X₈₁，X₈₂，X₈₄，X₈₅，X₈₇，X₈₈，X₈₉，X₉₃，X₉₆，X₉₇，X₉₉，X₁₀₁和X₁₀₈。In the present application, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise the light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:58. For example, compared with the sequence shown in SEQ ID NO: 58, the VL of the second targeting moiety in the second polypeptide chain of the bispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (eg, conservative amino acid substitutions, etc.): X₇ , X₉ , X₁₂ , X₁₄ , X₁₅ , X 18 , X₂₀ , X₄₁ , X₄₉ , X₆₄ , X₇₆ ,_{X 78}_, X₈₀ , X₈₁ , X₈₂ , X₈₄ , X₈₅ , X₈₇ , X₈₈ , X₈₉ , X₉₃ , X₉₆ , X₉₇ , X₉₉ , X₁₀₁ and X₁₀₈ .

DIVLTQX₇PX₉SLX₁₂VX₁₄X₁₅GQX₁₈AX₂₀ISCRASESVDNYGISFMNWFX₄₁QKPGQPPX₄₉LLIYAASNQGSGVPX₆₄RFSGSGSGTDFX₇₆LX₇₈IX₈₀X₈₁X₈₂EX₈₄X₈₅DX₈₇X₈₈X₈₉YFCX₉₃QSX₉₆X₉₇VX₉₉WX₁₀₁FGGGTKX₁₀₈EIK(SEQ ID NO：58)，其中，X₇可以是S或T，X₉可以是A或L，X₁₂可以是A或S，X₁₄可以是S或T，X₁₅可以是L或P，X₁₈可以是P或R，X₂₀可以是S或T，X₄₁可以是L或Q，X₄₉可以是K或Q，X₆₄可以是A或D，X₇₆可以是S或T，X₇₈可以是K或N，X₈₀可以是H或S，X₈₁可以是P或R，X₈₂可以是M或V，X₈₄可以是A或E，X₈₅可以是D或E，X₈₇可以是T或V，X₈₈可以是A或G，X₈₉可以是M或V，X₉₃可以是Q或S，X₉₆可以是K，L或S，X₉₇可以是E，H，K或R，X₉₉可以是N或P，X₁₀₁可以是S或T，X₁₀₈可以是L或V。DIVLTQX₇ PX₉ SLX₁₂ VX₁₄ X₁₅ GQX₁₈ AX₂₀ ISCRASESVDNYGISFMNWFX₄₁ QKPGQPPX₄₉ LLIYAASNQGSGVPX₆₄ RFSGSGSGTDFX₇₆ LX₇₈ IX₈₀ X₈₁ X₈₂ EX₈₄ X₈₅ DX₈₇ X₈₈ X₈₉ YFCX₉₃ QSX₉₆ X₉₇ VX₉₉ WX₁₀₁ FGGGTKX₁₀₈ EIK (SEQ ID NO:58), wherein, X₇ can be S or T, X₉ can be A or L, X₁₂ can be A or S, X₁₄ can be S or T, X₁₅ can be L Or P, X₁₈ can be P or R, X₂₀ can be S or T, X₄₁ can be L or Q, X₄₉ can be K or Q, X₆₄ can be A or D, X₇₆ can be S or T , X₇₈ can be K or N, X₈₀ can be H or S, X₈₁ can be P or R, X₈₂ can be M or V, X₈₄ can be A or E, X₈₅ can be D or E, X₈₇ can be T or V, X₈₈ can be A or G, X₈₉ can be M or V, X₉₃ can be Q or S, X₉₆ can be K, L or S, X₉₇ can be E, H, K Or R, X₉₉ can be N or P, X₁₀₁ can be S or T, X₁₀₈ can be L or V.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的轻链可变区可包含SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列。In the present application, the light chain variable region of the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise amino acids shown in any one of SEQ ID NO:32 to SEQ ID NO:38 sequence.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分可包含重链恒定区，所述重链恒定区可包括源自IgG的恒定区或源自IgY的恒定区。In the present application, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise a heavy chain constant region, which may comprise a constant region derived from IgG or a constant region derived from IgY. district.

例如，所述双特异性抗体第二多肽链中的第二靶向部分的重链恒定区可包含SEQID NO:112至SEQ ID NO:113所示的氨基酸序列。For example, the heavy chain constant region of the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequences shown in SEQ ID NO: 112 to SEQ ID NO: 113.

例如，所述第二多肽链可以包含SEQ ID NO:39至SEQ ID NO:40、SEQ ID NO:154至SEQ ID NO:160中任一项所示的氨基酸序列。For example, the second polypeptide chain may comprise the amino acid sequence shown in any one of SEQ ID NO:39 to SEQ ID NO:40, SEQ ID NO:154 to SEQ ID NO:160.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:95至SEQ ID NO:104、SEQ ID NO:105至SEQ ID NO:111中任一项所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:29至SEQ ID NO:31、SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise any one of SEQ ID NO: 95 to SEQ ID NO: 104, SEQ ID NO: 105 to SEQ ID NO: 111. Amino acid sequence, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise any one of SEQ ID NO:29 to SEQ ID NO:31, SEQ ID NO:32 to SEQ ID NO:38 Amino acid sequence shown.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:97所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:31所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 97, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:31.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:98所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:31所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 98, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:31.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:100所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:31所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 100, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:31.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:102所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:31所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 102, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:31.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:103所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:31所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 103, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:31.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:97所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:36所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 97, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:36.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:98所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:36所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 98, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:36.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:100所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:36所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 100, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:36.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:102所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:36所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 102, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:36.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:103所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:36所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 103, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:36.

例如，所述双特异性抗体可包含第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:146所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:40所示的氨基酸序列。For example, the bispecific antibody can comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:146, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:40.

例如，所述双特异性抗体可包含第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:147所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:40所示的氨基酸序列。For example, the bispecific antibody can comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:147, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:40.

例如，所述双特异性抗体可包含第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:149所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:40所示的氨基酸序列。For example, the bispecific antibody can comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:149, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:40.

例如，所述双特异性抗体可包含第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:151所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:40所示的氨基酸序列。For example, the bispecific antibody can comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:151, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:40.

例如，所述双特异性抗体可包含第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:152所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:40所示的氨基酸序列。For example, the bispecific antibody can comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:152, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:40.

例如，所述双特异性抗体可包含第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:146所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:158所示的氨基酸序列。For example, the bispecific antibody can comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:146, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:158.

例如，所述双特异性抗体可包含第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:147所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:158所示的氨基酸序列。For example, the bispecific antibody can comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:147, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:158.

例如，所述双特异性抗体可包含第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:149所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:158所示的氨基酸序列。For example, the bispecific antibody can comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:149, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:158.

例如，所述双特异性抗体可包含第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:151所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:158所示的氨基酸序列。For example, the bispecific antibody can comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:151, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:158.

例如，所述双特异性抗体可包含第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:152所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:158所示的氨基酸序列。For example, the bispecific antibody can comprise a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:152, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:158.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分可特异性结合PD-1蛋白，且所述双特异性抗体第二多肽链中的第二靶向部分可特异性结合PD-L1蛋白。In the present application, the first targeting moiety in the first polypeptide chain of the bispecific antibody can specifically bind to the PD-1 protein, and the second targeting moiety in the second polypeptide chain of the bispecific antibody Some can specifically bind PD-L1 protein.

例如，所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:51所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR3序列可根据Kabat编码系统定义。For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:51. For example, the HCDR3 sequence of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

例如，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR3与SEQ ID NO：51所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₁。For example, compared with the sequence shown in SEQ ID NO: 51, the HCDR3 of the first targeting part in the first polypeptide chain of the bispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (eg, conservative amino acid substitutions, etc.): X₁ .

X₁HYGTSPFVY(SEQ ID NO：51)，其中，X₁可以是D或E。X₁ HYGTSPFVY (SEQ ID NO: 51), wherein X₁ can be D or E.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR3可包含SEQID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR3序列可根据Kabat编码系统定义。In the present application, the HCDR3 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:9 and SEQ ID NO:10. For example, the HCDR3 sequence of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR2可包含SEQID NO:6所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR2序列可根据Kabat编码系统定义。In the present application, the HCDR2 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:6. For example, the HCDR2 sequence of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR1可包含SEQID NO:3所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR1序列可根据Kabat编码系统定义。In the present application, the HCDR1 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3. For example, the HCDR1 sequence of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

例如，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR1可包含SEQ IDNO:3所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:9所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4F1、hu19D4-25、19D4-25-1A3、19D4-25-1B3、19D4-25-1C2、19D4-25-1E4、19D4-25-2E10或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。For example, the HCDR1 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9. For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise antibodies 19D4F1, hu19D4-25, 19D4-25-1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25 - 1E4, 19D4-25-2E10 or an antigen-binding fragment thereof having the same HCDR3 (eg, having the same HCDR1-3).

例如，所述双特异性抗体第一多肽链中的第一靶向部分的HCDR1可包含SEQ IDNO:3所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:10所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4-25-3C11、19D4-25-1C2-3C11或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。For example, the HCDR1 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10. For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise antibody 19D4-25-3C11, 19D4-25-1C2-3C11 or have the same HCDR3 (eg, have the same HCDR1- 3) The antigen-binding fragment.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分可包含重链可变区，所述重链可变区可包SEQ ID NO:52所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分包含VH，所述VH与SEQ ID NO:52所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₁，X₅，X₁₃，X₁₆，X₁₇，X₂₀，X₃₇，X₄₈，X₆₇，X₆₈，X₇₃，X₇₆，X₇₉，X₈₂，X₈₃，X₈₅，X₈₆，X₈₇，X₈₈，X₉₂，X₉₈和X₁₁₈。In the present application, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:52 . For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody comprises VH. Compared with the sequence shown in SEQ ID NO:52, the VH has one or more amino acids selected from the group consisting of Amino acid substitutions (eg, conservative amino acid substitutions, etc.): X₁ , X₅ , X₁₃ , X₁₆ , X₁₇ , X 20 , X₃₇ , X₄₈ , X₆₇ ,_{X 68}_, X₇₃ , X₇₆ , X₇₉ , X₈₂ , X₈₃ , X₈₅ , X₈₆ , X₈₇ , X₈₈ , X₉₂ , X₉₈ and X₁₁₈ .

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的重链可变区可包含SEQ ID NO:29至SEQ ID NO:31中任一项所示的氨基酸序列。In the present application, the heavy chain variable region of the first targeting portion in the first polypeptide chain of the bispecific antibody may comprise amino acids shown in any one of SEQ ID NO:29 to SEQ ID NO:31 sequence.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分可以包含抗体轻链可变区VL中的至少一个CDR，所述VL可以包含SEQ ID NO:58所示的氨基酸序列。In the present application, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise at least one CDR in the antibody light chain variable region VL, and the VL may comprise SEQ ID NO:58 amino acid sequence.

例如，所述双特异性抗体第一多肽链中的第一靶向部分的VL可以包含SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列。在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR可以通过任何形式划分，只要VL与SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列相同，以任何形式划分得到的LCDR都可落入本申请的保护范围内。For example, the VL of the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38. In the present application, the LCDR of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be divided in any form, as long as VL is consistent with any one of SEQ ID NO:32 to SEQ ID NO:38 The amino acid sequences shown are the same, and the LCDR obtained by dividing in any form can fall within the scope of protection of the present application.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR3可包含SEQID NO:57所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR3可根据Kabat编号系统定义。In the present application, the LCDR3 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:57. For example, the LCDR3 of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

例如，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR3与SEQ ID NO:57所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₁，X₄，X₅，X₇和X₉。For example, compared with the sequence shown in SEQ ID NO: 57, the LCDR3 of the first targeting part in the first polypeptide chain of the bispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (eg, conservative amino acid substitutions, etc.): X₁ , X₄ , X₅ , X₇ and X₉ .

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR3可包含SEQID NO:21至SEQ ID NO:26中任一项所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR3可根据Kabat编号系统定义。In the present application, the LCDR3 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26. For example, the LCDR3 of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR2可包含SEQID NO:18所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR2可根据Kabat编号系统定义。In the present application, LCDR2 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:18. For example, LCDR2 of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的LCDR1可根据Kabat编号系统定义。In the present application, LCDR1 of the first targeting part in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:15. For example, LCDR1 of the first targeting moiety in the first polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

例如，本申请所述双特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:21所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4F1、hu19D4-25、19D4-25-3C11或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:21. For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise antibodies 19D4F1, hu19D4-25, 19D4-25-3C11 or antibodies having the same LCDR3 (eg, having the same LCDR1-3) Antigen-binding fragments.

例如，本申请所述双特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:22所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4-25-1A3或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:22. For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise antibody 19D4-25-1A3 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

例如，本申请所述双特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:23所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4-25-1B3或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:23. For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise antibody 19D4-25-1B3 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

例如，本申请所述双特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:24所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4-25-1C2、19D4-25-1C2-3C11或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:24. For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise antibody 19D4-25-1C2, 19D4-25-1C2-3C11 or have the same LCDR3 (e.g., have the same LCDR1-3 ) antigen-binding fragment.

例如，本申请所述双特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:25所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4-25-1E4或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:25. For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise antibody 19D4-25-1E4 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

例如，本申请所述双特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:26所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4-25-2E10或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:26. For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise antibody 19D4-25-2E10 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分可包含轻链可变区VL，所述VL可包含SEQ ID NO:58所示的氨基酸序列。例如，所述双特异性抗体第一多肽链中的第一靶向部分的VL与SEQ ID NO:58所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₇，X₉，X₁₂，X₁₄，X₁₅，X₁₈，X₂₀，X₄₁，X₄₉，X₆₄，X₇₆，X₇₈，X₈₀，X₈₁，X₈₂，X₈₄，X₈₅，X₈₇，X₈₈，X₈₉，X₉₃，X₉₆，X₉₇，X₉₉，X₁₀₁和X₁₀₈。In the present application, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:58. For example, compared with the sequence shown in SEQ ID NO: 58, the VL of the first targeting moiety in the first polypeptide chain of the bispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (eg, conservative amino acid substitutions, etc.): X₇ , X₉ , X₁₂ , X₁₄ , X₁₅ , X 18 , X₂₀ , X₄₁ , X₄₉ , X₆₄ , X₇₆ ,_{X 78}_, X₈₀ , X₈₁ , X₈₂ , X₈₄ , X₈₅ , X₈₇ , X₈₈ , X₈₉ , X₉₃ , X₉₆ , X₉₇ , X₉₉ , X₁₀₁ and X₁₀₈ .

在本申请中，所述双特异性抗体第一多肽链中的第一靶向部分的轻链可变区可包含SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列。In the present application, the light chain variable region of the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise amino acids shown in any one of SEQ ID NO:32 to SEQ ID NO:38 sequence.

例如，所述双特异性抗体第一多肽链中的第一靶向部分的轻链恒定区包含SEQ IDNO:114所示的氨基酸序列。For example, the light chain constant region of the first targeting moiety in the first polypeptide chain of the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:114.

例如，所述第一多肽链包含SEQ ID NO:141至143、SEQ ID NO:41至SEQ ID NO:46中任一项所示的氨基酸序列。在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR3可包含SEQ ID NO:76所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR3序列可根据Kabat编码系统定义。For example, the first polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NO: 141 to 143, SEQ ID NO: 41 to SEQ ID NO: 46. In the present application, the HCDR3 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:76. For example, the HCDR3 sequence of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR2可包含SEQID NO:70所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR2序列可根据Kabat编码系统定义。In the present application, the HCDR2 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:70. For example, the HCDR2 sequence of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR1可包含SEQID NO:61所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR1序列可根据Kabat编码系统定义。In the present application, the HCDR1 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:61. For example, the HCDR1 sequence of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat coding system.

例如，所述双特异性抗体第二多肽链中的第二靶向部分的HCDR1可包含SEQ IDNO:61所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:70所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:76所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分可包括抗体13H6D3、hu13H6D3、5BM、AH00228、AH00229、AH00230、AH00231、AH00232、AH00233、CBM或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。For example, the HCDR1 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:61; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:70; And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:76. For example, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM or have the same HCDR3 (e.g., have the same HCDR1-3) antigen-binding fragments.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分可包含重链可变区，所述重链可变区可包含SEQ ID NO:122所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分包含VH，所述VH与SEQ ID NO:122所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₅，X₇，X₁₀，X₁₁，X₁₂，X₁₆，X₂₀，X₃₇，X₃₈，X₄₀，X₄₆，X₄₈，X₆₇，X₆₈，X₆₉，X₇₅，X₈₁，X₈₆，X₉₀，X₁₁₅和X₁₁₆。In the present application, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:122 . For example, the second targeting moiety in the second polypeptide chain of the bispecific antibody comprises VH, and compared with the sequence shown in SEQ ID NO: 122, the VH has one or more amino acids selected from the group consisting of Amino acid substitutions (eg, conservative amino acid substitutions, etc.): X₅ , X₇ , X₁₀ , X₁₁ , X₁₂ , X₁₆ , X 20 , X₃₇ , X₃₈ ,_{X 40}_, X₄₆ , X₄₈ , X₆₇ , X₆₈ , X₆₉ , X₇₅ , X₈₁ , X₈₆ , X₉₀ , X₁₁₅ and X₁₁₆ .

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的重链可变区可包含SEQ ID NO:95至SEQ ID NO:104中任一项所示的氨基酸序列。In the present application, the heavy chain variable region of the second targeting portion in the second polypeptide chain of the bispecific antibody may comprise amino acids shown in any one of SEQ ID NO:95 to SEQ ID NO:104 sequence.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分可以包含抗体轻链可变区VL中的至少一个CDR，所述VL可以包含SEQ ID NO:123所示的氨基酸序列。In the present application, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise at least one CDR in the VL of the light chain variable region of the antibody, and the VL may comprise SEQ ID NO: 123 amino acid sequence.

例如，所述双特异性抗体第二多肽链中的第二靶向部分中的VL可以包含SEQ IDNO:105至SEQ ID NO:111中任一项所示的氨基酸序列。在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR可以通过任何形式划分，只要VL与SEQ ID NO:105至SEQID NO:111中任一项所示的氨基酸序列相同，以任何形式划分得到的LCDR都可落入本申请的保护范围内。For example, the VL in the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 105 to SEQ ID NO: 111. In this application, the LCDR of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be divided in any form, as long as VL is shown in any one of SEQ ID NO: 105 to SEQ ID NO: 111 The amino acid sequence is the same, and the LCDR obtained by dividing in any form can fall within the protection scope of the present application.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR3可包含SEQID NO:94所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR3可根据Kabat编号系统定义。In the present application, the LCDR3 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:94. For example, the LCDR3 of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR2可包含SEQID NO:87所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR2可根据Kabat编号系统定义。In the present application, the LCDR2 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:87. For example, LCDR2 of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:82所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的LCDR1可根据Kabat编号系统定义。In the present application, the LCDR1 of the second targeting part in the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:82. For example, LCDR1 of the second targeting moiety in the second polypeptide chain of the bispecific antibody can be defined according to the Kabat numbering system.

例如，本申请所述双特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:82所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:87所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:94所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分可包括抗体13H6D3、hu13H6D3、5BM、AH00228、AH00229、AH00230、AH00231、AH00232、AH00233、CBM或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the bispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:82; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:87 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:94. For example, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM or have the same LCDR3 (e.g., Antigen-binding fragments having the same LCDR1-3) as it.

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分可包含轻链可变区VL，所述VL可包含SEQ ID NO:123所示的氨基酸序列。例如，所述双特异性抗体第二多肽链中的第二靶向部分的VL与SEQ ID NO:123所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₇，X₈，X₁₅，X₂₂，X₄₁，X₄₂，X₄₃，X₄₄，X₄₆，X₆₀，X₇₁，X₇₂，X₇₇，X₇₉，X₈₀，X₈₃，X₈₇和X₁₀₄。In the present application, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise the light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:123. For example, compared with the sequence shown in SEQ ID NO: 123, the VL of the second targeting moiety in the second polypeptide chain of the bispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (e.g., conservative amino acid substitutions, etc.):_X7 ,_X8 ,_X15 ,_X22 ,_X41 ,_X42 ,_X43 , X44_,_X46 ,_X60 ,_X71 ,_X72 ,_X77 ,_X79 , X₈₀ , X₈₃ , X₈₇ and X₁₀₄ .

在本申请中，所述双特异性抗体第二多肽链中的第二靶向部分的轻链可变区可包含SEQ ID NO:105至SEQ ID NO:111中任一项所示的氨基酸序列。In the present application, the light chain variable region of the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise amino acids shown in any one of SEQ ID NO:105 to SEQ ID NO:111 sequence.

例如，所述双特异性抗体的第二多肽链可包含SEQ ID NO:124至133、SEQ ID NO:161至SEQ ID NO:167中任一项所示的氨基酸序列。For example, the second polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 124 to 133, SEQ ID NO: 161 to SEQ ID NO: 167.

在本申请中，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。例如，所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:29至SEQ ID NO:31、SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ IDNO:95至SEQ ID NO:104、SEQ ID NO:105至SEQ ID NO:111中任一项所示的氨基酸序列。In the present application, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise any one of SEQ ID NO:29 to SEQ ID NO:31, SEQ ID NO:32 to SEQ ID NO:38. The amino acid sequence shown, the second targeting moiety in the second polypeptide chain of the bispecific antibody may comprise any one of SEQ ID NO:95 to SEQ ID NO:104, SEQ ID NO:105 to SEQ ID NO:111 The amino acid sequence shown in item.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:31所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:97所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 31, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:97.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:31所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:98所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 31, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:98.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:31所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:100所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 31, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:100.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:31所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:102所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 31, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:102.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:31所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:103所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 31, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:103.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:36所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:97所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 36, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:97.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:36所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:98所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 36, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:98.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:36所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:100所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 36, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:100.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:36所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:102所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 36, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:102.

例如，所述双特异性抗体可包含双特异性抗体第一多肽链中的第一靶向部分和双特异性抗体第二多肽链中的第二靶向部分。所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:36所示的氨基酸序列，所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:103所示的氨基酸序列。For example, the bispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the bispecific antibody and a second targeting moiety in the second polypeptide chain of the bispecific antibody. The first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 36, and the second targeting moiety in the second polypeptide chain of the bispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:103.

在本申请中，所述双特异性抗体可包含两条前述第一多肽链和两条前述第二多肽链。例如，两条前述第一多肽链之间可通过二硫键连接，所述两条前述第一多肽链和两条前述第二多肽链可形成IgG样的抗体。例如，所述双特异性抗体可为同二聚体。In the present application, the bispecific antibody may comprise the two aforementioned first polypeptide chains and the two aforementioned second polypeptide chains. For example, the two aforementioned first polypeptide chains may be linked by a disulfide bond, and the two aforementioned first polypeptide chains and the two aforementioned second polypeptide chains may form an IgG-like antibody. For example, the bispecific antibody may be a homodimer.

例如，所述双特异性抗体可包含两条第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:143所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:126所示的氨基酸序列。For example, the bispecific antibody may comprise two first and second polypeptide chains. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:143, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:126.

例如，所述双特异性抗体可包含两条第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:143所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:127所示的氨基酸序列。For example, the bispecific antibody may comprise two first and second polypeptide chains. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:143, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:127.

例如，所述双特异性抗体可包含两条第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:143所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:129所示的氨基酸序列。For example, the bispecific antibody may comprise two first and second polypeptide chains. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:143, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:129.

例如，所述双特异性抗体可包含两条第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:143所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:131所示的氨基酸序列。For example, the bispecific antibody may comprise two first and second polypeptide chains. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:143, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:131.

例如，所述双特异性抗体可包含两条第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:143所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:132所示的氨基酸序列。For example, the bispecific antibody may comprise two first and second polypeptide chains. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:143, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:132.

例如，所述双特异性抗体可包含两条第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:44所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:126所示的氨基酸序列。For example, the bispecific antibody may comprise two first and second polypeptide chains. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:44, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:126.

例如，所述双特异性抗体可包含两条第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:44所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:127所示的氨基酸序列。For example, the bispecific antibody may comprise two first and second polypeptide chains. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:44, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:127.

例如，所述双特异性抗体可包含两条第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:44所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:129所示的氨基酸序列。For example, the bispecific antibody may comprise two first and second polypeptide chains. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:44, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:129.

例如，所述双特异性抗体可包含两条第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:44所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:131所示的氨基酸序列。For example, the bispecific antibody may comprise two first and second polypeptide chains. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:44, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:131.

例如，所述双特异性抗体可包含两条第一多肽链和第二多肽链。所述第一多肽链可包含SEQ ID NO:44所示的氨基酸序列，所述第二多肽链可包含SEQ ID NO:132所示的氨基酸序列。For example, the bispecific antibody may comprise two first and second polypeptide chains. The first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:44, and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:132.

例如，所述双特异性抗体第一多肽链中的第一靶向部分可特异性结合PD-1蛋白，且所述双特异性抗体第二多肽链中的第二靶向部分可特异性结合CD28或CD16a蛋白。例如，所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：31所示的氨基酸序列，且所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：176或SEQ ID NO：168所示的氨基酸序列。例如，所述双特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody can specifically bind PD-1 protein, and the second targeting moiety in the second polypeptide chain of the bispecific antibody can specifically bind Sexually binds CD28 or CD16a protein. For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 31, and the second targeting moiety in the second polypeptide chain of the bispecific antibody The direction part may comprise the amino acid sequence shown in SEQ ID NO: 176 or SEQ ID NO: 168. For example, the bispecific antibody may comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

例如，所述双特异性抗体第一多肽链中的第一靶向部分可特异性结合CD28或CD16a蛋白，且所述双特异性抗体第二多肽链中的第二靶向部分可特异性结合PD-1蛋白。例如，所述双特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：176或SEQ ID NO：168中任一项所示的氨基酸序列，且所述双特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：31所示的氨基酸序列。例如，所述双特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody can specifically bind CD28 or CD16a protein, and the second targeting moiety in the second polypeptide chain of the bispecific antibody can specifically bind Sexually binds to PD-1 protein. For example, the first targeting moiety in the first polypeptide chain of the bispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 176 or SEQ ID NO: 168, and the bispecific antibody The second targeting moiety in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31. For example, the bispecific antibody may comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

多特异性抗体multispecific antibody

另一方面，本申请提供了一种或多种多特异性抗体，其包含第一多肽链、第二多肽链，所述第一多肽链包含第一靶向部分，所述第二多肽链包含第二靶向部分，所述第一靶向部分和所述第二靶向部分各自独立地结合免疫细胞相关抗原，所述第一多肽链与所述第二多肽链通过二硫键连接。In another aspect, the application provides one or more multispecific antibodies comprising a first polypeptide chain, a second polypeptide chain, the first polypeptide chain comprising a first targeting moiety, the second The polypeptide chain comprises a second targeting moiety, the first targeting moiety and the second targeting moiety independently bind immune cell-associated antigens, the first polypeptide chain and the second polypeptide chain pass through Disulfide linkage.

例如，所述免疫细胞可包括T细胞、巨噬细胞、树突状细胞和/或NK细胞。例如，所述免疫细胞相关抗原可包括PD-1、PD-L1、CD3、CD16a和/或CD28。For example, the immune cells may include T cells, macrophages, dendritic cells and/or NK cells. For example, the immune cell-associated antigens may include PD-1, PD-L1, CD3, CD16a and/or CD28.

在本申请中，所述多特异性抗体的第一靶向部分可与前述双特异性抗体的第一靶向部分相同。在本申请中，所述多特异性抗体的第二靶向部分可与前述双特异性抗体的第二靶向部分相同。In the present application, the first targeting moiety of the multispecific antibody may be the same as the first targeting moiety of the aforementioned bispecific antibody. In the present application, the second targeting moiety of the multispecific antibody may be the same as the second targeting moiety of the aforementioned bispecific antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可特异性结合PD-L1蛋白，所述多特异性抗体第二多肽链中的第二靶向部分可特异性结合PD-1蛋白。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can specifically bind to PD-L1 protein, and the second targeting moiety in the second polypeptide chain of the multispecific antibody Can specifically bind PD-1 protein.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR3可包含SEQID NO:76所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR3序列可根据Kabat编码系统定义。In the present application, the HCDR3 of the first targeting portion in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:76. For example, the HCDR3 sequence of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR2可包含SEQID NO:70所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR2序列可根据Kabat编码系统定义。In the present application, the HCDR2 of the first targeting part in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:70. For example, the HCDR2 sequence of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR1可包含SEQID NO:61所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR1序列可根据Kabat编码系统定义。In the present application, the HCDR1 of the first targeting part in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:61. For example, the HCDR1 sequence of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

例如，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR1可包含SEQ IDNO:61所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:70所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:76所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包括抗体13H6D3、hu13H6D3、5BM、AH00228、AH00229、AH00230、AH00231、AH00232、AH00233、CBM或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。For example, the HCDR1 of the first targeting portion in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:61; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:70; And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:76. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM or have the same HCDR3 (e.g., have the same HCDR1-3) antigen-binding fragments.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可包含重链可变区，所述重链可变区可包含SEQ ID NO:122所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分包含VH，所述VH与SEQ ID NO:122所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₅，X₇，X₁₀，X₁₁，X₁₂，X₁₆，X₂₀，X₃₇，X₃₈，X₄₀，X₄₆，X₄₈，X₆₇，X₆₈，X₆₉，X₇₅，X₈₁，X₈₆，X₉₀，X₁₁₅和X₁₁₆。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:122 . For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody comprises VH, and compared with the sequence shown in SEQ ID NO: 122, the VH has one or more amino acids selected from the group consisting of Amino acid substitutions (eg, conservative amino acid substitutions, etc.): X₅ , X₇ , X₁₀ , X₁₁ , X₁₂ , X₁₆ , X 20 , X₃₇ , X₃₈ ,_{X 40}_, X₄₆ , X₄₈ , X₆₇ , X₆₈ , X₆₉ , X₇₅ , X₈₁ , X₈₆ , X₉₀ , X₁₁₅ and X₁₁₆ .

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的重链可变区可包含SEQ ID NO:95至SEQ ID NO:104中任一项所示的氨基酸序列。In the present application, the heavy chain variable region of the first targeting portion in the first polypeptide chain of the multispecific antibody may comprise amino acids shown in any one of SEQ ID NO:95 to SEQ ID NO:104 sequence.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可以包含轻链可变区VL中的至少一个CDR，所述VL可以包含SEQ ID NO:123所示的氨基酸序列。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise at least one CDR in the VL of the light chain variable region, and the VL may comprise the CDR shown in SEQ ID NO: 123. amino acid sequence.

例如，所述多特异性抗体第一多肽链中的第一靶向部分中的VL可以包含SEQ IDNO:105至SEQ ID NO:111中任一项所示的氨基酸序列。在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR可以通过任何形式划分，只要VL与SEQ ID NO:105至SEQID NO:111中任一项所示的氨基酸序列相同，以任何形式划分得到的LCDR都可落入本申请的保护范围内。For example, the VL in the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 105 to SEQ ID NO: 111. In this application, the LCDR of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be divided in any form, as long as VL is shown in any one of SEQ ID NO:105 to SEQ ID NO:111 The amino acid sequence is the same, and the LCDR obtained by dividing in any form can fall within the protection scope of the present application.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可包括轻链可变区VL，所述VL可包含LCDR1、LCDR2和LCDR3中的至少一个，至少两个或至少三个。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody may include a light chain variable region VL, and the VL may include at least one of LCDR1, LCDR2 and LCDR3, at least two or at least three.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR3可包含SEQID NO:94所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR3可根据Kabat编号系统定义。In the present application, the LCDR3 of the first targeting portion in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:94. For example, the LCDR3 of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR2可包含SEQID NO:87所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR2可根据Kabat编号系统定义。In the present application, LCDR2 of the first targeting portion in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:87. For example, LCDR2 of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:82所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR1可根据Kabat编号系统定义。In the present application, LCDR1 of the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:82. For example, LCDR1 of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

例如，本申请所述多特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:82所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:87所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:94所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包括抗体13H6D3、hu13H6D3、5BM、AH00228、AH00229、AH00230、AH00231、AH00232、AH00233、CBM或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:82; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:87 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:94. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody can comprise antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or have the same LCDR3 (e.g., Antigen-binding fragments having the same LCDR1-3) as it.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可包含轻链可变区VL，所述VL可包含SEQ ID NO:123所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的VL与SEQ ID NO:123所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₇，X₈，X₁₅，X₂₂，X₄₁，X₄₂，X₄₃，X₄₄，X₄₆，X₆₀，X₇₁，X₇₂，X₇₇，X₇₉，X₈₀，X₈₃，X₈₇和X₁₀₄。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:123. For example, compared with the sequence shown in SEQ ID NO: 123, the VL of the first targeting moiety in the first polypeptide chain of the multispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (e.g., conservative amino acid substitutions, etc.):_X7 ,_X8 ,_X15 ,_X22 ,_X41 ,_X42 ,_X43 , X44_,_X46 ,_X60 ,_X71 ,_X72 ,_X77 ,_X79 , X₈₀ , X₈₃ , X₈₇ and X₁₀₄ .

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的轻链可变区可包含SEQ ID NO:105至SEQ ID NO:111中任一项所示的氨基酸序列。In the present application, the light chain variable region of the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise amino acids shown in any one of SEQ ID NO:105 to SEQ ID NO:111 sequence.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可包含轻链恒定区，所述轻链恒定区可包括源自Igκ的恒定区或源自Igλ的恒定区。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise a light chain constant region, which may comprise a constant region derived from Igκ or a constant region derived from Igλ. district.

例如，所述多特异性抗体第一多肽链中的第一靶向部分的轻链恒定区包含SEQ IDNO:114中任一项所示的氨基酸序列。For example, the light chain constant region of the first targeting moiety in the first polypeptide chain of the multispecific antibody comprises the amino acid sequence shown in any one of SEQ ID NO:114.

例如，所述多特异性抗体的第一多肽链可包含SEQ ID NO:134至140、SEQ ID NO:144至153中任一项所示的氨基酸序列。For example, the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 134 to 140, SEQ ID NO: 144 to 153.

例如，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:51所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR3序列可根据Kabat编码系统定义。For example, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:51. For example, the HCDR3 sequence of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

例如，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR3与SEQ ID NO：51所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₁。For example, compared with the sequence shown in SEQ ID NO: 51, the HCDR3 of the second targeting part in the second polypeptide chain of the multispecific antibody has an amino acid substitution at one or more amino acids selected from the group (eg, conservative amino acid substitutions, etc.): X₁ .

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR3可包含SEQID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR3序列可根据Kabat编码系统定义。In the present application, the HCDR3 of the second targeting part in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:9 and SEQ ID NO:10. For example, the HCDR3 sequence of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR2可包含SEQID NO:6所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR2序列可根据Kabat编码系统定义。In the present application, the HCDR2 of the second targeting part in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:6. For example, the HCDR2 sequence of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR1可包含SEQID NO:3所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR1序列可根据Kabat编码系统定义。In the present application, the HCDR1 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3. For example, the HCDR1 sequence of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

例如，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR1可包含SEQ IDNO:3所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:9所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4F1、hu19D4-25、19D4-25-1A3、19D4-25-1B3、19D4-25-1C2、19D4-25-1E4、19D4-25-2E10或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。For example, the HCDR1 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9. For example, the second targeting moiety in the second polypeptide chain of the multispecific antibody can include antibodies 19D4F1, hu19D4-25, 19D4-25-1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25 - 1E4, 19D4-25-2E10 or an antigen-binding fragment thereof having the same HCDR3 (eg, having the same HCDR1-3).

例如，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR1可包含SEQ IDNO:3所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:10所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4-25-3C11、19D4-25-1C2-3C11或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。For example, the HCDR1 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10. For example, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise antibody 19D4-25-3C11, 19D4-25-1C2-3C11 or have the same HCDR3 (e.g., have the same HCDR1- 3) The antigen-binding fragment.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分可包含重链可变区，所述重链可变区可包SEQ ID NO:52所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分包含VH，所述VH与SEQ ID NO:52所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₁，X₅，X₁₃，X₁₆，X₁₇，X₂₀，X₃₇，X₄₈，X₆₇，X₆₈，X₇₃，X₇₆，X₇₉，X₈₂，X₈₃，X₈₅，X₈₆，X₈₇，X₈₈，X₉₂，X₉₈和X₁₁₈。In the present application, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:52 . For example, the second targeting moiety in the second polypeptide chain of the multispecific antibody comprises VH, and compared with the sequence shown in SEQ ID NO:52, the VH has one or more amino acids selected from the group consisting of Amino acid substitutions (eg, conservative amino acid substitutions, etc.): X₁ , X₅ , X₁₃ , X₁₆ , X₁₇ , X 20 , X₃₇ , X₄₈ , X₆₇ ,_{X 68}_, X₇₃ , X₇₆ , X₇₉ , X₈₂ , X₈₃ , X₈₅ , X₈₆ , X₈₇ , X₈₈ , X₉₂ , X₉₈ and X₁₁₈ .

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的重链可变区可包含SEQ ID NO:29至SEQ ID NO:31中任一项所示的氨基酸序列。In the present application, the heavy chain variable region of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise amino acids shown in any one of SEQ ID NO:29 to SEQ ID NO:31 sequence.

例如，所述多特异性抗体的第二多肽链包含SEQ ID NO:39和SEQ ID NO:40中任一项所示的氨基酸序列。For example, the second polypeptide chain of the multispecific antibody comprises the amino acid sequence shown in any one of SEQ ID NO:39 and SEQ ID NO:40.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分可以包含抗体轻链可变区VL中的至少一个CDR，所述VL可以包含SEQ ID NO:58所示的氨基酸序列。In the present application, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise at least one CDR in the VL of the light chain variable region of the antibody, and the VL may comprise SEQ ID NO:58 amino acid sequence.

例如，所述VL可以包含SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列。在本申请中，所述分离的多特异性抗体第二多肽链中的第二靶向部分的LCDR可以通过任何形式划分，只要VL与SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列相同，以任何形式划分得到的LCDR都可落入本申请的保护范围内。For example, the VL may comprise the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38. In the present application, the LCDR of the second targeting moiety in the second polypeptide chain of the isolated multispecific antibody can be divided in any form, as long as VL is related to any one of SEQ ID NO:32 to SEQ ID NO:38 The amino acid sequences shown in the items are the same, and the LCDR obtained by dividing in any form can fall within the protection scope of the present application.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分可包括轻链可变区VL，所述VL可包含LCDR1、LCDR2和LCDR3中的至少一个，至少两个或至少三个。In the present application, the second targeting moiety in the second polypeptide chain of the multispecific antibody may include a light chain variable region VL, and the VL may include at least one of LCDR1, LCDR2 and LCDR3, at least two or at least three.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR3可包含SEQID NO:57所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR3可根据Kabat编号系统定义。In the present application, the LCDR3 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:57. For example, the LCDR3 of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

例如，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR3与SEQ ID NO:57所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₁，X₄，X₅，X₇和X₉。For example, compared with the sequence shown in SEQ ID NO: 57, the LCDR3 of the second targeting portion in the second polypeptide chain of the multispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (eg, conservative amino acid substitutions, etc.): X₁ , X₄ , X₅ , X₇ and X₉ .

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR3可包含SEQID NO:21至SEQ ID NO:26中任一项所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR3可根据Kabat编号系统定义。In the present application, the LCDR3 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26. For example, the LCDR3 of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR2可包含SEQID NO:18所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR2可根据Kabat编号系统定义。In the present application, the LCDR2 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:18. For example, LCDR2 of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR1可根据Kabat编号系统定义。In the present application, the LCDR1 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:15. For example, LCDR1 of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

例如，本申请所述多特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:21所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4F1、hu19D4-25、19D4-25-3C11或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:21. For example, the second targeting moiety in the second polypeptide chain of the multispecific antibody can comprise antibody 19D4F1, hu19D4-25, 19D4-25-3C11 or an antibody having the same LCDR3 (e.g., having the same LCDR1-3) Antigen-binding fragments.

例如，本申请所述多特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:22所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4-25-1A3或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:22. For example, the second targeting moiety in the second polypeptide chain of the multispecific antibody can comprise antibody 19D4-25-1A3 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

例如，本申请所述多特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:23所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4-25-1B3或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:23. For example, the second targeting moiety in the second polypeptide chain of the multispecific antibody can comprise antibody 19D4-25-1B3 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

例如，本申请所述多特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:24所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4-25-1C2、19D4-25-1C2-3C11或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:24. For example, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise antibody 19D4-25-1C2, 19D4-25-1C2-3C11 or have the same LCDR3 (e.g., have the same LCDR1-3 ) antigen-binding fragment.

例如，本申请所述多特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:25所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4-25-1E4或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:25. For example, the second targeting moiety in the second polypeptide chain of the multispecific antibody can comprise antibody 19D4-25-1E4 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

例如，本申请所述多特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:26所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分可包括抗体19D4-25-2E10或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:26. For example, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise antibody 19D4-25-2E10 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分可包含轻链可变区VL，所述VL可包含SEQ ID NO:58所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的VL与SEQ ID NO:58所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₇，X₉，X₁₂，X₁₄，X₁₅，X₁₈，X₂₀，X₄₁，X₄₉，X₆₄，X₇₆，X₇₈，X₈₀，X₈₁，X₈₂，X₈₄，X₈₅，X₈₇，X₈₈，X₈₉，X₉₃，X₉₆，X₉₇，X₉₉，X₁₀₁和X₁₀₈。In the present application, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise the light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:58. For example, compared with the sequence shown in SEQ ID NO:58, the VL of the second targeting moiety in the second polypeptide chain of the multispecific antibody has an amino acid substitution at one or more amino acids selected from the group (eg, conservative amino acid substitutions, etc.): X₇ , X₉ , X₁₂ , X₁₄ , X₁₅ , X 18 , X₂₀ , X₄₁ , X₄₉ , X₆₄ , X₇₆ ,_{X 78}_, X₈₀ , X₈₁ , X₈₂ , X₈₄ , X₈₅ , X₈₇ , X₈₈ , X₈₉ , X₉₃ , X₉₆ , X₉₇ , X₉₉ , X₁₀₁ and X₁₀₈ .

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的轻链可变区可包含SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列。In the present application, the light chain variable region of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise amino acids shown in any one of SEQ ID NO:32 to SEQ ID NO:38 sequence.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分可包含重链恒定区，所述重链恒定区可包括源自IgG的恒定区或源自IgY的恒定区。In the present application, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise a heavy chain constant region, which may comprise a constant region derived from IgG or a constant region derived from IgY. district.

例如，所述多特异性抗体第二多肽链中的第二靶向部分的重链恒定区可包含SEQID NO:112至SEQ ID NO:113所示的氨基酸序列。For example, the heavy chain constant region of the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequences shown in SEQ ID NO: 112 to SEQ ID NO: 113.

例如，所述多特异性抗体的第二多肽链可以包含SEQ ID NO:39至SEQ ID NO:40、SEQ ID NO:154至SEQ ID NO:160中任一项所示的氨基酸序列。For example, the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:39 to SEQ ID NO:40, SEQ ID NO:154 to SEQ ID NO:160.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:95至SEQ ID NO:104、SEQ ID NO:105至SEQ ID NO:111中任一项所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:29至SEQ ID NO:31、SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise any one of SEQ ID NO:95 to SEQ ID NO:104, SEQ ID NO:105 to SEQ ID NO:111 Amino acid sequence, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise any one of SEQ ID NO:29 to SEQ ID NO:31, SEQ ID NO:32 to SEQ ID NO:38 Amino acid sequence shown.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:97所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:31所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 97, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:31.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:98所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:31所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 98, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:31.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:100所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:31所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 100, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:31.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:102所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:31所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 102, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:31.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:103所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:31所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 103, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:31.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:97所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:36所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 97, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:36.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:98所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:36所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 98, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:36.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:100所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:36所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 100, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:36.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:102所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:36所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 102, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:36.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:103所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:36所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 103, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:36.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可特异性结合PD-1蛋白，且所述多特异性抗体第二多肽链中的第二靶向部分可特异性结合PD-L1蛋白。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can specifically bind to the PD-1 protein, and the second targeting moiety in the second polypeptide chain of the multispecific antibody Some can specifically bind PD-L1 protein.

例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:51所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR3序列可根据Kabat编码系统定义。For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:51. For example, the HCDR3 sequence of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

例如，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR3与SEQ ID NO：51所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₁。For example, compared with the sequence shown in SEQ ID NO: 51, the HCDR3 of the first targeting part in the first polypeptide chain of the multispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (eg, conservative amino acid substitutions, etc.): X₁ .

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR3可包含SEQID NO:9和SEQ ID NO:10中任一项所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR3序列可根据Kabat编码系统定义。In the present application, the HCDR3 of the first targeting part in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:9 and SEQ ID NO:10. For example, the HCDR3 sequence of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR2可包含SEQID NO:6所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR2序列可根据Kabat编码系统定义。In the present application, the HCDR2 of the first targeting part in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:6. For example, the HCDR2 sequence of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR1可包含SEQID NO:3所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR1序列可根据Kabat编码系统定义。In the present application, the HCDR1 of the first targeting part in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3. For example, the HCDR1 sequence of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

例如，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR1可包含SEQ IDNO:3所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:9所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4F1、hu19D4-25、19D4-25-1A3、19D4-25-1B3、19D4-25-1C2、19D4-25-1E4、19D4-25-2E10或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。For example, the HCDR1 of the first targeting portion in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody can include antibodies 19D4F1, hu19D4-25, 19D4-25-1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25 - 1E4, 19D4-25-2E10 or an antigen-binding fragment thereof having the same HCDR3 (eg, having the same HCDR1-3).

例如，所述多特异性抗体第一多肽链中的第一靶向部分的HCDR1可包含SEQ IDNO:3所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:10所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4-25-3C11、19D4-25-1C2-3C11或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。For example, the HCDR1 of the first targeting portion in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise antibody 19D4-25-3C11, 19D4-25-1C2-3C11 or have the same HCDR3 (eg, have the same HCDR1- 3) The antigen-binding fragment.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可包含重链可变区，所述重链可变区可包SEQ ID NO:52所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分包含VH，所述VH与SEQ ID NO:52所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₁，X₅，X₁₃，X₁₆，X₁₇，X₂₀，X₃₇，X₄₈，X₆₇，X₆₈，X₇₃，X₇₆，X₇₉，X₈₂，X₈₃，X₈₅，X₈₆，X₈₇，X₈₈，X₉₂，X₉₈和X₁₁₈。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:52 . For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody comprises VH, and compared with the sequence shown in SEQ ID NO:52, the VH has one or more amino acids selected from the group consisting of Amino acid substitutions (eg, conservative amino acid substitutions, etc.): X₁ , X₅ , X₁₃ , X₁₆ , X₁₇ , X 20 , X₃₇ , X₄₈ , X₆₇ ,_{X 68}_, X₇₃ , X₇₆ , X₇₉ , X₈₂ , X₈₃ , X₈₅ , X₈₆ , X₈₇ , X₈₈ , X₉₂ , X₉₈ and X₁₁₈ .

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的重链可变区可包含SEQ ID NO:29至SEQ ID NO:31中任一项所示的氨基酸序列。In the present application, the heavy chain variable region of the first targeting portion in the first polypeptide chain of the multispecific antibody may comprise amino acids shown in any one of SEQ ID NO:29 to SEQ ID NO:31 sequence.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可以包含抗体轻链可变区VL中的至少一个CDR，所述VL可以包含SEQ ID NO:58所示的氨基酸序列。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise at least one CDR in the VL of the light chain variable region of the antibody, and the VL may comprise SEQ ID NO:58 amino acid sequence.

例如，所述多特异性抗体第一多肽链中的第一靶向部分的VL可以包含SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列。在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR可以通过任何形式划分，只要VL与SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列相同，以任何形式划分得到的LCDR都可落入本申请的保护范围内。For example, the VL of the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38. In the present application, the LCDR of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be divided in any form, as long as VL and any one of SEQ ID NO:32 to SEQ ID NO:38 The amino acid sequences shown are the same, and the LCDR obtained by dividing in any form can fall within the scope of protection of the present application.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR3可包含SEQID NO:57所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR3可根据Kabat编号系统定义。In the present application, the LCDR3 of the first targeting part in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:57. For example, the LCDR3 of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

例如，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR3与SEQ ID NO:57所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₁，X₄，X₅，X₇和X₉。For example, compared with the sequence shown in SEQ ID NO: 57, the LCDR3 of the first targeting part in the first polypeptide chain of the multispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (eg, conservative amino acid substitutions, etc.): X₁ , X₄ , X₅ , X₇ and X₉ .

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR3可包含SEQID NO:21至SEQ ID NO:26中任一项所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR3可根据Kabat编号系统定义。In the present application, the LCDR3 of the first targeting part in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26. For example, the LCDR3 of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR2可包含SEQID NO:18所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR2可根据Kabat编号系统定义。In the present application, LCDR2 of the first targeting portion in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:18. For example, LCDR2 of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的LCDR1可根据Kabat编号系统定义。In the present application, LCDR1 of the first targeting portion in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:15. For example, LCDR1 of the first targeting moiety in the first polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

例如，本申请所述多特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:21所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4F1、hu19D4-25、19D4-25-3C11或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:21. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise antibody 19D4F1, hu19D4-25, 19D4-25-3C11 or an antibody having the same LCDR3 (e.g., having the same LCDR1-3) Antigen-binding fragments.

例如，本申请所述多特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:22所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4-25-1A3或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:22. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody can comprise antibody 19D4-25-1A3 or an antigen-binding fragment having the same LCDR3 therewith (eg, having the same LCDR1-3 as it).

例如，本申请所述多特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:23所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4-25-1B3或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:23. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody can comprise antibody 19D4-25-1B3 or an antigen-binding fragment having the same LCDR3 therewith (eg, having the same LCDR1-3 as it).

例如，本申请所述多特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:24所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4-25-1C2、19D4-25-1C2-3C11或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:24. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise antibody 19D4-25-1C2, 19D4-25-1C2-3C11 or have the same LCDR3 (e.g., have the same LCDR1-3 ) antigen-binding fragment.

例如，本申请所述多特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:25所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4-25-1E4或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:25. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody can comprise antibody 19D4-25-1E4 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

例如，本申请所述多特异性抗体第一多肽链中的第一靶向部分的LCDR1可包含SEQID NO:15所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:18所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:26所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包括抗体19D4-25-2E10或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the first targeting moiety in the first polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:18 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:26. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody can comprise antibody 19D4-25-2E10 or an antigen-binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可包含轻链可变区VL，所述VL可包含SEQ ID NO:58所示的氨基酸序列。例如，所述多特异性抗体第一多肽链中的第一靶向部分的VL与SEQ ID NO:58所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₇，X₉，X₁₂，X₁₄，X₁₅，X₁₈，X₂₀，X₄₁，X₄₉，X₆₄，X₇₆，X₇₈，X₈₀，X₈₁，X₈₂，X₈₄，X₈₅，X₈₇，X₈₈，X₈₉，X₉₃，X₉₆，X₉₇，X₉₉，X₁₀₁和X₁₀₈。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:58. For example, compared with the sequence shown in SEQ ID NO: 58, the VL of the first targeting moiety in the first polypeptide chain of the multispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (eg, conservative amino acid substitutions, etc.): X₇ , X₉ , X₁₂ , X₁₄ , X₁₅ , X 18 , X₂₀ , X₄₁ , X₄₉ , X₆₄ , X₇₆ ,_{X 78}_, X₈₀ , X₈₁ , X₈₂ , X₈₄ , X₈₅ , X₈₇ , X₈₈ , X₈₉ , X₉₃ , X₉₆ , X₉₇ , X₉₉ , X₁₀₁ and X₁₀₈ .

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分的轻链可变区可包含SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列。In the present application, the light chain variable region of the first targeting portion in the first polypeptide chain of the multispecific antibody may comprise amino acids shown in any one of SEQ ID NO:32 to SEQ ID NO:38 sequence.

例如，所述多特异性抗体第一多肽链中的第一靶向部分的轻链恒定区包含SEQ IDNO:114所示的氨基酸序列。For example, the light chain constant region of the first targeting moiety in the first polypeptide chain of the multispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 114.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR3可包含SEQID NO:76所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR3序列可根据Kabat编码系统定义。In the present application, the HCDR3 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:76. For example, the HCDR3 sequence of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR2可包含SEQID NO:70所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR2序列可根据Kabat编码系统定义。In the present application, the HCDR2 of the second targeting part in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:70. For example, the HCDR2 sequence of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR1可包含SEQID NO:61所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR1序列可根据Kabat编码系统定义。In the present application, the HCDR1 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:61. For example, the HCDR1 sequence of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat coding system.

例如，所述多特异性抗体第二多肽链中的第二靶向部分的HCDR1可包含SEQ IDNO:61所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:70所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:76所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分可包括抗体13H6D3、hu13H6D3、5BM、AH00228、AH00229、AH00230、AH00231、AH00232、AH00233、CBM或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段。For example, the HCDR1 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:61; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:70; And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:76. For example, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM or have the same HCDR3 (e.g., have the same HCDR1-3) antigen-binding fragments.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分可包含重链可变区，所述重链可变区可包含SEQ ID NO:122所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分包含VH，所述VH与SEQ ID NO:122所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₅，X₇，X₁₀，X₁₁，X₁₂，X₁₆，X₂₀，X₃₇，X₃₈，X₄₀，X₄₆，X₄₈，X₆₇，X₆₈，X₆₉，X₇₅，X₈₁，X₈₆，X₉₀，X₁₁₅和X₁₁₆。In the present application, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:122 . For example, the second targeting moiety in the second polypeptide chain of the multispecific antibody comprises VH, compared with the sequence shown in SEQ ID NO: 122, the VH has one or more amino acids selected from the group consisting of Amino acid substitutions (eg, conservative amino acid substitutions, etc.): X₅ , X₇ , X₁₀ , X₁₁ , X₁₂ , X₁₆ , X 20 , X₃₇ , X₃₈ ,_{X 40}_, X₄₆ , X₄₈ , X₆₇ , X₆₈ , X₆₉ , X₇₅ , X₈₁ , X₈₆ , X₉₀ , X₁₁₅ and X₁₁₆ .

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的重链可变区可包含SEQ ID NO:95至SEQ ID NO:104中任一项所示的氨基酸序列。In the present application, the heavy chain variable region of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise amino acids shown in any one of SEQ ID NO:95 to SEQ ID NO:104 sequence.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分可以包含抗体轻链可变区VL中的至少一个CDR，所述VL可以包含SEQ ID NO:123所示的氨基酸序列。In the present application, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise at least one CDR in the VL of the light chain variable region of the antibody, and the VL may comprise SEQ ID NO: 123 amino acid sequence.

例如，所述多特异性抗体第二多肽链中的第二靶向部分中的VL可以包含SEQ IDNO:105至SEQ ID NO:111中任一项所示的氨基酸序列。在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR可以通过任何形式划分，只要VL与SEQ ID NO:105至SEQID NO:111中任一项所示的氨基酸序列相同，以任何形式划分得到的LCDR都可落入本申请的保护范围内。For example, the VL in the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 105 to SEQ ID NO: 111. In this application, the LCDR of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be divided in any form, as long as VL is shown in any one of SEQ ID NO:105 to SEQ ID NO:111 The amino acid sequence is the same, and the LCDR obtained by dividing in any form can fall within the protection scope of the present application.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR3可包含SEQID NO:94所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR3可根据Kabat编号系统定义。In the present application, the LCDR3 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:94. For example, the LCDR3 of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR2可包含SEQID NO:87所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR2可根据Kabat编号系统定义。In the present application, the LCDR2 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:87. For example, LCDR2 of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:82所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的LCDR1可根据Kabat编号系统定义。In the present application, the LCDR1 of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO:82. For example, LCDR1 of the second targeting moiety in the second polypeptide chain of the multispecific antibody can be defined according to the Kabat numbering system.

例如，本申请所述多特异性抗体第二多肽链中的第二靶向部分的LCDR1可包含SEQID NO:82所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:87所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:94所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分可包括抗体13H6D3、hu13H6D3、5BM、AH00228、AH00229、AH00230、AH00231、AH00232、AH00233、CBM或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段。For example, the LCDR1 of the second targeting moiety in the second polypeptide chain of the multispecific antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO:82; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:87 sequence; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:94. For example, the second targeting moiety in the second polypeptide chain of the multispecific antibody can comprise antibody 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or have the same LCDR3 (e.g., Antigen-binding fragments having the same LCDR1-3) as it.

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分可包含轻链可变区VL，所述VL可包含SEQ ID NO:123所示的氨基酸序列。例如，所述多特异性抗体第二多肽链中的第二靶向部分的VL与SEQ ID NO:123所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X₇，X₈，X₁₅，X₂₂，X₄₁，X₄₂，X₄₃，X₄₄，X₄₆，X₆₀，X₇₁，X₇₂，X₇₇，X₇₉，X₈₀，X₈₃，X₈₇和X₁₀₄。In the present application, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise the light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:123. For example, compared with the sequence shown in SEQ ID NO: 123, the VL of the second targeting moiety in the second polypeptide chain of the multispecific antibody has an amino acid substitution at one or more amino acids selected from the following group (e.g., conservative amino acid substitutions, etc.):_X7 ,_X8 ,_X15 ,_X22 ,_X41 ,_X42 ,_X43 , X44_,_X46 ,_X60 ,_X71 ,_X72 ,_X77 ,_X79 , X₈₀ , X₈₃ , X₈₇ and X₁₀₄ .

在本申请中，所述多特异性抗体第二多肽链中的第二靶向部分的轻链可变区可包含SEQ ID NO:105至SEQ ID NO:111中任一项所示的氨基酸序列。In the present application, the light chain variable region of the second targeting portion in the second polypeptide chain of the multispecific antibody may comprise amino acids shown in any one of SEQ ID NO:105 to SEQ ID NO:111 sequence.

在本申请中，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:29至SEQ ID NO:31、SEQ ID NO:32至SEQ ID NO:38中任一项所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ IDNO:95至SEQ ID NO:104、SEQ ID NO:105至SEQ ID NO:111中任一项所示的氨基酸序列。In the present application, the multispecific antibody may comprise a first targeting moiety in the first polypeptide chain of the multispecific antibody and a second targeting moiety in the second polypeptide chain of the multispecific antibody. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise any one of SEQ ID NO: 29 to SEQ ID NO: 31, SEQ ID NO: 32 to SEQ ID NO: 38. The amino acid sequence shown, the second targeting moiety in the second polypeptide chain of the multispecific antibody may comprise any of SEQ ID NO: 95 to SEQ ID NO: 104, SEQ ID NO: 105 to SEQ ID NO: 111 The amino acid sequence shown in item.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:31所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:97所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 31, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:97.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:31所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:98所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 31, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:98.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:31所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:100所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 31, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:100.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:31所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:102所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 31, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:102.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:31所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:103所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 31, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:103.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:36所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:97所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 36, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:97.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:36所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:98所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 36, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:98.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:36所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:100所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 36, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:100.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:36所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:102所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 36, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:102.

例如，所述多特异性抗体可包含多特异性抗体第一多肽链中的第一靶向部分和多特异性抗体第二多肽链中的第二靶向部分。所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:36所示的氨基酸序列，所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:103所示的氨基酸序列。For example, the multispecific antibody can comprise a first targeting moiety in a first polypeptide chain of the multispecific antibody and a second targeting moiety in a second polypeptide chain of the multispecific antibody. The first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 36, and the second targeting moiety in the second polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence shown in SEQ ID NO:103.

例如，所述多特异性抗体第一多肽链中的第一靶向部分可特异性结合PD-1蛋白，且所述多特异性抗体第二多肽链中的第二靶向部分可特异性结合CD28或CD16a蛋白。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：31所示的氨基酸序列，且所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：176或SEQ ID NO：168所示的氨基酸序列。For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody can specifically bind PD-1 protein, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can specifically bind Sexually binds CD28 or CD16a protein. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in SEQ ID NO: 31, and the second targeting moiety in the second polypeptide chain of the multispecific antibody The direction part may comprise the amino acid sequence shown in SEQ ID NO: 176 or SEQ ID NO: 168.

例如，所述多特异性抗体第一多肽链中的第一靶向部分可特异性结合CD28或CD16a蛋白，且所述多特异性抗体第二多肽链中的第二靶向部分可特异性结合PD-1蛋白。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：176或SEQ ID NO：168中任一项所示的氨基酸序列，且所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：31所示的氨基酸序列。For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody can specifically bind CD28 or CD16a protein, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can specifically bind Sexually binds to PD-1 protein. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 176 or SEQ ID NO: 168, and the multispecific antibody The second targeting moiety in the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO:31.

在本申请中，所述多特异性抗体的第一多肽链还可包含第三靶向部分。例如，所述多特异性抗体第一多肽链中的第三靶向部分可与所述多特异性抗体第一多肽链中的第一靶向部分的C端直接或间接地连接。例如，所述多特异性抗体第一多肽链中的第三靶向部分可与所述多特异性抗体第一多肽链中的第一靶向部分的C端通过连接子连接。例如，所述连接子可以是本领域常用的连接子。例如，所述连接子可以是柔性连接子。例如，所述连接子可以是G4S连接子。In the present application, the first polypeptide chain of the multispecific antibody may further comprise a third targeting moiety. For example, the third targeting moiety in the first polypeptide chain of the multispecific antibody can be directly or indirectly linked to the C-terminus of the first targeting moiety in the first polypeptide chain of the multispecific antibody. For example, the third targeting moiety in the first polypeptide chain of the multispecific antibody can be connected to the C-terminus of the first targeting moiety in the first polypeptide chain of the multispecific antibody through a linker. For example, the linker may be a linker commonly used in the art. For example, the linker can be a flexible linker. For example, the linker may be a G4S linker.

在本申请中，所述多特异性抗体第一多肽链中的所述第三靶向部分可特异性结合免疫细胞相关抗原。例如，所述免疫细胞相关抗原可包括PD-1、PD-L1、CD3、CD16a和/或CD28。例如，所述多特异性抗体第一多肽链中的所述第三靶向部分可特异性结合CD3或CD16a蛋白。In the present application, the third targeting moiety in the first polypeptide chain of the multispecific antibody can specifically bind to an immune cell-associated antigen. For example, the immune cell-associated antigens may include PD-1, PD-L1, CD3, CD16a and/or CD28. For example, the third targeting moiety in the first polypeptide chain of the multispecific antibody can specifically bind CD3 or CD16a protein.

在本申请中，所述多特异性抗体第一多肽链中的所述第三靶向部分可特异性结合肿瘤发生相关抗原。例如，所述多特异性抗体第一多肽链中的所述第三靶向部分可特异性结合EGFR和/或cMet。In the present application, the third targeting moiety in the first polypeptide chain of the multispecific antibody can specifically bind to an antigen associated with tumorigenesis. For example, the third targeting moiety in the first polypeptide chain of the multispecific antibody can specifically bind EGFR and/or cMet.

在本申请中，所述多特异性抗体第一多肽链中的所述第三靶向部分可特异性结合肿瘤相关抗原。例如，所述肿瘤相关抗原可包括PD-L1、HER2、CD47、CD19和/或CD20。In the present application, the third targeting moiety in the first polypeptide chain of the multispecific antibody can specifically bind to a tumor-associated antigen. For example, the tumor-associated antigens may include PD-L1, HER2, CD47, CD19 and/or CD20.

在本申请中，所述多特异性抗体的第二多肽链还可包含第四靶向部分。例如，所述多特异性抗体第二多肽链中的第四靶向部分可与所述多特异性抗体第二多肽链中的第二靶向部分的C端直接或间接地连接。例如，所述多特异性抗体第二多肽链中的第四靶向部分可与所述多特异性抗体第二多肽链中的第二靶向部分的C端通过连接子连接。例如，所述连接子可以是本领域常用的连接子。例如，所述连接子可以是柔性连接子。例如，所述连接子可以是G4S连接子。In the present application, the second polypeptide chain of the multispecific antibody may further comprise a fourth targeting moiety. For example, the fourth targeting moiety in the second polypeptide chain of the multispecific antibody can be directly or indirectly linked to the C-terminus of the second targeting moiety in the second polypeptide chain of the multispecific antibody. For example, the fourth targeting moiety in the second polypeptide chain of the multispecific antibody can be linked to the C-terminus of the second targeting moiety in the second polypeptide chain of the multispecific antibody through a linker. For example, the linker may be a linker commonly used in the art. For example, the linker can be a flexible linker. For example, the linker may be a G4S linker.

在本申请中，所述多特异性抗体第二多肽链中的所述第四靶向部分可特异性结合免疫细胞相关抗原。例如，所述免疫细胞相关抗原可包括PD-1、PD-L1、CD3、CD16a和/或CD28。例如，所述多特异性抗体第二多肽链中的所述第四靶向部分可特异性结合CD3或CD16a蛋白。In the present application, the fourth targeting moiety in the second polypeptide chain of the multispecific antibody can specifically bind to an immune cell-associated antigen. For example, the immune cell-associated antigens may include PD-1, PD-L1, CD3, CD16a and/or CD28. For example, the fourth targeting moiety in the second polypeptide chain of the multispecific antibody can specifically bind CD3 or CD16a protein.

在本申请中，所述多特异性抗体第二多肽链中的所述第四靶向部分可特异性结合肿瘤发生相关抗原。例如，所述多特异性抗体第二多肽链中的所述第四靶向部分可特异性结合EGFR和/或cMet。In the present application, the fourth targeting moiety in the second polypeptide chain of the multispecific antibody can specifically bind to an antigen associated with tumorigenesis. For example, the fourth targeting moiety in the second polypeptide chain of the multispecific antibody can specifically bind EGFR and/or cMet.

在本申请中，所述多特异性抗体第二多肽链中的所述第四靶向部分可特异性结合肿瘤相关抗原。例如，所述肿瘤相关抗原可包括PD-L1、HER2、CD47、CD19和/或CD20。In the present application, the fourth targeting moiety in the second polypeptide chain of the multispecific antibody can specifically bind to a tumor-associated antigen. For example, the tumor-associated antigens may include PD-L1, HER2, CD47, CD19 and/or CD20.

在本申请中，所述多特异性抗体可包含两条前述第一多肽链和两条前述第二多肽链。例如，两条前述第一多肽链之间可通过二硫键连接，所述两条前述第一多肽链和两条前述第二多肽链可形成IgG样的抗体。例如，所述多特异性抗体可为同二聚体。In the present application, the multispecific antibody may comprise two aforementioned first polypeptide chains and two aforementioned second polypeptide chains. For example, the two aforementioned first polypeptide chains may be linked by a disulfide bond, and the two aforementioned first polypeptide chains and the two aforementioned second polypeptide chains may form an IgG-like antibody. For example, the multispecific antibody can be a homodimer.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-L1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-L1，所述多特异性抗体第二多肽链中的第四靶向部分可靶向PD-1、CD3或CD16a。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：105-111中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：95-104中任一项所示的氨基酸序列；且所述第二多肽链中的第四靶向部分可包含SEQ ID NO：29-31、168和172中任一项所示的VH的氨基酸序列以及SEQ ID NO：32-38、169和173中任一项所示的VL的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-L1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target Towards PD-L1, the fourth targeting moiety in the second polypeptide chain of the multispecific antibody may target PD-1, CD3 or CD16a. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 105-111; the second polypeptide chain of the multispecific antibody The second targeting moiety in can comprise the amino acid sequence shown in any one of SEQ ID NO:95-104; And the 4th targeting moiety in the second polypeptide chain can comprise SEQ ID NO:29-31 The amino acid sequence of the VH shown in any one of , 168 and 172 and the amino acid sequence of the VL shown in any one of SEQ ID NO: 32-38, 169 and 173. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-1，所述多特异性抗体第二多肽链中的第四靶向部分可靶向PD-L1、CD3或CD16a。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：32-38中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：29-31中任一项所示的氨基酸序列；且所述第二多肽链中的第四靶向部分可包含SEQ ID NO：95-104、168和172中任一项所示的VH的氨基酸序列以及SEQ ID NO：105-111、169和173中任一项所示的VL的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target Towards PD-1, the fourth targeting moiety in the second polypeptide chain of the multispecific antibody may target PD-L1, CD3 or CD16a. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 32-38; the second polypeptide chain of the multispecific antibody The second targeting moiety in can comprise the amino acid sequence shown in any one of SEQ ID NO:29-31; And the 4th targeting moiety in the second polypeptide chain can comprise SEQ ID NO:95-104 The amino acid sequence of the VH shown in any one of , 168 and 172 and the amino acid sequence of the VL shown in any one of SEQ ID NO: 105-111, 169 and 173. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-L1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-1，所述多特异性抗体第一多肽链中的第三靶向部分可靶向CD3或CD16a。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:95-104和SEQ ID NO:105-111中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:29-31和SEQ ID NO:32-38中任一项所示的氨基酸序列；且所述第一多肽链中的第三靶向部分可包含SEQ ID NO：168和172中任一项所示的VH的氨基酸序列以及SEQ ID NO：169和173中任一项所示的VL的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-L1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target For PD-1, the third targeting moiety in the first polypeptide chain of the multispecific antibody may target CD3 or CD16a. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:95-104 and SEQ ID NO:105-111; The second targeting moiety in the second polypeptide chain of the specific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:29-31 and SEQ ID NO:32-38; and the first polypeptide chain The third targeting moiety in can comprise the amino acid sequence of the VH shown in any one of SEQ ID NOs: 168 and 172 and the amino acid sequence of the VL shown in any one of SEQ ID NOs: 169 and 173. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分靶向PD-1，所述多特异性抗体第二多肽链中的第二靶向部分靶向PD-L1，所述多特异性抗体第一多肽链中的第三靶向部分靶向CD3或CD16a。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:29-31和SEQ ID NO:32-38中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:95-104和SEQ ID NO:105-111中任一项所示的氨基酸序列；且所述第一多肽链中的第三靶向部分可包含SEQ ID NO：168和172中任一项所示的VH的氨基酸序列以及SEQ ID NO：169和173中任一项所示的VL的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody targets PD-1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody targets PD-1 -L1, the third targeting moiety in the first polypeptide chain of the multispecific antibody targets CD3 or CD16a. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:29-31 and SEQ ID NO:32-38; The second targeting moiety in the second polypeptide chain of the specific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:95-104 and SEQ ID NO:105-111; and the first polypeptide chain The third targeting moiety in can comprise the amino acid sequence of the VH shown in any one of SEQ ID NOs: 168 and 172 and the amino acid sequence of the VL shown in any one of SEQ ID NOs: 169 and 173. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向CD28或CD16a，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-1，所述多特异性抗体第二多肽链中的第四靶向部分可靶向PD-L1、HER2、CD47、CD19和/或CD20。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：168或SEQ ID NO：176中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:29-31中任一项所示的氨基酸序列；且所述第二多肽链中的第四靶向部分可包含SEQ ID NO：95-104、170、174和178中任一项所示的VH的氨基酸序列以及SEQ ID NO：105-111、171、175和179中任一项所示的VL的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target CD28 or CD16a, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target Towards PD-1, the fourth targeting moiety in the second polypeptide chain of the multispecific antibody may target PD-L1, HER2, CD47, CD19 and/or CD20. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 168 or SEQ ID NO: 176; The second targeting moiety in the two polypeptide chains may comprise the amino acid sequence shown in any one of SEQ ID NO:29-31; and the fourth targeting moiety in the second polypeptide chain may comprise SEQ ID NO The amino acid sequence of the VH shown in any one of: 95-104, 170, 174 and 178 and the amino acid sequence of the VL shown in any one of SEQ ID NO: 105-111, 171, 175 and 179. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分靶向PD-1，所述多特异性抗体第二多肽链中的第二靶向部分靶向CD28或CD16a，所述多特异性抗体第二多肽链中的第四靶向部分靶向PD-L1、HER2、CD47、CD19和/或CD20。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:29-31中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：168或SEQ ID NO：176中任一项所示的氨基酸序列；且所述第二多肽链中的第四靶向部分可包含SEQ ID NO：95-104、170、174和178中任一项所示的VH的氨基酸序列以及SEQ ID NO：105-111、171、175和179中任一项所示的VL的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody targets PD-1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody targets CD28 or CD16a, the fourth targeting moiety in the second polypeptide chain of the multispecific antibody targets PD-L1, HER2, CD47, CD19 and/or CD20. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 29-31; the second polypeptide chain of the multispecific antibody The second targeting moiety in may comprise the amino acid sequence shown in any one of SEQ ID NO: 168 or SEQ ID NO: 176; and the fourth targeting moiety in the second polypeptide chain may comprise SEQ ID NO The amino acid sequence of the VH shown in any one of: 95-104, 170, 174 and 178 and the amino acid sequence of the VL shown in any one of SEQ ID NO: 105-111, 171, 175 and 179. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向CD28或CD16a，所述多特异性抗体第二多肽链中的第二靶向部分靶向PD-1，所述多特异性抗体第一多肽链中的第三靶向部分靶向EGFR或cMET，所述多特异性抗体第二多肽链中的第四靶向部分靶向PD-L1、HER2、CD47、CD19和/或CD20。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：168或SEQ ID NO：176中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO:29-31中任一项所示的氨基酸序列；且所述第二多肽链中的第四靶向部分可包含SEQ ID NO：95-104、170、174和178中任一项所示的VH的氨基酸序列以及SEQ ID NO：105-111、171、175和179中任一项所示的VL的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target CD28 or CD16a, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target PD-1, the third targeting moiety in the first polypeptide chain of the multispecific antibody targets EGFR or cMET, and the fourth targeting moiety in the second polypeptide chain of the multispecific antibody targets PD- L1, HER2, CD47, CD19 and/or CD20. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 168 or SEQ ID NO: 176; The second targeting moiety in the two polypeptide chains may comprise the amino acid sequence shown in any one of SEQ ID NO:29-31; and the fourth targeting moiety in the second polypeptide chain may comprise SEQ ID NO The amino acid sequence of the VH shown in any one of: 95-104, 170, 174 and 178 and the amino acid sequence of the VL shown in any one of SEQ ID NO: 105-111, 171, 175 and 179. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向CD28或CD16a，所述多特异性抗体第一多肽链中的第三靶向部分可靶向PD-L1、HER2、CD47、CD19和/或CD20，所述多特异性抗体第二多肽链中的第四靶向部分可靶向EGFR或cMET。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO:29-31中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：168或SEQ ID NO：176中任一项所示的氨基酸序列；且所述多特异性抗体第一多肽链中的第三靶向部分可包含SEQ ID NO：95-104、170、174和178中任一项所示的VH的氨基酸序列以及SEQ ID NO：105-111、171、175和179中任一项所示的VL的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target To CD28 or CD16a, the third targeting moiety in the first polypeptide chain of the multispecific antibody may target PD-L1, HER2, CD47, CD19 and/or CD20, and the second polypeptide of the multispecific antibody A fourth targeting moiety in the chain can target EGFR or cMET. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 29-31; the second polypeptide chain of the multispecific antibody The second targeting moiety in may comprise the amino acid sequence shown in any one of SEQ ID NO: 168 or SEQ ID NO: 176; and the third targeting moiety in the first polypeptide chain of the multispecific antibody may be Comprising the amino acid sequence of the VH shown in any one of SEQ ID NO: 95-104, 170, 174 and 178 and the amino acid sequence of the VL shown in any one of SEQ ID NO: 105-111, 171, 175 and 179 . For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-1，且所述多特异性抗体的第二多肽链的C端可包含IL-2变体、IFNα-2b或IL-15。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：32-38中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：29-31中任一项所示的氨基酸序列；且所述多特异性抗体第二多肽链中的细胞因子可包含SEQ ID NO:180或SEQ ID NO:181所示的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target PD-1, and the C-terminus of the second polypeptide chain of the multispecific antibody may comprise IL-2 variants, IFNα-2b or IL-15. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 32-38; the second polypeptide chain of the multispecific antibody The second targeting moiety in may comprise the amino acid sequence shown in any one of SEQ ID NO: 29-31; and the cytokine in the second polypeptide chain of the multispecific antibody may comprise SEQ ID NO: 180 or Amino acid sequence shown in SEQ ID NO: 181. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-L1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-L1，且所述多特异性抗体的第二多肽链的C端可包含IL-2变体、IFNα-2b或IL-15。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：105-111中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：95-104中任一项所示的氨基酸序列；且所述多特异性抗体第二多肽链中的细胞因子可包含SEQ ID NO:180或SEQ ID NO:181所示的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-L1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target Towards PD-L1, and the C-terminus of the second polypeptide chain of the multispecific antibody may comprise IL-2 variants, IFNα-2b or IL-15. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 105-111; the second polypeptide chain of the multispecific antibody The second targeting moiety in may comprise the amino acid sequence shown in any one of SEQ ID NO: 95-104; and the cytokine in the second polypeptide chain of the multispecific antibody may comprise SEQ ID NO: 180 or Amino acid sequence shown in SEQ ID NO: 181. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-L1，所述多特异性抗体的第一多肽链的C端可包含IL-2、IFNα-2b或IL-15，所述多特异性抗体第二多肽链中的第四靶向部分可包含CD3或CD16a。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ IDNO：29-31中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：95-104中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的细胞因子可包含SEQ ID NO:180或SEQ ID NO:181所示的氨基酸序列；且所述第二多肽链中的第四靶向部分可包含SEQ ID NO：168和172中任一项所示的VH的氨基酸序列以及SEQ IDNO：169和173中任一项所示的VL的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target Towards PD-L1, the C-terminus of the first polypeptide chain of the multispecific antibody may comprise IL-2, IFNα-2b or IL-15, the fourth target in the second polypeptide chain of the multispecific antibody The targeting moiety may comprise CD3 or CD16a. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 29-31; The second targeting part of the can comprise the amino acid sequence shown in any one of SEQ ID NO:95-104; The cytokine in the second polypeptide chain of the multispecific antibody can comprise SEQ ID NO:180 or SEQ ID The amino acid sequence shown in NO:181; And the fourth targeting part in the second polypeptide chain can comprise the amino acid sequence of VH shown in any one of SEQ ID NO:168 and 172 and SEQ IDNO:169 and The amino acid sequence of the VL shown in any one of 173. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-L1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-1，所述多特异性抗体的第一多肽链的C端可包含IL-2、IFNα-2b或IL-15，第四靶向部分可包含CD3或CD16a。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：95-104中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：29-31中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的细胞因子可包含SEQ ID NO:180或SEQ IDNO:181所示的氨基酸序列；且所述第二多肽链中的第四靶向部分可包含SEQ ID NO：168和172中任一项所示的VH的氨基酸序列以及SEQ ID NO：169和173中任一项所示的VL的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-L1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target For PD-1, the C-terminus of the first polypeptide chain of the multispecific antibody may contain IL-2, IFNα-2b or IL-15, and the fourth targeting moiety may contain CD3 or CD16a. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 95-104; the second polypeptide chain of the multispecific antibody The second targeting moiety in can comprise the amino acid sequence shown in any one of SEQ ID NO:29-31; The cytokine in the second polypeptide chain of described multispecific antibody can comprise SEQ ID NO:180 or SEQ ID NO:180 or SEQ The amino acid sequence shown in IDNO:181; And the fourth targeting part in the second polypeptide chain can comprise the amino acid sequence of VH shown in any one of SEQ ID NO:168 and 172 and SEQ ID NO:169 and the amino acid sequence of the VL shown in any one of 173. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-L1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-1，所述多特异性抗体的第一多肽链的C端可包含IL-2、IFNα-2b或IL-15。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：95-104中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：29-31中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的细胞因子可包含SEQ ID NO:180或SEQ ID NO:181所示的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-L1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target For PD-1, the C-terminus of the first polypeptide chain of the multispecific antibody may contain IL-2, IFNα-2b or IL-15. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 95-104; the second polypeptide chain of the multispecific antibody The second targeting moiety in can comprise the amino acid sequence shown in any one of SEQ ID NO:29-31; The cytokine in the second polypeptide chain of described multispecific antibody can comprise SEQ ID NO:180 or SEQ ID NO:180 or SEQ Amino acid sequence shown in ID NO:181. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-L1，所述多特异性抗体的第一多肽链的C端可包含IL-2、IFNα-2b或IL-15。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：29-31中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：95-104中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的细胞因子可包含SEQ ID NO:180或SEQ ID NO:181所示的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target For PD-L1, the C-terminus of the first polypeptide chain of the multispecific antibody may contain IL-2, IFNα-2b or IL-15. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 29-31; the second polypeptide chain of the multispecific antibody The second targeting moiety in can comprise the amino acid sequence shown in any one of SEQ ID NO:95-104; The cytokine in the second polypeptide chain of described multispecific antibody can comprise SEQ ID NO:180 or SEQ ID NO:180 or SEQ ID NO:104; Amino acid sequence shown in ID NO:181. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-1，所述多特异性抗体的第一多肽链的C端可包含IL-2、IFNα-2b或IL-15，且多特异性抗体第二多肽链中的第四靶向部分可靶向PD-L1。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：32-38中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ IDNO：29-31中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的细胞因子可包含SEQ ID NO:180或SEQ ID NO:181所示的氨基酸序列；且所述第二多肽链中的第四靶向部分可包含SEQ ID NO：95-104中任一项所示的VH的氨基酸序列以及SEQ ID NO：105-111中任一项所示的VL的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target Towards PD-1, the C-terminus of the first polypeptide chain of the multispecific antibody may contain IL-2, IFNα-2b or IL-15, and the fourth target in the second polypeptide chain of the multispecific antibody Some can target PD-L1. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 32-38; the second polypeptide chain of the multispecific antibody The second targeting moiety in can comprise the amino acid sequence shown in any one of SEQ ID NO:29-31; The cytokine in the second polypeptide chain of described multispecific antibody can comprise SEQ ID NO:180 or SEQ ID The amino acid sequence shown in NO:181; and the fourth targeting part in the second polypeptide chain can comprise the amino acid sequence of VH shown in any one of SEQ ID NO:95-104 and SEQ ID NO:105 - the amino acid sequence of the VL shown in any one of 111. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-L1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-L1，所述多特异性抗体的第一多肽链的C端可包含IL-2、IFNα-2b或IL-15，且多特异性抗体第二多肽链中的第四靶向部分靶向PD-1。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：105-111中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ IDNO：95-104中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的细胞因子可包含SEQ ID NO:180或SEQ ID NO:181所示的氨基酸序列；且所述第二多肽链中的第四靶向部分可包含SEQ ID NO：29-31中任一项所示的VH的氨基酸序列以及SEQ ID NO：32-38中任一项所示的VL的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-L1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target Towards PD-L1, the C-terminus of the first polypeptide chain of the multispecific antibody may contain IL-2, IFNα-2b or IL-15, and the fourth target in the second polypeptide chain of the multispecific antibody Partially targets PD-1. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 105-111; the second polypeptide chain of the multispecific antibody The second targeting moiety in can comprise the amino acid sequence shown in any one of SEQ ID NO:95-104; The cytokine in the second polypeptide chain of the multispecific antibody can comprise SEQ ID NO:180 or SEQ ID The amino acid sequence shown in NO:181; and the fourth targeting part in the second polypeptide chain can comprise the amino acid sequence of VH shown in any one of SEQ ID NO:29-31 and SEQ ID NO:32 - the amino acid sequence of the VL shown in any one of 38. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-L1，所述多特异性抗体第一多肽链中的第三靶向部分可靶向CD3或CD16a，且所述多特异性抗体的第二多肽链的C端可包含IL-2、IFNα-2b或IL-15。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ IDNO：29-31中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：95-104中任一项所示的氨基酸序列；所述第一多肽链中的第三靶向部分可包含SEQ ID NO：168和172中任一项所示的VH的氨基酸序列以及SEQ ID NO：169和173中任一项所示的VL的氨基酸序列；且所述多特异性抗体第二多肽链中的细胞因子可包含SEQ IDNO:180或SEQ ID NO:181所示的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target To PD-L1, the third targeting moiety in the first polypeptide chain of the multispecific antibody may target CD3 or CD16a, and the C-terminus of the second polypeptide chain of the multispecific antibody may contain IL- 2. IFNα-2b or IL-15. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 29-31; The second targeting moiety can comprise the amino acid sequence shown in any one of SEQ ID NO:95-104; The third targeting moiety in the first polypeptide chain can comprise any of SEQ ID NO:168 and 172 The amino acid sequence of VH shown in one and the amino acid sequence of VL shown in any one of SEQ ID NO: 169 and 173; and the cytokine in the second polypeptide chain of the multispecific antibody may comprise SEQ ID NO: 180 or the amino acid sequence shown in SEQ ID NO: 181. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

在本申请中，所述多特异性抗体第一多肽链中的第一靶向部分可靶向PD-L1，所述多特异性抗体第二多肽链中的第二靶向部分可靶向PD-1，所述多特异性抗体第一多肽链中的第三靶向部分靶向CD3或CD16a，且所述多特异性抗体的第二多肽链的C端包含IL-2、IFNα-2b或IL-15。例如，所述多特异性抗体第一多肽链中的第一靶向部分可包含SEQ ID NO：95-104中任一项所示的氨基酸序列；所述多特异性抗体第二多肽链中的第二靶向部分可包含SEQ ID NO：29-31中任一项所示的氨基酸序列；所述第一多肽链中的第三靶向部分可包含SEQ ID NO：168和172中任一项所示的VH的氨基酸序列以及SEQ ID NO：169和173中任一项所示的VL的氨基酸序列；且所述多特异性抗体第二多肽链中的细胞因子可包含SEQ IDNO:180或SEQ ID NO:181所示的氨基酸序列。例如，所述多特异性抗体可包含两条第一多肽链和两条第二多肽链。例如，两条第一多肽链之间可通过二硫键连接，所述两条第一多肽链和两条第二多肽链可形成IgG样的抗体。In the present application, the first targeting moiety in the first polypeptide chain of the multispecific antibody can target PD-L1, and the second targeting moiety in the second polypeptide chain of the multispecific antibody can target To PD-1, the third targeting moiety in the first polypeptide chain of the multispecific antibody targets CD3 or CD16a, and the C-terminus of the second polypeptide chain of the multispecific antibody comprises IL-2, IFNα-2b or IL-15. For example, the first targeting moiety in the first polypeptide chain of the multispecific antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 95-104; the second polypeptide chain of the multispecific antibody The second targeting moiety in can comprise the amino acid sequence shown in any one of SEQ ID NO:29-31; The third targeting moiety in the first polypeptide chain can comprise SEQ ID NO:168 and 172 The amino acid sequence of VH shown in any one and the amino acid sequence of VL shown in any one of SEQ ID NO: 169 and 173; and the cytokine in the second polypeptide chain of the multispecific antibody may comprise SEQ ID NO : 180 or the amino acid sequence shown in SEQ ID NO: 181. For example, the multispecific antibody can comprise two first polypeptide chains and two second polypeptide chains. For example, two first polypeptide chains can be connected by a disulfide bond, and the two first polypeptide chains and two second polypeptide chains can form an IgG-like antibody.

多肽和免疫缀合物Peptides and Immunoconjugates

另一方面，本申请提供了一种或多种多肽，其可包含本申请的分离的抗原结合蛋白。例如，所述多肽可包括融合蛋白。例如，所述多肽可包括双特异性抗体。In another aspect, the application provides one or more polypeptides, which may comprise an isolated antigen binding protein of the application. For example, the polypeptide can include a fusion protein. For example, the polypeptide can include a bispecific antibody.

另一方面，本申请提供了一种或多种免疫缀合物，所述免疫缀合物可包含本申请的分离的抗原结合蛋白。在某些实施方式中，所述免疫缀合物还可包含药学上可接受的治疗剂、标记物和/或检测剂。In another aspect, the application provides one or more immunoconjugates, which may comprise an isolated antigen binding protein of the application. In certain embodiments, the immunoconjugate may further comprise a pharmaceutically acceptable therapeutic agent, label and/or detection agent.

核酸、载体、细胞和药物组合物Nucleic acids, vectors, cells and pharmaceutical compositions

另一方面，本申请还提供了分离的一种或多种核酸分子，所述一种或多种核酸分子可编码本申请所述分离的抗原结合蛋白。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述抗原结合蛋白，也可以编码其中的一部分(例如，HCDR1-3、重链可变区中的一种或多种)。In another aspect, the present application also provides one or more isolated nucleic acid molecules that encode the isolated antigen-binding protein described herein. For example, each of the one or more nucleic acid molecules may encode the entirety of the antigen binding protein, or may encode a portion thereof (e.g., HCDR1-3, one of the heavy chain variable regions, or variety).

例如，当核酸分子分别编码所述抗原结合蛋白的一部分时，核酸分子编码的产物合在一起可以形成有功能的(例如，可结合PD-L1和PD-1)的本申请的分离的抗原结合蛋白。For example, when the nucleic acid molecules respectively encode a part of the antigen binding protein, the products encoded by the nucleic acid molecules together can form a functional (for example, binding to PD-L1 and PD-1) isolated antigen-binding protein of the present application. protein.

本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。例如，所述分离的核酸可以是通过重组DNA技术制备的核酸分子。The nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified (iv) synthetic, for example by chemical synthesis. For example, the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA techniques.

在本申请中，可以通过本领域已知的多种方法来制备编码所述分离的抗原结合蛋白的核酸，这些方法包括但不限于，采用逆转录PCR和PCR获得本申请所述分离的抗原结合蛋白的核酸分子。In the present application, the nucleic acid encoding the isolated antigen-binding protein can be prepared by various methods known in the art, including but not limited to, using reverse transcription PCR and PCR to obtain the isolated antigen-binding protein described in the application. protein nucleic acid molecule.

另一方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动子可包括，例如用于SP6、T3和T7聚合酶的启动子、人U6RNA启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。In another aspect, the present application provides one or more vectors comprising one or more nucleic acid molecules described herein. Each vector may contain one or more such nucleic acid molecules. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may also contain expression control elements that permit proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like. In certain embodiments, the expression control sequences are regulatable elements. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA box, plus Cap sequence, CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence may comprise a promoter region which may comprise a promoter sequence for transcriptional control of the functionally linked nucleic acid. The expression control sequences may also include enhancer sequences or upstream activator sequences. In the present application, suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerases, human U6 RNA promoters, CMV promoters, and artificial hybrid promoters thereof (such as CMV), wherein the promoter's Portions may be fused to portions of gene promoters of other cellular proteins (eg, human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not contain additional introns. One or more nucleic acid molecules described herein can be operably linked to the expression control element.

所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体可为表达载体。例如，所述载体可为病毒载体。可以将病毒载体直接给予至患者(体内)或可以通过间接的形式，例如，在体外使用病毒处理细胞，然后将处理过的细胞给予至患者(离体)。病毒载体技术在本领域中是公知的，并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。常规的基于病毒的系统可以包括用于基因转移的逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关病毒载体以及单纯疱疹病毒载体。在某些情形中，可以用逆转录病毒、慢病毒和腺相关病毒的方法将基因转移整合进宿主基因组中，使插入的基因长期表达。慢病毒载体是能够转导或感染非分裂细胞并典型地产生较高病毒效价的逆转录病毒载体。慢病毒载体可包含长末端重复序列5’LTR和截短的3’LTR、RRE、rev应答元件(cPPT)、中央终止序列(CTS)和/或翻译后调控元件(WPRE)。本申请所述的载体可以被引入细胞。Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering. For example, the vector may be an expression vector. For example, the vector can be a viral vector. Viral vectors may be administered directly to the patient (in vivo) or may be indirect, for example, in vitro by treating cells with virus and then administering the treated cells to the patient (ex vivo). Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology. Conventional virus-based systems can include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex virus vectors for gene transfer. In some cases, retroviral, lentiviral, and adeno-associated viral methods can be used to transfer genes into the host genome, allowing long-term expression of the inserted gene. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing higher viral titers. Lentiviral vectors may contain long terminal repeat 5'LTR and truncated 3'LTR, RRE, rev response element (cPPT), central termination sequence (CTS) and/or post-translational regulatory element (WPRE). The vectors described herein can be introduced into cells.

另一方面，本申请提供了一种细胞。所述细胞可包含本申请所述的分离的抗原结合蛋白、所述的多肽、所述的免疫缀合物、一种或多种核酸分子和/或本申请所述的一种或多种载体。例如，每种或每个细胞可包含一个或一种本申请所述的核酸分子或载体。例如，每种或每个细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如原核细胞(例如，细菌细胞)、CHO细胞、NS/0细胞、HEK293T细胞、293F细胞或HEK293A细胞，或者其他真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。例如，所述细胞可以包括酵母细胞。例如，所述细胞可以包括大肠杆菌细胞。例如，所述细胞可以包括哺乳动物细胞。例如，所述细胞可以包括免疫细胞。In another aspect, the present application provides a cell. The cells may comprise the isolated antigen binding protein described herein, the polypeptide, the immunoconjugate, one or more nucleic acid molecules and/or one or more carriers described herein . For example, each or each cell may comprise one or more of the nucleic acid molecules or vectors described herein. For example, each or each cell may contain multiple (eg, 2 or more) or multiple (eg, 2 or more) nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into said host cells, such as prokaryotic cells (e.g., bacterial cells), CHO cells, NS/0 cells, HEK293T cells, 293F cells or HEK293A cells, or other eukaryotic cells, Such as cells from plants, fungal or yeast cells, etc. The vectors described in this application can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection and the like. For example, the cells can include yeast cells. For example, the cells may include E. coli cells. For example, the cells can include mammalian cells. For example, the cells can include immune cells.

所述细胞可以包括免疫细胞。在某些情形中，所述细胞可以包括免疫细胞。例如，所述细胞可包括T细胞、B细胞、天然杀伤(NK)细胞、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。The cells may include immune cells. In some instances, the cells may include immune cells. For example, the cells may include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells cell.

另一方面，本申请提供了一种药物组合物。所述药物组合物可包含本申请所述的分离的抗原结合蛋白、所述的多肽、所述的免疫缀合物、所述分离的核酸分子、所述的载体、所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。在本申请中，所述药学上可接受的佐剂可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂。除非与本申请所述的细胞不相容，否则任何常规介质或试剂均可以考虑用于本申请的药物组合物中。在本申请中，所述药学上可接受的赋形剂可以包括在药物制剂中除主药以外的附加物，也可称为辅料。例如，所述赋形剂可以包括片剂中的粘合剂、填充剂、崩解剂、润滑剂。例如，所述赋形剂可以包括中药丸剂中的酒、醋、药汁等。例如，所述赋形剂可以包括半固体制剂软膏剂、霜剂中的基质部分。例如，所述赋形剂可以包括液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂。In another aspect, the present application provides a pharmaceutical composition. The pharmaceutical composition may comprise the isolated antigen binding protein described herein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and/or Or pharmaceutically acceptable adjuvants and/or excipients. In this application, the pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and /or nonionic surfactants. Unless incompatible with the cells described herein, any conventional media or reagents are contemplated for use in the pharmaceutical compositions of the present application. In the present application, the pharmaceutically acceptable excipients may include additives other than the main drug in the pharmaceutical preparation, which may also be referred to as auxiliary materials. For example, the excipients may include binders, fillers, disintegrants, lubricants in tablets. For example, the excipients may include wine, vinegar, medicinal juice, etc. in traditional Chinese medicine pills. For example, the excipient may comprise the base part of a semi-solid formulation ointment, cream. For example, the excipients may include preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, colorants in liquid formulations.

用途和方法Uses and methods

另一方面，本申请提供了一种调节免疫应答的方法，其包括向有需要的受试者施用有效量的所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述分离的核酸分子，所述的载体，所述的细胞和/或所述的药物组合物，和/或药学上可接受的治疗剂。In another aspect, the present application provides a method for modulating an immune response, comprising administering to a subject in need an effective amount of the isolated antigen-binding protein, the polypeptide, the immunoconjugate, Said isolated nucleic acid molecule, said carrier, said cell and/or said pharmaceutical composition, and/or a pharmaceutically acceptable therapeutic agent.

在本申请中，所述调节免疫应答的方法可包括体外方法，离体方法，非诊断或非治疗目的的方法。In this application, the method for modulating immune response may include in vitro method, ex vivo method, non-diagnostic or non-therapeutic method.

另一方面，本申请提供了一种调节免疫应答的方法，其包括向有需要的受试者施用有效量的所述的药物组合，和/或药学上可接受的治疗剂。On the other hand, the present application provides a method for regulating immune response, which comprises administering an effective amount of the drug combination, and/or a pharmaceutically acceptable therapeutic agent to a subject in need.

另一方面，本申请提供了分离的抗原结合蛋白、所述的多肽、所述的免疫缀合物、所述分离的核酸分子、所述的载体、所述的药物组合物用于预防、缓解和/或治疗疾病或病症。In another aspect, the application provides the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, and the pharmaceutical composition for preventing, alleviating and/or treat a disease or condition.

在本申请中，所述疾病或病症可包括肿瘤。例如，所述肿瘤可包括非实体瘤。例如，所述肿瘤可包括与PD-L1的表达相关的肿瘤。术语“与PD-L1的表达相关的肿瘤”通常是指PD-L1表达改变导致疾病进展或逃避免疫监视而形成的肿瘤。例如，所述“与PD-L1的表达相关的肿瘤”可以是PD-L1表达量上调导致疾病进展或逃避免疫监视而形成的肿瘤。所述与PD-L1的蛋白表达相关的肿瘤可以是PD-L1阳性的肿瘤。在PD-L1阳性的肿瘤中，与正常细胞相比，肿瘤细胞表面或肿瘤微环境中的PD-L1的蛋白表达量高约1％，5％，10％，15％，20％，25％，30％，35％，40％，50％，60％，70％，80％或更高。In the present application, the disease or condition may include tumors. For example, the tumor can include a non-solid tumor. For example, the tumor can include a tumor associated with expression of PD-L1. The term "tumor associated with the expression of PD-L1" generally refers to a tumor formed due to changes in the expression of PD-L1 leading to disease progression or escape from immune surveillance. For example, the "tumor associated with the expression of PD-L1" may be a tumor formed by the upregulation of PD-L1 expression leading to disease progression or escape from immune surveillance. The tumors associated with the protein expression of PD-L1 may be PD-L1 positive tumors. In PD-L1-positive tumors, compared with normal cells, the protein expression of PD-L1 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25% higher , 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher.

另一方面，本申请提供了一种所述的分离的抗原结合蛋白、所述的多肽、所述的免疫缀合物、所述的分离的核酸分子、所述的载体，所述的细胞和/或所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗疾病或病症。In another aspect, the application provides a kind of said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said carrier, said cell and /or the use of the pharmaceutical composition in the preparation of medicaments for preventing, alleviating and/or treating diseases or conditions.

在本申请中，所述疾病或病症可包括肿瘤。例如，所述肿瘤可包括实体瘤。例如，所述肿瘤可包括非实体瘤。例如，所述肿瘤可包括与PD-L1的表达相关的肿瘤。术语“与PD-L1的表达相关的肿瘤”通常是指PD-L1表达改变导致疾病进展或逃避免疫监视而形成的肿瘤。例如，所述“与PD-L1的表达相关的肿瘤”可以是PD-L1表达量上调导致疾病进展或逃避免疫监视而形成的肿瘤。所述与PD-L1的蛋白表达相关的肿瘤可以是PD-L1阳性的肿瘤。在PD-L1阳性的肿瘤中，与正常细胞相比，肿瘤细胞表面或肿瘤微环境中的PD-L1的蛋白表达量高约1％，5％，10％，15％，20％，25％，30％，35％，40％，50％，60％，70％，80％或更高。In the present application, the disease or condition may include tumors. For example, the tumor can include a solid tumor. For example, the tumor can include a non-solid tumor. For example, the tumor can include a tumor associated with expression of PD-L1. The term "tumor associated with the expression of PD-L1" generally refers to a tumor formed due to changes in the expression of PD-L1 leading to disease progression or escape from immune surveillance. For example, the "tumor associated with the expression of PD-L1" may be a tumor formed by the upregulation of PD-L1 expression leading to disease progression or escape from immune surveillance. The tumors associated with the protein expression of PD-L1 may be PD-L1 positive tumors. In PD-L1-positive tumors, compared with normal cells, the protein expression of PD-L1 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25% higher , 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher.

另一方面，本申请提供了一种预防和/或治疗疾病或病症的方法，其包括向有需要的受试者施用所述的分离的抗原结合蛋白、所述的分离的核酸分子、所述的载体，所述的细胞、所述的药物组合物。In another aspect, the present application provides a method for preventing and/or treating a disease or disorder, comprising administering the isolated antigen-binding protein, the isolated nucleic acid molecule, the The carrier, the cell, the pharmaceutical composition.

在本申请中，所述疾病或病症可包括肿瘤。例如，所述肿瘤可包括非实体瘤。例如，所述肿瘤可包括与PD-L1的表达相关的肿瘤。术语“与PD-L1的表达相关的肿瘤”通常是指PD-L1表达改变导致疾病进展或逃避免疫监视而形成的肿瘤。例如，所述“与PD-L1的表达相关的肿瘤”可以是PD-L1表达量上调导致疾病进展或逃避免疫监视而形成的肿瘤。所述与PD-L1的蛋白表达相关的肿瘤可以是PD-L1阳性的肿瘤。在PD-L1阳性的肿瘤中，与正常细胞相比，肿瘤细胞表面或肿瘤微环境中的PD-L1的蛋白表达量高约1％，5％，10％，15％，20％，25％，30％，35％，40％，50％，60％，70％，80％或更高。本申请所述药物组合物及方法可与其他类型的癌症疗法结合使用，诸如化学疗法、手术、放射、基因疗法等。本申请中所描述的药物组合物及方法可用于其他依赖于免疫反应的疾病病状，诸如炎症、免疫疾病及感染性疾病。In the present application, the disease or condition may include tumors. For example, the tumor can include a non-solid tumor. For example, the tumor can include a tumor associated with expression of PD-L1. The term "tumor associated with the expression of PD-L1" generally refers to a tumor formed due to changes in the expression of PD-L1 leading to disease progression or escape from immune surveillance. For example, the "tumor associated with the expression of PD-L1" may be a tumor formed by the upregulation of PD-L1 expression leading to disease progression or escape from immune surveillance. The tumors associated with the protein expression of PD-L1 may be PD-L1 positive tumors. In PD-L1-positive tumors, compared with normal cells, the protein expression of PD-L1 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25% higher , 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher. The pharmaceutical compositions and methods described herein can be used in conjunction with other types of cancer therapy, such as chemotherapy, surgery, radiation, gene therapy, and the like. The pharmaceutical compositions and methods described in this application can be used in other disease conditions that depend on immune responses, such as inflammation, immune diseases and infectious diseases.

在本申请中，所述受试者可以包括人或非人动物。例如，所述非人动物可以选自下组：猴、鸡、鹅、猫、狗、小鼠和大鼠。此外，非人动物也可以包括任何除人以外的动物物种，例如家畜动物，或啮齿类动物，或灵长类动物，或家养动物，或家禽动物。所述人可以是高加索人、非洲人、亚洲人、闪族人，或其他种族，或各种种族的杂合体。又例如，所述人可以是老年、成年、青少年、儿童或者婴儿。In the present application, the subject may include humans or non-human animals. For example, the non-human animal can be selected from the group consisting of monkeys, chickens, geese, cats, dogs, mice and rats. In addition, non-human animals may also include any animal species other than humans, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals. The human can be Caucasian, African, Asian, Semitic, or other ethnicity, or a hybrid of various ethnicities. As another example, the human can be elderly, adult, adolescent, child or infant.

可以根据在实验动物中的有效量推测在人类中的有效量。例如，Freireich等人描述了动物和人的剂量的相互关系(基于每平方米身体表面的毫克数)(Freireich et al.，Cancer Chemother.Rep.50，219(1966))。身体表面积可以从患者的身高和体重近似确定。参见例如Scientific Tables，Geigy Pharmaceuticals，Ardsley，N.Y.，537(1970)。The effective amount in humans can be inferred from the effective amount in experimental animals. For example, Freireich et al. describe the correlation of doses in animals and humans (based on milligrams per square meter of body surface) (Freireich et al., Cancer Chemother. Rep. 50, 219 (1966)). Body surface area can be approximately determined from the patient's height and weight. See, eg, Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970).

不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的抗原结合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。Without intending to be limited by any theory, the following examples are only for explaining the antigen-binding protein of the present application, the preparation method and application, etc., and are not intended to limit the scope of the invention of the present application.

实施例Example

实施例1双特异性抗体载体的构建Example 1 Construction of bispecific antibody carrier

1.1 5BM和19D4-25-3C11单抗的获得1.1 Acquisition of 5BM and 19D4-25-3C11 monoclonal antibodies

采用杂交瘤技术获得抗原结合蛋白13H6D3和19D4F1。之后通过抗体的人源化设计、亲和力成熟获得13H6D3的人源化抗体hu13H6D3、5BM、AH00228、AH00229、AH00230、AH00231、AH00232、AH00233、CBM和19D4F1的人源化抗体hu19D4-25、19D4-25-1A3、19D4-25-1B3、19D4-25-1C2、19D4-25-1E4、19D4-25-2E10、19D4-25-3C11、19D4-25-1C2-3C11。杂交瘤的制备方法以及人源化设计的方法可根据本领域许多文献公示的方法进行。The antigen-binding proteins 13H6D3 and 19D4F1 were obtained by hybridoma technology. Afterwards, humanized antibodies to 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, and humanized antibodies to 19D4F1, hu19D4-25, 19D4-25- 1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25-1E4, 19D4-25-2E10, 19D4-25-3C11, 19D4-25-1C2-3C11. The preparation method of hybridoma and the method of humanized design can be carried out according to the methods published in many documents in this field.

1.2pCP-5BM-Lh载体构建1.2 Construction of pCP-5BM-Lh vector

使用下表1对应的引物Lh5BM-VL-S+Lh5BM-VL-AS，从抗体5BM重链载体pCP-5BM-H-agYTE(agYTE代表的是YTE突变和N297A突变)中扩增417bp片段。PCR酶及体系按Takara Bio

HS(Premix)试剂盒配置及使用。片段扩增后进行胶回收纯化待用。Using the primers Lh5BM-VL-S+Lh5BM-VL-AS corresponding to Table 1 below, a 417bp fragment was amplified from the antibody 5BM heavy chain vector pCP-5BM-H-agYTE (agYTE represents the YTE mutation and the N297A mutation). PCR enzyme and system according to Takara Bio

HS (Premix) kit configuration and use. After the fragments are amplified, they are gel-recovered and purified for use.

从含Kappa轻链恒定区的载体中使用PCP-CK1-S+PCP-CK1-AS引物扩增350bp轻链Kappa恒定区序列。片段扩增后进行胶回收纯化待用。A 350bp light chain Kappa constant region sequence was amplified from the vector containing the Kappa light chain constant region using PCP-CK1-S+PCP-CK1-AS primers. After the fragments are amplified, they are gel-recovered and purified for use.

对轻链表达载体pCP-L载体进行KpnI+NotI双酶切(KpnI和NotI来自Takara Bio)，切胶回收约5.5Kb大小的载体片段。The light chain expression vector pCP-L was digested with KpnI+NotI (KpnI and NotI were from Takara Bio), and the vector fragment of about 5.5Kb was recovered by gel cutting.

使用ClonExpress Ultra One Step Cloning Kit(Vazyme)将三片段进行同源重组构建，LB平板挑取10个阳性克隆，进行测序鉴定，选择其中一个构建正确的质粒进入后续Lh载体扩增制备流程。The three fragments were constructed by homologous recombination using the ClonExpress Ultra One Step Cloning Kit (Vazyme). 10 positive clones were picked from the LB plate for sequencing identification, and one of the correctly constructed plasmids was selected to enter the subsequent Lh vector amplification preparation process.

表1引物序列Table 1 Primer Sequence

1.3pCP-3C11-H-agYTE载体构建1.3 pCP-3C11-H-agYTE vector construction

使用下表2对应的引物3C11-VH-S+3C11-VH-AS，从抗体19D4-25-3C11 VH模板中(agYTE代表的是YTE突变和N297A突变)扩增411bp片段。PCR酶及体系按Takara Bio

HS(Premix)试剂盒配置及使用。片段扩增后进行胶回收纯化待用。Using the primers 3C11-VH-S+3C11-VH-AS corresponding to Table 2 below, a 411bp fragment was amplified from the antibody 19D4-25-3C11 VH template (agYTE represents the YTE mutation and the N297A mutation). PCR enzyme and system according to Takara Bio

对轻链表达载体pCP-H-agYTE载体进行KpnI+NheI双酶切(KpnI和NheI来自TakaraBio)，切胶回收约6.5Kb大小的载体片段。The light chain expression vector pCP-H-agYTE vector was digested with KpnI+NheI (KpnI and NheI were from TakaraBio), and the vector fragment of about 6.5Kb was recovered by gel cutting.

使用ClonExpress Ultra One Step Cloning Kit(Vazyme)将三片段进行同源重组构建，LB平板挑取10个阳性克隆，进行测序鉴定，选择其中一个构建正确的质粒进入后续pCP-3C11-H-agYTE扩增制备流程。Use the ClonExpress Ultra One Step Cloning Kit (Vazyme) to construct the three fragments by homologous recombination, pick 10 positive clones from the LB plate, perform sequencing identification, and select one of the correctly constructed plasmids for subsequent pCP-3C11-H-agYTE amplification Preparation process.

表2引物序列Table 2 Primer Sequence

实施例2双特异性抗体表征实验Example 2 Bispecific antibody characterization experiment

通过抗体表达质粒，将抗PDL1抗体5BMVH区域的序列(SEQ ID NO:97)接到人IgG1kappa链恒定区(CL)的N端，将抗PD1抗体19D4-25-3C11VH区域的序列(SEQ ID NO：31)连接到人IgG1重链第一恒定区(CH1)的N端。表达双特异性抗体的质粒转染HEK293细胞后培养，之后将培养液分离并提纯抗体。2xVH双抗抗体(5BMVH-3C11VH，也叫做Lh5BM-3C11)表达水平和一般抗体无异。为了对此分子进行表征研究，将高表达人PD1、PDL1或野生型的HEK293细胞和Lh5BM-3C11共孵育一个小时，洗涤后加荧光标记的抗人IgG1Fc并显色。结果表明(图2)，Lh5BM-3C11可高强度染色PD1或PDL1表达的细胞而对野生型HEK293没有任何染色，表明此双抗可结合PD1和PDL1。Through the antibody expression plasmid, the sequence of the anti-PDL1 antibody 5BMVH region (SEQ ID NO:97) was connected to the N-terminal of the human IgG1kappa chain constant region (CL), and the sequence of the anti-PD1 antibody 19D4-25-3C11VH region (SEQ ID NO: :31) to the N-terminus of the first constant region (CH1) of the human IgG1 heavy chain. HEK293 cells were transfected with the plasmid expressing the bispecific antibody and cultured, and then the culture medium was separated and the antibody was purified. The expression level of 2xVH double antibody (5BMVH-3C11VH, also called Lh5BM-3C11) is the same as that of general antibodies. In order to characterize this molecule, HEK293 cells highly expressing human PD1, PDL1 or wild type were co-incubated with Lh5BM-3C11 for one hour, after washing, fluorescently labeled anti-human IgG1Fc was added and developed. The results showed (Figure 2) that Lh5BM-3C11 could highly stain PD1 or PDL1 expressing cells without any staining on wild-type HEK293, indicating that this double antibody can bind PD1 and PDL1.

为了进一步表征Lh5BM-3C11而进行了体外PD1拮抗实验。ELISA板包被重组PDL-1-Fc作为捕获物，PD1-生物素标签，然后是辣根过氧化物酶HRP-链霉亲和素作为检测剂。抗PD1单抗(1C2-3C11-AgYTE，其指代的是19D4-25-1C2-3C11与YTE突变、N297突变的Fc融合后得到的抗原结合蛋白)和抗PD1/PDL1双特异性抗体(Lh5BM-3C11)均用作阻断剂。结果(图3)显示，抗PD1抗体可以剂量依赖性地降低PD1与包被的PDL-1的结合。双特异性抗体剂量依赖性地增加HRP信号，解释为双特异性抗PDL-1结合包被的PDL1，抗PD1臂结合被HRP-亲和素结合的PD1-生物素，因此，双特异性抗体剂量依赖性地增加了HRP信号。In order to further characterize Lh5BM-3C11, an in vitro PD1 antagonism experiment was performed. ELISA plates were coated with recombinant PDL-1-Fc as capture, PD1-biotin tag, and then horseradish peroxidase HRP-streptavidin as detection reagent. Anti-PD1 monoclonal antibody (1C2-3C11-AgYTE, which refers to the antigen-binding protein obtained by fusing Fc of 19D4-25-1C2-3C11 with YTE mutation and N297 mutation) and anti-PD1/PDL1 bispecific antibody (Lh5BM -3C11) were both used as blocking agents. The results ( FIG. 3 ) showed that the anti-PD1 antibody could dose-dependently reduce the binding of PD1 to the coated PDL-1. The bispecific antibody dose-dependently increases the HRP signal, interpreted as the bispecific anti-PDL-1 binds coated PDL1, and the anti-PD1 arm binds PD1-biotin bound by HRP-avidin, thus, the bispecific antibody Dose-dependently increased HRP signaling.

实施例3三特异性抗体(抗PD1/PDL1/CD16a的三特异性抗体)表征实验Example 3 Trispecific antibody (anti-PD1/PDL1/CD16a trispecific antibody) characterization experiment

通过抗体表达质粒，将抗PDL1抗体5BMVH区域的序列(SEQ ID NO:97)接到人IgG1kappa链恒定区(CL)的N端，将抗PD1抗体19D4-25-3C11VH区域的序列(SEQ ID NO：31)连接到人IgG1重链第一恒定区(CH1)的N端。将抗CD16a抗体的VH(SEQ ID NO:168)和VL(SEQID NO:169)用连接子(GGGGS)3相连并将VH的N端用连接子连接到Fc的C端。由此构成抗PD1/PDL1/CD16a的三特异性抗体。用表达PD1、PDL1、CD16a或野生型HEK293细胞进行染色。结果显示此三抗可特异性的染色PD1、PDL1或CD16a高表达的细胞而对野生型HEK293没有染色。Through the antibody expression plasmid, the sequence of the anti-PDL1 antibody 5BMVH region (SEQ ID NO:97) was connected to the N-terminal of the human IgG1kappa chain constant region (CL), and the sequence of the anti-PD1 antibody 19D4-25-3C11VH region (SEQ ID NO: :31) to the N-terminus of the first constant region (CH1) of the human IgG1 heavy chain. The VH (SEQ ID NO: 168) and VL (SEQ ID NO: 169) of the anti-CD16a antibody were connected with a linker (GGGGS)3 and the N-terminal of the VH was connected to the C-terminal of the Fc with a linker. Thus, a trispecific antibody against PD1/PDL1/CD16a was constructed. Staining was performed with HEK293 cells expressing PD1, PDL1, CD16a or wild type. The results showed that the third antibody could specifically stain the cells with high expression of PD1, PDL1 or CD16a, but did not stain the wild-type HEK293.

为了表征此三特异性抗体能同时连接PD1、PDL1和CD16a蛋白，Biacore实验显示用三特异性抗体包被芯片，按顺序加上PD1，PDL-1或CD16a后观察到峰值依次升高，说明此分子可同时结合PD1,PDL-1和CD16a。In order to characterize that this trispecific antibody can simultaneously connect to PD1, PDL1 and CD16a proteins, Biacore experiments showed that the chip was coated with trispecific antibodies, and the peaks were observed to increase sequentially after adding PD1, PDL-1 or CD16a in sequence, indicating that this The molecule can simultaneously bind PD1, PDL-1 and CD16a.

实施例4抗PD1/PDL1/IL-2的双特异性抗体表征实验Example 4 Characterization experiment of anti-PD1/PDL1/IL-2 bispecific antibody

通过抗体表达质粒，将抗PDL-1抗体5BMVH区域的序列(SEQ ID NO:97)接到人IgG1kappa链恒定区(CL)的N端，将抗PD1抗体19D4-25-3C11VH区域的序列(SEQ ID NO：31)连接到人IgG1重链第一恒定区(CH1)的N端。将IL-2(SEQ ID NO:180)表达基因用连接子(GGGGS)3连接到Fc的C端。由此构成抗PD1/PDL1/IL-2的双特异性抗体免疫细胞因子(Immunocytokines)。Biacore实验显示用抗PD1/PDL1/IL-2的双特异性抗体包被芯片，按顺序加上PD1，PDL-1或IL-2R后观察到峰值依次升高，说明此分子可同时结合PD1,PDL-1和IL-2R。Through the antibody expression plasmid, the sequence (SEQ ID NO:97) of the anti-PDL-1 antibody 5BMVH region was connected to the N-terminal of the human IgG1kappa chain constant region (CL), and the sequence of the anti-PD1 antibody 19D4-25-3C11VH region (SEQ ID NO:97) ID NO: 31) was linked to the N-terminus of the first constant region (CH1) of the human IgG1 heavy chain. The IL-2 (SEQ ID NO: 180) expression gene was connected to the C-terminal of the Fc with a linker (GGGGS) 3. Thus, anti-PD1/PDL1/IL-2 bispecific antibody immunocytokines (Immunocytokines) are constructed. Biacore experiments showed that the chip was coated with anti-PD1/PDL1/IL-2 bispecific antibody, and the peaks were observed to increase sequentially after adding PD1, PDL-1 or IL-2R in sequence, indicating that this molecule can bind PD1 at the same time, PDL-1 and IL-2R.

将抗PD1/PDL1/IL-2的双特异性抗体和人外周血PBMC一起孵育后，T细胞的增殖和细胞因子的产生明显高于PD1-PDL1双抗刺激的培养，说明了IL-2免疫增强作用。After incubating anti-PD1/PDL1/IL-2 bispecific antibodies with human peripheral blood PBMCs, the proliferation of T cells and the production of cytokines were significantly higher than those stimulated by PD1-PDL1 dual antibodies, indicating that IL-2 immunity Reinforcement.

实施例5抗PD1/PDL1/IFN-α的双特异性抗体的表征实验Example 5 Characterization experiment of anti-PD1/PDL1/IFN-α bispecific antibody

通过抗体表达质粒，将抗PDL-1抗体5BM VH区域的序列(SEQ ID NO:97)接到人IgG1kappa链恒定区(CL)的N端，将抗PD1抗体19D4-25-3C11 VH区域的序列(SEQ ID NO：31)连接到人IgG1重链第一恒定区(CH1)的N端。将IFN-a(SEQ ID NO:181)表达基因用连接子(GGGGS)3连接到Fc的C端。由此构成抗PD1/PDL1/IFN-α的双特异性抗体免疫细胞因子(Immunocytokines)。Biacore实验显示用抗PD1/PDL1/IFN-α的双特异性抗体包被芯片，按顺序加上PD1，PDL-1或IFNR后观察到峰值依次升高，说明此分子可同时结合PD1,PDL-1和IFNR。Through the antibody expression plasmid, the sequence of the anti-PDL-1 antibody 5BM VH region (SEQ ID NO:97) was connected to the N-terminal of the human IgG1 kappa chain constant region (CL), and the sequence of the anti-PD1 antibody 19D4-25-3C11 VH region (SEQ ID NO: 31) was linked to the N-terminus of the first constant region (CH1) of the human IgG1 heavy chain. The IFN-α (SEQ ID NO: 181) expression gene was connected to the C-terminal of the Fc with a linker (GGGGS)3. Thus, the anti-PD1/PDL1/IFN-α bispecific antibody immunocytokines (Immunocytokines) are constructed. Biacore experiments showed that the chip was coated with anti-PD1/PDL1/IFN-α bispecific antibody, and the peaks were observed to increase sequentially after adding PD1, PDL-1 or IFNR in sequence, indicating that this molecule can simultaneously bind PD1, PDL- 1 and IFNR.

将抗PD1/PDL1/IFN-α的双特异性抗体和人外周血PBMC一起孵育后，T细胞的增殖和细胞因子的产生明显高于PD1-PDL1双抗刺激的培养，说明了IFN-α免疫增强作用。After incubating anti-PD1/PDL1/IFN-α bispecific antibody with human peripheral blood PBMC, the proliferation of T cells and the production of cytokines were significantly higher than those stimulated by PD1-PDL1 double antibody culture, indicating that IFN-α immunity Reinforcement.

Claims

1. An isolated antigen binding protein comprising a first polypeptide chain comprising a first targeting moiety, a second polypeptide chain comprising a second targeting moiety, the first targeting moiety and the second targeting moiety each independently binding an immune cell associated antigen, the first polypeptide chain being linked to the second polypeptide chain by a disulfide bond.

2. The isolated antigen binding protein of claim 1, wherein the immune cells comprise T cells, macrophages, dendritic cells, and/or NK cells.

3. The isolated antigen binding protein of any one of claims 1-2, wherein the immune cell-associated antigen comprises PD-1, PD-L1, CD3, CD16a, and/or CD28.

4. The isolated antigen binding protein of any one of claims 1-3, wherein the first targeting moiety and second targeting moiety bind the same immune cell-related antigen.

5. The isolated antigen binding protein of any one of claims 1-4, wherein both the first targeting moiety and the second targeting moiety bind to a PD-1 protein or a PD-L1 protein.

6. The isolated antigen binding protein of any one of claims 1-5, wherein the first targeting moiety specifically binds to a PD-1 protein and the second targeting moiety specifically binds to a PD-L1 protein.

7. The isolated antigen binding protein of any one of claims 1-6, wherein the first targeting moiety specifically binds to a PD-L1 protein and the second targeting moiety specifically binds to a PD-1 protein.

8. The isolated antigen binding protein of any one of claims 1-7, wherein the first targeting moiety specifically binds to a PD-1 protein and the second targeting moiety specifically binds to a CD28 or CD16a protein.

9. The isolated antigen binding protein of any one of claims 1-8, wherein the first targeting moiety specifically binds CD28 or CD16a protein and the second targeting moiety specifically binds PD-1 protein.

10. The isolated antigen binding protein of any one of claims 1-9, wherein the first polypeptide chain further comprises a third targeting moiety.

11. The isolated antigen binding protein of claim 10, wherein the third targeting moiety in the first polypeptide chain is directly or indirectly linked to the C-terminus of the first targeting moiety.

12. The isolated antigen binding protein of any one of claims 10-11, wherein the third targeting moiety in the first polypeptide chain is linked to the C-terminus of the first targeting moiety by a linker.

13. The isolated antigen binding protein of any one of claims 10-12, wherein the third targeting moiety targets an immune cell-associated antigen.

14. The isolated antigen binding protein of claim 13, wherein the immune cell-associated antigen comprises PD-1, PD-L1, CD3, CD16a, and/or CD28.

15. The isolated antigen binding protein of claim 14, wherein the third targeting moiety targets CD3 or CD16a protein.

16. The isolated antigen binding protein of any one of claims 10-15, wherein the third targeting moiety targets a tumorigenesis related antigen.

17. The isolated antigen binding protein of claim 16, wherein the third targeting moiety targets EGFR and/or cMet.

18. The isolated antigen binding protein of any one of claims 10-17, wherein the third targeting moiety targets a tumor-associated antigen.

19. The isolated antigen binding protein of claim 18, wherein the tumor-associated antigen comprises PD-L1, HER2, CD47, CD19, and/or CD20.

20. The isolated antigen binding protein of any one of claims 1-19, wherein the second polypeptide chain further comprises a fourth targeting moiety.

21. The isolated antigen binding protein of claim 20, wherein a fourth targeting moiety in the second polypeptide chain is directly or indirectly linked to the C-terminus of the second targeting moiety.

22. The isolated antigen binding protein of any one of claims 20-21, wherein the fourth targeting moiety in the second polypeptide chain is linked to the C-terminus of the second targeting moiety by a linker.

23. The isolated antigen binding protein of any one of claims 20-22, wherein the fourth targeting moiety targets an immune cell-associated antigen.

24. The isolated antigen binding protein of claim 23, wherein the immune cell-associated antigen comprises PD-1, PD-L1, CD3, CD16a, and/or CD28.

25. The isolated antigen binding protein of any one of claims 20-24, wherein the fourth targeting moiety targets a tumorigenesis related antigen.

26. The isolated antigen binding protein of claim 25, wherein the fourth targeting moiety targets EGFR and/or cMet.

27. The isolated antigen binding protein of any one of claims 20-26, wherein the fourth targeting moiety targets a tumor-associated antigen.

28. The isolated antigen binding protein of claim 27, wherein the tumor-associated antigen comprises PD-L1, HER2, CD47, CD19, and/or CD20.

29. The isolated antigen binding protein of any one of claims 1-28, wherein the first polypeptide chain comprises a cytokine.

30. The isolated antigen binding protein of claim 29, wherein the cytokine comprises IL-2, ifnα, ifnβ, and/or IL-15.

31. The isolated antigen binding protein of any one of claims 1-30, wherein the second polypeptide chain comprises a cytokine.

32. The isolated antigen binding protein of claim 31, wherein the cytokine comprises IL-2, ifnα, ifnβ, and/or IL-15.

33. The isolated antigen binding protein of any one of claims 20-32, wherein the first targeting moiety targets PD-L1, the second targeting moiety targets PD-L1, and the fourth targeting moiety targets PD-1, CD3, or CD16a.

34. The isolated antigen binding protein of any one of claims 20-32, wherein the first targeting moiety targets PD-1, the second targeting moiety targets PD-1, and the fourth targeting moiety targets PD-L1, CD3, or CD16a.

35. The isolated antigen binding protein of any one of claims 10-32, wherein the first targeting moiety targets PD-L1, the second targeting moiety targets PD-1, and the third targeting moiety targets CD3 or CD16a.

36. The isolated antigen binding protein of any one of claims 10-32, wherein the first targeting moiety targets PD-1, the second targeting moiety targets PD-L1, and the third targeting moiety targets CD3 or CD16a.

37. The isolated antigen binding protein of any one of claims 20-32, wherein the first targeting moiety targets CD28 or CD16a, the second targeting moiety targets PD-1, and the fourth targeting moiety targets PD-L1, HER2, CD47, CD19, and/or CD20.

38. The isolated antigen binding protein of any one of claims 20-32, wherein the first targeting moiety targets PD-1, the second targeting moiety targets CD28 or CD16a, and the fourth targeting moiety targets PD-L1, HER2, CD47, CD19, and/or CD20.

39. The isolated antigen binding protein of any one of claims 20-32, wherein the first targeting moiety targets CD28 or CD16a, the second targeting moiety targets PD-1, the third targeting moiety targets EGFR or cMET, and the fourth targeting moiety targets PD-L1, HER2, CD47, CD19, and/or CD20.

40. The isolated antigen binding protein of any one of claims 20-32, wherein the first targeting moiety targets PD-1, the second targeting moiety targets CD28 or CD16a, the third targeting moiety targets PD-L1, HER2, CD47, CD19, and/or CD20, and the fourth targeting moiety targets EGFR or cMET.

41. The isolated antigen binding protein of any one of claims 1-40, wherein the first targeting moiety targets PD-1, the second targeting moiety targets PD-1, and the second polypeptide chain comprises IL-2 or IL-15.

42. The isolated antigen binding protein of any one of claims 1-40, wherein the first targeting moiety targets PD-L1, the second targeting moiety targets PD-L1, and the second polypeptide chain comprises IL-2 or IL-15.

43. The isolated antigen binding protein of any one of claims 20-42, wherein the first targeting moiety targets PD-1, the second targeting moiety targets PD-L1, the first polypeptide chain comprises IL-2 or IL-15, and the fourth targeting moiety comprises CD3 or CD16a.

44. The isolated antigen binding protein of any one of claims 20-42, wherein the first targeting moiety targets PD-L1, the second targeting moiety targets PD-1, the first polypeptide chain comprises IL-2 or IL-15, and the fourth targeting moiety comprises CD3 or CD16a.

45. The isolated antigen binding protein of any one of claims 1-44, wherein the first targeting moiety targets PD-L1, the second targeting moiety targets PD-1, and the first polypeptide chain comprises IL-2 or IL-15.

46. The isolated antigen binding protein of any one of claims 1-44, wherein the first targeting moiety targets PD-1, the second targeting moiety targets PD-L1, and the first polypeptide chain comprises IL-2 or IL-15.

47. The isolated antigen binding protein of any one of claims 20-46, wherein the first targeting moiety targets PD-1, the second targeting moiety targets PD-1, the first polypeptide chain comprises IL-2 or IL-15, and the fourth targeting moiety targets PD-L1.

48. The isolated antigen binding protein of any one of claims 20-46, wherein the first targeting moiety targets PD-L1, the second targeting moiety targets PD-L1, the first polypeptide chain comprises IL-2 or IL-15, and the fourth targeting moiety targets PD-1.

49. The isolated antigen binding protein of any one of claims 10-48, wherein the first targeting moiety targets PD-1, the second targeting moiety targets PD-L1, the third targeting moiety targets CD3 or CD16a, and the second polypeptide chain comprises IL-2 or IL-15.

50. The isolated antigen binding protein of any one of claims 10-48, wherein the first targeting moiety targets PD-L1, the second targeting moiety targets PD-1, the third targeting moiety targets CD3 or CD16a, and the second polypeptide chain comprises IL-2 or IL-15.

51. The isolated antigen binding protein of any one of claims 1-50, wherein the first targeting moiety comprises HCDR3, the HCDR3 comprising the amino acid sequence of SEQ ID No. 51.

52. The isolated antigen binding protein of claim 51, wherein said HCDR3 comprises an amino acid sequence of any one of SEQ ID No. 9 and SEQ ID No. 10.

53. The isolated antigen binding protein of any one of claims 1-52, wherein the first targeting moiety comprises HCDR2, the HCDR2 comprising the amino acid sequence of SEQ ID No. 6.

54. The isolated antigen binding protein of any one of claims 1-53, wherein the first targeting moiety comprises HCDR1, the HCDR1 comprising the amino acid sequence of SEQ ID NO: 3.

55. The isolated antigen binding protein of any one of claims 1-54, wherein the first targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in SEQ ID No. 52.

56. The isolated antigen binding protein of claim 55, wherein said first targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH of any one of SEQ ID NOs 29 to 31.

57. The isolated antigen binding protein of any one of claims 1-56, wherein the first targeting moiety comprises a heavy chain variable region VH comprising the HCDR1, HCDR2 and HCDR3, the HCDR3 comprising the amino acid sequence of SEQ ID No. 51; the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 6; and the HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 3.

58. The isolated antigen binding protein of claim 57, wherein said first targeting moiety comprises a heavy chain variable region VH comprising said HCDR1, HCDR2 and HCDR3, said HCDR3 comprising an amino acid sequence of any one of SEQ ID NOs 9 and 10; the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 6; and the HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 3.

59. The isolated antigen binding protein of any one of claims 57-58, wherein the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from any one of the group consisting of:

a) HCDR1: SEQ ID NO. 3, HCDR2: SEQ ID NO. 6 and HCDR3: SEQ ID NO. 9; and

b) HCDR1: SEQ ID NO. 3, HCDR2: SEQ ID NO. 6 and HCDR3: SEQ ID NO. 10.

60. The isolated antigen binding protein of any one of claims 1-59, wherein the first targeting moiety comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence of SEQ ID No. 47.

61. The isolated antigen binding protein of claim 60, wherein said H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID No. 1 and SEQ ID No. 2.

62. The isolated antigen binding protein of any one of claims 1-61, wherein the first targeting moiety comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence of SEQ ID NO: 48.

63. The isolated antigen binding protein of claim 62, wherein said H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID No. 4 and SEQ ID No. 5.

64. The isolated antigen binding protein of any one of claims 1-63, wherein the first targeting moiety comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence of SEQ ID No. 49.

65. The isolated antigen binding protein of claim 64, wherein said H-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NO. 7 and SEQ ID NO. 8.

66. The isolated antigen binding protein of any one of claims 1-65, wherein the first targeting moiety comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence of SEQ ID NO: 50.

67. The isolated antigen binding protein of claim 66, wherein said H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID No. 11 and SEQ ID No. 12.

68. The isolated antigen binding protein of any one of claims 1-67, wherein the first targeting moiety comprises H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR1 comprising the amino acid sequence set forth in SEQ ID No. 47; the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 48; the H-FR3 comprises an amino acid sequence shown in SEQ ID NO. 49; and the H-FR4 comprises the amino acid sequence shown as SEQ ID NO. 50.

69. The isolated antigen binding protein of claim 68, wherein said first targeting moiety comprises H-FR1, H-FR2, H-FR3, and H-FR4, said H-FR1 comprising the amino acid sequence set forth in any one of SEQ ID No. 1 and SEQ ID No. 2; the H-FR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 4 and SEQ ID NO. 5; the H-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO. 7 and SEQ ID NO. 8; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO. 11 and SEQ ID NO. 12.

70. The isolated antigen binding protein of any one of claims 68-69, wherein the H-FR1, H-FR2, H-FR3, and H-FR4 comprise an amino acid sequence selected from any one of the group consisting of:

a) H-FR1: SEQ ID NO:1, H-FR2: SEQ ID NO. 4, H-FR3: SEQ ID NO. 7 and H-FR4: SEQ ID NO. 11; and

b) H-FR1: SEQ ID NO. 2, H-FR2: 5,H-FR3 of SEQ ID NO: SEQ ID NO. 8 and H-FR4: SEQ ID NO. 12.

71. The isolated antigen binding protein of any one of claims 1-70, wherein the first targeting moiety comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID No. 52.

72. The isolated antigen binding protein of claim 71, wherein said VH comprises an amino acid sequence set forth in any one of SEQ ID NOs 29 to 31.

73. The isolated antigen binding protein of any one of claims 1-50, wherein the first targeting moiety comprises LCDR3, the LCDR3 comprising the amino acid sequence of SEQ ID NO: 57.

74. The isolated antigen binding protein of claim 73, wherein said LCDR3 comprises an amino acid sequence of any one of SEQ ID NOs 21-26.

75. The isolated antigen binding protein of any one of claims 1-50 and 73-74, wherein the first targeting moiety comprises LCDR2, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 18.

76. The isolated antigen binding protein of any one of claims 1-50 and 73-75, wherein the first targeting moiety comprises LCDR1, the LCDR1 comprising the amino acid sequence of SEQ ID No. 15.

77. The isolated antigen binding protein of any one of claims 1-50 and 73-76, wherein the first targeting moiety comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL shown in SEQ ID NO 58.

78. The isolated antigen binding protein of claim 77, wherein the first targeting moiety comprises LCDR1, LCDR2 and LCDR3 of a light chain variable region VL of any one of SEQ ID NOs 32 to 38.

79. The isolated antigen binding protein of any one of claims 1-50 and 73-78, wherein the first targeting moiety comprises a light chain variable region VL comprising the LCDR1, LCDR2 and LCDR3, the LCDR3 comprising the amino acid sequence of SEQ ID NO: 57; the LCDR2 comprises an amino acid sequence shown in SEQ ID NO. 18; and the LCDR1 comprises an amino acid sequence as shown in SEQ ID NO. 15.

80. The isolated antigen binding protein of claim 79, wherein the first targeting moiety comprises a light chain variable region VL comprising the LCDR1, LCDR2 and LCDR3, the LCDR3 comprising an amino acid sequence of any one of SEQ ID No. 21 to SEQ ID No. 26; the LCDR2 comprises an amino acid sequence shown in SEQ ID NO. 18; and the LCDR1 comprises an amino acid sequence as shown in SEQ ID NO. 15.

81. The isolated antigen binding protein of any one of claims 79-80, wherein the LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence selected from any one of the group consisting of:

a) LCDR1: SEQ ID NO. 15, LCDR2: SEQ ID NO. 18 and LCDR3: SEQ ID NO. 21;

b) LCDR1: SEQ ID NO. 15, LCDR2: SEQ ID NO. 18 and LCDR3: SEQ ID NO. 22;

c) LCDR1: SEQ ID NO. 15, LCDR2: SEQ ID NO. 18 and LCDR3: SEQ ID NO. 23;

d) LCDR1: SEQ ID NO. 15, LCDR2: SEQ ID NO. 18 and LCDR3: SEQ ID NO. 24;

e) LCDR1: SEQ ID NO. 15, LCDR2: SEQ ID NO. 18 and LCDR3: SEQ ID NO. 25; and

f) LCDR1: SEQ ID NO. 15, LCDR2: SEQ ID NO. 18 and LCDR3: SEQ ID NO. 26.

82. The isolated antigen binding protein of any one of claims 1-50 and 73-81, wherein the first targeting moiety comprises L-FR1, the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 comprises the amino acid sequence of SEQ ID NO: 53.

83. The isolated antigen binding protein of claim 82, wherein the L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID No. 13 and SEQ ID No. 14.

84. The isolated antigen binding protein of any one of claims 1-50 and 73-83, wherein the first targeting moiety comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence of SEQ ID NO: 54.

85. The isolated antigen binding protein of claim 84, wherein the L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID No. 16 and SEQ ID No. 17.

86. The isolated antigen binding protein of any one of claims 1-50 and 73-85, wherein the first targeting moiety comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence of SEQ ID NO: 55.

87. The isolated antigen binding protein of claim 86, wherein the L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID No. 19 and SEQ ID No. 20.

88. The isolated antigen binding protein of any one of claims 1-50 and 73-87, wherein the first targeting moiety comprises L-FR4, the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 56.

89. The isolated antigen binding protein of claim 88, wherein the L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID No. 27 and SEQ ID No. 28.

90. The isolated antigen binding protein of any one of claims 1-50 and 73-89, wherein the first targeting moiety comprises L-FR1, L-FR2, L-FR3, and L-FR4, the L-FR1 comprising the amino acid sequence set forth in SEQ ID No. 53; the L-FR2 comprises an amino acid sequence shown in SEQ ID NO. 54; the L-FR3 comprises an amino acid sequence shown as SEQ ID NO. 55; and the L-FR4 comprises the amino acid sequence shown as SEQ ID NO. 56.

91. The isolated antigen binding protein of claim 90, wherein the first targeting moiety comprises L-FR1, L-FR2, L-FR3, and L-FR4, the L-FR1 comprising the amino acid sequence set forth in any one of SEQ ID No. 13 and SEQ ID No. 14; the L-FR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 16 and SEQ ID NO. 17; the L-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO. 19 and SEQ ID NO. 20; and the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:27 and SEQ ID NO:28.

92. The isolated antigen binding protein of any one of claims 90-91, wherein the L-FR1, L-FR2, L-FR3, and L-FR4 comprise an amino acid sequence selected from any one of the group consisting of:

a) L-FR1: SEQ ID NO. 13, L-FR2: SEQ ID NO. 16, L-FR3: SEQ ID NO. 19 and L-FR4: SEQ ID NO. 27; and

b) L-FR1: SEQ ID NO. 14, L-FR2: SEQ ID NO. 17, L-FR3: SEQ ID NO. 20 and L-FR4: SEQ ID NO. 28.

93. The isolated antigen binding protein of any one of claims 1-50 and 73-92, wherein the first targeting moiety comprises a VL comprising an amino acid sequence set forth in any one of SEQ ID NOs 58.

94. The isolated antigen binding protein of claim 93, wherein the VL comprises an amino acid sequence shown in any one of SEQ ID NOs 32 to 38.

95. The isolated antigen binding protein of any one of claims 1-50, wherein the first targeting moiety comprises HCDR3, the HCDR3 comprising the amino acid sequence of SEQ ID No. 76.

96. The isolated antigen binding protein of any one of claims 1-50 and 95, wherein the first targeting moiety comprises HCDR2, the HCDR2 comprising the amino acid sequence of SEQ ID No. 70.

97. The isolated antigen binding protein of any one of claims 1-50 and 95-96, wherein the first targeting moiety comprises HCDR1, the HCDR1 comprising the amino acid sequence of SEQ ID No. 61.

98. The isolated antigen-binding protein of any one of claims 1-50 and 95-97, wherein the first targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown as SEQ ID No. 122.

99. The isolated antigen-binding protein of claim 98, wherein the first targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH set forth in any one of SEQ ID NOs 95 to 104.

100. The isolated antigen binding protein of any one of claims 1-50 and 95-99, wherein the first targeting moiety comprises a heavy chain variable region VH comprising the HCDR1, HCDR2 and HCDR3, the HCDR3 comprising the amino acid sequence of SEQ ID No. 76; the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 70; and the HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 61.

101. The isolated antigen binding protein of any one of claims 1-50 and 95-100, wherein the first targeting moiety comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence of SEQ ID NO: 115.

102. The isolated antigen binding protein of claim 101, wherein the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs 59 to 60.

103. The isolated antigen binding protein of any one of claims 1-50 and 95-102, wherein the first targeting moiety comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence of SEQ ID NO 116.

104. The isolated antigen binding protein of claim 103, wherein the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 62 to 69.

105. The isolated antigen binding protein of any one of claims 1-50 and 95-104, wherein the first targeting moiety comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence of SEQ ID NO: 117.

106. The isolated antigen binding protein of claim 105, wherein the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs 71 to 75.

107. The isolated antigen binding protein of any one of claims 1-50 and 95-106, wherein the first targeting moiety comprises H-FR4, the N-terminus of H-FR4 is directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence of SEQ ID NO: 118.

108. The isolated antigen binding protein of claim 107, wherein the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID No. 77 to SEQ ID No. 78.

109. The isolated antigen binding protein of any one of claims 1-50 and 95-108, wherein the first targeting moiety comprises H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR1 comprising the amino acid sequence set forth in SEQ ID NO 115; the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 116; the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 117; and the H-FR4 comprises the amino acid sequence shown as SEQ ID NO. 118.

110. The isolated antigen binding protein of claim 109, wherein the first targeting moiety comprises H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs 59 to 60; the H-FR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 62 to SEQ ID NO. 69; the H-FR3 comprises an amino acid sequence as set forth in any one of SEQ ID NO:71 to SEQ ID NO: 75; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:77 to SEQ ID NO: 78.

111. The isolated antigen binding protein of any one of claims 109-110, wherein the H-FR1, H-FR2, H-FR3, and H-FR4 comprise an amino acid sequence selected from any one of the group consisting of:

a) H-FR1: SEQ ID NO 59, H-FR2: SEQ ID NO. 62, H-FR3: SEQ ID NO:71 and H-FR4: SEQ ID NO. 77;

b) H-FR1: SEQ ID NO. 60, H-FR2: SEQ ID NO. 63, H-FR3: SEQ ID NO. 72 and H-FR4: SEQ ID NO. 78;

c) H-FR1: SEQ ID NO. 60, H-FR2: SEQ ID NO. 64, H-FR3: SEQ ID NO 73 and H-FR4: SEQ ID NO. 78;

d) H-FR1: SEQ ID NO. 60, H-FR2: SEQ ID NO. 65, H-FR3: SEQ ID NO 74 and H-FR4: SEQ ID NO. 78;

e) H-FR1: SEQ ID NO. 60, H-FR2: SEQ ID NO. 66, H-FR3: SEQ ID NO 75 and H-FR4: SEQ ID NO. 78;

f) H-FR1: SEQ ID NO. 60, H-FR2: SEQ ID NO. 67, H-FR3: SEQ ID NO. 72 and H-FR4: SEQ ID NO. 78;

g) H-FR1: SEQ ID NO. 60, H-FR2: SEQ ID NO. 65, H-FR3: SEQ ID NO. 72 and H-FR4: SEQ ID NO. 78;

h) H-FR1: SEQ ID NO. 60, H-FR2: SEQ ID NO. 64, H-FR3: SEQ ID NO 74 and H-FR4: SEQ ID NO. 78;

i) H-FR1: SEQ ID NO. 60, H-FR2: SEQ ID NO. 68, H-FR3: SEQ ID NO. 72 and H-FR4: SEQ ID NO. 78; and

j) H-FR1: SEQ ID NO. 60, H-FR2: SEQ ID NO:69, H-FR3: SEQ ID NO 73 and H-FR4: SEQ ID NO. 78.

112. The isolated antigen binding protein of any one of claims 1-50 and 95-111, wherein the first targeting moiety comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID No. 122.

113. The isolated antigen binding protein of claim 112, wherein said VH comprises an amino acid sequence set forth in any one of SEQ ID NOs 95 to 104.

114. The isolated antigen binding protein of any one of claims 1-50, wherein the first targeting moiety comprises LCDR3, the LCDR3 comprising the amino acid sequence of SEQ ID No. 94.

115. The isolated antigen binding protein of any one of claims 1-50 and 114, wherein the first targeting moiety comprises LCDR2, the LCDR2 comprising the amino acid sequence of SEQ ID No. 87.

116. The isolated antigen binding protein of any one of claims 1-50 and 114-115, wherein the first targeting moiety comprises LCDR1, the LCDR1 comprising the amino acid sequence of SEQ ID No. 82.

117. The isolated antigen binding protein of any one of claims 1-50 and 114-116, wherein the first targeting moiety comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL set forth in any one of SEQ ID NOs 123.

118. The isolated antigen binding protein of claim 117, wherein the first targeting moiety comprises LCDR1, LCDR2 and LCDR3 of a light chain variable region VL of any one of SEQ ID NOs 105 to 111.

119. The isolated antigen binding protein of any one of claims 1-50 and 114-118, wherein the first targeting moiety comprises a light chain variable region VL comprising the LCDR1, LCDR2 and LCDR3, the LCDR3 comprising the amino acid sequence of SEQ ID NO: 94; the LCDR2 comprises an amino acid sequence shown in SEQ ID NO. 87; and the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO. 82.

120. The isolated antigen binding protein of any one of claims 1-50 and 114-119, wherein the first targeting moiety comprises L-FR1, the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 comprises the amino acid sequence of SEQ ID NO: 119.

121. The isolated antigen binding protein of claim 120, wherein the L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs 79 to 81.

122. The isolated antigen binding protein of any one of claims 1-50 and 114-121, wherein the first targeting moiety comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence of SEQ ID NO: 120.

123. The isolated antigen binding protein of claim 122, wherein the L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID No. 83 to SEQ ID No. 86.

124. The isolated antigen binding protein of any one of claims 1-50 and 114-123, wherein the first targeting moiety comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence of SEQ ID NO: 121.

125. The isolated antigen binding protein of claim 124, wherein the L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID No. 88 to SEQ ID No. 93.

126. The isolated antigen binding protein of any one of claims 1-50 and 114-125, wherein the first targeting moiety comprises L-FR4, the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 56.

127. The isolated antigen binding protein of claim 126, wherein the L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs 27 to 28.

128. The isolated antigen binding protein of any one of claims 1-50 and 114-127, wherein the first targeting moiety comprises L-FR1, L-FR2, L-FR3, and L-FR4, the L-FR1 comprising the amino acid sequence set forth in SEQ ID No. 119; the L-FR2 comprises an amino acid sequence shown as SEQ ID NO. 120; the L-FR3 comprises an amino acid sequence shown as SEQ ID NO. 121; and the L-FR4 comprises the amino acid sequence shown as SEQ ID NO. 56.

129. The isolated antigen binding protein of claim 128, wherein the first targeting moiety comprises L-FR1, L-FR2, L-FR3, and L-FR4, the L-FR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs 79 to 81; the L-FR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 83 to SEQ ID NO. 86; the L-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO 88 to SEQ ID NO 93; and the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO 27 to SEQ ID NO 28.

130. The isolated antigen binding protein of any of claims 128-129, wherein the L-FR1, L-FR2, L-FR3, and L-FR4 comprise an amino acid sequence selected from any of the group consisting of:

a) L-FR1: SEQ ID NO. 79, L-FR2: SEQ ID NO. 83, L-FR3: SEQ ID NO 88 and L-FR4: SEQ ID NO. 27;

b) L-FR1: SEQ ID NO. 80, L-FR2: SEQ ID NO. 84, L-FR3: SEQ ID NO. 89 and L-FR4: SEQ ID NO. 28;

c) L-FR1: SEQ ID NO. 81, L-FR2: SEQ ID NO. 85, L-FR3: SEQ ID NO 90 and L-FR4: SEQ ID NO. 28;

d) L-FR1: SEQ ID NO. 81, L-FR2: SEQ ID NO. 85, L-FR3: SEQ ID NO 91 and L-FR4: SEQ ID NO. 28;

e) L-FR1: SEQ ID NO. 80, L-FR2: SEQ ID NO. 85, L-FR3: SEQ ID NO 90 and L-FR4: SEQ ID NO. 28;

f) L-FR1: SEQ ID NO. 80, L-FR2: SEQ ID NO. 85, L-FR3: SEQ ID NO. 92 and L-FR4: SEQ ID NO. 28; and

g) L-FR1: SEQ ID NO. 80, L-FR2: SEQ ID NO. 86, L-FR3: SEQ ID NO. 93 and L-FR4: SEQ ID NO. 28.

131. The isolated antigen binding protein of any one of claims 1-50 and 114-130, wherein the first targeting moiety comprises a VL comprising the amino acid sequence shown in SEQ ID No. 123.

132. The isolated antigen binding protein of claim 131, wherein the VL comprises an amino acid sequence depicted in any one of SEQ ID NOs 105 to 111.

133. The isolated antigen binding protein of any one of claims 1-50, wherein the first targeting moiety targets CD28, the first targeting moiety comprising a VH comprising the amino acid sequence of SEQ ID NO: 176.

134. The isolated antigen binding protein of any one of claims 1-50, wherein the first targeting moiety targets CD16a, the first targeting moiety comprising a VH comprising the amino acid sequence of SEQ ID NO: 168.

135. The isolated antigen binding protein of claims 1-134, wherein the second targeting moiety comprises HCDR3, the HCDR3 comprising the amino acid sequence of SEQ ID No. 51.

136. The isolated antigen binding protein of claim 135, wherein the second targeting moiety comprises HCDR3, the HCDR3 comprising an amino acid sequence of any one of SEQ ID No. 9 and SEQ ID No. 10.

137. The isolated antigen binding protein of any one of claims 1-136, wherein the second targeting moiety comprises HCDR2, the HCDR2 comprising the amino acid sequence of SEQ ID No. 6.

138. The isolated antigen binding protein of any one of claims 1-137, wherein the second targeting moiety comprises HCDR1, the HCDR1 comprising the amino acid sequence of SEQ ID No. 3.

139. The isolated antigen-binding protein of any one of claims 1-138, wherein the second targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in SEQ ID NO 52.

140. The isolated antigen-binding protein of claim 139, wherein the second targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH of any one of SEQ ID NOs 29 to 31.

141. The isolated antigen binding protein of any one of claims 1-140, wherein the second targeting moiety comprises a heavy chain variable region VH comprising the HCDR1, HCDR2 and HCDR3, the HCDR3 comprising the amino acid sequence of SEQ ID No. 51; the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 6; and

The HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 3.

142. The isolated antigen-binding protein of claim 141, wherein the second targeting moiety comprises a heavy chain variable region VH comprising the HCDR1, HCDR2, and HCDR3, the HCDR3 comprising an amino acid sequence of any one of SEQ ID NOs 9 and 10; the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 6; and the HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 3.

143. The isolated antigen binding protein of any one of claims 141-142, wherein the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from any one of the group consisting of:

a) HCDR1: SEQ ID NO. 3, HCDR2: SEQ ID NO. 6 and HCDR3: SEQ ID NO. 9; and

b) HCDR1: SEQ ID NO. 3, HCDR2: SEQ ID NO. 6 and HCDR3: SEQ ID NO. 10.

144. The isolated antigen binding protein of any one of claims 1-143, wherein the second targeting moiety comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence of SEQ ID No. 47.

145. The isolated antigen binding protein of claim 144, wherein said H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID No. 1 and SEQ ID No. 2.

146. The isolated antigen binding protein of any one of claims 1-145, wherein the second targeting moiety comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence of SEQ ID NO: 48.

147. The isolated antigen binding protein of claim 146, wherein said H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID No. 4 and SEQ ID No. 5.

148. The isolated antigen binding protein of any one of claims 1-147, wherein the second targeting moiety comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence of SEQ ID No. 49.

149. The isolated antigen binding protein of claim 148, wherein the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID No. 7 and SEQ ID No. 8.

150. The isolated antigen binding protein of any one of claims 1-149, wherein the second targeting moiety comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence of SEQ ID NO: 50.

151. The isolated antigen binding protein of claim 150, wherein the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID No. 11 and SEQ ID No. 12.

152. The isolated antigen binding protein of any one of claims 1-151, wherein the second targeting moiety comprises H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR1 comprising the amino acid sequence set forth in SEQ ID No. 47; the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 48; the H-FR3 comprises an amino acid sequence shown in SEQ ID NO. 49; and the H-FR4 comprises the amino acid sequence shown as SEQ ID NO. 50.

153. The isolated antigen binding protein of claim 152, wherein the second targeting moiety comprises H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR1 comprising the amino acid sequence set forth in any one of SEQ ID No. 1 and SEQ ID No. 2; the H-FR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 4 and SEQ ID NO. 5; the H-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO. 7 and SEQ ID NO. 8; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO. 11 and SEQ ID NO. 12.

154. The isolated antigen binding protein of any of claims 152-153, wherein the H-FR1, H-FR2, H-FR3, and H-FR4 comprise an amino acid sequence selected from any one of the group consisting of:

a) H-FR1: SEQ ID NO:1, H-FR2: SEQ ID NO. 4, H-FR3: SEQ ID NO. 7 and H-FR4: SEQ ID NO. 11;

155. The isolated antigen binding protein of any one of claims 1-154, wherein the second targeting moiety comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID No. 52.

156. The isolated antigen-binding protein of claim 155, wherein the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs 29 to 31.

157. The isolated antigen binding protein of any one of claims 1-134, wherein the second targeting moiety comprises LCDR3, the LCDR3 comprising the amino acid sequence of SEQ ID NO: 57.

158. The isolated antigen binding protein of claim 157, wherein the second targeting moiety comprises LCDR3, the LCDR3 comprising an amino acid sequence of any one of SEQ ID NOs 21 to 26.

159. The isolated antigen binding protein of any of claims 1-134 and 157-158, wherein the second targeting moiety comprises LCDR2, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 18.

160. The isolated antigen binding protein of any one of claims 1-134 and 157-159, wherein the second targeting moiety comprises LCDR1, the LCDR1 comprising the amino acid sequence of SEQ ID NO: 15.

161. The isolated antigen binding protein of any one of claims 1-134 and 157-160, wherein the second targeting moiety comprises LCDR1, LCDR2, and LCDR3 of the light chain variable region VL shown in SEQ ID NO 58.

162. The isolated antigen-binding protein of claim 161, wherein the second targeting moiety comprises LCDR1, LCDR2, and LCDR3 of a light chain variable region VL of any one of SEQ ID NOs 32-38.

163. The isolated antigen binding protein of any one of claims 1-134 and 157-162, wherein the second targeting moiety comprises a light chain variable region VL comprising the LCDR1, LCDR2 and LCDR3, the LCDR3 comprising the amino acid sequence of SEQ ID NO: 57; the LCDR2 comprises an amino acid sequence shown in SEQ ID NO. 18; and the LCDR1 comprises an amino acid sequence as shown in SEQ ID NO. 15.

164. The isolated antigen binding protein of claim 163, wherein the second targeting moiety comprises a light chain variable region VL comprising the LCDR1, LCDR2 and LCDR3, the LCDR3 comprising an amino acid sequence of any one of SEQ ID NOs 21 to 26; the LCDR2 comprises an amino acid sequence shown in SEQ ID NO. 18; and the LCDR1 comprises an amino acid sequence as shown in SEQ ID NO. 15.

165. The isolated antigen binding protein of any one of claims 163-164, wherein the LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence selected from any one of the group consisting of:

a) LCDR1: SEQ ID NO. 15, LCDR2: SEQ ID NO. 18 and LCDR3: SEQ ID NO. 21;

b) LCDR1: SEQ ID NO. 15, LCDR2: SEQ ID NO. 18 and LCDR3: SEQ ID NO. 22;

c) LCDR1: SEQ ID NO. 15, LCDR2: SEQ ID NO. 18 and LCDR3: SEQ ID NO. 23;

d) LCDR1: SEQ ID NO. 15, LCDR2: SEQ ID NO. 18 and LCDR3: SEQ ID NO. 24;

e) LCDR1: SEQ ID NO. 15, LCDR2: SEQ ID NO. 18 and LCDR3: SEQ ID NO. 25; and

f) LCDR1: SEQ ID NO. 15, LCDR2: SEQ ID NO. 18 and LCDR3: SEQ ID NO. 26.

166. The isolated antigen binding protein of any one of claims 1-134 and 157-165, wherein the second targeting moiety comprises L-FR1, the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 comprises the amino acid sequence of SEQ ID NO: 53.

167. The isolated antigen binding protein of claim 166, wherein the L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID No. 13 and SEQ ID No. 14.

168. The isolated antigen binding protein of any one of claims 1-134 and 157-167, wherein the second targeting moiety comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence of SEQ ID NO: 54.

169. The isolated antigen binding protein of claim 168, wherein the L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID No. 16 and SEQ ID No. 17.

170. The isolated antigen binding protein of any one of claims 1-134 and 157-169, wherein the second targeting moiety comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence of SEQ ID NO: 55.

171. The isolated antigen binding protein of claim 170, wherein the L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID No. 19 and SEQ ID No. 20.

172. The isolated antigen binding protein of any one of claims 1-134 and 157-171, wherein the second targeting moiety comprises L-FR4, the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 56.

173. The isolated antigen binding protein of claim 172, wherein the L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID No. 27 and SEQ ID No. 28.

174. The isolated antigen binding protein of any one of claims 1-134 and 157-173, wherein the second targeting moiety comprises L-FR1, L-FR2, L-FR3, and L-FR4, the L-FR1 comprising the amino acid sequence set forth in SEQ ID No. 53; the L-FR2 comprises an amino acid sequence shown in SEQ ID NO. 54; the L-FR3 comprises an amino acid sequence shown as SEQ ID NO. 55; and the L-FR4 comprises the amino acid sequence shown as SEQ ID NO. 56.

175. The isolated antigen binding protein of claim 174, wherein the second targeting moiety comprises L-FR1, L-FR2, L-FR3, and L-FR4, the L-FR1 comprising the amino acid sequence set forth in any one of SEQ ID No. 13 and SEQ ID No. 14; the L-FR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 16 and SEQ ID NO. 17; the L-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO. 19 and SEQ ID NO. 20; and the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:27 and SEQ ID NO:28.

176. The isolated antigen binding protein of any of claims 174-175, wherein the L-FR1, L-FR2, L-FR3, and L-FR4 comprise an amino acid sequence selected from any one of the group consisting of:

177. The isolated antigen binding protein of any one of claims 1-134 and 157-176, wherein the second targeting moiety comprises a VL comprising an amino acid sequence set forth in any one of SEQ ID NOs 58.

178. The isolated antigen binding protein of claim 177, wherein the VL comprises an amino acid sequence depicted in any one of SEQ ID NOs 32 to 38.

179. The isolated antigen binding protein of claims 1-134, wherein the second targeting moiety comprises HCDR3, the HCDR3 comprising the amino acid sequence of SEQ ID No. 76.

180. The isolated antigen binding protein of any one of claims 1-134 and 179, wherein the second targeting moiety comprises HCDR2, the HCDR2 comprising the amino acid sequence of SEQ ID No. 70.

181. The isolated antigen binding protein of any one of claims 1-134 and 179-180, wherein the second targeting moiety comprises HCDR1, the HCDR1 comprising the amino acid sequence of SEQ ID No. 61.

182. The isolated antigen-binding protein of any one of claims 1-134 and 179-181, wherein the second targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in SEQ ID No. 122.

183. The isolated antigen-binding protein of claim 182, wherein the second targeting moiety comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH of any one of SEQ ID NOs 95 to 104.

184. The isolated antigen binding protein of any one of claims 1-134 and 179-183, wherein the second targeting moiety comprises a heavy chain variable region VH comprising the HCDR1, HCDR2 and HCDR3, the HCDR3 comprising the amino acid sequence of SEQ ID No. 76; the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 70; and the HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 61.

185. The isolated antigen binding protein of any one of claims 1-134 and 179-184, wherein the second targeting moiety comprises H-FR1, the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence of SEQ ID NO: 115.

186. The isolated antigen binding protein of claim 185, wherein the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs 59 to 60.

187. The isolated antigen binding protein of any one of claims 1-134 and 179-186, wherein the second targeting moiety comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence of SEQ ID NO 116.

188. The isolated antigen binding protein of claim 187, wherein the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID No. 62 to SEQ ID No. 69.

189. The isolated antigen binding protein of any one of claims 1-134 and 179-188, wherein the second targeting moiety comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence of SEQ ID NO: 117.

190. The isolated antigen binding protein of claim 189, wherein the H-FR3 comprises an amino acid sequence set forth in any one of SEQ ID No. 71 to SEQ ID No. 75.

191. The isolated antigen binding protein of any one of claims 1-134 and 179-190, wherein the second targeting moiety comprises H-FR4, the N-terminus of H-FR4 is directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence of SEQ ID NO: 118.

192. The isolated antigen binding protein of claim 191, wherein the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID No. 77 to SEQ ID No. 78.

193. The isolated antigen binding protein of any one of claims 1-134 and 179-192, wherein the second targeting moiety comprises H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR1 comprising the amino acid sequence set forth in SEQ ID NO 115; the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 116; the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 117; and the H-FR4 comprises the amino acid sequence shown as SEQ ID NO. 118.

194. The isolated antigen binding protein of claim 193, wherein the second targeting moiety comprises H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs 59 to 60; the H-FR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 62 to SEQ ID NO. 69; the H-FR3 comprises an amino acid sequence as set forth in any one of SEQ ID NO:71 to SEQ ID NO: 75; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:77 to SEQ ID NO: 78.

195. The isolated antigen binding protein of any one of claims 193-194, wherein the H-FR1, H-FR2, H-FR3, and H-FR4 comprise an amino acid sequence selected from any one of the group consisting of:

196. The isolated antigen binding protein of any one of claims 1-134 and 179-195, wherein the second targeting moiety comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID No. 122.

197. The isolated antigen binding protein of claim 196, wherein said VH comprises an amino acid sequence set forth in any one of SEQ ID NOs 95 to 104.

198. The isolated antigen binding protein of any one of claims 1-134, wherein the second targeting moiety comprises LCDR3, the LCDR3 comprising the amino acid sequence of SEQ ID No. 94.

199. The isolated antigen binding protein of any one of claims 1-134 and 198, wherein the second targeting moiety comprises LCDR2, the LCDR2 comprising the amino acid sequence of SEQ ID No. 87.

200. The isolated antigen binding protein of any of claims 1-134 and 198-199, wherein the second targeting moiety comprises LCDR1, the LCDR1 comprising the amino acid sequence shown as SEQ ID No. 82.

201. The isolated antigen binding protein of any one of claims 1-134 and 198-200, wherein the second targeting moiety comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL of any one of SEQ ID NOs 123.

202. The isolated antigen-binding protein of claim 201, wherein the second targeting moiety comprises LCDR1, LCDR2, and LCDR3 of a light chain variable region VL of any one of SEQ ID NOs 105-111.

203. The isolated antigen binding protein of any one of claims 1-134 and 198-202, wherein the second targeting moiety comprises a light chain variable region VL comprising the LCDR1, LCDR2 and LCDR3, the LCDR3 comprising the amino acid sequence of SEQ ID NO: 94; the LCDR2 comprises an amino acid sequence shown in SEQ ID NO. 87; and the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO. 82.

204. The isolated antigen binding protein of any one of claims 1-134 and 198-203, wherein the second targeting moiety comprises L-FR1, the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 comprises the amino acid sequence of SEQ ID NO: 119.

205. The isolated antigen binding protein of claim 204, wherein the L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID No. 79 to SEQ ID No. 81.

206. The isolated antigen binding protein of any one of claims 1-134 and 198-205, wherein the second targeting moiety comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence of SEQ ID NO: 120.

207. The isolated antigen binding protein of claim 206, wherein the L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID No. 83 to SEQ ID No. 86.

208. The isolated antigen binding protein of any one of claims 1-134 and 198-207, wherein the second targeting moiety comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence of SEQ ID NO: 121.

209. The isolated antigen binding protein of claim 208, wherein the L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs 88 to 93.

210. The isolated antigen binding protein of any one of claims 1-134 and 198-209, wherein the second targeting moiety comprises L-FR4, the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 56.

211. The isolated antigen binding protein of claim 210, wherein the L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs 27 to 28.

212. The isolated antigen binding protein of any one of claims 1-134 and 198-211, wherein the second targeting moiety comprises L-FR1, L-FR2, L-FR3, and L-FR4, the L-FR1 comprising the amino acid sequence set forth in SEQ ID NO 119; the L-FR2 comprises an amino acid sequence shown as SEQ ID NO. 120; the L-FR3 comprises an amino acid sequence shown as SEQ ID NO. 121; and the L-FR4 comprises the amino acid sequence shown as SEQ ID NO. 56.

213. The isolated antigen binding protein of claim 212, wherein the second targeting moiety comprises L-FR1, L-FR2, L-FR3, and L-FR4, the L-FR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs 79 to 81; the L-FR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 83 to SEQ ID NO. 86; the L-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO 88 to SEQ ID NO 93; and the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO 27 to SEQ ID NO 28.

214. The isolated antigen binding protein of any of claims 212-213, wherein the L-FR1, L-FR2, L-FR3, and L-FR4 comprise an amino acid sequence selected from any one of the group consisting of:

215. The isolated antigen binding protein of any one of claims 1-134 and 198-214, wherein the second targeting moiety comprises a VL comprising the amino acid sequence shown in SEQ ID No. 123.

216. The isolated antigen binding protein of claim 215, wherein the VL comprises an amino acid sequence depicted in any one of SEQ ID NOs 105 to 111.

217. The isolated antigen binding protein of any one of claims 1-216, wherein the second targeting moiety targets CD28, the second targeting moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 176.

218. The isolated antigen binding protein of any one of claims 1-216, wherein the second targeting moiety targets CD16a, the second targeting moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 168.

219. The isolated antigen binding protein of any one of claims 1-218, comprising a first targeting moiety comprising SEQ ID No. 29 to SEQ ID No. 31, SEQ ID No. 32 to SEQ ID No. 38, SEQ ID No. 95 to SEQ ID No. 104, SEQ ID No. 105 to SEQ ID No. 111, SEQ ID NO:168 and SEQ ID NO:176, and a sequence of any one of the amino acids set forth in seq id no; the second targeting moiety comprises SEQ ID NO. 29 to SEQ ID NO. 31, SEQ ID NO. 32 to SEQ ID NO. 38, SEQ ID NO. 95 to SEQ ID NO. 104 and SEQ ID NO. 105 to SEQ ID NO. 111, SEQ ID NO:168 and SEQ ID NO:176, and a sequence of any one of the amino acids set forth in seq id no.

220. The isolated antigen binding protein of any one of claims 1-219, wherein the first targeting moiety and the second targeting moiety comprise an amino acid sequence selected from any one of the group consisting of:

a) A first targeting moiety: SEQ ID NO 97 and a second targeting moiety: SEQ ID NO. 31;

b) A first targeting moiety: 98 and a second targeting moiety: SEQ ID NO. 31;

c) A first targeting moiety: SEQ ID NO. 100 and a second targeting moiety: SEQ ID NO. 31;

d) A first targeting moiety: SEQ ID NO. 102 and a second targeting moiety: SEQ ID NO. 31;

e) A first targeting moiety: SEQ ID NO. 103 and a second targeting moiety: SEQ ID NO. 31;

f) A first targeting moiety: SEQ ID NO. 31 and a second targeting moiety: SEQ ID NO. 97;

g) A first targeting moiety: SEQ ID NO. 31 and a second targeting moiety: SEQ ID NO. 98;

h) A first targeting moiety: SEQ ID NO. 31 and a second targeting moiety: SEQ ID NO. 100;

i) A first targeting moiety: SEQ ID NO. 31 and a second targeting moiety: SEQ ID NO. 102;

j) A first targeting moiety: SEQ ID NO. 31 and a second targeting moiety: SEQ ID NO. 103;

k) A first targeting moiety: SEQ ID NO 97 and a second targeting moiety: SEQ ID NO. 36;

l) a first targeting moiety: 98 and a second targeting moiety: SEQ ID NO. 36;

m) a first targeting moiety: SEQ ID NO. 100 and a second targeting moiety: SEQ ID NO. 36;

n) a first targeting moiety: SEQ ID NO. 102 and a second targeting moiety: SEQ ID NO. 36;

o) a first targeting moiety: SEQ ID NO. 103 and a second targeting moiety: SEQ ID NO. 36;

p) a first targeting moiety: SEQ ID NO. 36 and a second targeting moiety: SEQ ID NO. 97;

q) a first targeting moiety: SEQ ID NO. 36 and a second targeting moiety: SEQ ID NO. 98;

r) a first targeting moiety: SEQ ID NO. 36 and a second targeting moiety: SEQ ID NO. 100;

s) a first targeting moiety: SEQ ID NO. 36 and a second targeting moiety: SEQ ID NO. 102;

t) a first targeting moiety: SEQ ID NO. 36 and a second targeting moiety: SEQ ID NO. 103;

u) a first targeting moiety: SEQ ID NO. 31 and a second targeting moiety: 176 of SEQ ID NO;

v) a first targeting moiety: SEQ ID NO. 31 and a second targeting moiety: 168 of SEQ ID NO;

w) a first targeting moiety: 176 and a second targeting moiety: SEQ ID NO. 31;

x) a first targeting moiety: 168 and a second targeting moiety: SEQ ID NO. 31.

221. The isolated antigen binding protein of any one of claims 1-220, wherein the second polypeptide chain comprises a heavy chain constant region, and the heavy chain constant region comprises an IgG-derived constant region or an IgY-derived constant region.

222. The isolated antibody binding protein of claim 221, wherein the heavy chain constant region comprises an IgG-derived constant region.

223. The isolated antigen binding protein of any one of claims 221-222, wherein the heavy chain constant region comprises a constant region derived from IgG1, igG2, igG3, or IgG 4.

224. The isolated antigen binding protein of any one of claims 221-223, wherein the heavy chain constant region comprises an Fc region derived from IgG 1.

225. The isolated antigen binding protein of claim 224, wherein the Fc region in the heavy chain constant region comprises an N297A mutation, residues numbered according to the Kabat system.

226. The isolated antigen binding protein of any one of claims 224-225, wherein the Fc region in the heavy chain constant region comprises M252Y, S T and T256E mutations, residues numbered according to the Kabat system.

227. The isolated antigen binding protein of any one of claims 221-226, wherein the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID No. 112 to SEQ ID No. 113.

228. The isolated antigen binding protein of any one of claims 1-227, wherein the first polypeptide chain comprises a light chain constant region, and the light chain constant region comprises an igκ -derived constant region or an igλ -derived constant region.

229. The isolated antigen binding protein of claim 228, wherein the light chain constant region comprises a constant region derived from human igκ.

230. The isolated antigen binding protein of any one of claims 228-229, wherein the light chain constant region comprises the amino acid sequence set forth in SEQ ID No. 114.

231. The isolated antigen binding protein of any one of claims 10-230, wherein the third targeting moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 168. 170, 172, 174, 176 and 178.

232. The isolated antigen binding protein of any one of claims 10-231, wherein the third targeting moiety comprises a VL comprising the amino acid sequence of SEQ ID NO: 169. 171, 173, 175, 177 and 179.

233. The isolated antigen binding protein of any one of claims 10-232, wherein the third targeting moiety comprises an antibody or antigen binding fragment.

234. The isolated antigen binding protein of claim 233, wherein the antigen binding fragment is selected from the group consisting of: fab, fab ', F (ab) 2, fv fragments, F (ab') 2, scFv, di-scFv, VHH and dAb.

235. The isolated antigen binding protein of claim 234, wherein the third targeting moiety is a scFv.

236. The isolated antigen binding protein of any one of claims 231-235, wherein the C-terminus of VH in the third targeting moiety is directly or indirectly linked to the N-terminus of VL.

237. The isolated antigen binding protein of any one of claims 231-235, wherein the C-terminus of VL in the third targeting moiety is directly or indirectly linked to the N-terminus of the VH.

238. The isolated antigen-binding protein of any one of claims 20-237, wherein the fourth targeting moiety comprises a VH comprising the amino acid sequence of SEQ ID NO:29-31, 95-104, 168, 170, 172, 174, 176 and 178.

239. The isolated antigen binding protein of any one of claims 20-238, wherein the fourth targeting moiety comprises a VL comprising the amino acid sequence of SEQ ID NO:32-38, 105-111, 169, 171, 173, 175, 177, and 179.

240. The isolated antigen binding protein of any one of claims 20-239, wherein the fourth targeting moiety comprises an antibody or antigen binding fragment.

241. The isolated antigen binding protein of claim 240, wherein the antigen binding fragment is selected from the group consisting of: fab, fab ', F (ab) 2, fv fragments, F (ab') 2, scFv, di-scFv, VHH and dAb.

242. The isolated antigen binding protein of claim 241, wherein the fourth targeting moiety is a scFv.

243. The isolated antigen binding protein of any one of claims 238-242, wherein the C-terminus of VH in the fourth targeting moiety is directly or indirectly linked to the N-terminus of VL.

244. The isolated antigen binding protein of any one of claims 238-242, wherein the C-terminus of VL in the fourth targeting moiety is directly or indirectly linked to the N-terminus of the VH.

245. The isolated antigen binding protein of any one of claims 1-244, comprising two first polypeptide chains and two second polypeptide chains, the two first polypeptide chains being linked by a disulfide bond, the two first polypeptide chains and the two second polypeptide chains forming an IgG-like antibody.

246. The isolated antigen binding protein of any one of claims 1-245, comprising a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, a multispecific antibody, a bispecific antibody, and/or a fully human antibody.

247. A polypeptide comprising the isolated antigen binding protein of any one of claims 1-246.

248. An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-246 or the polypeptide of claim 247.

249. An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-246, or the polypeptide of claim 247.

250. A vector comprising the isolated nucleic acid molecule of claim 249.

251. A cell comprising the isolated antigen binding protein of any one of claims 1-246, the polypeptide of claim 247, the immunoconjugate of claim 248, the isolated nucleic acid molecule of claim 249, and/or the vector of claim 250.

252. A method of making the isolated antigen binding protein of any one of claims 1-246 or the polypeptide of claim 247, the method comprising culturing the cell of claim 251 under conditions that allow expression of the isolated antigen binding protein of any one of claims 1-246 or the polypeptide of claim 247.

253. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-246, the polypeptide of claim 247, the immunoconjugate of claim 248, the isolated nucleic acid molecule of claim 249, the vector of claim 250, the cell of claim 251, and/or a pharmaceutically acceptable adjuvant and/or excipient.

254. A method of inhibiting the interaction of PD-1 with PD-L1, comprising administering to a subject in need thereof an effective amount of the isolated antigen-binding protein of any one of claims 1-246, the polypeptide of claim 247, the immunoconjugate of claim 248, the isolated nucleic acid molecule of claim 249, the vector of claim 250, and/or the cell of claim 251.

255. Use of the isolated antigen binding protein of any one of claims 1-246, the polypeptide of claim 247, the immunoconjugate of claim 248, the isolated nucleic acid molecule of claim 249, the vector of claim 250, the cell of claim 251 and/or the pharmaceutical composition of claim 253 in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder.

256. The use of claim 255, wherein the disease or condition comprises a tumor.

257. The use of claim 256, wherein the tumor comprises a solid tumor.

258. The use of any one of claims 256-257, wherein the tumor comprises a non-solid tumor.

259. The use of any one of claims 256-258, wherein the tumor comprises breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and/or liver cancer.

260. The isolated antigen binding protein of any one of claims 1-246, the polypeptide of claim 247, the immunoconjugate of claim 248, the isolated nucleic acid molecule of claim 249, the vector of claim 250, the cell of claim 251, and/or the pharmaceutical composition of claim 253 for use in preventing, alleviating, and/or treating a disease or disorder.

261. The use of claim 260, wherein the disease or disorder comprises a tumor.

262. The use of claim 261, wherein the tumor comprises a solid tumor.

263. The use of any one of claims 261-262, wherein the tumor comprises a non-solid tumor.

264. The use of any one of claims 261-263, wherein the tumor comprises breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and/or liver cancer.

265. A method of preventing and/or treating a disease or disorder comprising administering to a subject in need thereof an effective amount of the isolated antigen binding protein of any one of claims 1-246, the polypeptide of claim 247, the immunoconjugate of claim 248, the isolated nucleic acid molecule of claim 249, the vector of claim 250, the cell of claim 251, and/or the pharmaceutical composition of claim 253.

266. The method of claim 265, wherein the disease or disorder comprises a tumor.

267. The method of claim 266, wherein the tumor comprises a solid tumor.

268. The method of any of claims 266-267, wherein the tumor comprises a non-solid tumor.

269. The method of any one of claims 266-268, wherein the tumor comprises breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and/or liver cancer.