CN113166264B - Isolated antigen binding proteins and uses thereof - Google Patents

Abstract

An isolated antigen binding protein is provided comprising at least one CDR in a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID No. 16 and at least one CDR in a light chain variable region VL comprising the amino acid sequence shown in SEQ ID No. 15. Also provided are nucleic acid molecules encoding the isolated antigen binding proteins, vectors comprising the isolated antigen binding proteins, cells comprising the nucleic acid molecules or the vectors, methods of preparing the isolated antigen binding proteins, pharmaceutical compositions, and uses of the isolated antigen binding proteins.

Description

Translated fromChinese

一种分离的抗原结合蛋白及其用途An isolated antigen-binding protein and its use

技术领域Technical field

本申请涉及生物医药领域，具体的涉及一种分离的抗原结合蛋白及其用途。This application relates to the field of biomedicine, specifically to an isolated antigen-binding protein and its use.

背景技术Background technique

肿瘤是一种严重威胁人类健康的疾病，近年来，免疫治疗作为一种新疗法，在肿瘤治疗中显示出了巨大的潜力。Tumor is a disease that seriously threatens human health. In recent years, immunotherapy, as a new therapy, has shown great potential in tumor treatment.

T细胞免疫球蛋白及黏蛋白分子3(T cell immunoglobulin and mucin-domaincontaining molecule 3，Tim-3)是近年来发现的重要免疫检查点分子，不同于其他“免疫检查点”分子，其主要诱导性表达于炎症和肿瘤微环境中的T细胞表面。它是一种I型膜表面分子，高表达于Th1细胞，并产生抑制信号从而导致Th1细胞的凋亡。在癌症与慢性病毒感染过程中，Tim-3与其配体半乳糖凝集素9(galectin 9，Gal-9)、磷脂酰丝氨酸(PtdSer)、高迁移率族蛋白1(high mobility group box 1 protein，HMGB1)、癌胚抗原相关细胞黏附分子1(carcino-embryonic antigen related cellular adhesion molecule 1，CEACAM1)相结合，继而引起T细胞的衰竭和凋亡，是导致肿瘤细胞发生免疫逃逸的重要原因之一。同时，选择性激活Tim-3从而导致免疫逃逸是PD-1/PD-L1抗体免疫治疗过程中发生耐药的主要机制。T cell immunoglobulin and mucin-domaincontaining molecule 3 (Tim-3) is an important immune checkpoint molecule discovered in recent years. Unlike other "immune checkpoint" molecules, its main inducible Expressed on the surface of T cells in inflammation and tumor microenvironments. It is a type I membrane surface molecule that is highly expressed in Th1 cells and produces inhibitory signals that lead to Th1 cell apoptosis. In the process of cancer and chronic viral infection, Tim-3 and its ligands galectin 9 (Gal-9), phosphatidylserine (PtdSer), high mobility group box 1 protein, HMGB1) and carcino-embryonic antigen related cellular adhesion molecule 1 (CEACAM1) combine to cause T cell exhaustion and apoptosis, which is one of the important reasons for immune escape of tumor cells. At the same time, selective activation of Tim-3 leading to immune evasion is the main mechanism of drug resistance during PD-1/PD-L1 antibody immunotherapy.

发明内容Contents of the invention

一方面，本申请提供一种分离的抗原结合蛋白，其包括重链可变区VH中的至少一个CDR和轻链可变区VL中的至少一个CDR，其中所述VH包含SEQ ID NO：16所示的氨基酸序列，所述VL包含SEQ ID NO：15所示的氨基酸序列。In one aspect, the application provides an isolated antigen-binding protein comprising at least one CDR in the heavy chain variable region VH and at least one CDR in the light chain variable region VL, wherein the VH comprises SEQ ID NO: 16 The amino acid sequence shown in SEQ ID NO: 15, the VL includes the amino acid sequence shown in SEQ ID NO: 15.

在某些实施方式中，本申请所述的分离的抗原结合蛋白具有Tim-3结合能力。In certain embodiments, an isolated antigen-binding protein described herein has Tim-3 binding ability.

在某些实施方式中，本申请所述的分离的抗原结合蛋白，其具有下述性质中的一种或多种：In certain embodiments, the isolated antigen-binding protein described in the present application has one or more of the following properties:

1)能够以10nM或更低的KD值结合源自人的Tim-3，其中所述KD值通过表面等离子体共振法测定；1) Able to bind human-derived Tim-3 with a KD value of 10 nM or less, wherein the KD value is determined by surface plasmon resonance;

2)在FACS测定中，能够特异性结合人的Tim-3而不结合小鼠的Tim-3；2) In FACS assay, it can specifically bind human Tim-3 but not mouse Tim-3;

3)在ELISA测定中，能够抑制Tim-3与PtdSer的结合；和3) Able to inhibit the binding of Tim-3 to PtdSer in ELISA assay; and

4)促进IFN-γ和/或TNF-α的分泌。4) Promote the secretion of IFN-γ and/or TNF-α.

根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。The isolated antigen-binding protein according to any one of claims 1-3, which includes an antibody or an antigen-binding fragment thereof.

在某些实施方式中，所述抗体包括人源化和鼠源抗体。In certain embodiments, the antibodies include humanized and murine antibodies.

在某些实施方式中，所述抗原结合片段包括Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb。In certain embodiments, the antigen-binding fragments include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.

在某些实施方式中，所述VH包含HCDR1，HCDR2和HCDR3，其中所述HCDR3包含SEQ IDNO：6所示的氨基酸序列。In certain embodiments, the VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6.

在某些实施方式中，所述HCDR1包含SEQ ID NO：4所示的氨基酸序列。In certain embodiments, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:4.

在某些实施方式中，所述HCDR2包含SEQ ID NO：5所示的氨基酸序列。In certain embodiments, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5.

在某些实施方式中，所述VL包含LCDR1，LCDR2和LCDR3，其中所述LCDR1包含SEQ IDNO：1所示的氨基酸序列。In certain embodiments, the VL comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1.

在某些实施方式中，所述LCDR2包含SEQ ID NO：2所示的氨基酸序列。In certain embodiments, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2.

在某些实施方式中，所述LCDR3包含SEQ ID NO：3所示的氨基酸序列。In certain embodiments, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:3.

在某些实施方式中，本申请所述的分离的抗原结合蛋白与参比抗体竞争结合所述Tim-3蛋白，其中所述参比抗体包含轻链可变区和重链可变区，所述参比抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO：1所示的氨基酸序列；所述LCDR2包含SEQ ID NO：2所示的氨基酸序列；所述LCDR3包含SEQ ID NO：3所示的氨基酸序列，所述参比抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO：4所示的氨基酸序列；所述HCDR2包含SEQ ID NO：5所示的氨基酸序列；所述HCDR3包含SEQ ID NO：6所示的氨基酸序列。In certain embodiments, an isolated antigen-binding protein described herein competes with a reference antibody for binding to the Tim-3 protein, wherein the reference antibody includes a light chain variable region and a heavy chain variable region, so The light chain variable region of the reference antibody includes LCDR1, LCDR2 and LCDR3. The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 1; the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 2; the LCDR3 Contains the amino acid sequence shown in SEQ ID NO: 3, the heavy chain variable region of the reference antibody includes HCDR1, HCDR2 and HCDR3, the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 4; the HCDR2 includes SEQ The amino acid sequence shown in ID NO: 5; the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 6.

在某些实施方式中，所述VL包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。In certain embodiments, the VL includes framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

在某些实施方式中，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO：7所示的氨基酸序列。In certain embodiments, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 includes the amino acid sequence shown in SEQ ID NO: 7.

在某些实施方式中，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：8所示的氨基酸序列。In certain embodiments, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 includes the amino acid sequence shown in SEQ ID NO: 8.

在某些实施方式中，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：9所示的氨基酸序列。In certain embodiments, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 9.

在某些实施方式中，所述L-FR4的N末端与所述LCDR3的C末端直接或间接相连，且所述L-FR4包含SEQ ID NO：10所示的氨基酸序列。In certain embodiments, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 10.

在某些实施方式中，所述VL包含SEQ ID NO：15所示的氨基酸序列。In certain embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 15.

在某些实施方式中，本申请所述的分离的抗原结合蛋白包括抗体轻链恒定区，且所述抗体轻链恒定区包括人Igκ恒定区。In certain embodiments, an isolated antigen-binding protein described herein includes an antibody light chain constant region, and the antibody light chain constant region includes a human Igκ constant region.

在某些实施方式中，所述抗体轻链恒定区包含SEQ ID NO：17中所示的氨基酸序列。In certain embodiments, the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 17.

在某些实施方式中，本申请所述的分离的抗原结合蛋白包含抗体轻链LC，且所述LC包含SEQ ID NO：19所示的氨基酸序列。In certain embodiments, the isolated antigen-binding protein described herein comprises an antibody light chain LC, and the LC comprises the amino acid sequence set forth in SEQ ID NO: 19.

在某些实施方式中，所述VH包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。In certain embodiments, the VH includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

在某些实施方式中，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO：11所示的氨基酸序列。In certain embodiments, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 includes the amino acid sequence shown in SEQ ID NO: 11.

在某些实施方式中，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：12所示的氨基酸序列。In certain embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 12.

在某些实施方式中，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：13所示的氨基酸序列。In certain embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 13.

在某些实施方式中，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO：14所示的氨基酸序列。In certain embodiments, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 14.

在某些实施方式中，所述VH包含SEQ ID NO：16所示的氨基酸序列。In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 16.

在某些实施方式中，本申请所述的分离的抗原结合蛋白包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG恒定区。In certain embodiments, an isolated antigen-binding protein described herein includes an antibody heavy chain constant region, and the antibody heavy chain constant region includes a human IgG constant region.

在某些实施方式中，所述抗体重链恒定区包含SEQ ID NO：18中所示的氨基酸序列。In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 18.

在某些实施方式中，本申请所述的分离的抗原结合蛋白包含抗体重链HC，且所述HC包含SEQ ID NO：20所示的氨基酸序列。In certain embodiments, the isolated antigen-binding protein described herein comprises an antibody heavy chain HC, and the HC comprises the amino acid sequence set forth in SEQ ID NO: 20.

另一方面，本申请还提供分离的一种或多种核酸分子，其编码本申请所述的分离的抗原结合蛋白。In another aspect, the present application also provides isolated one or more nucleic acid molecules encoding the isolated antigen-binding proteins described herein.

另一方面，本申请还提供载体，其包含本申请所述的核酸分子，或，表达本申请所述的抗原结合蛋白。On the other hand, the present application also provides a vector, which contains the nucleic acid molecule described in the present application, or expresses the antigen-binding protein described in the present application.

另一方面，本申请还提供细胞，其包含本申请所述的核酸分子或本申请所述的载体。On the other hand, the present application also provides cells comprising the nucleic acid molecules described in the present application or the vectors described in the present application.

另一方面，本申请还提供制备本申请所述的分离的抗原结合蛋白的方法，所述方法包括在使得本申请所述的分离的抗原结合蛋白表达的条件下，培养本申请所述的细胞。On the other hand, the present application also provides a method for preparing the isolated antigen-binding protein described in the present application, the method comprising culturing the cells described in the present application under conditions that allow the expression of the isolated antigen-binding protein described in the present application. .

另一方面，本申请还提供药物组合物，其包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载体。On the other hand, the present application also provides a pharmaceutical composition comprising the isolated antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application and/or the cell described in the present application, and Optionally a pharmaceutically acceptable carrier.

另一方面，本申请还提供所述的分离的抗原结合蛋白、所述的核酸分子、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/或治疗肿瘤。On the other hand, this application also provides the use of the isolated antigen-binding protein, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition in the preparation of medicines, Medications are used to prevent, alleviate, and/or treat tumors.

在某些实施方式中，所述肿瘤包括实体瘤和/或血液肿瘤。In certain embodiments, the tumors include solid tumors and/or hematological tumors.

另一方面，本申请还提供所述分离的抗原结合蛋白、所述的核酸分子、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Tim-3相关的疾病。On the other hand, this application also provides the use of the isolated antigen-binding protein, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in the preparation of medicines. For the prevention, mitigation and/or treatment of Tim-3 related diseases.

另一方面，本申请还提供抑制Tim-3与PtdSer结合的方法，所述方法包括施用本申请所述的分离的抗原结合蛋白。On the other hand, the present application also provides a method of inhibiting the binding of Tim-3 to PtdSer, the method comprising administering the isolated antigen-binding protein described in the present application.

另一方面，本申请还提供预防、缓解和/或治疗肿瘤的方法，所述方法包括向有需要的受试者施用本申请所述的分离的抗原结合蛋白或本申请所述的药物组合物。On the other hand, the present application also provides a method for preventing, alleviating and/or treating tumors, the method comprising administering to a subject in need thereof an isolated antigen-binding protein described herein or a pharmaceutical composition described herein .

在某些实施方式中，所述肿瘤包括实体瘤和/或血液肿瘤。In certain embodiments, the tumors include solid tumors and/or hematological tumors.

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 will readily appreciate other aspects and advantages of the present application from the detailed description below. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will realize, the contents of this application enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention covered by this application. Accordingly, the drawings and descriptions of the present application are illustrative only and not 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。对附图简要说明书如下：The specific 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 by reference to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

图1显示本申请所述的分离的抗原结合蛋白Z1与表达人Tim-3细胞株的结合情况；Figure 1 shows the binding situation between the isolated antigen-binding protein Z1 described in the present application and the human Tim-3 expressing cell line;

图2显示本申请所述的分离的抗原结合蛋白Z1与表达小鼠Tim-3细胞株的结合情况；Figure 2 shows the binding of the isolated antigen-binding protein Z1 described in this application to the mouse Tim-3 expressing cell line;

图3显示对比例抗体与表达小鼠Tim-3细胞株的结合情况；Figure 3 shows the binding of the comparative example antibody to the mouse Tim-3 expressing cell line;

图4A-4C显示本申请所述的分离的抗原结合蛋白Z1阻断人Tim-3与配体PtdSer的结合情况。Figures 4A-4C show that the isolated antigen-binding protein Z1 described in the present application blocks the binding of human Tim-3 to the ligand PtdSer.

具体实施方式Detailed ways

本发明首次提供了一种分离的抗原结合蛋白，其包括重链可变区VH中的至少一个CDR和轻链可变区VL中的至少一个CDR，其中所述VH包含SEQ ID NO：16所示的氨基酸序列，所述VL包含SEQ ID NO：15所示的氨基酸序列。本申请所述分离的抗原结合蛋白能够以10nM或更低的KD值结合源自人的Tim-3，此外，其还能够特异性结合人的Tim-3而不结合小鼠的Tim-3。另外，本申请所述分离的抗原结合蛋白能够抑制Tim-3与PtdSer的结合，还可以促进IFN-γ和/或TNF-α的分泌。The present invention provides for the first time an isolated antigen-binding protein, which includes at least one CDR in the heavy chain variable region VH and at least one CDR in the light chain variable region VL, wherein the VH includes SEQ ID NO: 16 The amino acid sequence shown in SEQ ID NO: 15, the VL includes the amino acid sequence shown in SEQ ID NO: 15. The isolated antigen-binding protein described in the present application can bind to human-derived Tim-3 with a KD value of 10 nM or lower. In addition, it can also specifically bind to human Tim-3 without binding to mouse Tim-3. In addition, the isolated antigen-binding protein described in this application can inhibit the binding of Tim-3 to PtdSer, and can also promote the secretion of IFN-γ and/or TNF-α.

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 below with specific examples. Those familiar with this technology can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

以下对本申请做进一步描述：在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。The present application is further described below: In the present invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used in this article are terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

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 this application, the term "isolated" generally means obtained from the natural state by artificial means. If an "isolated" substance or ingredient occurs in nature, it may be that the natural environment in which it is located has changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called isolation. of. The term "isolated" does not exclude the admixture of artificial or synthetic substances or the presence of other impure substances which do not affect the activity of the substance.

在本申请中，术语“分离的抗原结合蛋白”通常指从天然状态下经人工手段获得的具有抗原结合能力的蛋白。该“分离的抗原结合蛋白”可以包含结合抗原的部分和任选地，允许抗原结合部分采用促进所述抗原结合部分结合抗原的构象的支架或构架部分。抗原结合蛋白可以包含例如抗体来源的蛋白支架或具有移植的CDR或CDR衍生物的备选蛋白支架或人工支架。此类支架包括，但不限于包含被引入例如以稳定抗原结合蛋白的三维结构的突变的抗体来源的支架以及包含例如生物相容性聚合物的完全合成的支架。参见例如Korndorfer等，2003，Proteins：Structure，Function，andBioinformatics，53(1)：121-129(2003)；Roque等，Biotechnol.Prog.20：639-654(2004)。此外，肽抗体模拟物(″PAMs″)以及利用纤连蛋白组分基于抗体模拟物的支架可以用作支架。In this application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability obtained from its natural state through artificial means. The "isolated antigen-binding protein" may comprise an antigen-binding portion and, optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding portion by the antigen-binding portion. The antigen-binding protein may comprise, for example, an antibody-derived protein scaffold or an alternative protein scaffold with grafted CDRs or CDR derivatives or an artificial scaffold. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing mutations introduced, eg, to stabilize the three-dimensional structure of the antigen-binding protein, as well as fully synthetic scaffolds containing, eg, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Additionally, peptide antibody mimetics ("PAMs") as well as antibody mimetics-based scaffolds utilizing fibronectin components can be used as scaffolds.

在本申请中，术语“KD”(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(K_dissoc)除以结合速率常数(K_assoc)所获得的值。使用结合速率常数(K_assoc)、解离速率常数(K_dissoc)和平衡解离常数(KD)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Tim-3)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，可以通过表面等离子体共振法测定所述K_D值，也可以通过Octet测定所述K_D值，也可以使用其他实验途径和仪器例如BIAcore(生物分子相互作用分析)测定(例如，可以从BIAcoreIntemationalAB，aGEHealthcarecompany，Uppsala，瑞典获得的仪器)。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。In this application, the term "KD" (similarly, "_KD " or "_KD ") generally refers to the "affinity constant" or "equilibrium dissociation constant" and refers to the value in a titration measurement at equilibrium, or by The value obtained by dividing the dissociation rate constant (K_dissoc ) by the association rate constant (K_assoc ). The association rate constant (K_assoc ), the dissociation rate constant (K_dissoc ), and the equilibrium dissociation constant (KD) are used to represent the binding protein (eg, an isolated antigen-binding protein described herein) to an antigen (eg, Tim-3). Binding affinity. 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 K_D value can be measured by surface plasmon resonance method, the K_D value can also be measured by Octet, or can be measured using other experimental approaches and instruments such as BIAcore (Biomolecule Interaction Analysis) (for example, it can be measured from Instruments obtained from BIAcore International AB, aGE Healthcare company, Uppsala, Sweden). Alternatively, the K_D value can be determined using KinExA (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, Idaho).

在本申请中，氨基酸或核苷酸序列的同源性可与术语“同一性”相互交换地使用。术语“同一性”通常是指配对的相同残基的百分比。“序列同一性百分数”是这样计算的：将两个进行最佳比对的序列在特定区域范围内进行比较；确定两个序列中出现相同碱基或氨基酸的位置的数量，以得到相匹配位置的数量；将这种位置的数量除以被比较区段中的位置总数，所得的商再乘以100。如上所述，用于确定同源性(或同一性)的程序在氨基酸-对-氨基酸的基础上比较被比对的序列，并且所述程序可对该比较设定严格性的不同水平(例如相同氨基酸、保守氨基酸置换等)。如本文中所使用的术语，如果所讨论的两个氨基酸各自属于相同的化学类别(即酸性、非极性/疏水性、不带电荷的极性和碱性)，则被认为是彼此的“保守置换”。非限制实例为，属于非极性氨基酸的两个不同的氨基酸将被认定是彼此的“保守置换”，即使这两个氨基酸不相同，而一方面为非极性氨基酸、与另一方面为碱性氨基酸时将不被认为是彼此的“保守置换”。Alberts、Johnson、Lewis、Raff、Roberts和Walter的《MolecularBi ology of the Cell》第4版(2002)第3.1栏，将氨基酸分成四个主要组别：酸性、非极性、不带电荷的极性和碱性。此分组在本发明的上下文中可用于确定特定氨基酸对所讨论的另一个氨基酸是否为保守取代的目的。上述主要组别可进一步再分类为例如小的非极性和大的非极性氨基酸、大的芳香族氨基酸等。术语“保守的氨基酸置换”还指对给定氨基酸残基的任一个氨基酸置换，其中该置换残基与给定的残基在化学上是如此的相近而在多肽功能(例如结合)结果方面无实质减少。In this application, amino acid or nucleotide sequence homology may be used interchangeably with the term "identity." The term "identity" generally refers to the percentage of identical residues in a pair. "Percent sequence identity" is calculated by comparing two optimally aligned sequences within a specific region; determining the number of positions where the same base or amino acid occurs in the two sequences to obtain matching positions number; divide the number of such positions by the total number of positions in the segment being compared, and multiply the quotient by 100. As noted above, programs used to determine homology (or identity) compare aligned sequences on an amino acid-for-amino acid basis, and the programs can set different levels of stringency for the comparison (e.g., Identical amino acids, conservative amino acid substitutions, etc.). As the term is used in this article, two amino acids in question are considered "to each other" if they each belong to the same chemical class (i.e., acidic, nonpolar/hydrophobic, uncharged polar, and basic). Conservative replacement". As a non-limiting example, two different amino acids that are non-polar amino acids will be considered "conservative substitutions" of each other even though the two amino acids are not identical and are non-polar on the one hand and basic on the other. Amino acids will not be considered "conservative substitutions" for each other. Column 3.1 of "Molecular Biology of the Cell" 4th Edition (2002) by Alberts, Johnson, Lewis, Raff, Roberts, and Walter divides amino acids into four main groups: acidic, nonpolar, and uncharged polar and alkaline. This grouping may be used in the context of the present invention for the purpose of determining whether a particular amino acid is a conservative substitution for another amino acid in question. The above major groups can be further subdivided into, for example, small non-polar and large non-polar amino acids, large aromatic amino acids, etc. The term "conservative amino acid substitution" also refers to any amino acid substitution for a given amino acid residue in which the substitution residue is so chemically close to the given residue that it has no consequences in terms of polypeptide function (e.g., binding). Substantial reduction.

在本申请中，术语“Tim-3”是“T细胞免疫球蛋白及黏蛋白分子3(T cellimmunoglobulin and mucin-domain containing molecule 3)”的缩写，也称作TIM-3，HAVCR2，KIM-3，TIMD3，和FLJ14428，其通常是指一种T辅助细胞I型特异性细胞表面蛋白，其调节巨噬细胞活化和炎性状况的严重程度。Tim-3还与癌症，特别是癌干细胞有关，其主要诱导性表达于炎症和肿瘤微环境中的T细胞表面，高表达于Th1细胞，并产生抑制信号从而导致Th1细胞的凋亡。在癌症与慢性病毒感染过程中，Tim-3与其配体半乳糖凝集素9(galectin 9，Gal-9)、磷脂酰丝氨酸(PtdSer)、高迁移率族蛋白1(high mobility groupbox 1 protein，HMGB1)、癌胚抗原相关细胞黏附分子1(carcino-embryonic antigenrelated cellular adhesion molecule 1，CEACAM1)相结合，继而引起T细胞的衰竭和凋亡，是导致肿瘤细胞发生免疫逃逸的重要原因之一。同时，选择性激活Tim-3从而导致免疫逃逸是PD-1/PD-L1抗体免疫治疗过程中发生耐药的主要机制。Tim-3可以包括人Tim-3的变体，同等型，物种同系物，和与Tim-3具有至少一个共同表位的类似物。例如，源自人的Tim-3的氨基酸序列如SEQ ID NO：25所示，源自小鼠的Tim-3的氨基酸序列如SEQ ID NO：26所示。In this application, the term "Tim-3" is the abbreviation of "T cell immunoglobulin and mucin-domain containing molecule 3 (T cell immunoglobulin and mucin-domain containing molecule 3)", also known as TIM-3, HAVCR2, KIM-3 , TIMD3, and FLJ14428, which generally refers to a T helper cell type I-specific cell surface protein that regulates macrophage activation and the severity of inflammatory conditions. Tim-3 is also related to cancer, especially cancer stem cells. It is mainly inducibly expressed on the surface of T cells in inflammation and tumor microenvironment. It is highly expressed on Th1 cells and produces inhibitory signals that lead to the apoptosis of Th1 cells. In the process of cancer and chronic viral infection, Tim-3 and its ligands galectin 9 (Gal-9), phosphatidylserine (PtdSer), high mobility group box 1 protein (HMGB1) ) and carcino-embryonic antigen related cellular adhesion molecule 1 (CEACAM1) combine, which in turn causes T cell exhaustion and apoptosis, which is one of the important reasons for immune escape of tumor cells. At the same time, selective activation of Tim-3 leading to immune evasion is the main mechanism of drug resistance during PD-1/PD-L1 antibody immunotherapy. Tim-3 can include variants, isoforms, species homologues, and analogs of human Tim-3 that share at least one common epitope with Tim-3. For example, the amino acid sequence of human-derived Tim-3 is shown in SEQ ID NO: 25, and the amino acid sequence of mouse-derived Tim-3 is shown in SEQ ID NO: 26.

在本申请中，术语“PtdSer”通常是指磷脂酰丝氨酸，为Tim-3的配体。Tim-3与PtdSer结合，会导致细胞凋亡或者引发自身免疫性疾病、过敏性疾病和病毒感染相关性疾病。In this application, the term "PtdSer" generally refers to phosphatidylserine, a ligand for Tim-3. The combination of Tim-3 and PtdSer can cause cell apoptosis or cause autoimmune diseases, allergic diseases and viral infection-related diseases.

在本申请中，术语“特异性结合”或“特异性的”通常指可测量的和可再现的相互作用，比如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况可决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标的抗体。在一个实施方案中，抗体结合无关靶标的程度小于抗体对靶标的结合的约10％，如例如通过放射免疫分析(RIA)测量的。例如，在本申请中，所述分离的抗原结合蛋白能够以10nM或更低的KD值与源自人的Tim-3相结合。又例如，所述分离的抗原结合蛋白能够特异性结合人的Tim-3而不结合小鼠的Tim-3。在某些实施方案中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在另一个实施方案中，特异性结合可以包括但不要求排他性地结合。In this application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can occur in a heterogeneous population of molecules, including biomolecules. The presence of the situation determines the presence of the target. For example, an antibody that specifically binds a target (which may be an epitope) is one that binds to that target with greater affinity, avidity, more readily, and/or for a greater duration than it binds to other targets. In one embodiment, the antibody binds the unrelated target to an extent that is less than about 10% of the antibody's binding to the target, as measured, for example, by radioimmunoassay (RIA). For example, in the present application, the isolated antigen-binding protein is capable of binding to human-derived Tim-3 with a KD value of 10 nM or less. For another example, the isolated antigen-binding protein can specifically bind to human Tim-3 but not to mouse Tim-3. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding may include, but does not require, exclusive binding.

在本申请中，术语“抑制”通常指降低细胞的生长速率或者细胞的数量。例如，本申请所述的分离的抗原结合蛋白，其能够抑制肿瘤生长和/或肿瘤细胞增殖。又例如，本申请所述的分离的抗原结合蛋白能够抑制Tim-3与PtdSer的结合。In this application, the term "inhibition" generally refers to reducing the growth rate of cells or the number of cells. For example, the isolated antigen-binding proteins described herein are capable of inhibiting tumor growth and/or tumor cell proliferation. For another example, the isolated antigen-binding protein described in the present application can inhibit the binding of Tim-3 to PtdSer.

在本申请中，术语“IFN-γ”是干扰素(IFN)的一种类型，IFN-γ是II型干扰素并且结合II型干扰素抗体。IFN-γ调控多种生物学功能，如抗病毒反应、细胞生长、免疫反应和肿瘤抑制。In this application, the term "IFN-γ" is a type of interferon (IFN), which is a type II interferon and binds type II interferon antibodies. IFN-γ regulates a variety of biological functions, such as antiviral responses, cell growth, immune responses, and tumor suppression.

在本申请中，术语“TNF-α”通常是指肿瘤坏死因子α(也称为恶病质素)，其是由许多细胞类型(包括应答内毒素或其他刺激的单核细胞和巨噬细胞)产生的天然存在的哺乳动物细胞因子。TNF-α是炎症性、免疫学和病理生理反应的主要介质(Grell，M.等人，(1995)Cell[细胞]，83：793-802)。在本申请中，TNF-α可以包括来自各种物种(例如人、小鼠和猴)的野生型TNF-α、TNF-α的多态性变体和TNF-α的功能等效物。In this application, the term "TNF-alpha" generally refers to tumor necrosis factor alpha (also known as cachexin), which is produced by many cell types, including monocytes and macrophages in response to endotoxins or other stimuli. of naturally occurring mammalian cytokines. TNF-α is a major mediator of inflammatory, immunological and pathophysiological responses (Grell, M. et al. (1995) Cell, 83:793-802). In this application, TNF-α may include wild-type TNF-α, polymorphic variants of TNF-α, and functional equivalents of TNF-α from various species (eg, humans, mice, and monkeys).

在本申请中，术语“肿瘤”通常指由异常细胞生长形成的赘生物或实体病变。在本申请中，肿瘤可以是实体瘤或血液瘤肿瘤。As used herein, the term "tumor" generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In this application, the tumor may be a solid tumor or a hematological tumor.

在本申请中，术语“可变结构域”通常指抗体重链或轻链的氨基末端结构域。重链和轻链的可变结构域可以分别称为“VH”和“VL”。这些结构域通常是抗体的变化最大的部分(相对于相同类型的其它抗体)，且包含抗原结合位点。In this application, the term "variable domain" generally refers to the amino-terminal domain of an antibody heavy or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL" respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

在本申请中，术语“可变”通常指在抗体之间可变结构域的某些区段在序列上存在很大差异的事实。V结构域介导抗原结合并决定特定抗体对其特定抗原的特异性。然而，可变性并非在整个可变结构域范围内均匀分布。相反，它集中在轻链和重链可变结构域中称为高变区(CDR或HVR)的三个区段中。可变结构域的更高度保守的部分称为框架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区，大多数采用β-折叠构型，通过三个CDR连接，其形成环形连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDR通过FR区紧密靠近地保持在一起，并且来自另一条链的CDR一同促进抗体的抗原结合位点的形成(参见Kabat et al，Sequences of Immunological Interest，Fifth Edition，NationalInstitute of Health，Bethesda，Md.(1991))。恒定结构域不直接参与抗体与抗原的结合，但显示出各种效应子功能，例如抗体参与抗体依赖性细胞毒性。In this application, the term "variable" generally refers to the fact that certain segments of the variable domains differ significantly in sequence between antibodies. The V domain mediates antigen binding and determines the specificity of a given antibody for its particular antigen. However, variability is not evenly distributed across the entire variable domain. Instead, it is concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). The variable domains of native heavy and light chains each contain four FR regions, most adopting a β-sheet configuration, connected by three CDRs, which form a circular link and in some cases form part of the β-sheet structure . The CDRs in each chain are held in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antibody's antigen-binding site (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health , Bethesda, Md. (1991)). The constant domain is not directly involved in the binding of the antibody to the antigen, but displays various effector functions, such as the involvement of the antibody in antibody-dependent cellular cytotoxicity.

在本申请中，术语“抗体”通常指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，为了本发明的目的，术语“抗体”也包括抗体片段，比如Fab、F(ab′)₂、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合Tim-3)的其它抗体片段。通常，这样的片段应当包括抗原结合结构域。基本的4链抗体单元是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般为约150,000道尔顿。每个L链通过一个共价二硫键与H链连接，而两个H链通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还具有规则间隔的链内二硫化桥键。每个H链在N末端具有可变结构域(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端具有可变结构域(VL)，在其另一端具有恒定结构域。VL与VH对应，且CL与重链的第一恒定结构域(CH1)相对应。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。VH和VL配对一起形成单个抗原结合位点。对于不同类别抗体的结构和性质，参见例如Basic and ClinicalImmunology，8th Edition，Daniel P.Sties，Abba I.Terr and Tristram G.Parsolw(eds)，Appleton & Lange，Norwalk，Conn.，1994，第71页和第6章。来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。取决于其重链(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。基于CH序列和功能方面的相对小的差异，将γ和α类进一步分成亚类，例如，人表达下述亚类：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1和IgK1。In this application, the term "antibody" generally refers to an immunoglobulin or fragment thereof or derivative thereof, encompassing any polypeptide including an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid , mutated and transplanted antibodies. Unless otherwise modified by the term "intact", as in "intact antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, F(ab')₂ , Fv, scFv, Fd, dAbs and other antibody fragments that retain antigen-binding functionality (e.g., specifically bind Tim-3). Typically, such fragments should include an antigen-binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of 5 basic heterotetramer units and another polypeptide called J chain, and contain 10 antigen-binding sites, while IgA antibodies include 2-5 that can combine with the J chain and polymerize to form a multivalent Combined basic 4-chain unit. For IgG, a 4-chain unit is typically about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, while the two H chains are connected to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isoforms. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see for example Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and Chapter 6. L chains from any vertebrate species can be classified into one of two distinct types, termed kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of their heavy chain (CH) constant domain, immunoglobulins can be divided into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains named alpha, delta, epsilon, gamma and mu respectively. The gamma and alpha classes are further divided into subclasses based on relatively small differences in CH sequence and function, for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgK1.

在本申请中，术语“CDR”通常指抗体可变结构域的区域，其序列是高度可变的和/或形成结构定义环。通常，抗体包括六个CDR；在VH中三个(HCDR1、HCDR2、HCDR3)，和在VL中三个(LCDR1、LCDR2、LCDR3)。在天然抗体中，HCDR3和LCDR3显示所述六个CDR的大多数多样性，并且特别地HCDR3被认为在赋予抗体的精细特异性方面起独特作用。参见，例如Xu etal，Immunity 13：37-45(2000)；Johnson and Wu，in Methods in Molecular Biology248：1-25(Lo，ed.，Human Press，Totowa，N.J.，2003)。实际上，仅由重链组成的天然存在的骆驼抗体在缺乏轻链的情况功能正常且稳定。参见，例如，Hamers-Casterman et al.，Nature 363：446-448(1993)；Sheriff et al，Nature Struct.Biol.3：733-736(1996)。In this application, the term "CDR" generally refers to a region of an antibody variable domain whose sequence is highly variable and/or forms a structurally defined loop. Typically, antibodies include six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). Among natural antibodies, HCDR3 and LCDR3 display most of the diversity of the six CDRs, and HCDR3 in particular is thought to play a unique role in conferring fine specificity to antibodies. See, eg, Xu et al., Immunity 13: 37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camel antibodies, consisting only of heavy chains, are functional and stable in the absence of light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

在本申请中，术语“FR”通常指抗体可变结构域的更高度保守的部分，其被称为框架区。通常，天然重链和轻链的可变结构域各自包含四个FR区，即在VH中四个(H-FR1，H-FR2，H-FR3，和H-FR4)，和在VL中四个(L-FR1，L-FR2，L-FR3，和L-FR4)。例如，本申请所述的分离的抗原结合蛋白的VL可以包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。本申请所述的分离的抗原结合蛋白的VH可以包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。In this application, the term "FR" generally refers to the more highly conserved portions of antibody variable domains, which are known as framework regions. Typically, the variable domains of native heavy and light chains each contain four FR regions, namely four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL. (L-FR1, L-FR2, L-FR3, and L-FR4). For example, the VL of an isolated antigen-binding protein described herein may include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4. The VH of the isolated antigen-binding protein described herein may include the framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

在本申请中，术语“抗原结合片段”通常指具有抗原结合活性的片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab)₂、Fv片段、F(ab’)₂，scFv，di-scFv和/或dAb。In this application, the term "antigen-binding fragment" generally refers to a fragment having antigen-binding activity. In this application, the antigen-binding fragment may include Fab, Fab', F(ab)₂ , Fv fragment, F(ab')₂ , scFv, di-scFv and/or dAb.

在本申请中，术语“竞争结合”通常指抗体或其片段通过对另一种抗体(例如，参比抗体)进行变构调节，干扰另一种抗体直接地或间接地结合靶标/抗原(例如，Tim-3)的能力。例如，在本申请中，所述分离的抗原结合蛋白可以与参比抗体竞争结合Tim-3。此外，抗体或其片段能够干扰另一个抗体或其片段结合靶标的程度，且因此无论其是否可以被认为是根据本发明的阻断或竞争，都可以使用竞争性结合试验来确定。一种特别合适的定量竞争试验使用基于FACS或基于AlphaScreen方法测量标记的(例如，His标记的、生物素化的或放射性标记的)抗体或其片段和另一抗体或其片段之间在结合靶标方面的竞争。通常，竞争抗体或其片段为例如一种下述的：在竞争试验中结合靶标，使得在试验期间和在第二抗体或其片段的存在下，本发明的分离的抗原结合蛋白的所记录的取代达到由以给定量存在的所检测的潜在阻断抗体或其片段得到的最大理论取代(例如，由需要被阻断的冷(例如，未标记的)抗体或其片段取代)的至多100％(例如，在基于FACS的竞争试验中)。优选地，竞争抗体或其片段具有在10％至100％之间，比如在50％至100％之间的所记录的取代。In this application, the term "competing binding" generally refers to an antibody or fragment thereof that interferes with the direct or indirect binding of another antibody (e.g., a reference antibody) to a target/antigen (e.g., a reference antibody) by allosterically modulating another antibody (e.g., a reference antibody). , Tim-3) ability. For example, in this application, the isolated antigen binding protein may compete with a reference antibody for binding to Tim-3. Furthermore, the extent to which an antibody or fragment thereof is able to interfere with the binding of another antibody or fragment thereof to a target, and therefore whether this can be considered blocking or competing according to the present invention, can be determined using competitive binding assays. A particularly suitable quantitative competition assay uses a FACS-based or AlphaScreen-based method to measure the binding to the target between a labeled (e.g., His-tagged, biotinylated, or radiolabeled) antibody or fragment thereof and another antibody or fragment thereof aspects of competition. Typically, the competing antibody or fragment thereof is, for example, one that binds the target in a competition assay such that during the assay and in the presence of a second antibody or fragment thereof, the recorded activity of the isolated antigen-binding protein of the invention is Displacement reaches up to 100% of the maximum theoretical displacement (e.g., by a cold (e.g., unlabeled) antibody or fragment thereof that needs to be blocked) by the detected potential blocking antibody or fragment thereof present in a given amount. (e.g., in FACS-based competition experiments). Preferably, the competing antibody or fragment thereof has between 10% and 100%, such as between 50% and 100% of the recorded substitutions.

在本申请中，术语“直接相连”与术语“间接相连”相对，术语“直接相连”通常是指直接连接。例如，所述直接相连可以为物质间没有间隔子而直接相连的情况。所述间隔子可以是连接子。例如，所述连接子可以为肽连接子。术语“间接相连”通常是指物质间不直接相连的情况。例如，所述间接相连可以为通过间隔子而连接的情况。例如，在本申请所述的分离的抗原结合蛋白中，所述L-FR1的C末端与所述LCDR1的N末端可以直接或间接相连。In this application, the term "directly connected" is contrasted with the term "indirectly connected," which generally refers to a direct connection. For example, the direct connection may be a case where substances are directly connected without a spacer. The spacer may be a linker. For example, the linker may be a peptide linker. The term "indirectly connected" generally refers to situations where substances are not directly connected. For example, the indirect connection may be a connection through a spacer. For example, in the isolated antigen-binding protein described in the present application, the C-terminus of the L-FR1 and the N-terminus of the LCDR1 can be directly or indirectly connected.

在本申请中，术语“分离的核酸分子”通常指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，或从其天然环境分离的或人工合成的类似物。In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of a nucleotide, deoxyribonucleotide or ribonucleotide, of any length, or analog thereof isolated from its natural environment or artificially synthesized.

在本申请中，术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。举例来说，载体包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed. The vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries are expressed in the host cell. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 phage and animal viruses, etc. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. Viruses (such as SV40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication site. Vectors may also contain components that facilitate entry into cells, such as viral particles, liposomes, or protein coats, but they are not the only ones.

在本申请中，术语“细胞”通常指可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物，其包括本发明所述的核酸分子或本发明所述的载体。细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。细胞可包括用本发明所述的载体在体外转染的细胞。细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，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。在某些实施方案中，细胞为哺乳动物细胞。在某些实施方案中，哺乳动物细胞为HEK293细胞。In this application, the term "cell" generally refers to an individual cell, cell line or cell culture that may be or has been the recipient of a subject's plasmid or vector, which includes a nucleic acid molecule as described herein or a nucleic acid molecule as described herein. Carrier. Cells can include the descendants of a single cell. Due to natural, accidental or intentional mutations, the offspring may not necessarily be identical (either in morphology of the total DNA complement or in genome) to the original parent cell. Cells may include cells transfected in vitro with the vectors of the invention. The cells can be bacterial cells (eg, E. coli), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells. In certain embodiments, the cells are mammalian cells. In certain embodiments, the mammalian cell is a HEK293 cell.

在本申请中，术语“药物组合物”通常指涉及适合施用于患者、优选人患者的组合物。例如，本申请所述的药物组合物，其可以包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载体。此外，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。In this application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, preferably a human patient. For example, the pharmaceutical composition described in the present application may comprise the isolated antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application and/or the cells described in the present application, and Optionally a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition may also contain one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. preparations. Acceptable ingredients of the composition are preferably non-toxic to the recipient at the doses and concentrations used. Pharmaceutical compositions of the invention include, but are not limited to, liquid, frozen and lyophilized compositions.

在本申请中，术语“药学上可接受的载体”通常指与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂等，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。As used herein, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc. that are compatible with drug administration, are generally safe, non-toxic, and is neither biologically nor otherwise undesirable.

在本申请中，术语“Tim-3相关的疾病”通常是指与表达Tim-3的细胞相关的疾病。例如癌症、自体免疫性疾病和过敏性疾病。在本申请中，Tim-3相关的疾病可以是肿瘤和/或白血病。In this application, the term "Tim-3-related disease" generally refers to a disease associated with cells expressing Tim-3. Examples include cancer, autoimmune diseases, and allergic diseases. In this application, Tim-3 related diseases may be tumors and/or leukemias.

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 a human or non-human animal including, but not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

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。In this application, the term "comprising" generally means the inclusion of explicitly specified features, but not the exclusion of other elements.

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 variation within the range of 0.5% to 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%.

分离的抗原结合蛋白isolated antigen binding protein

一方面，本申请提供一种分离的抗原结合蛋白，其包括重链可变区VH中的至少一个CDR和轻链可变区VL中的至少一个CDR，其中所述VH包含SEQ ID NO：16所示的氨基酸序列，所述VL包含SEQ ID NO：15所示的氨基酸序列。In one aspect, the application provides an isolated antigen-binding protein comprising at least one CDR in the heavy chain variable region VH and at least one CDR in the light chain variable region VL, wherein the VH comprises SEQ ID NO: 16 The amino acid sequence shown in SEQ ID NO: 15, the VL includes the amino acid sequence shown in SEQ ID NO: 15.

例如，在本申请中，所述分离的抗原结合蛋白可以包含氨基酸序列如SEQ ID NO：16所示的VH中的HCDR1。例如，在本申请中，所述分离的抗原结合蛋白可以包含氨基酸序列如SEQ ID NO：16所示的VH中的HCDR2。例如，在本申请中，所述分离的抗原结合蛋白可以包含氨基酸序列如SEQ ID NO：16所示的VH中的HCDR3。又例如，在本申请中，所述分离的抗原结合蛋白可以包含氨基酸序列如SEQ ID NO：15所示的VL中的LCDR1。例如，在本申请中，所述分离的抗原结合蛋白可以包含氨基酸序列如SEQ ID NO：15所示的VL中的LCDR2。例如，在本申请中，所述分离的抗原结合蛋白可以包含氨基酸序列如SEQ ID NO：15所示的VL中的LCDR3。For example, in the present application, the isolated antigen-binding protein may comprise HCDR1 in VH with an amino acid sequence as shown in SEQ ID NO: 16. For example, in the present application, the isolated antigen-binding protein may comprise HCDR2 in the VH with the amino acid sequence shown in SEQ ID NO: 16. For example, in the present application, the isolated antigen-binding protein may comprise HCDR3 in VH with an amino acid sequence as shown in SEQ ID NO: 16. For another example, in the present application, the isolated antigen-binding protein may comprise LCDR1 in VL whose amino acid sequence is as shown in SEQ ID NO: 15. For example, in the present application, the isolated antigen-binding protein may comprise LCDR2 in VL with an amino acid sequence as shown in SEQ ID NO: 15. For example, in the present application, the isolated antigen-binding protein may comprise LCDR3 in VL with an amino acid sequence as shown in SEQ ID NO: 15.

在本申请中，所述分离的抗原结合蛋白还包括与SEQ ID NO：16所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的重链可变区VH中的至少一个CDR，以及与SEQ ID NO：15所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的轻链可变区VL中的至少一个CDR。在本申请中，所述分离的抗原结合蛋白具有Tim-3结合能力。In this application, the isolated antigen-binding protein also includes at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence shown in SEQ ID NO: 16 Identity to at least one CDR in the heavy chain variable region VH, and at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, to the amino acid sequence shown in SEQ ID NO: 15 At least one CDR in the light chain variable region VL that is 99% identical. In the present application, the isolated antigen-binding protein has Tim-3 binding ability.

在本申请中，所述分离的抗原结合蛋白可以结合与SEQ ID NO：25所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的Tim-3片段内的表位。In the present application, the isolated antigen-binding protein can bind to the amino acid sequence shown in SEQ ID NO: 25 with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% Identity of the epitope within the Tim-3 fragment.

所述分离的抗原结合蛋白的性质Properties of the isolated antigen-binding protein

在本申请中，所述分离的抗原结合蛋白可以具有下述性质中的一种或多种：In this application, the isolated antigen-binding protein may have one or more of the following properties:

1)能够以10nM或更低的KD值结合源自人的Tim-3，其中所述KD值通过表面等离子体共振法测定；1) Able to bind human-derived Tim-3 with a KD value of 10 nM or less, wherein the KD value is determined by surface plasmon resonance;

2)在FACS测定中，能够特异性结合人的Tim-3而不结合小鼠的Tim-3；2) In FACS assay, it can specifically bind human Tim-3 but not mouse Tim-3;

3)在ELISA测定中，能够抑制Tim-3与PtdSer的结合；和3) Able to inhibit the binding of Tim-3 to PtdSer in ELISA assay; and

4)促进IFN-γ和/或TNF-α的分泌。4) Promote the secretion of IFN-γ and/or TNF-α.

在本申请中，所述分离的抗原结合蛋白能够以10nM或更低的KD值结合源自人的Tim-3，其中所述KD值可以通过表而等离子体共振法测定。例如，本申请所述的分离的抗原结合蛋白结合源自人的Tim-3的KD值可以为≤10nM、≤9.5nM、≤9nM、≤8.9nM、≤8.8nM、≤8.7nM、≤8.6nM、≤8.5nM、≤8.4nM、≤8.3nM、≤8.2nM、≤8.1nM、≤8nM、≤7.9nM、≤7.8nM、≤7.7nM、≤7.6nM、≤7.5nM、≤7.4nM、≤7.3nM、≤7.2nM、≤7.1nM、≤7nM、≤6.9nM、≤6.5nM、≤6nM、≤5.5nM、≤5nM、≤4.5nM、≤4nM、≤3.5nM、≤3nM、≤2.9nM、≤2.8nM、≤2.7nM、≤2.6nM、≤2.5nM、≤2.4nM、≤2.3nM、≤2.2nM、≤2.1nM、≤2Nm、≤1.5nM、≤1nM。In the present application, the isolated antigen-binding protein is capable of binding human-derived Tim-3 with a KD value of 10 nM or lower, wherein the KD value can be determined by surface plasmon resonance. For example, the KD value of the isolated antigen-binding protein described in the present application for binding to human-derived Tim-3 can be ≤10 nM, ≤9.5 nM, ≤9 nM, ≤8.9 nM, ≤8.8 nM, ≤8.7 nM, ≤8.6 nM , ≤8.5nM, ≤8.4nM, ≤8.3nM, ≤8.2nM, ≤8.1nM, ≤8nM, ≤7.9nM, ≤7.8nM, ≤7.7nM, ≤7.6nM, ≤7.5nM, ≤7.4nM, ≤7.3 nM, ≤7.2nM, ≤7.1nM, ≤7nM, ≤6.9nM, ≤6.5nM, ≤6nM, ≤5.5nM, ≤5nM, ≤4.5nM, ≤4nM, ≤3.5nM, ≤3nM, ≤2.9nM, ≤ 2.8nM, ≤2.7nM, ≤2.6nM, ≤2.5nM, ≤2.4nM, ≤2.3nM, ≤2.2nM, ≤2.1nM, ≤2Nm, ≤1.5nM, ≤1nM.

在本申请中，所述KD值还可以通过FACS、ELISA、竞争ELISA或BIACORE或KINEXA进行测定。In this application, the KD value can also be determined by FACS, ELISA, competitive ELISA or BIACORE or KINEXA.

在本申请中，所述分离的抗原结合蛋白能够特异性结合人的Tim-3而不结合小鼠的Tim-3，所述特异性结合可以通过FACS测定。例如，可以通过FACS测定中的半最大效应浓度(EC50)来反映本申请所述分离的抗原结合蛋白特异性结合人的Tim-3的情况，例如，半最大效应浓度(EC50)越低说明特异性结合越好。In the present application, the isolated antigen-binding protein is capable of specifically binding to human Tim-3 but not to mouse Tim-3, and the specific binding can be determined by FACS. For example, the half-maximum effect concentration (EC50) in the FACS assay can be used to reflect the specific binding of the isolated antigen-binding protein to human Tim-3. For example, the lower the half-maximum effect concentration (EC50), the lower the specificity. The better the sexual union.

在本申请中，所述人的Tim-3可以包含如SEQ ID NO：25所示的氨基酸序列，所述小鼠的Tim-3可以包含如SEQ ID NO：26所示的氨基酸序列。In the present application, the human Tim-3 may comprise the amino acid sequence shown in SEQ ID NO: 25, and the mouse Tim-3 may comprise the amino acid sequence shown in SEQ ID NO: 26.

在本申请中，所述分离的抗原结合蛋白能够抑制Tim-3与PtdSer的结合，所述抑制情况可以通过流式细胞仪来测定。In this application, the isolated antigen-binding protein can inhibit the binding of Tim-3 to PtdSer, and the inhibition can be measured by flow cytometry.

在本申请中，所述分离的抗原结合蛋白能够促进IFN-γ和/或TNF-α的分泌。例如，本申请所述分离的抗原结合蛋白使IFN-γ和/或TNF-α的数量增加。In the present application, the isolated antigen-binding protein is capable of promoting the secretion of IFN-γ and/or TNF-α. For example, the isolated antigen-binding proteins described herein increase the amount of IFN-γ and/or TNF-α.

所述分离的抗原结合蛋白的种类The type of isolated antigen-binding protein

在本申请中，所述分离的抗原结合蛋白可以包括抗体或其抗原结合片段。例如，本申请所述的分离的抗原结合蛋白可以包括但不限于重组抗体、单克隆抗体、人抗体、人源化抗体、嵌合抗体、双特异性抗体、单链抗体、双抗体、三抗体、四抗体、Fv片段、scFv片段、Fab片段、Fab′片段、F(ab′)2片段和骆驼化单结构域抗体。In this application, the isolated antigen-binding protein may include an antibody or an antigen-binding fragment thereof. For example, the isolated antigen-binding proteins described herein may include, but are not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, diabodies, tribodies , tetrabodies, Fv fragments, scFv fragments, Fab fragments, Fab′ fragments, F(ab′)2 fragments and camelized single domain antibodies.

在本申请中，所述抗体可以包括鼠源抗体。例如，制备所述鼠源抗体时可以用Tim-3注射试验对象(例如小鼠)，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在某些实施方案中，所述鼠源抗体或其抗原结合片段，还可以包含鼠源κ、λ链或其变体的轻链恒定区，或包含鼠源IgGl，IgG2，IgG3或IgG4或其变体的重链恒定区。In this application, the antibodies may include murine antibodies. For example, when preparing the murine antibody, a test subject (eg, a mouse) can be injected with Tim-3, and then hybridomas expressing antibodies with desired sequences or functional properties can be isolated. In certain embodiments, the murine antibody or antigen-binding fragment thereof may also comprise a light chain constant region of a murine kappa or lambda chain or a variant thereof, or a murine IgGl, IgG2, IgG3 or IgG4 or its variant. Variant heavy chain constant regions.

在本申请中，所述抗体可以为人源化抗体。换句话说，本申请所述的分离的抗原结合蛋白，其可以为免疫特异性结合至相关抗原(例如人类Tim-3)且包含基本上具有人类抗体的氨基酸序列的框架(FR)区及基本上具有非人类抗体的氨基酸序列的互补决定区(CDR)的抗体或其变异体、衍生物、类似物或片段。此处的“基本上”在CDR的情况下是指CDR的氨基酸序列与非人类抗体CDR的氨基酸序列至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一。所述人源化抗体基本上可以包含所有至少一个且通常两个可变域(Fab、Fab′、F(ab′)2、FabC、Fv)，其中所有或基本上所有CDR区对应于非人类免疫球蛋白(即抗体)的CDR区且所有或基本上所有框架区为具有人类免疫球蛋白共有序列的框架区。优选地，人源化抗体还包含至少一部分免疫球蛋白恒定区(例如，Fc)，通常为人类免疫球蛋白的恒定区。在一些实施例中，人源化抗体含有轻链以及重链的至少可变域。抗体还可包括重链的CH1、铰链、CH2、CH3及CH4区。在一些实施例中，人源化抗体仅含人源化轻链。在一些实施例中，人源化抗体仅含人源化重链。在特定实施例中，人源化抗体仅含轻链和/或人源化重链的人源化可变域。In this application, the antibody may be a humanized antibody. In other words, the isolated antigen-binding protein described in the present application can be immunospecifically bound to a related antigen (such as human Tim-3) and comprise a framework (FR) region that essentially has the amino acid sequence of a human antibody and a basic An antibody or a variant, derivative, analog or fragment thereof having a complementarity determining region (CDR) of an amino acid sequence of a non-human antibody. "Substantially" here in the context of a CDR means that the amino acid sequence of the CDR is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the non-human antibody CDR . The humanized antibody may comprise essentially all of at least one and typically two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to non-human The CDR regions and all or substantially all of the framework regions of an immunoglobulin (i.e., an antibody) are framework regions with human immunoglobulin consensus sequences. Preferably, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (eg, Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains a light chain and at least the variable domain of a heavy chain. Antibodies may also include the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only humanized light chains. In some embodiments, the humanized antibody contains only humanized heavy chains. In specific embodiments, the humanized antibody contains only the humanized variable domain of the light chain and/or the humanized heavy chain.

在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab)₂、Fv片段、F(ab’)₂，scFv，di-scFv和/或dAb。In this application, the antigen-binding fragment may include Fab, Fab', F(ab)₂ , Fv fragment, F(ab')₂ , scFv, di-scFv and/or dAb.

参比抗体reference antibody

在本申请中，所述分离的抗原结合蛋白可以与参比抗体竞争结合所述Tim-3蛋白，其中所述参比抗体可以包含轻链可变区和重链可变区，所述参比抗体的轻链可变区可以包含LCDR1、LCDR2和LCDR3，所述LCDR1可以包含SEQ ID NO：1所示的氨基酸序列；所述LCDR2可以包含SEQ ID NO：2所示的氨基酸序列；所述LCDR3可以包含SEQ ID NO：3所示的氨基酸序列，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ IDNO：4所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO：5所示的氨基酸序列；所述HCDR3可以包含SEQ ID NO：6所示的氨基酸序列。In the present application, the isolated antigen-binding protein can compete with a reference antibody for binding to the Tim-3 protein, wherein the reference antibody can comprise a light chain variable region and a heavy chain variable region, and the reference antibody can compete with a reference antibody for binding to the Tim-3 protein. The light chain variable region of the antibody may include LCDR1, LCDR2 and LCDR3. The LCDR1 may include the amino acid sequence shown in SEQ ID NO: 1; the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 2; the LCDR3 It may include the amino acid sequence shown in SEQ ID NO: 3, the heavy chain variable region of the reference antibody may include HCDR1, HCDR2 and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 4; the HCDR2 The HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 5; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6.

CDRCDR

在本申请中，对于所述分离的抗原结合蛋白的可变区序列(即所述VH或VL的序列)，可以根据Kabat定义或者Chothia定义来确定VH和VL序列中CDR区序列(参阅例如Kabat，“Sequences of Proteins of ImmunologicalInterest”，National Institutes ofHealth，Bethesda，Md.(1991)；A1-Lazikani etal.，J.Mol.Biol.273：927-948(1997)；以及Martin et al.，Proc.Natl.Acad.Sci.USA86：9268-9272(1989))。In this application, for the variable region sequence of the isolated antigen-binding protein (ie, the sequence of the VH or VL), the CDR region sequence in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition (see, for example, Kabat , "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989)).

在本申请中，对于所述分离的抗原结合蛋白的可变区序列(即所述VH或VL的序列)，还可以根据包含了Kabat定义和Chothia定义的Combined定义规则来确定VH和VL序列中CDR区序列。In this application, for the variable region sequence of the isolated antigen-binding protein (ie, the sequence of the VH or VL), the VH and VL sequences can also be determined according to the Combined definition rules that include the Kabat definition and the Chothia definition. CDR region sequence.

在本申请中，所述VH可以包含HCDR1，HCDR2和HCDR3，其中所述HCDR3可以包含SEQID NO：6所示的氨基酸序列。In the present application, the VH may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6.

在本申请中，所述HCDR3可以包含与SEQ ID NO：6所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the HCDR3 may comprise amino acids having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 6 sequence.

在本申请中，所述HCDR1可以包含SEQ ID NO：4所示的氨基酸序列。In the present application, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 4.

在本申请中，所述HCDR1可以包含与SEQ ID NO：4所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the HCDR1 may comprise amino acids having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 4 sequence.

在本申请中，所述HCDR2可以包含SEQ ID NO：5所示的氨基酸序列。In the present application, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5.

在本申请中，所述HCDR2可以包含与SEQ ID NO：5所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the HCDR2 may comprise amino acids having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 5 sequence.

例如，本申请所述的分离的抗原结合蛋白的HCDR1可包含SEQ ID NO：4所示的氨基酸序列，HCDR2可包含SEQ ID NO：5所示的氨基酸序列，HCDR3可包含SEQ ID NO：6所示的氨基酸序列。For example, HCDR1 of the isolated antigen-binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 4, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 5, and HCDR3 may include the amino acid sequence shown in SEQ ID NO: 6. The amino acid sequence shown.

在本申请中，所述VL可以包含LCDR1，LCDR2和LCDR3，其中所述LCDR1可以包含SEQID NO：1所示的氨基酸序列。In this application, the VL may include LCDR1, LCDR2 and LCDR3, wherein the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 1.

在本申请中，所述LCDR1可以包含与SEQ ID NO：1所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the LCDR1 may comprise amino acids having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 1 sequence.

在本申请中，所述LCDR2可以包含SEQ ID NO：2所示的氨基酸序列。In this application, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2.

在本申请中，所述LCDR2可以包含与SEQ ID NO：2所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the LCDR2 may comprise amino acids having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 2 sequence.

在本申请中，所述LCDR3可以包含SEQ ID NO：3所示的氨基酸序列。In this application, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3.

在本申请中，所述LCDR3可以包含与SEQ ID NO：3所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the LCDR3 may comprise amino acids having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 3 sequence.

例如，本申请所述的分离的抗原结合蛋白的LCDR1可包含SEQ ID NO：1所示的氨基酸序列，LCDR2可包含SEQ ID NO：2所示的氨基酸序列，LCDR3可包含SEQ ID NO：3所示的氨基酸序列。For example, LCDR1 of the isolated antigen-binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 1, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 2, and LCDR3 may include the amino acid sequence shown in SEQ ID NO: 3. The amino acid sequence shown.

又例如，本申请所述的分离的抗原结合蛋白的HCDR1可包含SEQ ID NO：4所示的氨基酸序列，HCDR2可包含SEQ ID NO：5所示的氨基酸序列，HCDR3可包含SEQ ID NO：6所示的氨基酸序列，且LCDR1可包含SEQ ID NO：1所示的氨基酸序列，LCDR2可包含SEQ ID NO：2所示的氨基酸序列，LCDR3可包含SEQ ID NO：3所示的氨基酸序列。For another example, HCDR1 of the isolated antigen-binding protein described in the present application may comprise the amino acid sequence shown in SEQ ID NO: 4, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5, and HCDR3 may comprise SEQ ID NO: 6 The amino acid sequence shown is, and LCDR1 can include the amino acid sequence shown in SEQ ID NO: 1, LCDR2 can include the amino acid sequence shown in SEQ ID NO: 2, and LCDR3 can include the amino acid sequence shown in SEQ ID NO: 3.

FRFR

在本申请中，所述分离的抗原结合蛋白的VL可以包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。In the present application, the VL of the isolated antigen-binding protein may include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

在本申请中，所述L-FR1可以包含SEQ ID NO：7所示的氨基酸序列。In the present application, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 7.

在本申请中，所述L-FR1可以包含与SEQ ID NO：7所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the L-FR1 may comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 7 amino acid sequence.

例如，所述L-FR1的C末端可以与所述LCDR1的N末端直接或间接相连，且所述L-FR1可以包含SEQ ID NO：7所示的氨基酸序列。For example, the C-terminus of the L-FR1 can be directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 can comprise the amino acid sequence shown in SEQ ID NO: 7.

在本申请中，所述L-FR2可以包含SEQ ID NO：8所示的氨基酸序列。In the present application, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 8.

在本申请中，所述L-FR2可以包含与SEQ ID NO：8所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the L-FR2 may comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 8 amino acid sequence.

例如，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2可以包含SEQ IDNO：8所示的氨基酸序列。For example, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 8.

在本申请中，所述L-FR3可以包含SEQ ID NO：9所示的氨基酸序列。In the present application, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 9.

在本申请中，所述L-FR3可以包含与SEQ ID NO：9所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the L-FR3 may comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 9 amino acid sequence.

例如，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3可以包含SEQ IDNO：9所示的氨基酸序列。For example, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 9.

在本申请中，所述L-FR4可以包含SEQ ID NO：10所示的氨基酸序列。In the present application, the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 10.

在本申请中，所述L-FR4可以包含与SEQ ID NO：10所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the L-FR4 may comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 10 amino acid sequence.

例如，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4可以包含SEQ IDNO：10所示的氨基酸序列。For example, the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 10.

又例如，本申请所述的分离的抗原结合蛋白的L-FR1可包含SEQ ID NO：7所示的氨基酸序列，L-FR2可包含SEQ ID NO：8所示的氨基酸序列，L-FR3可包含SEQ ID NO：9所示的氨基酸序列，L-FR4可包含SEQ ID NO：10所示的氨基酸序列。For another example, L-FR1 of the isolated antigen-binding protein described in the present application may comprise the amino acid sequence shown in SEQ ID NO: 7, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 8, and L-FR3 may Comprising the amino acid sequence shown in SEQ ID NO: 9, L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 10.

在本申请中，所述分离的抗原结合蛋白的所述VH可以包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。In the present application, the VH of the isolated antigen-binding protein may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

在本申请中，所述H-FR1可以包含SEQ ID NO：11所示的氨基酸序列。In the present application, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11.

在本申请中，所述H-FR1可以包含与SEQ ID NO：11所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the H-FR1 may comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 11 amino acid sequence.

例如，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1可以包含SEQ ID NO：11所示的氨基酸序列。For example, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11.

在本申请中，所述H-FR2可以包含SEQ ID NO：12所示的氨基酸序列。In the present application, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12.

在本申请中，所述H-FR2可以包含与SEQ ID NO：12所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the H-FR2 may comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 12 amino acid sequence.

例如，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2可以包含SEQ IDNO：12所示的氨基酸序列。For example, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12.

在本申请中，所述H-FR3可以包含SEQ ID NO：13所示的氨基酸序列。In the present application, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13.

在本申请中，所述H-FR3可以包含与SEQ ID NO：13所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the H-FR3 may comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 13 amino acid sequence.

例如，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3可以包含SEQ IDNO：13所示的氨基酸序列。For example, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13.

在本申请中，所述H-FR4可以包含SEQ ID NO：14所示的氨基酸序列。In the present application, the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14.

在本申请中，所述H-FR4可以包含与SEQ ID NO：14所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In the present application, the H-FR4 may comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 14 amino acid sequence.

例如，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4可以包含SEQ IDNO：14所示的氨基酸序列。For example, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14.

例如，本申请所述的分离的抗原结合蛋白的H-FR1可包含SEQ ID NO：11所示的氨基酸序列，H-FR2可包含SEQ ID NO：12所示的氨基酸序列，H-FR3可包含SEQ ID NO：13所示的氨基酸序列，H-FR4可包含SEQ ID NO：14所示的氨基酸序列。For example, H-FR1 of the isolated antigen-binding protein described in the present application may comprise the amino acid sequence shown in SEQ ID NO: 11, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12, and H-FR3 may comprise The amino acid sequence shown in SEQ ID NO: 13, H-FR4 may include the amino acid sequence shown in SEQ ID NO: 14.

又例如，本申请所述的分离的抗原结合蛋白的L-FR1可包含SEQ ID NO：7所示的氨基酸序列，L-FR2可包含SEQ ID NO：8所示的氨基酸序列，L-FR3可包含SEQ ID NO：9所示的氨基酸序列，L-FR4可包含SEQ ID NO：10所示的氨基酸序列，且H-FR1可包含SEQ ID NO：11所示的氨基酸序列，H-FR2可包含SEQ ID NO：12所示的氨基酸序列，H-FR3可包含SEQ IDNO：13所示的氨基酸序列，H-FR4可包含SEQ ID NO：14所示的氨基酸序列。For another example, L-FR1 of the isolated antigen-binding protein described in the present application may comprise the amino acid sequence shown in SEQ ID NO: 7, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 8, and L-FR3 may Comprising the amino acid sequence shown in SEQ ID NO: 9, L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 10, and H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11, H-FR2 may comprise The amino acid sequence shown in SEQ ID NO: 12, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 13, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 14.

VL和VHVL and VH

本申请所述的分离的抗原结合蛋白可包含抗体轻链可变区VL和抗体重链可变区VH。例如，所述VL可包含SEQ ID NO：15所示的氨基酸序列，所述VH可包含SEQ ID NO：16所示的氨基酸序列。The isolated antigen-binding proteins described herein may comprise an antibody light chain variable region VL and an antibody heavy chain variable region VH. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO: 15, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 16.

例如，所述VL可包含SEQ ID NO：15所示的氨基酸序列，且所述VH可包含SEQ IDNO：16所示的氨基酸序列。For example, the VL may comprise the amino acid sequence shown in SEQ ID NO: 15, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 16.

轻链和重链light chain and heavy chain

在本申请中，所述分离的抗原结合蛋白可以包括抗体轻链恒定区，且所述抗体轻链恒定区可以包括人Igκ恒定区。In the present application, the isolated antigen binding protein may comprise an antibody light chain constant region, and the antibody light chain constant region may comprise a human Igκ constant region.

在本申请中，所述分离的抗原结合蛋白可以包括抗体轻链恒定区，且所述抗体轻链恒定区可以包括人Igλ恒定区。In this application, the isolated antigen binding protein may comprise an antibody light chain constant region, and the antibody light chain constant region may comprise a human Igλ constant region.

在本申请中，所述抗体轻链恒定区可以包含SEQ ID NO：17所示的氨基酸序列。In the present application, the antibody light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 17.

在本申请中，所述抗体轻链恒定区可以包含与SEQ ID NO：17所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In this application, the antibody light chain constant region may comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence shown in SEQ ID NO: 17 Identity of the amino acid sequence.

在本申请中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG重链恒定区。在某些实施方式中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG1重链恒定区。在某些实施方式中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG2重链恒定区。在某些实施方式中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG3重链恒定区。在某些实施方式中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG4重链恒定区。In the present application, the isolated antigen-binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In certain embodiments, the isolated antigen binding protein can include an antibody heavy chain constant region, and the antibody heavy chain constant region can be derived from a human IgG1 heavy chain constant region. In certain embodiments, the isolated antigen binding protein can include an antibody heavy chain constant region, and the antibody heavy chain constant region can be derived from a human IgG2 heavy chain constant region. In certain embodiments, the isolated antigen binding protein can include an antibody heavy chain constant region, and the antibody heavy chain constant region can be derived from a human IgG3 heavy chain constant region. In certain embodiments, the isolated antigen binding protein can include an antibody heavy chain constant region, and the antibody heavy chain constant region can be derived from a human IgG4 heavy chain constant region.

在本申请中，所述抗体重链恒定区可以包含SEQ ID NO：18所示的氨基酸序列。In this application, the antibody heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 18.

在本申请中，所述抗体重链恒定区可以包含与SEQ ID NO：18所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In this application, the antibody heavy chain constant region may comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence shown in SEQ ID NO: 18 Identity of the amino acid sequence.

在本申请中，所述分离的抗原结合蛋白可以包含抗体轻链LC，且所述LC可以包含SEQ ID NO：19所示的氨基酸序列。In the present application, the isolated antigen-binding protein may comprise an antibody light chain LC, and the LC may comprise the amino acid sequence shown in SEQ ID NO: 19.

在本申请中，所述LC可以包含与SEQ ID NO：19所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In this application, the LC may comprise amino acids having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 19 sequence.

在本申请中，所述分离的抗原结合蛋白可以包含抗体重链HC，且所述HC可以包含SEQ ID NO：20所示的氨基酸序列。In the present application, the isolated antigen-binding protein may comprise an antibody heavy chain HC, and the HC may comprise the amino acid sequence shown in SEQ ID NO: 20.

在本申请中，所述HC可以包含与SEQ ID NO：20所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。In this application, the HC may comprise an amino acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 20 sequence.

本申请所述的分离的抗原结合蛋白可以包含抗体轻链和抗体重链。The isolated antigen-binding proteins described herein may comprise an antibody light chain and an antibody heavy chain.

例如，所述轻链可包含SEQ ID NO：19所示的氨基酸序列，且所述重链可包含SEQID NO：20所示的氨基酸序列。For example, the light chain may comprise the amino acid sequence shown in SEQ ID NO: 19, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 20.

在本申请中，所述分离的抗原结合蛋白的轻链可包含SEQ ID NO：19所示的氨基酸序列，且重链可包含SEQ ID NO：20所示的氨基酸序列。其中，所述分离的抗原结合蛋白的HCDR1可包含SEQ ID NO：4所示的氨基酸序列，HCDR2可包含SEQ ID NO：5所示的氨基酸序列，HCDR3可包含SEQ ID NO：6所示的氨基酸序列，且LCDR1可包含SEQ ID NO：1所示的氨基酸序列，LCDR2可包含SEQ ID NO：2所示的氨基酸序列，LCDR3可包含SEQ ID NO：3所示的氨基酸序列。其中，所述分离的抗原结合蛋白的L-FR1可包含SEQ ID NO：7所示的氨基酸序列，L-FR2可包含SEQ ID NO：8所示的氨基酸序列，L-FR3可包含SEQ ID NO：9所示的氨基酸序列，L-FR4可包含SEQ ID NO：10所示的氨基酸序列，且H-FR1可包含SEQ ID NO：11所示的氨基酸序列，H-FR2可包含SEQ ID NO：12所示的氨基酸序列，H-FR3可包含SEQ ID NO：13所示的氨基酸序列，H-FR4可包含SEQ ID NO：14所示的氨基酸序列。所述VL可包含SEQ ID NO：15所示的氨基酸序列，且所述VH可包含SEQ ID NO：16所示的氨基酸序列。例如，所述分离的抗原结合蛋白可以为Z1。In the present application, the light chain of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 19, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 20. Wherein, HCDR1 of the isolated antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 4, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 5, and HCDR3 may include the amino acid sequence shown in SEQ ID NO: 6 sequence, and LCDR1 may include the amino acid sequence shown in SEQ ID NO: 1, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 2, and LCDR3 may include the amino acid sequence shown in SEQ ID NO: 3. Wherein, L-FR1 of the isolated antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 7, L-FR2 may include the amino acid sequence shown in SEQ ID NO: 8, and L-FR3 may include SEQ ID NO. : The amino acid sequence shown in 9, L-FR4 may include the amino acid sequence shown in SEQ ID NO: 10, and H-FR1 may include the amino acid sequence shown in SEQ ID NO: 11, and H-FR2 may include SEQ ID NO: For the amino acid sequence shown in 12, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 13, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 14. The VL may comprise the amino acid sequence shown in SEQ ID NO: 15, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 16. For example, the isolated antigen binding protein can be Z1.

核酸分子、载体、细胞、制备方法和药物组合物Nucleic acid molecules, vectors, cells, preparation methods 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，其可以编码本申请所述的分离的抗原结合蛋白。本申请所述的分离的一种或多种核酸分子可以为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，或从其天然环境分离的或人工合成的类似物，但可以编码本申请所述的分离的抗原结合蛋白。此外，考虑到同一种氨基酸具有两个或更多个密码子，即密码子的简并性，因此，编码同一种本申请所述的分离的抗原结合蛋白的核酸分子不是唯一的。In another aspect, the present application also provides isolated one or more nucleic acid molecules that can encode the isolated antigen-binding proteins described herein. The isolated nucleic acid molecule(s) described herein may be an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs isolated from their natural environment or artificially synthesized , but may encode an isolated antigen-binding protein as described herein. In addition, considering that the same amino acid has two or more codons, that is, the degeneracy of codons, the nucleic acid molecule encoding the same isolated antigen-binding protein described in the present application is not unique.

另一方面，本申请还提供了载体，其可以包含本申请所述的核酸分子，或，表达本申请所述的抗原结合蛋白。所述载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。例如，载体可以包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。又例如，所述载体可以含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，所述载体还可以含有复制起始位点。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。On the other hand, the present application also provides a vector, which can comprise the nucleic acid molecule described in the present application, or express the antigen-binding protein described in the present application. The vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries are expressed in the host cell. For example, vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 phage; and Animal viruses, etc. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. Viruses (such as SV40). For another example, the vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain an origin of replication site. In addition, the vector may also include components that facilitate its entry into cells, such as virus particles, liposomes or protein coats, but is not limited to these substances.

另一方面，本申请还提供了细胞，其可以包含本申请所述的核酸分子或本申请所述的载体。所述细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。在某些实施方式中，所述细胞还可以包括用本发明所述的载体在体外转染的细胞。在某些实施方式中，所述细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，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。在某些实施方案中，所述细胞可以为哺乳动物细胞。在某些实施方案中，所述哺乳动物细胞可以为HEK293细胞。蛋白质的纯化分离方法可以为盐析法、等电点沉淀法、低温有机溶剂沉淀法、透析与超滤、凝胶过滤法、电泳法、离子交换层析法或亲和色谱法。On the other hand, the present application also provides cells, which may contain the nucleic acid molecules described in the present application or the vectors described in the present application. The cells may include progeny of a single cell. Due to natural, accidental or intentional mutations, the offspring may not necessarily be identical (either in morphology of the total DNA complement or in genome) to the original parent cell. In certain embodiments, the cells may also include cells transfected in vitro with the vectors of the invention. In certain embodiments, the cells can be bacterial cells (eg, E. coli), yeast cells, or other eukaryotic cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells or myeloma cells. In certain embodiments, the cells can be mammalian cells. In certain embodiments, the mammalian cells can be HEK293 cells. Protein purification and separation methods can be salting out, isoelectric precipitation, low-temperature organic solvent precipitation, dialysis and ultrafiltration, gel filtration, electrophoresis, ion exchange chromatography or affinity chromatography.

另一方面，本申请还提供了制备本申请所述的分离的抗原结合蛋白的方法，所述方法可以包括在使得本申请所述的分离的抗原结合蛋白表达的条件下，培养本申请所述的细胞。On the other hand, the present application also provides a method for preparing the isolated antigen-binding protein described in the present application. The method may include culturing the isolated antigen-binding protein described in the present application under conditions that allow expression of the isolated antigen-binding protein described in the present application. Cell.

在某些实施方式中，可以通过外源基因的诱导(例如IPTG诱导)，使本申请所述的分离的抗原结合蛋白进行表达。In certain embodiments, the isolated antigen-binding proteins described herein can be expressed through induction of exogenous genes (eg, IPTG induction).

另一方面，本申请还提供了药物组合物，其可以包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载体。On the other hand, the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application and/or the cell described in the present application. , and optionally a pharmaceutically acceptable carrier.

在某些实施方案中，所述药物组合物还可以包含一种或多种(安全有效量的)药学上可接受的载体，例如稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。In certain embodiments, the pharmaceutical composition may also include one or more (safe and effective amounts) pharmaceutically acceptable carriers, such as stabilizers, excipients, diluents, solubilizers, surfactants , emulsifiers and/or preservatives. Acceptable ingredients of the composition are preferably non-toxic to the recipient at the doses and concentrations used. Pharmaceutical compositions of the invention include, but are not limited to, liquid, frozen and lyophilized compositions.

在某些实施方案中，所述药学上可接受的载体可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。In certain embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with drug administration, are generally safe, non-toxic, and is neither biologically nor otherwise undesirable.

在某些实施方案中，所述药物组合物可以包含胃肠内施用和/或肠胃外施用途径，例如皮下、透皮、腔内、静脉内、动脉内、鞘内、瘤内、腹膜内、和/或鼻内施用或直接注射到组织中。例如，所述药物组合物可以通过输注或注射施用于患者或者受试者。在某些实施方案中，所述药物组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。在某些实施方案中，所述药物组合物可以不间断施用。所述不间断(或连续)施用可以通过患者佩戴的小泵系统来实现，以测量流入患者体内的治疗剂，如WO2015/036583所述。In certain embodiments, the pharmaceutical compositions may comprise intragastric and/or parenteral routes of administration, such as subcutaneous, transdermal, intracavitary, intravenous, intraarterial, intrathecal, intratumoral, intraperitoneal, and/or administered intranasally or injected directly into tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, administration of the pharmaceutical composition can be by various means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition can be administered without interruption. The uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the flow of therapeutic agent into the patient's body, as described in WO2015/036583.

用途和应用Purpose and application

另一方面，本申请还提供了本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物在制备药物中的用途，所述药物可以用于预防、缓解和/或治疗肿瘤。On the other hand, the present application also provides the isolated antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cells described in the present application and/or the medicine described in the present application. Use of the composition in the preparation of a medicament that can be used to prevent, alleviate and/or treat tumors.

另一方面，本申请还提供了本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物在制备药物中的用途，所述药物可以用于预防、缓解和/或治疗Tim-3相关的疾病。On the other hand, the present application also provides the isolated antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cells described in the present application and/or the medicine described in the present application. Use of the composition in the preparation of a medicament that can be used to prevent, alleviate and/or treat Tim-3 related diseases.

另一方面，本申请还提供了预防、缓解和/或治疗肿瘤的方法，所述方法可以包括向有需要的受试者施用本申请所述的分离的抗原结合蛋白。在本申请中，所述施用可以通过不同的方式进行，例如静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。In another aspect, the present application also provides methods for preventing, alleviating and/or treating tumors, which methods may comprise administering to a subject in need thereof an isolated antigen-binding protein described herein. In this application, the administration may be carried out by different means, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

另一方面，本申请还提供了预防、缓解和/或治疗Tim-3相关的疾病的方法，所述方法可以包括向有需要的受试者施用本申请所述的分离的抗原结合蛋白。在本申请中，所述施用可以通过不同的方式进行，例如静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。On the other hand, the present application also provides methods for preventing, alleviating and/or treating Tim-3 related diseases, which methods may include administering the isolated antigen-binding protein described in the present application to a subject in need thereof. In this application, the administration may be carried out by different means, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

另一方面，本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物，其可以用于预防、缓解或治疗肿瘤。On the other hand, the isolated antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application and/or the pharmaceutical composition described in the present application can be For the prevention, relief or treatment of tumors.

另一方面，本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物，其可以用于预防、缓解或治疗Tim-3相关的疾病。On the other hand, the isolated antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application and/or the pharmaceutical composition described in the present application can be For the prevention, relief or treatment of Tim-3 related diseases.

在本申请中，所述肿瘤可以包括实体瘤和/或血液肿瘤。例如，所述肿瘤可以为白血病。In this application, the tumor may include solid tumors and/or hematological tumors. For example, the tumor may be leukemia.

在本申请中，所述Tim-3相关的疾病可以包括与表达Tim-3的细胞相关的疾病。例如癌症、自体免疫性疾病和过敏性疾病。在本申请中，Tim-3相关的疾病可以是白血病和/或肿瘤。In this application, the Tim-3-related diseases may include diseases related to cells expressing Tim-3. Examples include cancer, autoimmune diseases, and allergic diseases. In this application, the Tim-3 related disease may be leukemia and/or tumor.

在本申请中，所述受试者可以包括人类和非人类动物。例如，所述受试者可以包括但不限于猫、狗、马、猪、牛、羊、兔、小鼠、大鼠或猴。In this application, the subjects may include humans and non-human animals. For example, the subject may include, but is not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

另一方面，本申请还提供了抑制Tim-3与PtdSer结合的方法，所述方法包括施用本申请所述的分离的抗原结合蛋白。在本申请中，所述施用可以通过不同的方式进行，例如静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。On the other hand, the present application also provides a method of inhibiting the binding of Tim-3 to PtdSer, the method comprising administering the isolated antigen-binding protein described in the present application. In this application, the administration may be carried out by different means, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

在某些实施方式中，本申请所述的分离的抗原结合蛋白还可以与一种或多种有效量的治疗剂给与所需要的对象，所述一种或多种有效量的治疗剂可以包括化疗剂、细胞毒制剂、免疫抑制剂、类固醇、止吐药、癌症疫苗、镇痛药或另一种抗体。In certain embodiments, the isolated antigen-binding proteins described herein can also be administered to a subject in need with one or more effective amounts of therapeutic agents, which can Includes chemotherapeutic agents, cytotoxic agents, immunosuppressants, steroids, antiemetics, cancer vaccines, analgesics, or another antibody.

在某些实施方式中，本申请所述的分离的抗原结合蛋白还可以与毒素融合，以产生免疫缀合物，从而在特异性结合表达Tim-3蛋白的细胞后，在该细胞上发挥细胞毒性活性，特异性杀死癌细胞。In certain embodiments, the isolated antigen-binding proteins described herein can also be fused with toxins to generate immunoconjugates, thereby exerting cellular effects on cells expressing the Tim-3 protein after specifically binding to the cells. Toxic activity, specifically kills cancer cells.

在某些实施方式中，本申请所述的分离的抗原结合蛋白还可以制成溶瘤病毒，从而通过细胞表面分子入侵到肿瘤细胞中，然后以在肿瘤细胞中过度表达的特异性受体为靶向，将病毒入侵到肿瘤细胞中并行使后续的各项功能。In certain embodiments, the isolated antigen-binding proteins described in the present application can also be made into oncolytic viruses, thereby invading tumor cells through cell surface molecules, and then using specific receptors overexpressed in tumor cells as Targeting, the virus invades tumor cells and performs subsequent functions.

在某些实施方式中，本申请所述的分离的抗原结合蛋白还可以与能够特异性结合其他抗原(即除了Tim-3之外)的抗体融合，以产生双特异性抗体，从而同时特异性结合两种不同的抗原，以达到更好的肿瘤治疗效果。In certain embodiments, the isolated antigen-binding proteins described herein can also be fused to antibodies capable of specifically binding other antigens (i.e., in addition to Tim-3) to generate bispecific antibodies that simultaneously specifically bind Combine two different antigens to achieve better tumor treatment effects.

本申请所述的分离的抗原结合蛋白至少具有一种下述的有益效果：The isolated antigen-binding protein described in the present application has at least one of the following beneficial effects:

1)本申请所述的分离的抗原结合蛋白能够以10nM或更低的KD值结合源自人的Tim-3，其中所述KD值通过表面等离子体共振法测定；1) The isolated antigen-binding protein described in the present application is capable of binding human-derived Tim-3 with a KD value of 10 nM or lower, wherein the KD value is determined by surface plasmon resonance method;

2)在FACS测定中，本申请所述的分离的抗原结合蛋白能够特异性结合人的Tim-3而不结合小鼠的Tim-3，从而具有种属特异性；2) In the FACS assay, the isolated antigen-binding protein described in the present application can specifically bind to human Tim-3 but not to mouse Tim-3, thus having species specificity;

3)在ELISA测定中，本申请所述的分离的抗原结合蛋白能够抑制Tim-3与PtdSer的结合，阻断Tim-3信号通路，从而抑制肿瘤细胞生长；3) In the ELISA assay, the isolated antigen-binding protein described in the present application can inhibit the binding of Tim-3 to PtdSer, block the Tim-3 signaling pathway, and thus inhibit the growth of tumor cells;

4)本申请所述的分离的抗原结合蛋白能够促进IFN-γ和/或TNF-α的分泌，从而可以产生免疫刺激，进而抑制或消除肿瘤。4) The isolated antigen-binding protein described in this application can promote the secretion of IFN-γ and/or TNF-α, thereby producing immune stimulation and thereby inhibiting or eliminating tumors.

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蛋白质分子、制备方法和用途等，而不用于限制本申请发明的范围。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。Without intending to be limited by any theory, the following examples are only to illustrate the protein molecules, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application. The examples do not include a detailed description of traditional methods, such as those used to construct vectors and plasmids, insert genes encoding proteins into such vectors and plasmids, or introduce plasmids into host cells.

实施例Example

实施例1.本申请所述的分离的抗原结合蛋白Z1的结构Example 1. Structure of the isolated antigen-binding protein Z1 described in the present application

本申请所述的分离的抗原结合蛋白Z1的轻链包含SEQ ID NO：19所示的氨基酸序列，且重链包含SEQ ID NO：20所示的氨基酸序列。其中，所述分离的抗原结合蛋白Z1的HCDR1包含SEQ ID NO：4所示的氨基酸序列，HCDR2包含SEQ ID NO：5所示的氨基酸序列，HCDR3包含SEQ ID NO：6所示的氨基酸序列，且LCDR1包含SEQ ID NO：1所示的氨基酸序列，LCDR2包含SEQ ID NO：2所示的氨基酸序列，LCDR3包含SEQ ID NO：3所示的氨基酸序列。此外，所述分离的抗原结合蛋白Z1的L-FR1包含SEQ ID NO：7所示的氨基酸序列，L-FR2包含SEQ ID NO：8所示的氨基酸序列，L-FR3包含SEQ ID NO：9所示的氨基酸序列，L-FR4包含SEQID NO：10所示的氨基酸序列，且H-FR1包含SEQ ID NO：11所示的氨基酸序列，H-FR2包含SEQID NO：12所示的氨基酸序列，H-FR3包含SEQ ID NO：13所示的氨基酸序列，H-FR4包含SEQID NO：14所示的氨基酸序列。此外，所述分离的抗原结合蛋白Z1的VL包含SEQ ID NO：15所示的氨基酸序列，且VH包含SEQ ID NO：16所示的氨基酸序列。The light chain of the isolated antigen-binding protein Z1 described in the present application includes the amino acid sequence shown in SEQ ID NO: 19, and the heavy chain includes the amino acid sequence shown in SEQ ID NO: 20. Wherein, HCDR1 of the isolated antigen-binding protein Z1 includes the amino acid sequence shown in SEQ ID NO: 4, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 5, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 6, And LCDR1 includes the amino acid sequence shown in SEQ ID NO: 1, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 2, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 3. In addition, L-FR1 of the isolated antigen-binding protein Z1 includes the amino acid sequence shown in SEQ ID NO:7, L-FR2 includes the amino acid sequence shown in SEQ ID NO:8, and L-FR3 includes SEQ ID NO:9 The amino acid sequence shown is that L-FR4 includes the amino acid sequence shown in SEQ ID NO: 10, H-FR1 includes the amino acid sequence shown in SEQ ID NO: 11, and H-FR2 includes the amino acid sequence shown in SEQ ID NO: 12, H-FR3 includes the amino acid sequence shown in SEQ ID NO: 13, and H-FR4 includes the amino acid sequence shown in SEQ ID NO: 14. In addition, the VL of the isolated antigen-binding protein Z1 includes the amino acid sequence shown in SEQ ID NO: 15, and the VH includes the amino acid sequence shown in SEQ ID NO: 16.

编码本申请所述的分离的抗原结合蛋白Z1的VL的核苷酸序列如SEQ ID NO：21所示，编码本申请所述的分离的抗原结合蛋白Z1的VH的核苷酸序列如SEQ ID NO：22所示，编码本申请所述的分离的抗原结合蛋白Z1的轻链恒定区的核苷酸序列如SEQ ID NO：23所示，编码本申请所述的分离的抗原结合蛋白Z1的重链恒定区的核苷酸序列如SEQ ID NO：24所示。The nucleotide sequence encoding the VL of the isolated antigen-binding protein Z1 described in the present application is shown in SEQ ID NO: 21, and the nucleotide sequence encoding the VH of the isolated antigen-binding protein Z1 described in the present application is shown in SEQ ID NO. As shown in NO: 22, the nucleotide sequence encoding the light chain constant region of the isolated antigen-binding protein Z1 described in the application is shown in SEQ ID NO: 23, and the nucleotide sequence encoding the isolated antigen-binding protein Z1 described in the application is shown in SEQ ID NO: 23. The nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO: 24.

实施例2.本申请所述的分离的抗原结合蛋白Z1与人Tim-3的结合亲和力检测Example 2. Detection of binding affinity between the isolated antigen-binding protein Z1 and human Tim-3 described in this application

2.1 Protein A的偶联2.1 Coupling of Protein A

以1x HBS-EP+(GE Healthcare#BR-1006-69)为运行缓冲液，流速10μl/min，在CM5的4个通道上偶联Protein A(Thermo Fisher #21181)：1)设置注入时间800s，将50mM NHS和200mM EDC以1∶1体积比混合后注入4个通道；2)用pH4.5的醋酸钠将Protein A稀释至20μg/ml，注入800s；3)注入1M乙醇胺800s，以封闭芯片表面剩余的活性羧基。封闭后继续用1xHBS-EP+缓冲液平衡仪器两小时，Protein A最终偶联量约为2000RU。Use 1x HBS-EP+ (GE Healthcare #BR-1006-69) as the running buffer, flow rate 10μl/min, couple Protein A (Thermo Fisher #21181) on the 4 channels of CM5: 1) Set the injection time to 800s, Mix 50mM NHS and 200mM EDC at a volume ratio of 1:1 and inject into 4 channels; 2) Dilute Protein A to 20μg/ml with sodium acetate at pH 4.5 and inject for 800s; 3) Inject 1M ethanolamine for 800s to seal the chip remaining active carboxyl groups on the surface. After blocking, continue to equilibrate the instrument with 1xHBS-EP+ buffer for two hours. The final coupling amount of Protein A is approximately 2000RU.

2.2动力学测试2.2 Kinetic test

设置多循环动力学模式，每个循环包括抗体的捕获、分析物的结合以及芯片的再生。将抗体均稀释至1μg/ml，以10μl/min的流速注入2、3、4通道40s，由预先偶联的ProteinA捕获各抗体，捕获量约为200RU。分别将人Tim-3-hIg Fc融合蛋白(即人的Tim-3蛋白，美国R&D system公司，货号2365-TM-050)依次按照0nM、1.25nM、2.5nM、5nM、10nM、20nM、40nM的浓度梯度注入四个通道，流速为30ul/min，设置注入时间180s，解离时间900s。其中，人的Tim-3蛋白的氨基酸序列如SEQ ID NO：25所示。最后以同样流速注入甘氨酸(10mM，pH1.5)30s，以再生芯片。Set up a multi-cycle kinetic mode, with each cycle including antibody capture, analyte binding, and chip regeneration. Dilute the antibodies to 1 μg/ml and inject them into channels 2, 3, and 4 at a flow rate of 10 μl/min for 40 seconds. Each antibody is captured by pre-coupled ProteinA, and the capture volume is approximately 200RU. The human Tim-3-hIg Fc fusion protein (i.e., human Tim-3 protein, American R&D system company, product number 2365-TM-050) was sequentially divided into 0nM, 1.25nM, 2.5nM, 5nM, 10nM, 20nM, and 40nM. Concentration gradient was injected into four channels, the flow rate was 30ul/min, the injection time was set to 180s, and the dissociation time was 900s. Among them, the amino acid sequence of human Tim-3 protein is shown in SEQ ID NO: 25. Finally, glycine (10mM, pH1.5) was injected at the same flow rate for 30s to regenerate the chip.

2.3数据分析2.3 Data analysis

用Biacore T200分析软件2.0对实验结果进行分析，1通道作为参比通道扣除，分析模型选用1∶1动力学拟合模型。The experimental results were analyzed using Biacore T200 analysis software 2.0. Channel 1 was subtracted as the reference channel, and the 1:1 kinetic fitting model was selected as the analysis model.

结果如表1所示。实验结果表明，本申请所述的分离的抗原结合蛋白Z1与人Tim-3有较好的结合。The results are shown in Table 1. Experimental results show that the isolated antigen-binding protein Z1 described in this application has good binding to human Tim-3.

表1抗原结合蛋白Z1与人Tim-3结合的结合亲和力Table 1 Binding affinity of antigen binding protein Z1 to human Tim-3

抗原结合蛋白antigen binding proteinK_assoc(1/Ms)K_assoc (1/Ms)K_dissoc(1/s)K_dissoc (1/s)KD(nM)KD(nM)同种型(Isotype)IsotypeZ1Z12.3×10⁴2.3×10⁴1.9×10^-41.9×^10-48.18.1IgG2b，κIgG2b,kappa

表1中，K_assoc：结合速率常数；K_dissoc：解离速率常数；KD：亲和常数，等于K_dissoc/K_assoc。In Table 1, K_assoc : association rate constant; K_dissoc : dissociation rate constant; KD: affinity constant, equal to K_dissoc /K_assoc .

实施例3.本申请所述的分离的抗原结合蛋白Z1与细胞表面Tim-3的结合检测Example 3. Detection of binding of the isolated antigen-binding protein Z1 described in this application to Tim-3 on the cell surface

通过流式细胞荧光分选技术(FACS)，使用iQue Screener流式仪(购自IntelliCyt公司)，用含有0.1％BSA的PBS作为缓冲液进行细胞表面靶抗原(Tim-3，包括源自人的Tim-3和源自小鼠的Tim-3)与抗体(即本申请所述的分离的抗原结合蛋白Z1或对比例抗体)结合亲和力检测，其中，人的Tim-3的氨基酸序列如SEQ ID NO：25所示，小鼠的Tim-3的氨基酸序列如SEQ ID NO：26所示。对比例抗体用Anti-mTim-3Ab表示，其为商用抗体(Rat IgG2aClone #215008，购自R&D)。具体检测过程如下：Cell surface target antigens (Tim-3, including human-derived Tim-3 and Tim-3 derived from mice) and the antibody (i.e., the isolated antigen-binding protein Z1 described in the present application or the comparative antibody) binding affinity detection, wherein the amino acid sequence of human Tim-3 is as SEQ ID NO: 25 is shown, and the amino acid sequence of mouse Tim-3 is shown in SEQ ID NO: 26. The comparative antibody is represented by Anti-mTim-3Ab, which is a commercial antibody (Rat IgG2aClone #215008, purchased from R&D). The specific detection process is as follows:

1.使用缓冲液配置浓度为1*10⁶cells/ml的靶细胞(即表达人Tim-3的转基因K562细胞，或表达小鼠Tim-3的转基因K562细胞)，加入96孔尖底板(corning 3894)，每孔30μl；1. Use buffer to prepare target cells (i.e., transgenic K562 cells expressing human Tim-3, or transgenic K562 cells expressing mouse Tim-3) with a concentration of 1*10⁶ cells/ml, and add them to a 96-well sharp bottom plate (corning 3894), 30 μl per well;

2.使用缓冲液配置检测抗体浓度为3μg/ml，并按3倍比稀释抗体，形成8个浓度梯度；2. Use buffer to configure the detection antibody concentration to 3 μg/ml, and dilute the antibody 3 times to form 8 concentration gradients;

3.将配置好的不同浓度的抗体按30μl/孔加入已铺好的靶细胞中混匀；3. Add 30 μl/well of prepared antibodies of different concentrations into the spread target cells and mix well;

4. 4℃冰箱孵育1小时；4. Incubate in 4℃ refrigerator for 1 hour;

5.每孔加150μl缓冲液，300g离心5分钟，弃上清后将细胞震松散；5. Add 150μl buffer to each well, centrifuge at 300g for 5 minutes, discard the supernatant and shake the cells loose;

6.重复步骤5；6. Repeat step 5;

7.使用缓冲液按1∶200配比配置荧光二抗(ab98593)，每孔30μl加入细胞中混匀，4°冰箱孵育30分钟；7. Use buffer to prepare fluorescent secondary antibody (ab98593) at a ratio of 1:200, add 30 μl per well to the cells, mix well, and incubate in the refrigerator at 4° for 30 minutes;

8.每孔加150μl缓冲液，300g离心5分钟，弃上清后将细胞震松散；8. Add 150μl buffer to each well, centrifuge at 300g for 5 minutes, discard the supernatant and shake the cells loose;

9.重复步骤8；9. Repeat step 8;

10.每孔加35μl缓冲液混匀后用流式仪器检测；10. Add 35μl buffer to each well and mix well before detecting with a flow cytometer;

11.用Graphpad软件对数据进行分析。11. Use Graphpad software to analyze the data.

流式亲和力结合实验的分析结果如图1-图3所示，其中，图1显示本申请所述的分离的抗原结合蛋白Z1与表达人Tim-3细胞株的结合情况，图2显示本申请所述的分离的抗原结合蛋白Z1与表达小鼠Tim-3细胞株的结合情况，图3显示对比例抗体与表达小鼠Tim-3细胞株的结合情况。此外，图1-图3中的IgG表示实验中的同种型对照抗体小鼠IgG2b，其为商用抗体(Mouse IgG2b Clone#133303，购自R&D)。The analysis results of the flow cytometry affinity binding experiment are shown in Figures 1 to 3. Figure 1 shows the binding of the isolated antigen-binding protein Z1 described in the present application to the human Tim-3 expressing cell line, and Figure 2 shows the present application. The binding of the isolated antigen-binding protein Z1 to the mouse Tim-3-expressing cell line, Figure 3 shows the binding of the comparative antibody to the mouse Tim-3-expressing cell line. In addition, IgG in Figures 1 to 3 represents the isotype control antibody mouse IgG2b in the experiment, which is a commercial antibody (Mouse IgG2b Clone #133303, purchased from R&D).

从图1-图3可以看出，抗原结合蛋白Z1具有特异性结合人Tim-3的生物学活性。As can be seen from Figures 1 to 3, antigen-binding protein Z1 has the biological activity of specifically binding human Tim-3.

实施例4.本申请所述的分离的抗原结合蛋白Z1阻断人Tim-3与PtdSer的结合检测Example 4. The isolated antigen-binding protein Z1 described in this application blocks the detection of binding of human Tim-3 to PtdSer

磷脂酰丝氨酸(PtdSer)是Tim-3的另一配体，通常暴露于凋亡细胞胞膜表面，与Tim-3IgV结构域结合，介导凋亡细胞吞噬，促进凋亡小体清除和树突状细胞(DCs)抗原交叉提呈。本申请将Jurkat细胞稀释成5×10⁵细胞/ml并加入5μM Camptothecin，在37℃共培养5小时以诱导细胞凋亡，表达磷脂酰丝氨酸。之后，用PBS加2％FSA洗涤2次。最后将凋亡的Jurkat细胞以每孔1×10⁵个加入96孔板。将本申请所述的分离的抗原结合蛋白Z1和同种型对照抗体小鼠IgG2b(Mouse IgG2b Clone #133303，购自R&D)用PBS稀释成20μg/ml，或Tim-3-hIg Fc融合蛋白(即人的Tim-3蛋白，美国R&D system公司，货号2365-TM-050)用PBS稀释成4μg/ml，并分别让Tim-3-hIg Fc融合蛋白单独或Z1和Tim-3-hIg Fc融合蛋白共同混合与上述细胞在室温中共培养30分钟。培养结束后，离心，再用PBS加2％FSA洗涤2次。加入二抗APC-Conjugated anti-hIgG Fc Ab(R&D系统，#FAB110A)100μl，洗涤3次后进行流式细胞上机测试。检测结果如图4A-4C所示，从图4A-4C可以看出，只有在本申请所述的分离的抗原结合蛋白Z1存在的情况下，Tim-3-hIg Fc融合蛋白(即人的Tim-3蛋白)测不到凋亡细胞表达的相应受体磷脂酰丝氨酸(PtdSer)，而同种型对照小鼠抗体则无此效果。故本申请所述的分离的抗原结合蛋白Z1可以阻断人Tim-3与PtdSer的结合。Phosphatidylserine (PtdSer) is another ligand of Tim-3, which is usually exposed on the surface of apoptotic cell membranes, binds to the Tim-3 IgV domain, mediates phagocytosis of apoptotic cells, and promotes the clearance of apoptotic bodies and dendrites. Antigen cross-presentation by DCs. In this application, Jurkat cells were diluted to 5×10⁵ cells/ml and 5 μM Camptothecin was added, and co-cultured at 37°C for 5 hours to induce apoptosis and express phosphatidylserine. Afterwards, wash twice with PBS plus 2% FSA. Finally, apoptotic Jurkat cells were added to the 96-well plate at 1×10⁵ per well. The isolated antigen-binding protein Z1 and isotype control antibody mouse IgG2b (Mouse IgG2b Clone #133303, purchased from R&D) described in this application were diluted to 20 μg/ml with PBS, or Tim-3-hIg Fc fusion protein ( That is, human Tim-3 protein, American R&D system company, product number 2365-TM-050) was diluted to 4 μg/ml with PBS, and the Tim-3-hIg Fc fusion protein was used alone or Z1 and Tim-3-hIg Fc were fused. The protein was mixed with the above cells and incubated at room temperature for 30 minutes. After the culture, centrifuge and wash twice with PBS plus 2% FSA. Add 100 μl of secondary antibody APC-Conjugated anti-hIgG Fc Ab (R&D Systems, #FAB110A), wash 3 times, and perform flow cytometry testing. The detection results are shown in Figures 4A-4C. It can be seen from Figures 4A-4C that only in the presence of the isolated antigen-binding protein Z1 described in this application, the Tim-3-hIg Fc fusion protein (i.e. human Tim -3 protein) cannot detect the corresponding receptor phosphatidylserine (PtdSer) expressed by apoptotic cells, while the isotype control mouse antibody has no such effect. Therefore, the isolated antigen-binding protein Z1 described in this application can block the binding of human Tim-3 to PtdSer.

前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。The foregoing detailed description is provided by way of explanation and example, and is not intended to limit the scope of the appended claims. Various modifications to the embodiments described herein will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

序列表 sequence list

<110> 上药生物治疗（香港）有限公司;上海医药集团生物治疗技术有限公司<110> Shanghai Pharmaceuticals Biotherapeutics (Hong Kong) Co., Ltd.; Shanghai Pharmaceutical Group Biotherapeutics Technology Co., Ltd.

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

<220><220>

<223> Z1 VL<223> Z1 VL

<400> 15<400> 15

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro AlaMet Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala

1 5 10 151 5 10 15

Ser Ser Ser Glu Val Phe Met Thr Gln Thr Pro Leu Ser Leu Pro ValSer Ser Ser Glu Val Phe Met Thr Gln Thr Pro Leu Ser Leu Pro Val

20 25 30 20 25 30

Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Leu IleSer Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Leu Ile

35 40 45 35 40 45

Val His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys ProVal His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro

50 55 60 50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe PheGly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Phe

65 70 75 8065 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrGly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95 85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr CysLeu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys

100 105 110 100 105 110

Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys LeuPhe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125 115 120 125

Glu Ile Lys ArgGlu Ile Lys Arg

130 130

<210> 16<210> 16

<211> 128<211> 128

<212> PRT<212> PRT

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

<220><220>

<223> Z1 VH<223> Z1 VH

<400> 16<400> 16

Met Lys Trp Ser Trp Val Ile Leu Phe Leu Met Ala Val Val Thr GlyMet Lys Trp Ser Trp Val Ile Leu Phe Leu Met Ala Val Val Thr Gly

1 5 10 151 5 10 15

Val Asn Ser Glu Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Val LysVal Asn Ser Glu Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Val Lys

20 25 30 20 25 30

Pro Gly Ala Ser Val Ile Leu Ser Cys Thr Ala Ser Gly Phe Asn IlePro Gly Ala Ser Val Ile Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile

35 40 45 35 40 45

Lys Val Ala Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly LeuLys Val Ala Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu

50 55 60 50 55 60

Glu Trp Ile Gly Ser Ile Asp Pro Ala His Gly Glu Thr Arg Tyr AspGlu Trp Ile Gly Ser Ile Asp Pro Ala His Gly Glu Thr Arg Tyr Asp

65 70 75 8065 70 75 80

Ser Lys Phe Gln Asp Lys Ala Thr Met Thr Ala Asp Pro Ser Ser AsnSer Lys Phe Gln Asp Lys Ala Thr Met Thr Ala Asp Pro Ser Ser Asn

85 90 95 85 90 95

Thr Ala Phe Leu Gln Leu Ile Ser Leu Thr Ser Glu Asp Thr Ala ValThr Ala Phe Leu Gln Leu Ile Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110 100 105 110

Tyr Tyr Cys Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser ValTyr Tyr Cys Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val

115 120 125 115 120 125

<210> 17<210> 17

<211> 107<211> 107

<212> PRT<212> PRT

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

<220><220>

<223> Z1轻链恒定区<223> Z1 light chain constant region

<400> 17<400> 17

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser GluArg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 151 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn PheGln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30 20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu ArgTyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45 35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp SerGln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60 50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr GluThr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 8065 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr SerArg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95 85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu CysPro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105 100 105

<210> 18<210> 18

<211> 404<211> 404

<212> PRT<212> PRT

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

<220><220>

<223> Z1重链恒定区<223> Z1 heavy chain constant region

<400> 18<400> 18

Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly AspLys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp

1 5 10 151 5 10 15

Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr PheThr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe

20 25 30 20 25 30

Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser SerPro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser

35 40 45 35 40 45

Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met SerVal His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met Ser

50 55 60 50 55 60

Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val ThrSer Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr

65 70 75 8065 70 75 80

Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys LeuCys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys Leu

85 90 95 85 90 95

Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys LysGlu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys Lys

100 105 110 100 105 110

Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser ValGlu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val

115 120 125 115 120 125

Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu ThrPhe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu Thr

130 135 140 130 135 140

Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro AspPro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp

145 150 155 160145 150 155 160

Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala GlnVal Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln

165 170 175 165 170 175

Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val SerThr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser

180 185 190 180 185 190

Thr Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe LysThr Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys

195 200 205 195 200 205

Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr IleCys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile

210 215 220 210 215 220

Ser Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu ProSer Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu Pro

225 230 235 240225 230 235 240

Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys LeuPro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys Leu

245 250 255 245 250 255

Val Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser AsnVal Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser Asn

260 265 270 260 265 270

Gly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp SerGly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp Ser

275 280 285 275 280 285

Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser LysAsp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser Lys

290 295 300 290 295 300

Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly LeuTrp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu

305 310 315 320305 310 315 320

Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Leu AspLys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Leu Asp

325 330 335 325 330 335

Leu Asp Asp Ile Cys Ala Glu Ala Lys Asp Gly Glu Leu Asp Gly LeuLeu Asp Asp Ile Cys Ala Glu Ala Lys Asp Gly Glu Leu Asp Gly Leu

340 345 350 340 345 350

Trp Thr Thr Ile Thr Ile Phe Ile Ser Leu Phe Leu Leu Ser Val CysTrp Thr Thr Ile Thr Ile Phe Ile Ser Leu Phe Leu Leu Ser Val Cys

355 360 365 355 360 365

Tyr Ser Ala Ser Val Thr Leu Phe Lys Val Lys Trp Ile Phe Ser SerTyr Ser Ala Ser Val Thr Leu Phe Lys Val Lys Trp Ile Phe Ser Ser

370 375 380 370 375 380

Val Val Glu Leu Lys Gln Lys Ile Ser Pro Asp Tyr Arg Asn Met IleVal Val Glu Leu Lys Gln Lys Ile Ser Pro Asp Tyr Arg Asn Met Ile

385 390 395 400385 390 395 400

Gly Gln Gly AlaGly Gln Gly Ala

<210> 19<210> 19

<211> 239<211> 239

<212> PRT<212> PRT

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

<220><220>

<223> Z1轻链<223> Z1 light chain

<400> 19<400> 19

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro AlaMet Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala

1 5 10 151 5 10 15

Ser Ser Ser Glu Val Phe Met Thr Gln Thr Pro Leu Ser Leu Pro ValSer Ser Ser Glu Val Phe Met Thr Gln Thr Pro Leu Ser Leu Pro Val

20 25 30 20 25 30

Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Leu IleSer Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Leu Ile

35 40 45 35 40 45

Val His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys ProVal His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro

50 55 60 50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe PheGly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Phe

65 70 75 8065 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrGly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95 85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr CysLeu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys

100 105 110 100 105 110

Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys LeuPhe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125 115 120 125

Glu Ile Lys Arg Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe ProGlu Ile Lys Arg Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro

130 135 140 130 135 140

Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys PhePro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe

145 150 155 160145 150 155 160

Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile AspLeu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp

165 170 175 165 170 175

Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln AspGly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp

180 185 190 180 185 190

Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr LysSer Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys

195 200 205 195 200 205

Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His LysAsp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys

210 215 220 210 215 220

Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu CysThr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235225 230 235

<210> 20<210> 20

<211> 532<211> 532

<212> PRT<212> PRT

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

<220><220>

<223> Z1重链<223> Z1 heavy chain

<400> 20<400> 20

Met Lys Trp Ser Trp Val Ile Leu Phe Leu Met Ala Val Val Thr GlyMet Lys Trp Ser Trp Val Ile Leu Phe Leu Met Ala Val Val Thr Gly

1 5 10 151 5 10 15

Val Asn Ser Glu Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Val LysVal Asn Ser Glu Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Val Lys

20 25 30 20 25 30

Pro Gly Ala Ser Val Ile Leu Ser Cys Thr Ala Ser Gly Phe Asn IlePro Gly Ala Ser Val Ile Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile

35 40 45 35 40 45

Lys Val Ala Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly LeuLys Val Ala Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu

50 55 60 50 55 60

Glu Trp Ile Gly Ser Ile Asp Pro Ala His Gly Glu Thr Arg Tyr AspGlu Trp Ile Gly Ser Ile Asp Pro Ala His Gly Glu Thr Arg Tyr Asp

65 70 75 8065 70 75 80

Ser Lys Phe Gln Asp Lys Ala Thr Met Thr Ala Asp Pro Ser Ser AsnSer Lys Phe Gln Asp Lys Ala Thr Met Thr Ala Asp Pro Ser Ser Asn

85 90 95 85 90 95

Thr Ala Phe Leu Gln Leu Ile Ser Leu Thr Ser Glu Asp Thr Ala ValThr Ala Phe Leu Gln Leu Ile Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110 100 105 110

Tyr Tyr Cys Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser ValTyr Tyr Cys Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val

115 120 125 115 120 125

Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly AspLys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp

130 135 140 130 135 140

Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr PheThr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe

145 150 155 160145 150 155 160

Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser SerPro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser

165 170 175 165 170 175

Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met SerVal His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met Ser

180 185 190 180 185 190

Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val ThrSer Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr

195 200 205 195 200 205

Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys LeuCys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys Leu

210 215 220 210 215 220

Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys LysGlu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys Lys

225 230 235 240225 230 235 240

Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser ValGlu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val

245 250 255 245 250 255

Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu ThrPhe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu Thr

260 265 270 260 265 270

Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro AspPro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp

275 280 285 275 280 285

Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala GlnVal Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln

290 295 300 290 295 300

Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val SerThr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser

305 310 315 320305 310 315 320

Thr Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe LysThr Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys

325 330 335 325 330 335

Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr IleCys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile

340 345 350 340 345 350

Ser Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu ProSer Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu Pro

355 360 365 355 360 365

Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys LeuPro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys Leu

370 375 380 370 375 380

Val Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser AsnVal Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser Asn

385 390 395 400385 390 395 400

Gly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp SerGly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp Ser

405 410 415 405 410 415

Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser LysAsp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser Lys

420 425 430 420 425 430

Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly LeuTrp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu

435 440 445 435 440 445

Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Leu AspLys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Leu Asp

450 455 460 450 455 460

Leu Asp Asp Ile Cys Ala Glu Ala Lys Asp Gly Glu Leu Asp Gly LeuLeu Asp Asp Ile Cys Ala Glu Ala Lys Asp Gly Glu Leu Asp Gly Leu

465 470 475 480465 470 475 480

Trp Thr Thr Ile Thr Ile Phe Ile Ser Leu Phe Leu Leu Ser Val CysTrp Thr Thr Ile Thr Ile Phe Ile Ser Leu Phe Leu Leu Ser Val Cys

485 490 495 485 490 495

Tyr Ser Ala Ser Val Thr Leu Phe Lys Val Lys Trp Ile Phe Ser SerTyr Ser Ala Ser Val Thr Leu Phe Lys Val Lys Trp Ile Phe Ser Ser

500 505 510 500 505 510

Val Val Glu Leu Lys Gln Lys Ile Ser Pro Asp Tyr Arg Asn Met IleVal Val Glu Leu Lys Gln Lys Ile Ser Pro Asp Tyr Arg Asn Met Ile

515 520 525 515 520 525

Gly Gln Gly AlaGly Gln Gly Ala

530 530

<210> 21<210> 21

<211> 396<211> 396

<212> DNA<212> DNA

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

<220><220>

<223> 编码 Z1 VL<223> Encoding Z1 VL

<400> 21<400> 21

atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagtagtgaa 60atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagtagtgaa 60

gtttttatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120gtttttatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120

tcttgcagat ctagtcagct cattgtacat agtaatggaa acacctattt acaatggtac 180tcttgcagat ctagtcagct cattgtacat agtaatggaa acacctattt acaatggtac 180

ctgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccaa tcgatttttt 240ctgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccaa tcgatttttt 240

ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300

agagtggagg ctgaggatct gggagtttat tactgctttc aaggatcaca tgttccgtac 360agagtggagg ctgaggatct gggagtttat tactgctttc aaggatcaca tgttccgtac 360

acgttcggag gggggaccaa gctggaaata aaacgg 396acgttcggag gggggaccaa gctggaaata aaacgg 396

<210> 22<210> 22

<211> 384<211> 384

<212> DNA<212> DNA

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

<220><220>

<223> 编码 Z1 VH<223> Encoding Z1 VH

<400> 22<400> 22

atgaaatgga gctgggttat tctcttcctg atggcagtgg ttacaggggt caattcagag 60atgaaatgga gctgggttat tctcttcctg atggcagtgg ttacaggggt caattcagag 60

gttcagctgc agcagtctgg gtcagagctt gtgaagccag gggcctcagt cattttgtcc 120gttcagctgc agcagtctgg gtcagagctt gtgaagccag gggcctcagt cattttgtcc 120

tgcacagctt ctggcttcaa cattaaagtc gcctatattc actgggtgaa gcagaggcct 180tgcacagctt ctggcttcaa cattaaagtc gcctatattc actgggtgaa gcagaggcct 180

gaacagggcc tggagtggat tggaagtatt gatcctgcgc atggtgaaac tagatatgac 240gaacagggcc tggagtggat tggaagtatt gatcctgcgc atggtgaaac tagatatgac 240

tcgaagttcc aggacaaggc cactatgaca gcggacccat cctccaacac agccttcctg 300tcgaagttcc aggacaaggc cactatgaca gcggacccat cctccaacac agccttcctg 300

cagctcatta gcctgacatc tgaggacact gccgtctatt actgtactac ggctatggac 360cagctcatta gcctgacatc tgaggacact gccgtctatt actgtactac ggctatggac 360

tactggggtc aaggaacctc agtc 384tactggggtc aaggaacctc agtc 384

<210> 23<210> 23

<211> 321<211> 321

<212> DNA<212> DNA

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

<220><220>

<223> 编码 Z1 轻链恒定区<223> Encoding Z1 light chain constant region

<400> 23<400> 23

cgcgcggatg cggcgccgac cgtgagcatt tttccgccga gcagcgaaca gctgaccagc 60cgcgcggatg cggcgccgac cgtgagcatt tttccgccga gcagcgaaca gctgaccagc 60

ggcggcgcga gcgtggtgtg ctttctgaac aacttttatc cgaaagatat taacgtgaaa 120ggcggcgcga gcgtggtgtg ctttctgaac aacttttatc cgaaagatat taacgtgaaa 120

tggaaaattg atggcagcga acgccagaac ggcgtgctga acagctggac cgatcaggat 180tggaaaattg atggcagcga acgccagaac ggcgtgctga acagctggac cgatcaggat 180

agcaaagata gcacctatag catgagcagc accctgaccc tgaccaaaga tgaatatgaa 240agcaaagata gcacctatag catgagcagc accctgaccc tgaccaaaga tgaatatgaa 240

cgccataaca gctatacctg cgaagcgacc cataaaacca gcaccagccc gattgtgaaa 300cgccataaca gctatacctg cgaagcgacc cataaaacca gcaccagccc gattgtgaaa 300

agctttaacc gcaacgaatg c 321agctttaacc gcaacgaatg c 321

<210> 24<210> 24

<211> 1212<211> 1212

<212> DNA<212> DNA

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

<220><220>

<223> 编码 Z1 重链恒定区<223> Encoding Z1 heavy chain constant region

<400> 24<400> 24

aaaaccaccc cgccgagcgt gtatccgctg gcgccgggct gcggcgatac caccggcagc 60aaaaccaccc cgccgagcgt gtatccgctg gcgccgggct gcggcgatac caccggcagc 60

agcgtgaccc tgggctgcct ggtgaaaggc tattttccgg aaagcgtgac cgtgacctgg 120agcgtgaccc tgggctgcct ggtgaaaggc tattttccgg aaagcgtgac cgtgacctgg 120

aacagcggca gcctgagcag cagcgtgcat acctttccgg cgctgctgca gagcggcctg 180aacagcggca gcctgagcag cagcgtgcat acctttccgg cgctgctgca gagcggcctg 180

tataccatga gcagcagcgt gaccgtgccg agcagcacct ggccgagcca gaccgtgacc 240tataccatga gcagcagcgt gaccgtgccg agcagcacct ggccgagcca gaccgtgacc 240

tgcagcgtgg cgcatccggc gagcagcacc accgtggata aaaaactgga accgagcggc 300tgcagcgtgg cgcatccggc gagcagcacc accgtggata aaaaactgga accgagcggc 300

ccgattagca ccattaaccc gtgcccgccg tgcaaagaat gccataaatg cccggcgccg 360ccgattagca ccattaaccc gtgcccgccg tgcaaagaat gccataaatg cccggcgccg 360

aacctggaag gcggcccgag cgtgtttatt tttccgccga acattaaaga tgtgctgatg 420aacctggaag gcggcccgag cgtgtttattttccgccga acattaaaga tgtgctgatg 420

attagcctga ccccgaaagt gacctgcgtg gtggtggatg tgagcgaaga tgatccggat 480attagcctga ccccgaaagt gacctgcgtg gtggtggatg tgagcgaaga tgatccggat 480

gtgcagatta gctggtttgt gaacaacgtg gaagtgcata ccgcgcagac ccagacccat 540gtgcagatta gctggtttgt gaacaacgtg gaagtgcata ccgcgcagac ccagacccat 540

cgcgaagatt ataacagcac cattcgcgtg gtgagcaccc tgccgattca gcatcaggat 600cgcgaagatt ataacagcac cattcgcgtg gtgagcaccc tgccgattca gcatcaggat 600

tggatgagcg gcaaagaatt taaatgcaaa gtgaacaaca aagatctgcc gagcccgatt 660tggatgagcg gcaaagaatt taaatgcaaa gtgaacaaca aagatctgcc gagcccgatt 660

gaacgcacca ttagcaaaat taaaggcctg gtgcgcgcgc cgcaggtgta tattctgccg 720gaacgcacca ttagcaaaat taaaggcctg gtgcgcgcgc cgcaggtgta tattctgccg 720

ccgccggcgg aacagctgag ccgcaaagat gtgagcctga cctgcctggt ggtgggcttt 780ccgccggcgg aacagctgag ccgcaaagat gtgagcctga cctgcctggt ggtgggcttt 780

aacccgggcg atattagcgt ggaatggacc agcaacggcc ataccgaaga aaactataaa 840aacccgggcg atattagcgt ggaatggacc agcaacggcc ataccgaaga aaactataaa 840

gataccgcgc cggtgctgga tagcgatggc agctatttta tttatagcaa actgaacatg 900gataccgcgc cggtgctgga tagcgatggc agctatttta tttatagcaa actgaacatg 900

aaaaccagca aatgggaaaa aaccgatagc tttagctgca acgtgcgcca tgaaggcctg 960aaaaccagca aatgggaaaa aaccgatagc tttagctgca acgtgcgcca tgaaggcctg 960

aaaaactatt atctgaaaaa aaccattagc cgcagcccgg gcctggatct ggatgatatt 1020aaaaactatt atctgaaaaa aaccattagc cgcagcccgg gcctggatct ggatgatatt 1020

tgcgcggaag cgaaagatgg cgaactggat ggcctgtgga ccaccattac catttttatt 1080tgcgcggaag cgaaagatgg cgaactggat ggcctgtgga ccaccatac catttttatt 1080

agcctgtttc tgctgagcgt gtgctatagc gcgagcgtga ccctgtttaa agtgaaatgg 1140agcctgtttc tgctgagcgt gtgctatagc gcgagcgtga ccctgtttaa agtgaaatgg 1140

atttttagca gcgtggtgga actgaaacag aaaattagcc cggattatcg caacatgatt 1200atttttagca gcgtggtgga actgaaacag aaaattagcc cggattatcg caacatgatt 1200

ggccagggcg cg 1212ggccagggcg cg 1212

<210> 25<210> 25

<211> 179<211> 179

<212> PRT<212> PRT

<213> Homo sapiens<213> Homo sapiens

<400> 25<400> 25

Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln Asn Ala Tyr Leu ProSer Glu Val Glu Tyr Arg Ala Glu Val Gly Gln Asn Ala Tyr Leu Pro

1 5 10 151 5 10 15

Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu Val Pro Val Cys TrpCys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu Val Pro Val Cys Trp

20 25 30 20 25 30

Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly Asn Val Val Leu ArgGly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly Asn Val Val Leu Arg

35 40 45 35 40 45

Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser Arg Tyr Trp Leu AsnThr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser Arg Tyr Trp Leu Asn

50 55 60 50 55 60

Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr Ile Glu Asn Val ThrGly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr Ile Glu Asn Val Thr

65 70 75 8065 70 75 80

Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg Ile Gln Ile Pro Gly IleLeu Ala Asp Ser Gly Ile Tyr Cys Cys Arg Ile Gln Ile Pro Gly Ile

85 90 95 85 90 95

Met Asn Asp Glu Lys Phe Asn Leu Lys Leu Val Ile Lys Pro Ala LysMet Asn Asp Glu Lys Phe Asn Leu Lys Leu Val Ile Lys Pro Ala Lys

100 105 110 100 105 110

Val Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe Thr Ala Ala Phe ProVal Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe Thr Ala Ala Phe Pro

115 120 125 115 120 125

Arg Met Leu Thr Thr Arg Gly His Gly Pro Ala Glu Thr Gln Thr LeuArg Met Leu Thr Thr Arg Gly His Gly Pro Ala Glu Thr Gln Thr Leu

130 135 140 130 135 140

Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile Ser Thr Leu Ala AsnGly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile Ser Thr Leu Ala Asn

145 150 155 160145 150 155 160

Glu Leu Arg Asp Ser Arg Leu Ala Asn Asp Leu Arg Asp Ser Gly AlaGlu Leu Arg Asp Ser Arg Leu Ala Asn Asp Leu Arg Asp Ser Gly Ala

165 170 175 165 170 175

Thr Ile ArgThr Ile Arg

<210> 26<210> 26

<211> 281<211> 281

<212> PRT<212> PRT

<213> Mus musculus<213> Mus musculus

<400> 26<400> 26

Met Phe Ser Gly Leu Thr Leu Asn Cys Val Leu Leu Leu Leu Gln LeuMet Phe Ser Gly Leu Thr Leu Asn Cys Val Leu Leu Leu Leu Gln Leu

1 5 10 151 5 10 15

Leu Leu Ala Arg Ser Leu Glu Asp Gly Tyr Lys Val Glu Val Gly LysLeu Leu Ala Arg Ser Leu Glu Asp Gly Tyr Lys Val Glu Val Gly Lys

20 25 30 20 25 30

Asn Ala Tyr Leu Pro Cys Ser Tyr Thr Leu Pro Thr Ser Gly Thr LeuAsn Ala Tyr Leu Pro Cys Ser Tyr Thr Leu Pro Thr Ser Gly Thr Leu

35 40 45 35 40 45

Val Pro Met Cys Trp Gly Lys Gly Phe Cys Pro Trp Ser Gln Cys ThrVal Pro Met Cys Trp Gly Lys Gly Phe Cys Pro Trp Ser Gln Cys Thr

50 55 60 50 55 60

Asn Glu Leu Leu Arg Thr Asp Glu Arg Asn Val Thr Tyr Gln Lys SerAsn Glu Leu Leu Arg Thr Asp Glu Arg Asn Val Thr Tyr Gln Lys Ser

65 70 75 8065 70 75 80

Ser Arg Tyr Gln Leu Lys Gly Asp Leu Asn Lys Gly Asp Val Ser LeuSer Arg Tyr Gln Leu Lys Gly Asp Leu Asn Lys Gly Asp Val Ser Leu

85 90 95 85 90 95

Ile Ile Lys Asn Val Thr Leu Asp Asp His Gly Thr Tyr Cys Cys ArgIle Ile Lys Asn Val Thr Leu Asp Asp His Gly Thr Tyr Cys Cys Arg

100 105 110 100 105 110

Ile Gln Phe Pro Gly Leu Met Asn Asp Lys Lys Leu Glu Leu Lys LeuIle Gln Phe Pro Gly Leu Met Asn Asp Lys Lys Leu Glu Leu Lys Leu

115 120 125 115 120 125

Asp Ile Lys Ala Ala Lys Val Thr Pro Ala Gln Thr Ala His Gly AspAsp Ile Lys Ala Ala Lys Val Thr Pro Ala Gln Thr Ala His Gly Asp

130 135 140 130 135 140

Ser Thr Thr Ala Ser Pro Arg Thr Leu Thr Thr Glu Arg Asn Gly SerSer Thr Thr Ala Ser Pro Arg Thr Leu Thr Thr Glu Arg Asn Gly Ser

145 150 155 160145 150 155 160

Glu Thr Gln Thr Leu Val Thr Leu His Asn Asn Asn Gly Thr Lys IleGlu Thr Gln Thr Leu Val Thr Leu His Asn Asn Asn Gly Thr Lys Ile

165 170 175 165 170 175

Ser Thr Trp Ala Asp Glu Ile Lys Asp Ser Gly Glu Thr Ile Arg ThrSer Thr Trp Ala Asp Glu Ile Lys Asp Ser Gly Glu Thr Ile Arg Thr

180 185 190 180 185 190

Ala Ile His Ile Gly Val Gly Val Ser Ala Gly Leu Thr Leu Ala LeuAla Ile His Ile Gly Val Gly Val Ser Ala Gly Leu Thr Leu Ala Leu

195 200 205 195 200 205

Ile Ile Gly Val Leu Ile Leu Lys Trp Tyr Ser Cys Lys Lys Lys LysIle Ile Gly Val Leu Ile Leu Lys Trp Tyr Ser Cys Lys Lys Lys Lys

210 215 220 210 215 220

Leu Ser Ser Leu Ser Leu Ile Thr Leu Ala Asn Leu Pro Pro Gly GlyLeu Ser Ser Leu Ser Leu Ile Thr Leu Ala Asn Leu Pro Pro Gly Gly

225 230 235 240225 230 235 240

Leu Ala Asn Ala Gly Ala Val Arg Ile Arg Ser Glu Glu Asn Ile TyrLeu Ala Asn Ala Gly Ala Val Arg Ile Arg Ser Glu Glu Asn Ile Tyr

245 250 255 245 250 255

Thr Ile Glu Glu Asn Val Tyr Glu Val Glu Asn Ser Asn Glu Tyr TyrThr Ile Glu Glu Asn Val Tyr Glu Val Glu Asn Ser Asn Glu Tyr Tyr

260 265 270 260 265 270

Cys Tyr Val Asn Ser Gln Gln Pro SerCys Tyr Val Asn Ser Gln Gln Pro Ser

275 280 275 280

Claims (29)

Translated fromChinese

1.分离的抗原结合蛋白，其包括重链可变区VH的HCDR1、HCDR2、HCDR3，和轻链可变区VL的LCDR1，LCDR2和LCDR3；其中，所述HCDR1的氨基酸序列如SEQ ID NO:4所示，所述HCDR2的氨基酸序列如SEQ ID NO:5所示，所述HCDR3的氨基酸序列如SEQ ID NO:6所示，所述LCDR1的氨基酸序列如SEQ ID NO:1所示，所述LCDR2的氨基酸序列如SEQ ID NO:2所示，所述LCDR3的氨基酸序列如SEQ ID NO:3所示；所述分离的抗原结合蛋白为抗体或其抗原结合片段，且所述分离的抗原结合蛋白具有Tim-3蛋白结合能力。1. An isolated antigen-binding protein, which includes HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH, and LCDR1, LCDR2, and LCDR3 of the light chain variable region VL; wherein, the amino acid sequence of the HCDR1 is such as SEQ ID NO: 4, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:5, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:6, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:1, so The amino acid sequence of LCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 3; the isolated antigen-binding protein is an antibody or an antigen-binding fragment thereof, and the isolated antigen The binding protein has Tim-3 protein binding ability.2.根据权利要求1所述的分离的抗原结合蛋白，其中所述抗体包括人源化和鼠源抗体。2. The isolated antigen-binding protein of claim 1, wherein the antibodies include humanized and murine antibodies.3.根据权利要求1所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，和/或di-scFv。3. The isolated antigen-binding protein of claim 1, wherein the antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, and/or di- scFv.4.根据权利要求1所述的分离的抗原结合蛋白，其中所述VL包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。4. The isolated antigen-binding protein of claim 1, wherein the VL includes framework regions L-FR1, L-FR2, L-FR3, and L-FR4.5.根据权利要求4所述的分离的抗原结合蛋白，其中所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1具有SEQ ID NO:7所示的氨基酸序列。5. The isolated antigen-binding protein according to claim 4, wherein the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 has SEQ ID NO:7 amino acid sequence.6.根据权利要求4所述的分离的抗原结合蛋白，其中所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2具有SEQ ID NO:8所示的氨基酸序列。6. The isolated antigen-binding protein according to claim 4, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 has the amino acid sequence shown in SEQ ID NO:8.7.根据权利要求4所述的分离的抗原结合蛋白，其中所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3具有SEQ ID NO:9所示的氨基酸序列。7. The isolated antigen-binding protein according to claim 4, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 has the amino acid sequence shown in SEQ ID NO: 9.8.根据权利要求4所述的分离的抗原结合蛋白，其中所述L-FR4的N末端与所述LCDR3的C末端直接或间接相连，且所述L-FR4具有SEQ ID NO:10所示的氨基酸序列。8. The isolated antigen-binding protein according to claim 4, wherein the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 has SEQ ID NO: 10 amino acid sequence.9.根据权利要求1所述的分离的抗原结合蛋白，其中所述VL的氨基酸序列如SEQ IDNO:15所示。9. The isolated antigen-binding protein of claim 1, wherein the amino acid sequence of the VL is as shown in SEQ ID NO: 15.10.根据权利要求1所述的分离的抗原结合蛋白，其包括抗体轻链恒定区，且所述抗体轻链恒定区包括人Igκ恒定区。10. The isolated antigen binding protein of claim 1, comprising an antibody light chain constant region, and the antibody light chain constant region comprises a human Igκ constant region.11.根据权利要求10所述的分离的抗原结合蛋白，其中所述抗体轻链恒定区具有SEQID NO:17中所示的氨基酸序列。11. The isolated antigen-binding protein of claim 10, wherein the antibody light chain constant region has the amino acid sequence set forth in SEQ ID NO: 17.12.根据权利要求1所述的分离的抗原结合蛋白，其包含抗体轻链LC，且所述LC具有SEQID NO:19所示的氨基酸序列。12. The isolated antigen-binding protein according to claim 1, which comprises an antibody light chain LC, and the LC has the amino acid sequence shown in SEQ ID NO: 19.13.根据权利要求1所述的分离的抗原结合蛋白，其中所述VH包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。13. The isolated antigen-binding protein of claim 1, wherein the VH includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4.14.根据权利要求13所述的分离的抗原结合蛋白，其中所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1具有SEQ ID NO:11所示的氨基酸序列。14. The isolated antigen-binding protein according to claim 13, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 has SEQ ID NO: 11 amino acid sequence.15.根据权利要求13所述的分离的抗原结合蛋白，其中所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2具有SEQ ID NO:12所示的氨基酸序列。15. The isolated antigen-binding protein of claim 13, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 has the amino acid sequence shown in SEQ ID NO: 12.16.根据权利要求13所述的分离的抗原结合蛋白，其中所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3具有SEQ ID NO:13所示的氨基酸序列。16. The isolated antigen-binding protein of claim 13, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 has the amino acid sequence shown in SEQ ID NO: 13.17.根据权利要求13所述的分离的抗原结合蛋白，其中所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4具有SEQ ID NO:14所示的氨基酸序列。17. The isolated antigen-binding protein according to claim 13, wherein the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 has SEQ ID NO: 14 amino acid sequence.18.根据权利要求1所述的分离的抗原结合蛋白，其中所述VH的氨基酸序列如SEQ IDNO:16所。18. The isolated antigen-binding protein according to claim 1, wherein the amino acid sequence of the VH is as shown in SEQ ID NO: 16.19.根据权利要求1所述的分离的抗原结合蛋白，其包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG恒定区。19. The isolated antigen binding protein of claim 1, comprising an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG constant region.20.根据权利要求19所述的分离的抗原结合蛋白，其中所述抗体重链恒定区具有SEQID NO:18中所示的氨基酸序列。20. The isolated antigen-binding protein of claim 19, wherein the antibody heavy chain constant region has the amino acid sequence set forth in SEQ ID NO: 18.21.根据权利要求1所述的分离的抗原结合蛋白，其包含抗体重链HC，且所述HC具有SEQID NO:20所示的氨基酸序列。21. The isolated antigen-binding protein of claim 1, which comprises an antibody heavy chain HC, and the HC has the amino acid sequence shown in SEQ ID NO: 20.22.分离的核酸分子，其编码权利要求1-21中任一项所述的分离的抗原结合蛋白。22. An isolated nucleic acid molecule encoding the isolated antigen-binding protein of any one of claims 1-21.23.载体，其包含根据权利要求22所述的核酸分子，或，表达权利要求1-21中任一项所述的抗原结合蛋白。23. A vector comprising the nucleic acid molecule according to claim 22, or expressing the antigen-binding protein according to any one of claims 1-21.24.细胞，其包含根据权利要求22所述的核酸分子或根据权利要求23所述的载体。24. A cell comprising a nucleic acid molecule according to claim 22 or a vector according to claim 23.25.制备权利要求1-21中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得权利要求1-21中任一项所述的分离的抗原结合蛋白表达的条件下，培养根据权利要求24所述的细胞。25. A method of preparing the isolated antigen-binding protein of any one of claims 1-21, said method comprising expressing the isolated antigen-binding protein of any one of claims 1-21. , culturing the cells according to claim 24.26.药物组合物，其包含权利要求1-21中任一项所述的分离的抗原结合蛋白、权利要求22所述的核酸分子、权利要求23所述的载体和/或权利要求24所述的细胞，以及任选地药学上可接受的载体。26. A pharmaceutical composition comprising the isolated antigen-binding protein of any one of claims 1-21, the nucleic acid molecule of claim 22, the vector of claim 23, and/or the vector of claim 24 cells, and optionally a pharmaceutically acceptable carrier.27.权利要求1-21中任一项所述的分离的抗原结合蛋白、权利要求22所述的核酸分子、权利要求23所述的载体、权利要求24所述的细胞和/或权利要求26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/或治疗肿瘤。27. The isolated antigen-binding protein of any one of claims 1-21, the nucleic acid molecule of claim 22, the vector of claim 23, the cell of claim 24 and/or claim 26 The use of the pharmaceutical composition in the preparation of medicines for preventing, alleviating and/or treating tumors.28.根据权利要求27所述的用途，其中所述肿瘤包括实体瘤和/或血液肿瘤。28. Use according to claim 27, wherein said tumors comprise solid tumors and/or hematological tumors.29.权利要求1-21中任一项所述的分离的抗原结合蛋白、权利要求22所述的核酸分子、权利要求23所述的载体、权利要求24所述的细胞和/或权利要求26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Tim-3相关的疾病。29. The isolated antigen-binding protein of any one of claims 1-21, the nucleic acid molecule of claim 22, the vector of claim 23, the cell of claim 24 and/or claim 26 The use of the pharmaceutical composition in the preparation of medicines for preventing, alleviating and/or treating Tim-3 related diseases.