WO2024146419A1 - Specific antibody for claudin-18.2 and use thereof - Google Patents

Abstract

The present application relates to the technical field of antibodies, and specifically relates to a specific antibody for Claudin-18.2 and the use thereof. The antibody is used as a specific antibody for the detection of the Claudin-18.2 antigen, and can be used for quantitative detection of expressed Claudin-18.2, or for the accompanying diagnosis and detection of preclinical and clinical samples.

Description

一种Claudin-18.2特异性抗体及其应用Claudin-18.2 specific antibody and its application

相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS

本申请要求于2023年8月10日提交中国专利局的申请号为202311006962.3、名称为“一种Claudin-18.2特异性抗体及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims priority to the Chinese patent application with application number 202311006962.3 filed with the Chinese Patent Office on August 10, 2023, entitled “A Claudin-18.2-specific antibody and its application”, the entire contents of which are incorporated by reference into this application.

技术领域Technical Field

本申请涉及抗体技术领域，具体涉及Claudin-18.2特异性抗体及其应用。The present application relates to the field of antibody technology, and in particular to Claudin-18.2 specific antibodies and applications thereof.

背景技术Background technique

Claudin-18.2蛋白是紧密连接蛋白家族成员Claudin 18的一种亚型，是一种高度选择性的生物标志物，在正常组织中表达有限，在各种原发恶性肿瘤如胃癌/胃食管结合部(GC/GEJ)癌、乳腺癌、结肠癌、肝癌、头颈癌、支气管癌和非小细胞肺癌的发生发展过程中经常出现异常表达。Claudin-18.2在肿瘤的发展和转移中起着重要的作用，参与肿瘤细胞的增殖、分化和迁移等生物学过程。由于Claudin-18.2在肿瘤中的表达特异性较高，因此它被认为是肿瘤治疗的潜在靶点。Claudin-18.2 protein is a subtype of Claudin 18, a member of the tight junction protein family. It is a highly selective biomarker with limited expression in normal tissues. Abnormal expression often occurs during the development and progression of various primary malignant tumors such as gastric cancer/gastroesophageal junction (GC/GEJ) cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer. Claudin-18.2 plays an important role in the development and metastasis of tumors and is involved in biological processes such as tumor cell proliferation, differentiation, and migration. Due to the high specificity of Claudin-18.2 expression in tumors, it is considered a potential target for tumor treatment.

最近研究发现，Claudin-18.2表达也可作为肿瘤疾病诊断的潜在特异性标记物，有鉴于此，提出本申请。Recent studies have found that Claudin-18.2 expression can also be used as a potential specific marker for the diagnosis of tumor diseases. In view of this, the present application is proposed.

发明概述SUMMARY OF THE INVENTION

针对上述技术问题，本申请通过以体外构建的Claudin-18.2蛋白作为免疫原进行小鼠免疫，经细胞融合与筛选、获得表达抗体的杂交瘤细胞株；通过实验验证发现，该抗体能够特异性识别Claudin-18.2抗原识别位点，效果明显优于目前商品化的抗体，可应用于ELISA、流式和IHC等多种检测方法，因此本申请至少包括如下目的：In response to the above technical problems, the present application immunizes mice with the Claudin-18.2 protein constructed in vitro as an immunogen, obtains a hybridoma cell line expressing the antibody through cell fusion and screening; experimental verification shows that the antibody can specifically recognize the Claudin-18.2 antigen recognition site, and the effect is significantly better than the currently commercialized antibodies, and can be applied to a variety of detection methods such as ELISA, flow cytometry and IHC. Therefore, the present application includes at least the following purposes:

本申请第一目的是提供一种抗Claudin-18.2的特异性抗体或其抗原结合片段，及其相应产品；The first purpose of the present application is to provide a specific antibody or antigen-binding fragment thereof against Claudin-18.2, and corresponding products;

本申请第二目的是提供一种上述抗体或抗原结合片段在检测方面方面的应用，包括在临床前后的定性或定量检测等。The second purpose of the present application is to provide an application of the above-mentioned antibody or antigen-binding fragment in detection, including qualitative or quantitative detection before and after clinical practice.

为了实现上述目的，本申请具体采用以下技术方案：In order to achieve the above objectives, this application specifically adopts the following technical solutions:

本申请首先提供一种抗Claudin-18.2的抗体或其抗原结合片段，包含SEQ ID NO.7所示重链可变区氨基酸序列中的3个CDR和SEQ ID NO.8所示的轻链可变区氨基酸序列中的3个CDR；The present application first provides an anti-Claudin-18.2 antibody or an antigen-binding fragment thereof, comprising three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO.7 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO.8;

或者，与上述轻重链CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸保守性变化的变体。 Alternatively, the variants may have single or multiple CDRs with no more than 2 conservative amino acid changes in each CDR region compared to the above light and heavy chain CDR regions.

进一步的，当按照Kabat编码规则对抗体HCDRs编码时，所述抗体或抗原结合片段具体包含如下CDR序列：Further, when the antibody HCDRs are encoded according to the Kabat coding rules, the antibody or antigen-binding fragment specifically comprises the following CDR sequences:

i.重链可变区的互补决定区HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO.1、2、3所示；i. The amino acid sequences of the complementary determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown in SEQ ID NO. 1, 2, and 3, respectively;

ii.轻链可变区的互补决定区LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO.4、5、6所示；ii. The amino acid sequences of the complementary determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO. 4, 5 and 6 respectively;

或者，与上述i-ii的6个CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸保守性变化的变体。Alternatively, a variant having a single or multiple CDRs with no more than 2 conservative amino acid changes in each CDR region compared to the 6 CDR regions of i-ii above.

进一步的，所述抗体或抗原结合片段包含重链可变区和轻链可变区，序列选自如下：Furthermore, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the sequences of which are selected from the following:

a)所述重链可变区的氨基酸序列如SEQ ID NO.7所示，或与SEQ ID NO.7具有至少70％、80％、90％、95％或99％序列同一性；a) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.7, or has at least 70%, 80%, 90%, 95% or 99% sequence identity with SEQ ID NO.7;

b)所述轻链可变区的氨基酸序列如SEQ ID NO.8所示，或与SEQ ID NO.8具有至少70％、80％、90％、95％或99％序列同一性。b) The amino acid sequence of the light chain variable region is as shown in SEQ ID NO.8, or has at least 70%, 80%, 90%, 95% or 99% sequence identity with SEQ ID NO.8.

进一步的，所述抗体或抗原结合片段连接有偶联部分，所述偶联部分选自放射性核素、药物、毒素、细胞因子、酶、荧光素、载体蛋白、脂类、和生物素中的一种或多种，其中所述抗体或抗原结合片段与所述偶联部分可选择性通过连接子相连；优选的，所述连接子为肽或多肽。Furthermore, the antibody or antigen-binding fragment is connected to a coupling portion, and the coupling portion is selected from one or more of radionuclides, drugs, toxins, cytokines, enzymes, fluorescein, carrier proteins, lipids, and biotin, wherein the antibody or antigen-binding fragment and the coupling portion can be selectively connected via a linker; preferably, the linker is a peptide or polypeptide.

进一步的，所述抗体或抗原结合片段选自单克隆抗体、多克隆抗体、抗血清、嵌合抗体、人源化抗体和人抗体；优选的，所述抗体选自多特异性抗体、单链Fv(scFv)、单链抗体、抗独特型(抗-Id)抗体、双抗体、微型抗体、纳米抗体、单结构域抗体、Fab片段、F(ab’)片段、二硫化物连接的双特异性Fv(sdFv)和胞内抗体。Further, the antibody or antigen-binding fragment is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antisera, chimeric antibodies, humanized antibodies and human antibodies; preferably, the antibody is selected from the group consisting of multispecific antibodies, single-chain Fv (scFv), single-chain antibodies, anti-idiotypic (anti-Id) antibodies, diabodies, miniantibodies, nanoantibodies, single domain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fv (sdFv) and intracellular antibodies.

本申请还提供一种分离的多核苷酸，所述多核苷酸编码上述任一所述的抗体或抗原结合片段。The present application also provides an isolated polynucleotide, which encodes any of the antibodies or antigen-binding fragments described above.

本申请还提供一种重组载体，其包含上述所述的多核苷酸，以及任选的调控序列；The present application also provides a recombinant vector, which comprises the polynucleotide described above, and an optional regulatory sequence;

进一步优选的，所述重组载体为克隆载体或表达载体；Further preferably, the recombinant vector is a cloning vector or an expression vector;

更进一步优选的，所述调控序列选自前导序列、多聚腺苷酸化序列、前肽序列、启动子、信号序列、转录终止子，或其任何组合。Still further preferred, the regulatory sequence is selected from a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, a transcription terminator, or any combination thereof.

本申请还提供一种产品，包含上述任一所述的抗体或抗原结合片段、多核苷酸或重组载体中的一种或更多种，并容纳于合适的容器中。The present application also provides a product, comprising one or more of any of the above-mentioned antibodies or antigen-binding fragments, polynucleotides or recombinant vectors, and contained in a suitable container.

进一步的，所述产品为试剂盒形式；Further, the product is in the form of a kit;

进一步优选的，所述试剂盒包括但不限于：ELISA、流式分选和IHC试剂盒等。Further preferably, the kit includes but is not limited to: ELISA, flow cytometry and IHC kits and the like.

本申请提供上述任一所述的抗体或抗原结合片段的如下任一应用：The present application provides any of the following applications of any of the above-mentioned antibodies or antigen-binding fragments:

(1)在Claudin-18.2蛋白的检测、鉴定或筛选中的应用，或在制备Claudin-18.2的检测、鉴定或筛选产品中的应用； (1) Application in the detection, identification or screening of Claudin-18.2 protein, or in the preparation of products for the detection, identification or screening of Claudin-18.2;

优选的，所述检测包括定量检测或定性检测；Preferably, the detection includes quantitative detection or qualitative detection;

更优选的，所述检测包括免疫原性检测；More preferably, the detection comprises immunogenicity detection;

进一步优选的，所述检测包括但不限于流式检测、ELISA检测或IHC检测；Further preferably, the detection includes but is not limited to flow cytometry, ELISA detection or IHC detection;

(2)在肿瘤的诊断或伴随诊断中的应用，或在制备肿瘤的诊断或伴随诊断试剂盒中的应用；(2) Use in the diagnosis or companion diagnosis of tumors, or in the preparation of diagnostic or companion diagnosis kits for tumors;

(3)在制备用Claudin-18.2的临床前及临床检测试剂的应用。(3) Application of Claudin-18.2 in the preparation of preclinical and clinical detection reagents.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1抗体Claudin18.2(3B10)敏感性、特异性和交叉反应性的IHC检测结果；Figure 1 IHC test results of sensitivity, specificity and cross-reactivity of antibody Claudin18.2 (3B10);

图2不同抗体敏感性和特异性的IHC检测结果。Fig. 2 IHC test results of different antibody sensitivity and specificity.

发明详述DETAILED DESCRIPTION OF THE INVENTION

本申请公开了一种分离抗体或其抗原结合片段，本领域技术人员可以参考本文内容，实现其应用，特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本申请内。本申请的制备方法及应用已经通过较佳的实施例进行了描述，相关人员明显能在不脱离本申请内容、精神和范围内对本文制备方法和应用进行改动或适当变更与组合，来实现和应用本申请技术。除非另外定义，否则本文使用的所有技术和科学术语具有本申请所属领域的普通技术人员通常所理解的相同的含义。The present application discloses a separation antibody or an antigen-binding fragment thereof. Those skilled in the art can refer to the contents of this article to realize its application. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The preparation method and application of this application have been described through preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the preparation method and application of this article without departing from the content, spirit and scope of this application to realize and apply the technology of this application. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the field to which this application belongs.

以下术语或定义仅仅是为了帮助理解本申请而提供。这些定义不应被理解为具有小于本领域技术人员所理解的范围。The following terms or definitions are provided only to help understand the present application. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

除非在下文中另有定义，本申请具体实施方式中所用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。虽然相信以下术语对于本领域技术人员很好理解，但仍然阐述以下定义以更好地解释本申请。Unless otherwise defined below, the meaning of all technical terms and scientific terms used in the specific embodiments of the present application is intended to be the same as that commonly understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present application.

如本申请中所使用，术语“包括”、“包含”、“具有”、“含有”或“涉及”为包含性的(inclusive)或开放式的，且不排除其它未列举的元素或方法步骤。术语“由…组成”被认为是术语“包含”的优选实施方案。如果在下文中某一组被定义为包含至少一定数目的实施方案，这也应被理解为揭示了一个优选地仅由这些实施方案组成的组。As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

在提及单数形式名词时使用的不定冠词或定冠词例如“一个”或“一种”，“所述”，包括该名词的复数形式。When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" or "an" is used, this includes a plural of that noun.

本申请中的术语“大约”、“大体”表示本领域技术人员能够理解的仍可保证论及特征的技术效果的准确度区间。该术语通常表示偏离指示数值的±10％，优选±5％。The terms "approximately" and "substantially" in this application represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the characteristic in question. The term usually represents ±10%, preferably ±5%, of the deviation from the indicated value.

术语“或更多”、“至少”、“超过”等，例如“至少一种”应当理解为包括但不限于至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19 20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、 61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100或200、300、400、500、600、700、800、900、1000、2000、3000、4000、5000或超过所述值。还包括其间任何更大的数字或分数。47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Also included are any larger numbers or fractions therebetween.

相反地，术语“不超过”包括小于所述值的每个值。例如，“不超过100个核苷酸”包括100、99、98、97、96、95、94、93、92、91、90、89、88、87、86、85、84、83、82、81、80、79、78、77、76、75、74、73、72、71、70、69、68、67、66、65、64、63、62、61、60、59、58、57、56、55、54、53、52、51、50、49、48、47、46、45、44、43、42、41、40、39、38、37、36、35、34、33、32、31、30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2、1和0个核苷酸。还包括其间任何更小的数字或分数。Conversely, the term "no more than" includes every value less than the stated value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 56, 57, 58, 59 ... 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included are any smaller numbers or fractions therebetween.

术语“多个”、“至少两个”、“两个或更多个”、“至少第二个”等应当理解为包括但不限于至少2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19 20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100或200、300、400、500、600、700、800、900、1000、2000、3000、4000、5000或更多。还包括其间任何更大的数字或分数。43,44,45,46,47,48,49,50,51,52,53,54,55 , 50, 50, 60, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Also included are any larger numbers or fractions therebetween.

术语定义：Definition of Terms:

如本文中所使用的，术语“抗体”是指能够非共价地、可逆地并以特异性方式结合对应抗原的免疫球蛋白家族的多肽。例如，天然存在的IgG抗体是一种包括通过二硫键互连的至少两个重(H)链和两个轻(L)链的四聚体。每个重链包含重链可变区(在此缩写为VH)和重链恒定区。重链恒定区包含三个结构域CH1、CH2和CH3。每个轻链包含轻链可变区(在此缩写为VL)和轻链恒定区。轻链恒定区包含一个结构域CL。VH区和VL区可以进一步细分为被称作互补决定区(CDR)的超变区，所述超变区散布有更保守的被称作框架区(FR)的区。每个VH和VL由按照以下顺序从氨基末端到羧基末端布置的三个CDR和四个FR构成：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，包含免疫***的不同细胞(例如，效应细胞)以及经典补体***的第一组分(Clq)。“抗体”包含但不限于单克隆抗体、人抗体、人源化抗体、骆驼科抗体、嵌合抗体和抗独特型(抗Id)抗体(包含例如针对本公开的抗体的抗Id抗体)。抗体可以属于任何同种型/种类(例如，IgG、IgE、IgM、IgD、IgA和IgY)或子类(例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)。As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that can non-covalently, reversibly and in a specific manner bind to the corresponding antigen. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by a disulfide bond. Each heavy chain comprises a heavy chain variable region (abbreviated as VH here) and a heavy chain constant region. The heavy chain constant region comprises three domains CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated as VL here) and a light chain constant region. The light chain constant region comprises a domain CL. The VH region and the VL region can be further subdivided into hypervariable regions referred to as complementary determining regions (CDRs), which are interspersed with more conservative regions referred to as framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminal to the carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of an antibody can mediate the binding of an immunoglobulin to a host tissue or factor, including different cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. "Antibody" includes but is not limited to monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies for antibodies of the present disclosure). Antibodies can belong to any isotype/category (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

抗体包含称为“结构域”的重链或轻链多肽的球状区域。结构域可包含肽环，通常为3至4个环，其例如通过β折叠和/或链内二硫键得以稳定。基于在“恒定”域的情况下， 不同类别成员的结构域内相对缺乏序列变异，或者在“可变”域的情况下，不同类别成员的结构域内的显著变异，结构域通常称为“恒定”或“可变”。抗体或多肽“结构域”在本领域中通常可互换地称为抗体或多肽“区域”。Antibodies contain globular regions of heavy or light chain polypeptides called "domains". Domains may contain peptide loops, typically 3 to 4 loops, which are stabilized, for example, by beta sheets and/or intrachain disulfide bonds. Based on the case of "constant" domains, The relative lack of sequence variation within a domain of different class members, or, in the case of a "variable" domain, the significant variation within a domain of different class members, domains are often referred to as "constant" or "variable." Antibody or polypeptide "domains" are often referred to interchangeably in the art as antibody or polypeptide "regions."

术语“单克隆抗体”是指具有单一氨基酸组成的抗体分子的制备物，不涉及其制备的方法。单克隆抗体或其免疫活性片段可以通过杂交瘤技术、重组技术、噬菌体展示技术、合成技术等或其它本领域已知的生产技术来产生，本申请中涉及单克隆抗体制备的方法包括杂交瘤细胞体外培养制备或通过DNA重组技术制备。单克隆抗体是高度特异性的，针对单个抗原位点。每种单克隆抗体针对抗原上的单一决定簇。The term "monoclonal antibody" refers to a preparation of an antibody molecule having a single amino acid composition, and does not refer to the method of its preparation. Monoclonal antibodies or immunologically active fragments thereof can be produced by hybridoma technology, recombinant technology, phage display technology, synthetic technology, or other production technologies known in the art. The methods for preparing monoclonal antibodies in this application include preparation by hybridoma cell culture in vitro or preparation by DNA recombination technology. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Each monoclonal antibody is directed against a single determinant cluster on the antigen.

术语“抗原”是免疫球蛋白或抗体(或其抗原结合片段)特异性结合的实体(例如，蛋白实体或肽)。The term "antigen" is an entity (eg, a protein entity or a peptide) to which an immunoglobulin or antibody (or antigen-binding fragment thereof) specifically binds.

术语“片段”是指抗体或抗体链的一部分或部分，其包含的氨基酸残基比完整或完全抗体或抗体链少，其中该部分优选保留当该部分存在于完整抗体中时与该部分正常相关的功能中的至少一个，优选大部分或全部的功能。片段可以通过化学或酶促处理完整或完全抗体或抗体链而获得。片段还可以通过重组方式获得。The term "fragment" refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain, wherein the portion preferably retains at least one, preferably most or all, of the functions normally associated with the portion when the portion is present in an intact antibody. Fragments can be obtained by chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means.

术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。The term "variable" means that some parts of the variable region of an antibody differ in sequence, which contributes to the binding and specificity of each specific antibody for its specific antigen. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-folded configuration and are connected by three CDRs that form a connecting loop, which in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as involvement in antibody-dependent cellular toxicity of antibodies.

“互补决定结构域”或“互补决定区”(“CDR”)可互换地指VL和VH的超变区。CDR是携带此种靶蛋白的特异性的抗体链的靶蛋白结合位点。每个人VL或VH中存在三个CDR(CDR1-3，按顺序从N末端编号)，总计占可变结构域的约15-20％。CRD可以由其区和顺序指代。例如，“VHCDR1”或“HCDR1”两者指代重链可变区的第一CDR。CDR与靶蛋白的表位在结构上互补并且因此直接负责结合特异性。VL或VH的其余延伸段(所谓的框架区)在氨基酸序列中展现出较少变化(Kuby,《免疫学(Immunology)》,第4版,第4章W.H.弗里曼公司(W.H.Freeman&Co.),纽约,2000)。"Complementarity determining domain" or "complementarity determining region" ("CDR") refers interchangeably to the hypervariable regions of VL and VH. CDR is the target protein binding site of the antibody chain that carries the specificity of such a target protein. There are three CDRs (CDR1-3, numbered sequentially from the N-terminus) in each human VL or VH, totaling about 15-20% of the variable domain. CRD can be referred to by its region and order. For example, "VHCDR1" or "HCDR1" both refer to the first CDR of the heavy chain variable region. CDR is structurally complementary to the epitope of the target protein and is therefore directly responsible for binding specificity. The remaining extensions of VL or VH (the so-called framework regions) show less variation in the amino acid sequence (Kuby, Immunology, 4th edition, Chapter 4 W.H. Freeman & Co., New York, 2000).

在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派***的任一种或其组合确定，所述指派***包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等 人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat (Kabat et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the variability of antibody sequences, and the topology of the CDR loops. Human, Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT), and North CDR definitions based on affinity propagation clustering using a large number of crystal structures.

然而，应该注意，基于不同的指派***获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派***下定义的同一抗体可变区的CDR序列有所不同。例如，对使用Kabat、AbM、Chothia、IMGT、Contact编号的CDR区域在不同指派***定义下的残基范围如下表所示。However, it should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. For example, the residue ranges of the CDR regions using Kabat, AbM, Chothia, IMGT, and Contact numbering under different assignment systems are shown in the following table.

不同指派***定义下的CDR残基范围
CDR residue ranges defined by different assignment systems

因此，在涉及用本申请定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派***规则或组合)而导致其所声称的CDR边界与本申请所定义的具体CDR边界不同。Therefore, when referring to antibodies defined by specific CDR sequences defined in this application, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose declared CDR boundaries are different from the specific CDR boundaries defined in this application due to the application of a different scheme (e.g., a different assignment system rule or combination).

本申请抗体的CDR可以根据本领域的任何方案或其组合人工地评估确定边界。除非另有说明，否则在本申请中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。即，本申请中的CDR序列包括SEQ ID NO.7所示的重链可变区氨基酸序列中的任一基于上表定义的3个CDR和SEQ ID NO.8所示轻链可变区氨基酸序列中的任一基于上表定义的3个CD。比如，在本发明的一些特定的实施方式中，当按照Kabat编码规则对抗体CDRs编码时，CDR序列为：i.重链可变区的互补决定区HCDR1、HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.1、2、3所示；ii.轻链可变区的互补决定区LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、5、6所示。The CDR of the antibody of the present application can be manually evaluated and determined according to any scheme or combination of schemes in the art. Unless otherwise specified, in the present application, the term "CDR" or "CDR sequence" covers CDR sequences determined in any of the above ways. That is, the CDR sequence in the present application includes any of the three CDRs defined in the above table in the heavy chain variable region amino acid sequence shown in SEQ ID NO.7 and any of the three CDs defined in the above table in the light chain variable region amino acid sequence shown in SEQ ID NO.8. For example, in some specific embodiments of the present invention, when the antibody CDRs are encoded according to the Kabat coding rules, the CDR sequences are: i. The amino acid sequences of the complementary determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown in SEQ ID NO.1, 2, and 3, respectively; ii. The amino acid sequences of the complementary determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region are shown in SEQ ID NO.4, 5, and 6, respectively.

抗体可以包括例如，单克隆抗体、重组产生的抗体、单特异性抗体、多特异性抗体(包括双特异性抗体)、人抗体、工程化抗体、人源化抗体、嵌合抗体、免疫球蛋白、合成抗体、包含两个重链和两个轻链分子的四聚体抗体、抗体轻链单体、抗体重链单体、抗体轻链二聚体、抗体重链二聚体、抗体轻链-抗体重链对、胞内抗体、抗体融合物(本 文有时称为“抗体缀合物”)、异缀合抗体、单结构域抗体、单价抗体、单链抗体或单链Fv(scFv)、骆驼源化抗体、亲和体、Fab片段、F(ab’)2片段、二硫键连接的Fv(sdFv)、抗独特型(抗Id)抗体(包括例如，抗-抗Id抗体)、微抗体、结构域抗体、合成抗体(本文有时称为“抗体模拟物”)和以上任何的抗原结合片段。The antibodies may include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (the present invention) In some embodiments, the present invention relates to antibodies comprising: a) antibodies, wherein the antibodies are antigen-binding fragments of the present invention; b) antibodies, wherein the antibodies are antigen-binding fragments of the present invention; c) antibodies, wherein the antibodies are antigen-binding fragments of the present invention; and d) antibodies, wherein the antibodies are antigen-binding fragments of the present invention.

术语“抗原结合片段”是指保留与抗原的表位特异性地相互作用(例如，通过结合、位阻、稳定化/去稳定化、空间分布)的能力的抗体的一个或多个部分。结合片段的实例包含但不限于：单链Fv(scFv)、二硫键连接的Fv(sdFv)、Fab片段、F(ab')片段(即由VL、VH、CL和CH1结构域组成的单价片段)；F(ab)2片段(即包括在铰链区处由二硫桥连接的两个Fab片段的双价片段)；由VH和CH1结构域组成的Fd片段；由抗体单臂的VL和VH结构域组成的Fv片段；由VH结构域组成的dAb片段(Ward等人,《自然》341:544-546,1989)；以及分离的互补决定区(CDR)或抗体的其它表位结合片段。此外，虽然Fv片段的两个结构域VL和VH由单独的基因编码，但是这两个结构域可以使用重组方法通过使这两个结构域能够成为单个蛋白链的合成连接子来连接，在所述单个蛋白链中，VL区和VH区对用于形成单价分子(被称为单链Fv(“scFv”))；参见例如，Bird等人,《科学(Science)》242:423-426,1988；以及Huston等人,《美国国家科学院院刊》85:5879-5883,1988)。此类单链抗体也旨在涵盖于术语“抗原结合片段”内。使用本领域技术人员已知的常规技术获得这些抗原结合片段，并且以与完整抗体相同的方式筛选所述片段的实用性。The term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution). Examples of binding fragments include, but are not limited to: single-chain Fv (scFv), disulfide-linked Fv (sdFv), Fab fragments, F(ab') fragments (i.e., monovalent fragments consisting of VL, VH, CL, and CH1 domains); F(ab)2 fragments (i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region); Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); and isolated complementary determining regions (CDRs) or other epitope-binding fragments of antibodies. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, the two domains can be connected using recombinant methods by a synthetic linker that enables the two domains to become a single protein chain, in which the VL region and the VH region are used to form a monovalent molecule (referred to as a single-chain Fv ("scFv"); see, for example, Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment". These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as intact antibodies.

抗原结合片段还可以并入到单结构域抗体、大抗体、微抗体、纳米抗体、胞内抗体、双抗体、三抗体、四抗体、v-NAR和双-scFv中(参见例如，Hollinger和Hudson,《自然生物技术(Nature Biotechnology)》23:1126-1136,2005)。抗原结合片段可以基于如纤连蛋白III型(Fn3)等多肽接枝到支架中(参见美国专利第6,703,199号，所述美国专利描述了纤连蛋白多肽单抗)。抗原结合片段可以并入到包括与互补轻链多肽一起形成一对抗原结合区的一对串联Fv区段(VH-CH1-VH-CH1)的单链分子中(Zapata等人,《蛋白质工程(Protein Eng.)》8:1057-1062,1995；以及美国专利第5,641,870号。Antigen binding fragments can also be incorporated into single domain antibodies, large antibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen binding fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monoclonal antibodies). Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) that form a pair of antigen binding regions together with complementary light chain polypeptides (Zapata et al., Protein Eng. 8: 1057-1062, 1995; and U.S. Patent No. 5,641,870).

术语“人源化抗体”是指包含来自非人HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在一些实施方案中，人源化抗体将包含基本上所有的至少一个、通常两个可变结构域，其中所有或基本上所有的HVR(例如，CDR)对应于非人抗体的序列，并且所有或基本上所有的FR对应于人抗体的序列。The term "humanized antibody" refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the sequence of a non-human antibody, and all or substantially all of the FRs correspond to the sequence of a human antibody.

术语“亲和力”是指抗体与抗原之间在单个抗原位点处的相互作用的强度。在每个抗原位点内，抗体“臂”的可变区通过弱非共价力与抗原在许多位点处相互作用；相互作用越多，亲和力越强。The term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" interact with the antigen at many sites through weak non-covalent forces; the more interactions, the stronger the affinity.

本文中术语“竞争”指用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：在所述测定法中，待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原的特异性结合。 The term "competition" herein, when referring to the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, means competition between antigen binding proteins, which is determined by the following assay: in the assay, the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen.

如本文所用，术语“变体”是指已经修饰至少一个，例如1、2或3个氨基酸取代、缺失或添加的重链可变区或轻链可变区，其中包含重链或轻链变体的经修饰的抗原结合蛋白基本上保留修饰前抗原结合蛋白的生物学特征。在一个实施方案中，含有变体重链可变区或轻链可变区序列的抗原结合蛋白保留修饰前抗原结合蛋白的70％、80％、90％、100％生物学特征。应当理解，可以单独或在与另一个重链可变区或轻链可变区组合修饰每个重链可变区或轻链可变区。本公开的抗原结合蛋白包含与本文描述的重链可变区氨基酸序列70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同源的重链可变区氨基酸序列。本公开的抗原结合蛋白包括与本文描述的轻链可变区氨基酸序列70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同源的轻链可变区氨基酸序列。同源性百分比可以在整个重链可变区和/或整个轻链可变区上，或者百分比同源性可以限于构架区，而对应于CDR的序列与重链可变区和/或轻链可变区内本文中公开的CDR具有100％同一性。如本文所用，术语“CDR变体”是指已经修饰至少一个，例如1、2或3个氨基酸取代、缺失或添加的CDR，其中包含CDR变体的经修饰的抗原结合蛋白基本上保留修饰前抗原结合蛋白的生物学特征。在一个实施方案中，含有变体CDR的抗原结合蛋白保留修饰前抗原结合蛋白的60％、70％、80％、90％、100％生物学特征。As used herein, the term "variant" refers to a heavy chain variable region or light chain variable region that has been modified with at least one, such as 1, 2 or 3 amino acid substitutions, deletions or additions, wherein the modified antigen-binding protein comprising a heavy chain or light chain variant substantially retains the biological characteristics of the antigen-binding protein before modification. In one embodiment, the antigen-binding protein containing the variant heavy chain variable region or light chain variable region sequence retains 70%, 80%, 90%, 100% biological characteristics of the antigen-binding protein before modification. It should be understood that each heavy chain variable region or light chain variable region can be modified alone or in combination with another heavy chain variable region or light chain variable region. The antigen-binding proteins of the present disclosure include heavy chain variable region amino acid sequences that are 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequences described herein. The antigen-binding proteins disclosed herein include light chain variable region amino acid sequences that are 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequences described herein. The homology percentage can be over the entire heavy chain variable region and/or the entire light chain variable region, or the percentage homology can be limited to the framework region, and the sequence corresponding to the CDR has 100% identity with the CDR disclosed herein in the heavy chain variable region and/or the light chain variable region. As used herein, the term "CDR variant" refers to a CDR that has been modified by at least one, such as 1, 2 or 3 amino acid substitutions, deletions or additions, wherein the modified antigen-binding protein containing the CDR variant substantially retains the biological characteristics of the antigen-binding protein before modification. In one embodiment, the antigen-binding protein containing the variant CDR retains 60%, 70%, 80%, 90%, 100% biological characteristics of the antigen-binding protein before modification.

上述氨基酸同源性中的氨基酸变化，优选为保守性变化，可以包括氨基酸的取代、***或缺失。优选的，本文所述的氨基酸变化为氨基酸取代，优选地保守性取代。保守性取代是指一个氨基酸经相同类别内的另一氨基酸取代，例如一个酸性氨基酸经另一酸性氨基酸取代，一个碱性氨基酸经另一碱性氨基酸取代，或一个中性氨基酸经另一中性氨基酸取代。示例性的取代如下表所示(氨基酸取代)。

The amino acid changes in the above amino acid homology are preferably conservative changes, and may include substitutions, insertions or deletions of amino acids. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. Conservative substitutions refer to substitutions of one amino acid by another amino acid in the same class, such as substitutions of one acidic amino acid by another acidic amino acid, substitutions of one basic amino acid by another basic amino acid, or substitutions of one neutral amino acid by another neutral amino acid. Exemplary substitutions are shown in the following table (amino acid substitutions).

在优选的实施方案中，本申请所述的氨基酸变化发生在CDR外的区域(例如在FR中)。更优选地，本申请所述的氨基酸变化发生在Fc区。In a preferred embodiment, the amino acid changes described herein occur in regions outside of the CDRs (eg, in the FRs). More preferably, the amino acid changes described herein occur in the Fc region.

术语“载体”在本文中使用时是指能够通过转化扩增与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其可操作相连的核酸的表达。这样的载体在本文中被称为“表达载体”。The term "vector" as used herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked by transformation. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

术语“宿主细胞”是指引入外源核酸的细胞，包括这种细胞的后代。并能够表达外源核酸于细胞内或细胞膜或释放至胞外。The term "host cell" refers to a cell into which an exogenous nucleic acid is introduced, including the progeny of such a cell, and is capable of expressing the exogenous nucleic acid in the cell or cell membrane or releasing it to the outside of the cell.

术语“受试者”包含人和非人动物。非人动物包含所有脊椎动物，例如哺乳动物和非哺乳动物，如非人灵长类动物、绵羊、狗、牛、鸡、两栖动物和爬行动物。除在指出时之外，术语“患者”或“受试者”在本文中可互换使用。The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Except when indicated, the terms "patient" or "subject" are used interchangeably herein.

1、本申请的针对Claudin-18.2的特异性抗体或抗原结合片段1. The specific antibody or antigen-binding fragment against Claudin-18.2 of the present application

术语“针对Claudin-18.2的特异性抗体”、“抗Claudin-18.2的特异性抗体”、“特异性针对Claudin-18.2的抗体”、“Claudin-18.2特异性抗体”或“结合Claudin-18.2的特异性抗体”在本文中可互换地使用，是指这样的本申请的抗体或抗原结合片段，所述抗体或抗原结合片段能够以特异性方式结合Claudin-18.2蛋白或抗原，由此所述抗体抗原结合片段可以用于Claudin-18.2的检测、鉴定或筛选等。 The terms "specific antibody against Claudin-18.2", "specific antibody against Claudin-18.2", "antibody specific to Claudin-18.2", "Claudin-18.2-specific antibody" or "specific antibody that binds to Claudin-18.2" are used interchangeably herein and refer to the antibody or antigen-binding fragment of the present application, which can bind to Claudin-18.2 protein or antigen in a specific manner, so that the antibody antigen-binding fragment can be used for the detection, identification or screening of Claudin-18.2, etc.

本申请的抗体和抗原结合片段以高亲和力特异性结合Claudin-18.2蛋白，在一些实施方案中，根据上述定义中关于CDR的不同分析方法可知，本申请的特异性抗体或抗原结合片段是这样一类序列：The antibodies and antigen-binding fragments of the present application specifically bind to the Claudin-18.2 protein with high affinity. In some embodiments, according to the different analysis methods for CDR in the above definition, the specific antibodies or antigen-binding fragments of the present application are such a sequence:

包含SEQ ID NO.7所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO.8所示轻链可变区氨基酸序列中的3个CDR；或者与上述轻重链CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸保守性变化的变体。It comprises the three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO.7 and the three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO.8; or a variant having a single or multiple CDRs with no more than two conservative amino acid changes in each CDR region compared with the above-mentioned light and heavy chain CDR regions.

在一些实施方案中，当按照Kabat编码规则对抗体CDRs编码时，所述抗体或抗原结合片段包含如下CDR序列：In some embodiments, when the antibody CDRs are encoded according to the Kabat numbering rules, the antibody or antigen-binding fragment comprises the following CDR sequence:

i.重链可变区的互补决定区HCDR1、HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.1、2、3所示；i. The amino acid sequences of the complementary determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown in SEQ ID NO. 1, 2, and 3, respectively;

ii.轻链可变区的互补决定区LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、5、6所示；ii. The amino acid sequences of the complementary determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO. 4, 5 and 6 respectively;

或者，与上述i-ii的6个CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸保守性变化的变体。Alternatively, a variant having a single or multiple CDRs with no more than 2 conservative amino acid changes in each CDR region compared to the 6 CDR regions of i-ii above.

在一些实施方案中，所述抗体或抗原结合片段包含重链可变区和轻链可变区，序列选自如下：In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the sequence of which is selected from the following:

a)所述重链可变区的氨基酸序列如SEQ ID NO.7所示，或与SEQ ID NO.7具有至少70％、80％、90％、95％或99％序列同一性；a) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.7, or has at least 70%, 80%, 90%, 95% or 99% sequence identity with SEQ ID NO.7;

b)所述轻链可变区的氨基酸序列如SEQ ID NO.8所示，或与SEQ ID NO.8具有至少70％、80％、90％、95％或99％序列同一性；b) the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.8, or has at least 70%, 80%, 90%, 95% or 99% sequence identity with SEQ ID NO.8;

在一些具体的实施方案中，所述抗体或抗原结合片段还包含如SEQ ID NO.9所示的重链恒定区，和SEQ ID NO.10所示的轻链恒定区；In some specific embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region as shown in SEQ ID NO.9, and a light chain constant region as shown in SEQ ID NO.10;

在一些更具体的实施方式中，所述抗体或抗原结合片段的重链全长序列如SEQ ID NO.10所示，轻链全长序列如SEQ ID NO.11所示。In some more specific embodiments, the full-length sequence of the heavy chain of the antibody or antigen-binding fragment is shown as SEQ ID NO.10, and the full-length sequence of the light chain is shown as SEQ ID NO.11.

在一些实施方案中，所述抗体或抗原结合片段可进一步连接至偶联部分，所述偶联部分选自放射性核素、药物、毒素、细胞因子、酶、荧光素、载体蛋白、脂类、和生物素中的一种或多种，其中所述多肽或抗体与所述偶联部分可选择性通过连接子相连，优选所述连接子为肽或多肽。In some embodiments, the antibody or antigen-binding fragment may be further linked to a coupling portion, which is selected from one or more of radionuclides, drugs, toxins, cytokines, enzymes, fluorescein, carrier proteins, lipids, and biotin, wherein the polypeptide or antibody and the coupling portion may be selectively connected via a linker, preferably the linker is a peptide or polypeptide.

在一些实施方案中，所述抗体或抗原结合片段可选自单克隆抗体、多克隆抗体、抗血清、嵌合抗体、人源化抗体和人抗体；更优选的，所述抗体选自多特异性抗体、单链Fv(scFv)、单链抗体、抗独特型(抗-Id)抗体、双抗体、微型抗体、纳米抗体、单结构域抗体、Fab片段、F(ab’)片段、二硫化物连接的双特异性Fv(sdFv)和胞内抗体。In some embodiments, the antibody or antigen-binding fragment can be selected from a monoclonal antibody, a polyclonal antibody, an antiserum, a chimeric antibody, a humanized antibody and a human antibody; more preferably, the antibody is selected from a multispecific antibody, a single-chain Fv (scFv), a single-chain antibody, an anti-idiotypic (anti-Id) antibody, a diabody, a minibody, a nanobody, a single domain antibody, a Fab fragment, a F(ab') fragment, a disulfide-linked bispecific Fv (sdFv) and an intrabody.

在一些实施方案中，本申请所述的抗体或其抗原结合片段可以通过重组表达产生。可以将编码任选与恒定区连接的轻链和重链可变区的上述核酸***表达载体中。包含编码本文所述抗体的核酸的载体本身是本申请的一方面。轻链和重链可以克隆到相同或不同的表达载体中。编码本文所述抗体链的核酸可以与表达载体中的一个或多个控制序列 可操作地连接，以确保抗体链的表达。表达控制序列包括但不限于启动子(例如，天然相关或异源启动子)、信号序列、增强子元件和转录终止序列。优选地，表达控制序列是能够转化或转染真核宿主细胞的载体中的真核启动子***。此类载体可并入合适的宿主中，由此宿主维持在适合于高水平表达核苷酸序列以及收集和纯化抗体的条件下。In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be produced by recombinant expression. The above-mentioned nucleic acids encoding the light chain and heavy chain variable regions, optionally linked to constant regions, can be inserted into an expression vector. The vectors containing nucleic acids encoding the antibodies described herein are themselves one aspect of the present application. The light chain and heavy chain can be cloned into the same or different expression vectors. The nucleic acids encoding the antibody chains described herein can be linked to one or more control sequences in the expression vector. Operably connected to ensure the expression of the antibody chain. Expression control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Such vectors can be incorporated into a suitable host, whereby the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of the antibody.

2、本申请的核酸、相应载体和宿主细胞等2. Nucleic acids, corresponding vectors, host cells, etc. of the present application

本申请提供了编码以上任何Claudin-18.2抗体或其抗原结合片段或其任一条链的核酸，提供包含所述核酸的载体，提供包含所述核酸或所述载体的宿主细胞。The present application provides nucleic acids encoding any of the above Claudin-18.2 antibodies or antigen-binding fragments thereof or any of their chains, vectors comprising the nucleic acids, and host cells comprising the nucleic acids or the vectors.

本申请涉及的核酸是编码抗Claudin-18.2蛋白抗体或其抗原结合片段，或其VH或VL结构域的核酸；可以理解，但凡能够编码上述抗体或其抗原结合片段，或其VH或VL结构域的核酸都在本申请的范围内。The nucleic acid involved in the present application is a nucleic acid encoding an anti-Claudin-18.2 protein antibody or its antigen-binding fragment, or its VH or VL domain; it can be understood that any nucleic acid that can encode the above-mentioned antibody or its antigen-binding fragment, or its VH or VL domain is within the scope of the present application.

当然，本申请的核酸也可以是与上述任一具有密码子兼并性的核酸序列，比如与上述序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。Of course, the nucleic acid of the present application can also be a nucleic acid sequence having codon degeneracy with any of the above-mentioned sequences, such as a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above-mentioned sequences.

本申请的包含上述一种或多种编码本文所述抗体的核酸的载体，载体可以是克隆载体或表达载体，在此不作限制。The vector of the present application comprising one or more nucleic acids encoding the antibodies described herein may be a cloning vector or an expression vector, which is not limited herein.

在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)等。In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. The vector includes, but is not limited to, a virus, a plasmid, a cosmid, a lambda phage, or a yeast artificial chromosome (YAC).

在一个实施方案中，载体包含任选的调控序列；在一些实施方式中，所述调控序列可以选自前导序列、多聚腺苷酸化序列、前肽序列、启动子、信号序列、转录终止子，或其任何组合，不作限制。In one embodiment, the vector comprises an optional regulatory sequence; in some embodiments, the regulatory sequence can be selected from a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, a transcription terminator, or any combination thereof, without limitation.

本申请的包含所述表达载体的宿主细胞，比如宿主细胞为酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。在一些实施方案中，合适的宿主细胞包括原核微生物，如大肠杆菌。宿主细胞还可以是真核微生物如丝状真菌或酵母，或各种真核细胞，例如昆虫细胞等。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用的哺乳动物宿主细胞系的例子包括SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK293或293F细胞)、293细胞、幼仓鼠肾细胞(BHK)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人***细胞(HELA)、犬肾细胞(MDCK)、布法罗大鼠肝脏细胞(BRL 3A)、人肺细胞(W138)、人肝脏细胞(Hep G2)、中国仓鼠卵巢细胞(CHO细胞)、CHOS细胞、NSO细胞、骨髓瘤细胞系如Y0、NS0、P3X63和Sp2/0等。适于产生蛋白质的哺乳动物宿主细胞系的综述参见例如Yazaki和Wu，Methods in Molecular Biology，第248卷(B.K.C.Lo编著，Humana Press，Totowa，NJ)，第255-268页(2003)。在一个优选的实施方案中，所述宿主细胞是CHO细胞或293细胞。The host cell comprising the expression vector of the present application, such as host cell is yeast cell, mammalian cell or other cells suitable for preparing antibody or its antigen-binding fragment. In some embodiments, suitable host cell includes prokaryotic microorganism, such as Escherichia coli. Host cell can also be eukaryotic microorganism such as filamentous fungi or yeast, or various eukaryotic cells, such as insect cells etc. Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2/0, etc. For a review of mammalian host cell lines suitable for producing proteins, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or a 293 cell.

可以通过熟知的方法将本文描述的包含感兴趣的多核苷酸序列(例如，重链和轻链编码序列和表达控制序列)的载体转移到宿主细胞中，该方法根据细胞宿主的类型而有所不同。例如，氯化钙转染通常用于原核细胞，而磷酸钙处理、电穿孔、脂质转染、生 物射弹或基于病毒的转染可用于其它细胞宿主。(通常参见Green和Sambrook，分子克隆：实验室手册(Molecular Cloning:ALaboratory Manual)(冷泉港出版社，第4版，2012年)。用于转化哺乳动物细胞的其它方法包括使用聚凝胺、原生质体融合、脂质体、电穿孔和显微注射(通常参见，Sambrook等.,同上)。为了产生转基因动物，可以将转基因显微注射到受精***中，或者可以将其整合到胚胎干细胞的基因组中，并且这些细胞的细胞核被转移到去核***中。The vectors described herein containing the polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biotinylation, and the like are commonly used for prokaryotic cells. Biolistic or virus-based transfection can be used for other cellular hosts. (See generally Green and Sambrook, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 4th edition, 2012). Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al., supra). To produce transgenic animals, the transgene can be microinjected into fertilized oocytes, or it can be integrated into the genome of embryonic stem cells, and the nuclei of these cells are transferred into enucleated oocytes.

3、本申请的抗体或抗原结合片段的制备、生产和纯化方法3. Preparation, production and purification methods of the antibodies or antigen-binding fragments of the present application

本申请提供了制备Claudin-18.2抗体或抗原结合片段的方法，其中所述方法包括在适于表达编码所述抗体或抗原结合片段的核酸的条件下培养包含编码所述抗体的核酸或包含所述核酸的表达载体的宿主细胞，以及任选地分离所述抗体或抗原结合片段。在某个实施方案中，所述方法还包括从所述宿主细胞(或宿主细胞培养基)回收纯化相应抗体或抗原结合片段。The present application provides a method for preparing a Claudin-18.2 antibody or antigen-binding fragment, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody or an expression vector comprising the nucleic acid under conditions suitable for expressing the nucleic acid encoding the antibody or antigen-binding fragment, and optionally isolating the antibody or antigen-binding fragment. In a certain embodiment, the method further comprises recovering and purifying the corresponding antibody or antigen-binding fragment from the host cell (or host cell culture medium).

在一些实施方案中，该方法可包括将包含一种或多种编码如上所述抗Claudin-18.2蛋白抗体或其抗原结合片段或其抗体链的核酸的载体转移到如本文所述的宿主细胞中，在允许表达核酸的条件下培养宿主细胞培养物，回收表达的抗Claudin-18.2蛋白抗体或其抗原结合片段。可以采用本领域已知的任何合适的方法。In some embodiments, the method may include transferring a vector comprising one or more nucleic acids encoding an anti-Claudin-18.2 protein antibody or its antigen-binding fragment or its antibody chain as described above into a host cell as described herein, culturing the host cell culture under conditions that allow expression of the nucleic acid, and recovering the expressed anti-Claudin-18.2 protein antibody or its antigen-binding fragment. Any suitable method known in the art may be used.

在一些实施方案中，本文所述制备的抗体或抗原结合片段可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。可以通过多种熟知分析方法中的任一种方法确定本申请的抗体的纯度，所述熟知分析方法包括大小排阻层析、凝胶电泳、高效液相色谱等。In some embodiments, the antibodies or antigen-binding fragments prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and these are obvious to those skilled in the art. The purity of the antibodies of the present application can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

在一些实施方案中，可以通过本领域中已知的多种测定法对本文中提供的Claudin-18.2抗体鉴定、筛选或表征其物理/化学特性和/或生物学活性。一方面，对本申请的抗体测试其抗原结合活性，例如通过已知的方法诸如ELISA，Western印迹、IHC等来进行。可使用本领域已知方法来测定对Claudin-18.2蛋白的结合，在一些实施方案中，可以使用SPR或生物膜层干涉测定本申请的Claudin-18.2抗体对Claudin-18.2蛋白的结合。In some embodiments, the Claudin-18.2 antibodies provided herein can be identified, screened or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. On the one hand, the antibodies of the present application are tested for their antigen binding activity, for example, by known methods such as ELISA, Western blotting, IHC, etc. The binding to the Claudin-18.2 protein can be determined using methods known in the art. In some embodiments, the binding of the Claudin-18.2 antibody of the present application to the Claudin-18.2 protein can be determined using SPR or biofilm interferometry.

4、本申请的抗体或其抗原结合片段的检测和诊断用途4. Detection and diagnostic uses of the antibodies or antigen-binding fragments thereof of the present application

本申请提供的抗体或抗原结合片段可以用于检测Claudin-18.2在生物样品中的存在或含量，即，既可定性检测，也可定量检测。进而可用于临床前中后的分析，比如免疫评价、免疫原性分析等，甚至用于疾病的诊断或伴随诊断方面。The antibodies or antigen-binding fragments provided in the present application can be used to detect the presence or content of Claudin-18.2 in biological samples, that is, both qualitative and quantitative detection. They can then be used for preclinical, mid-term and post-clinical analysis, such as immune evaluation, immunogenicity analysis, and even for disease diagnosis or companion diagnosis.

本申请提供的Claudin-18.2抗体或其抗原结合片段能方便地用于试剂盒中，体内或体外的生物流体内或组织上的Claudin-18.2可被本申请提供的抗体或其抗原结合片段检测出来。可用于任何含有可检测量的Claudin-18.2蛋白的样品；样本可以是临床前后的任何阶段，这里不做限制。 The Claudin-18.2 antibody or antigen-binding fragment thereof provided in the present application can be conveniently used in a kit, and Claudin-18.2 in biological fluids or on tissues in vivo or in vitro can be detected by the antibody or antigen-binding fragment thereof provided in the present application. It can be used for any sample containing a detectable amount of Claudin-18.2 protein; the sample can be at any stage before or after clinical trials, and there is no limitation here.

本申请所述的检测样品可以包括但不限于液体类，比如尿、唾液、脑脊髓液、血液、血清及类似物，或者本申请所述的检测样品可以是固体或半固体类，比如组织、粪便及类似物，或者，可以是如那些常用于组织学诊断的固体组织。The test samples described in the present application may include but are not limited to liquids, such as urine, saliva, cerebrospinal fluid, blood, serum and the like, or the test samples described in the present application may be solid or semi-solid, such as tissue, feces and the like, or may be solid tissues such as those commonly used in histological diagnosis.

术语“试剂盒”用于指有助于样品分析的试剂和其它材料的组合。在一些实施方式中，本文所述的免疫测定试剂盒包括合适的抗原、包含可检测部分的结合剂和检测试剂。用于放大由可检测部分产生的信号的***也可以包括或不包括在试剂盒中。此外，在其它实施方式中，试剂盒包括但不限于诸如用于样品收集的装置、样品管、支架、托盘、架子、盘子、板、试剂盒用户的说明书、溶液或其它化学试剂等组件，以及用于标准化、归一化的样品和/或对照样品。The term "kit" is used to refer to a combination of reagents and other materials that aid in sample analysis. In some embodiments, the immunoassay kits described herein include suitable antigens, binding agents comprising detectable moieties, and detection reagents. Systems for amplifying the signals generated by the detectable moieties may or may not be included in the kit. In addition, in other embodiments, the kit includes, but is not limited to, components such as devices for sample collection, sample tubes, racks, trays, shelves, plates, instructions for kit users, solutions or other chemical reagents, and samples and/or control samples for standardization and normalization.

术语“检测”用于本文中时，包括了定量或定性检测，示例性的检测方法可以涉及免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法。在一些实施方式中，本申请的Claudin-18.2抗体或其抗原结合片段可以偶联荧光素酶、生物素酶等可检测标记，在液相或固相中用于FACS、IHC、ELISA等直接或间接的免疫测定，包括竞争性或非竞争性等等。The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, and ELISA assays. In some embodiments, the Claudin-18.2 antibody or antigen-binding fragment thereof of the present application may be coupled to a detectable marker such as luciferase or biotinidase, and used in a liquid or solid phase for direct or indirect immunoassays such as FACS, IHC, ELISA, including competitive or non-competitive, etc.

示例性的，本申请提供检测Claudin-18.2在生物样品中的存在的方法，所述方法包括检测Claudin-18.2蛋白在生物样品中的存在。在某些实施方案中，所述方法包括将生物样品与如本文所述的Claudin-18.2抗体或抗原结合片段在允许Claudin-18.2抗体或抗原结合片段与Claudin-18.2蛋白结合的条件下接触，并检测在Claudin-18.2抗体或抗原结合片段和Claudin-18.2蛋白之间是否形成复合物。复合物的形成表示存在Claudin-18.2。该方法可以是体外或体内方法。Exemplarily, the present application provides a method for detecting the presence of Claudin-18.2 in a biological sample, the method comprising detecting the presence of Claudin-18.2 protein in the biological sample. In certain embodiments, the method comprises contacting the biological sample with a Claudin-18.2 antibody or antigen-binding fragment as described herein under conditions that allow the Claudin-18.2 antibody or antigen-binding fragment to bind to the Claudin-18.2 protein, and detecting whether a complex is formed between the Claudin-18.2 antibody or antigen-binding fragment and the Claudin-18.2 protein. The formation of a complex indicates the presence of Claudin-18.2. The method can be an in vitro or in vivo method.

本申请的检测用于相关疾病(比如各类肿瘤疾病或癌症：胃癌/胃食管结合部(GC/GEJ)癌、乳腺癌、结肠癌、肝癌、头颈癌、支气管癌和非小细胞肺癌等与Claudin-18.2密切相关的肿瘤疾病)的诊断，包括例如使用本申请的Claudin-18.2抗体或抗原结合片段接触从患者获得的样品，其中使用可检测的标记或报告分子标记该抗体或抗原结合片段或用作捕获配体以选择性地从患者样品分离Claudin-18.2。可检测的标记或报告分子可以是放射性同位素、如3H、14C、32P、35S或125I；荧光或化学发光的部分如荧光素异硫氰酸酯或罗丹明，或酶如碱性磷酸酶、β-半乳糖苷酶、辣根过氧化物酶或荧光素酶。可用于检测或测量样品中Claudin-18.2的具体的示例性测定包括酶连接的免疫吸附测定(ELISA)、放射免疫测定(RIA)、免疫组化(IHC)和荧光活化细胞分选(FACS)等。The detection of the present application is used for the diagnosis of related diseases (such as various tumor diseases or cancers: gastric cancer/gastroesophageal junction (GC/GEJ) cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer and non-small cell lung cancer, etc., which are closely related to Claudin-18.2), including, for example, contacting a sample obtained from a patient with the Claudin-18.2 antibody or antigen-binding fragment of the present application, wherein the antibody or antigen-binding fragment is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively separate Claudin-18.2 from the patient sample. The detectable label or reporter molecule can be a radioactive isotope, such as 3H, 14C, 32P, 35S or 125I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure Claudin-18.2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC), and fluorescence activated cell sorting (FACS), among others.

可在根据本申请的疾病诊断测定中使用的样品包括从患者可获得的任何生物样品，其包含在正常或生理条件下可检测量的Claudin-18.2蛋白或其片段。在一些实施方案中，包括但不限于液体如尿、唾液、脑脊髓液、血液、血清及类似物，或者样品可以是固体或半固体如组织、粪便及类似物，或者，可以是如那些常用于组织学诊断的固体组织。一般而言，将测量从健康患者获得的特定样品中的Claudin-18.2蛋白水平以初始性地建 立基线或标准水平。该基线或标准水平可随后与从疑似具有相关疾病的个体获得的样品中测量Claudin-18.2蛋白水平进行比较。Samples that can be used in disease diagnosis assays according to the present application include any biological sample obtainable from a patient that contains a detectable amount of Claudin-18.2 protein or fragments thereof under normal or physiological conditions. In some embodiments, including but not limited to liquids such as urine, saliva, cerebrospinal fluid, blood, serum, and the like, or the sample can be solid or semi-solid such as tissue, feces, and the like, or, can be solid tissues such as those commonly used for histological diagnosis. In general, the level of Claudin-18.2 protein in a specific sample obtained from a healthy patient will be measured to initially establish This baseline or standard level can then be compared to the level of Claudin-18.2 protein measured in a sample obtained from an individual suspected of having the disease in question.

5、本申请的抗体或其抗原结合片段的其他方面应用5. Other applications of the antibodies or antigen-binding fragments thereof of the present application

可以理解，基于抗体的基础抗原抗体结合活性，本申请的抗体或其抗原结合片段还可以应用于筛选、纯化或制备Claudin-18.2蛋白等。It can be understood that based on the basic antigen-antibody binding activity of the antibody, the antibody or antigen-binding fragment thereof of the present application can also be used to screen, purify or prepare Claudin-18.2 protein, etc.

下面将结合实施例对本申请的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。The technical solution of the present application will be described clearly and completely in conjunction with the embodiments below. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

实施例1特异性Claudin-18.2抗体的制备Example 1 Preparation of specific Claudin-18.2 antibody

本实施例中，按照以下方法制备Claudin-18.2特异性抗体：In this example, Claudin-18.2-specific antibodies were prepared according to the following method:

1)Claudin-18.2蛋白免疫原制备：通过基因合成方法构建可表达人源的Claudin-18.2蛋白、His、Twin-Strep Tag的表达质粒，利用Invitrogen Lipofectamine 2000转染试剂将表达质粒转染到HEK293细胞中，48小时后收获培养上清，之后通过亲和层析的方法获得纯化的Claudin-18.2(DDM)蛋白。1) Preparation of Claudin-18.2 protein immunogen: An expression plasmid expressing human Claudin-18.2 protein, His, and Twin-Strep Tag was constructed by gene synthesis. The expression plasmid was transfected into HEK293 cells using Invitrogen Lipofectamine 2000 transfection reagent. The culture supernatant was harvested after 48 hours, and purified Claudin-18.2 (DDM) protein was obtained by affinity chromatography.

2)小鼠免疫：以步骤1制备的Claudin-18.2(DDM)蛋白作为免疫原，用Claudin-18.2(DDM)蛋白免疫3只Balb/c小鼠，采用锰佐剂免疫手段。免疫结束后，用ELISA方法检测免疫动物血清，以此确定免疫应答的水平。常规免疫结束后，如果免疫动物能够达到针对免疫原的免疫应答水平(OD值>1.0，效价达到1：8,000)，可进行细胞融合。2) Mouse immunization: Use the Claudin-18.2 (DDM) protein prepared in step 1 as an immunogen, and immunize three Balb/c mice with the Claudin-18.2 (DDM) protein, using manganese adjuvant immunization. After the immunization, the immunized animal serum is tested by ELISA to determine the level of immune response. After the routine immunization, if the immunized animal can reach the level of immune response to the immunogen (OD value>1.0, titer reaches 1:8,000), cell fusion can be performed.

3)细胞融合与铺板：采用电融合方法进行1次细胞融合。融合的一半细胞铺到半固体培养基中，另一半融合细胞进行冻存。3) Cell fusion and plating: Cell fusion is performed once using the electrofusion method. Half of the fused cells are plated in semi-solid culture medium, and the other half of the fused cells are frozen.

4)挑取单克隆细胞株：挑取半固体培养基中培养的单个细胞团至96孔培养板中进行培养。4) Picking monoclonal cell lines: Picking single cell clusters cultured in semi-solid culture medium and culturing them in 96-well culture plates.

5)筛选：用ELISA方法筛选融合细胞的上清液，挑选出同时对Claudin-18.2(DDM)和Claudin-18.2(VLP)蛋白结合呈阳性的细胞。5) Screening: The supernatant of the fused cells was screened by ELISA to select cells that were positive for both Claudin-18.2 (DDM) and Claudin-18.2 (VLP) protein binding.

6)克隆扩大培养及复测：将阳性克隆细胞转到48孔板扩大培养，每个扩大培养的克隆收集0.2毫升上清，用于间接ELISA方法进行检测。6) Clone expansion and retesting: The positive clone cells were transferred to a 48-well plate for expansion and culture. 0.2 ml of supernatant was collected from each expanded clone for detection by indirect ELISA method.

7)克隆扩大培养及复测：将阳性克隆细胞转到12孔板扩大培养，每个扩大培养的克隆收集1毫升上清，用于间接ELISA方法进行检测。在抗原识别确认的基础上，本申请筛选到2个稳定的细胞系(克隆号3B10和12C11)进行低温保存。低温冻存前每个克隆收集5毫升上清，并对所有的克隆进行亚型鉴定和保存。7) Clone expansion and retest: The positive clone cells were transferred to a 12-well plate for expansion and culture, and 1 ml of supernatant was collected from each clone for indirect ELISA detection. Based on the confirmation of antigen recognition, this application screened 2 stable cell lines (clone numbers 3B10 and 12C11) for cryopreservation. Before cryopreservation, 5 ml of supernatant was collected from each clone, and all clones were subtyped and stored.

8、杂交瘤细胞抗体基因测序：提取杂交瘤细胞总RNA，通过RT-PCR反应将RNA反转录成cDNA。克隆抗体轻链和重链序列，将抗体轻链和重链序列构建到T载体上， 之后进行DNA测序分析获得抗体基因序列。8. Hybridoma cell antibody gene sequencing: Extract total RNA from hybridoma cells and reverse transcribe RNA into cDNA through RT-PCR reaction. Clone antibody light chain and heavy chain sequences and construct antibody light chain and heavy chain sequences onto T vector. DNA sequencing analysis was then performed to obtain the antibody gene sequence.

通过测序分析，按照Kabat编码规则，克隆号3B10抗体的重链可变区的互补决定区CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：1、2、3所示；轻链可变区的互补决定区CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：4、5、6所示；重链可变区的氨基酸序列如SEQ ID NO：7所示；轻链可变区的氨基酸序列如SEQ ID NO：8所示。Through sequencing analysis, according to the Kabat coding rules, the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the clone 3B10 antibody are as shown in SEQ ID NOs: 1, 2, and 3, respectively; the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are as shown in SEQ ID NOs: 4, 5, and 6, respectively; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8.

具体序列如下表：

The specific sequence is as follows:

9)抗体生产与纯化：将步骤8获得的抗体基因序列转染到HEK293细胞中，并进行扩大培养，采用蛋白质A/G亲和层析方法纯化抗体，用透析方法将纯化抗体保存在磷酸盐缓冲液(PBS)中。9) Antibody production and purification: The antibody gene sequence obtained in step 8 was transfected into HEK293 cells and cultured amplified. The antibody was purified by protein A/G affinity chromatography and stored in phosphate buffered saline (PBS) by dialysis.

实施例2 Claudin-18.2抗体3B10特异性分析Example 2 Specificity analysis of Claudin-18.2 antibody 3B10

本实施例中，按照上述实施例1中的方法获得的单克隆抗体3B10，并采用酶联免 疫吸附试验对该抗体进行特异性分析，分析方法如下：In this example, the monoclonal antibody 3B10 was obtained according to the method in Example 1 above, and ELISA was used to The specificity of the antibody was analyzed by immunoabsorption test, and the analysis method was as follows:

1)用PBS将Claudin-18.2(DDM)、Claudin-18.2(VLP)、Claudin-18.1、Claudin-6的蛋白稀释至5μg/mL，加入酶标板孔中，每孔100μL。用封板膜封板，放置4℃过夜(约16h)。1) Dilute the proteins of Claudin-18.2 (DDM), Claudin-18.2 (VLP), Claudin-18.1, and Claudin-6 to 5 μg/mL with PBS and add 100 μL to each well of the ELISA plate. Seal the plate with a sealing film and place at 4°C overnight (about 16 hours).

2)弃去孔中液体，拍干酶标板，用PBST洗液洗板，300μL/孔浸泡30S，拍干酶标板，进行下一次清洗，共洗板3次。2) Discard the liquid in the wells, pat the ELISA plate dry, wash the plate with PBST solution, 300 μL/well, soak for 30 seconds, pat the ELISA plate dry, and proceed to the next wash, washing the plate 3 times in total.

3)每孔加入100μL封闭剂(含有5％BSA的PBST洗液),用封板膜封板，放置37℃孵育1.5h。3) Add 100 μL of blocking agent (PBST containing 5% BSA) to each well, seal the plate with a sealing film, and incubate at 37°C for 1.5 h.

4)重复步骤2洗板3次。4) Repeat step 2 and wash the plate 3 times.

5)将上述Claudin-18.2抗体用样品稀释液(含有0.5％BSA的PBST洗液)稀释至1μg/mL。加入酶标板中，每孔100μL。用封板膜封板，放置37℃孵育1.0h。5) Dilute the above Claudin-18.2 antibody to 1 μg/mL with sample diluent (PBST washing solution containing 0.5% BSA). Add 100 μL to each well of the ELISA plate. Seal the plate with a sealing film and incubate at 37°C for 1.0 h.

6)重复步骤2洗板3次。6) Repeat step 2 and wash the plate 3 times.

7)用样品稀释液将HRP-Goat-Anti-Mouse IgG稀释至1:20000倍，每孔加入100μL，用封板膜封板，放置37℃孵育1.0h。7) Dilute HRP-Goat-Anti-Mouse IgG to 1:20000 with sample diluent, add 100 μL to each well, seal the plate with sealing film, and incubate at 37°C for 1.0 h.

8)重复步骤2洗板3次。8) Repeat step 2 and wash the plate 3 times.

9)每孔加入100μL显色液.用封板膜封板，放置37℃避光孵育20min。9) Add 100 μL of color development solution to each well. Seal the plate with a sealing film and incubate at 37°C in the dark for 20 min.

10)每孔加入50μL中止液，轻轻震荡酶标板至显色均匀。10) Add 50 μL of stop solution to each well and gently shake the ELISA plate until the color is evenly developed.

11)用酶标仪读取450nm和630nm的吸光度值，用OD₄₅₀扣减OD₆₃₀值得到吸光度值(OD值)。11) Read the absorbance values at 450 nm and 630 nm using a microplate reader, and subtract the OD ₆₃₀ value from the OD ₄₅₀ value to obtain the absorbance value (OD value).

单克隆抗体的吸光度值(OD值)检测结果如表1所示。The absorbance value (OD value) detection results of the monoclonal antibody are shown in Table 1.

表1抗体的ELISA检测OD值
Table 1 OD values of antibodies detected by ELISA

实验结果显示，上述抗体针对Claudin18.2蛋白的特异性最好、活性最高且与Claudin18.1和Claudin 6蛋白都没有交叉反应。Experimental results showed that the above antibodies had the best specificity and highest activity against Claudin18.2 protein and had no cross-reaction with Claudin18.1 and Claudin 6 proteins.

实施例3 Claudin-18.2抗体3B10性能分析(敏感性、特异性、交叉反应性)Example 3 Performance Analysis of Claudin-18.2 Antibody 3B10 (Sensitivity, Specificity, Cross-Reactivity)

将抗体Claudin-18.2(3B10)按照一定比例使用稀释液进行稀释，制备成一抗抗体工作液。The antibody Claudin-18.2 (3B10) was diluted with a diluent according to a certain ratio to prepare a primary antibody working solution.

1)将FFPE正常胃组织***固定，石蜡包埋，切片样本恢复至室温，备用。1) Formalin fixation of FFPE normal gastric tissue, paraffin embedding, and sectioning samples are restored to room temperature for later use.

2)对组织切片进行脱蜡，水化及抗原修复。2) Dewax, hydrate and perform antigen retrieval on tissue sections.

3)加入150μL内源性过氧化物酶阻断剂直到完全覆盖组织，室温孵育5分钟后， PBS水冲洗3次，3) Add 150 μL of endogenous peroxidase blocker until the tissue is completely covered, and incubate at room temperature for 5 minutes. Rinse with PBS water 3 times.

4)加入150μL一抗抗体工作液，室温孵育30分钟后，PBS冲洗3次。4) Add 150 μL of primary antibody working solution, incubate at room temperature for 30 minutes, and then rinse with PBS three times.

5)加入150μL二抗，室温孵育8分钟；PBS冲洗3次，每次2分钟，纯化水冲洗1次。5) Add 150 μL of secondary antibody and incubate at room temperature for 8 minutes; rinse with PBS 3 times, 2 minutes each time, and rinse once with purified water.

7)加入150μLDAB显色液完全覆盖组织，室温孵育10分钟，纯化水冲洗3次。7) Add 150 μL of DAB colorimetric solution to completely cover the tissue, incubate at room temperature for 10 minutes, and rinse with purified water three times.

8)加入150μL苏木素，室温孵育5分钟，纯化水冲洗1次，PBS冲洗1次，纯化水冲洗1次。8) Add 150 μL of hematoxylin, incubate at room temperature for 5 minutes, rinse once with purified water, once with PBS, and once with purified water.

依次浸泡梯度酒精85％，95％，100％各一次，每次3分钟；二甲苯透明两次，每次15分钟。Soak in gradient alcohol 85%, 95%, and 100% once each for 3 minutes; transparentize in xylene twice for 15 minutes each time.

9)用中性树胶对样品进行封片。9) Seal the samples with neutral gum.

10)免疫组化检测结果要由有资质的病理医生在显微镜下对染色后的切片进行观察和判断。在显微镜下，抗原对应的特定细胞位置可观察到深棕色着色，且无背景染色。10) The results of immunohistochemistry testing should be observed and judged by a qualified pathologist under a microscope on the stained sections. Under the microscope, dark brown staining can be observed at the specific cell locations corresponding to the antigen, with no background staining.

各单克隆抗体免疫组织化学染色结果如图1所示。The results of immunohistochemical staining with each monoclonal antibody are shown in Figure 1 .

实验结果显示，敏感性和特异性方面，Claudin18.2(3B10)一抗染色胃腺细胞细胞质和/或细胞膜强阳性染色，而其他组织细胞阴性染色，证明上述单克隆抗体针对Claudin18.2蛋白具有非常高的敏感性和特异性。而且在交叉反应性上，考虑到肺组织为CLDN18.1阳性反应而上述克隆抗体染色肺组织并没有产生阳性信号，证明其与Claudin18.1蛋白没有交叉反应。The experimental results showed that in terms of sensitivity and specificity, the Claudin18.2 (3B10) primary antibody stained the cytoplasm and/or cell membrane of gastric gland cells with strong positive staining, while other tissue cells stained negatively, proving that the above monoclonal antibody has very high sensitivity and specificity for the Claudin18.2 protein. In terms of cross-reactivity, considering that lung tissue is CLDN18.1 positive, the above cloned antibody did not produce a positive signal when staining lung tissue, proving that it has no cross-reaction with the Claudin18.1 protein.

实施例4与商品化抗体性能比较Example 4 Comparison of performance with commercial antibodies

将本申请抗体Claudin-18.2(3B10)和ABCAM公司的商品化的抗体Claudin-18.2(ABCAM EPR19202)分别按照比例使用稀释液进行稀释，制备成一抗抗体工作液(使用时按同等浓度使用)，分别按照如下步骤进行测试。The antibody Claudin-18.2 (3B10) of the present application and the commercialized antibody Claudin-18.2 (ABCAM EPR19202) of ABCAM were diluted with diluents in proportion to prepare primary antibody working solutions (used at the same concentrations), and tested according to the following steps.

1)将FFPE正常胃组织***固定，石蜡包埋，切片样本恢复至室温，备用。1) Formalin fixation of FFPE normal gastric tissue, paraffin embedding, and sectioning samples are restored to room temperature for later use.

2)对组织切片进行脱蜡，水化及抗原修复。2) Dewax, hydrate and perform antigen retrieval on tissue sections.

3)加入150μL内源性过氧化物酶阻断剂直到完全覆盖组织，室温孵育5分钟后，PBS水冲洗3次，3) Add 150 μL of endogenous peroxidase blocker until the tissue is completely covered, incubate at room temperature for 5 minutes, and then rinse with PBS water 3 times.

4)加入150μL一抗抗体工作液，室温孵育30分钟后，PBS冲洗3次。4) Add 150 μL of primary antibody working solution, incubate at room temperature for 30 minutes, and then rinse with PBS three times.

5)加入150μL二抗，室温孵育8分钟；PBS冲洗3次，每次2分钟，纯化水冲洗1次。5) Add 150 μL of secondary antibody and incubate at room temperature for 8 minutes; rinse with PBS 3 times, 2 minutes each time, and rinse once with purified water.

7)加入150μLDAB显色液完全覆盖组织，室温孵育10分钟，纯化水冲洗3次。7) Add 150 μL of DAB colorimetric solution to completely cover the tissue, incubate at room temperature for 10 minutes, and rinse with purified water three times.

8)加入150μL苏木素，室温孵育5分钟，纯化水冲洗1次，PBS冲洗1次，纯化水冲洗1次。8) Add 150 μL of hematoxylin, incubate at room temperature for 5 minutes, rinse once with purified water, once with PBS, and once with purified water.

依次浸泡梯度酒精85％，95％，100％各一次，每次3分钟；二甲苯透明两次，每 次15分钟。Soak in gradient alcohol 85%, 95%, and 100% once, each for 3 minutes; transparentize with xylene twice, each time 15 minutes each time.

9)用中性树胶对样品进行封片。9) Seal the samples with neutral gum.

10)免疫组化检测结果要由有资质的病理医生在显微镜下对染色后的切片进行观察和判断。在显微镜下，抗原对应的特定细胞位置可观察到深棕色着色，且无背景染色。10) The results of immunohistochemistry testing should be observed and judged by a qualified pathologist under a microscope on the stained sections. Under the microscope, dark brown staining can be observed at the specific cell locations corresponding to the antigen, with no background staining.

对上述两支抗体染色结果进行比对分析，各单克隆抗体免疫组织化学染色结果如图2所示。The staining results of the above two antibodies were compared and analyzed, and the immunohistochemical staining results of each monoclonal antibody are shown in Figure 2.

实验结果显示，上述2个克隆抗体针对Claudin18.2蛋白检测的的特异性都满足抗体使用需求。不过在敏感性方面吗，本申请抗体Caudin18.2(3B10)的阳性信号远强于商品化抗体Claudin18.2(EPR19202)，敏感性性能上，抗体Caudin18.2(3B10)比较抗体Claudin18.2(EPR19202)有着明显且预料之外的优势。The experimental results show that the specificity of the above two cloned antibodies for detecting Claudin18.2 protein meets the requirements for antibody use. However, in terms of sensitivity, the positive signal of the antibody Caudin18.2 (3B10) of the present application is much stronger than that of the commercial antibody Claudin18.2 (EPR19202). In terms of sensitivity performance, the antibody Caudin18.2 (3B10) has obvious and unexpected advantages over the antibody Claudin18.2 (EPR19202).

前述对本申请的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本申请限定为所公开的精确形式，并且很显然，根据上述教导，可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本申请的特定原理及其实际应用，从而使得本领域的技术人员能够实现并利用本申请的各种不同的示例性实施方案以及各种不同的选择和改变。本申请的范围意在由权利要求书及其等同形式所限定。 The foregoing description of the specific exemplary embodiments of the present application is for the purpose of illustration and illustration. These descriptions are not intended to limit the present application to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present application and its practical application, so that those skilled in the art can realize and utilize the various exemplary embodiments of the present application and various selections and changes. The scope of the present application is intended to be limited by the claims and their equivalents.

Claims (10)

1. 一种抗Claudin-18.2的抗体或其抗原结合片段，其特征在于，包含SEQ ID NO.7所示重链可变区氨基酸序列中的3个CDR，和SEQ ID NO.8所示轻链可变区氨基酸序列中的3个CDR；An anti-Claudin-18.2 antibody or an antigen-binding fragment thereof, characterized in that it comprises three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO.7, and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO.8;

   或者与上述轻重链CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸保守性变化的变体。Or variants having single or multiple CDRs with no more than 2 conservative amino acid changes in each CDR region compared to the above light and heavy chain CDR regions.
2. 根据权利要求1所述的抗体或抗原结合片段，其特征在于，当按照Kabat编码规则对抗体CDRs编码时，所述抗体或抗原结合片段包含如下CDR序列：The antibody or antigen-binding fragment according to claim 1, characterized in that when the antibody CDRs are encoded according to the Kabat coding rules, the antibody or antigen-binding fragment comprises the following CDR sequence:

   i.重链可变区的互补决定区HCDR1、HCDR2、HCDR3氨基酸序列分别如SEQ ID NO.1、2、3所示；i. The amino acid sequences of the complementary determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown in SEQ ID NO. 1, 2, and 3, respectively;

   ii.轻链可变区的互补决定区LCDR1、LCDR2、LCDR3氨基酸序列分别如SEQ ID NO.4、5、6所示；ii. The amino acid sequences of the complementary determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO. 4, 5 and 6 respectively;

   或者，与上述i-ii的6个CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸保守性变化的变体。Alternatively, a variant having a single or multiple CDRs with no more than 2 conservative amino acid changes in each CDR region compared to the 6 CDR regions of i-ii above.
3. 根据权利要求1-2任一所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含重链可变区和轻链可变区，序列选自如下：The antibody or antigen-binding fragment according to any one of claims 1-2, characterized in that the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the sequence is selected from the following:

   a)所述重链可变区的氨基酸序列如SEQ ID NO.7所示，或与SEQ ID NO.7具有至少70％、80％、90％、95％或99％序列同一性；a) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.7, or has at least 70%, 80%, 90%, 95% or 99% sequence identity with SEQ ID NO.7;

   b)所述轻链可变区的氨基酸序列如SEQ ID NO.8所示，或与SEQ ID NO.8具有至少70％、80％、90％、95％或99％序列同一性。b) The amino acid sequence of the light chain variable region is as shown in SEQ ID NO.8, or has at least 70%, 80%, 90%, 95% or 99% sequence identity with SEQ ID NO.8.
4. 根据权利要求1-3任一所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段连接有偶联部分，所述偶联部分选自放射性核素、药物、毒素、细胞因子、酶、荧光素、载体蛋白、脂类、和生物素中的一种或多种；The antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that the antibody or antigen-binding fragment is connected to a coupling portion, and the coupling portion is selected from one or more of radionuclides, drugs, toxins, cytokines, enzymes, fluorescein, carrier proteins, lipids, and biotin;

   优选的，所述抗体或抗原结合片段与所述偶联部分可选择性通过连接子相连；Preferably, the antibody or antigen-binding fragment and the coupling moiety can be selectively connected via a linker;

   更优选的，所述连接子为肽或多肽。More preferably, the linker is a peptide or polypeptide.
5. 根据权利要求1-4任一所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段选自单克隆抗体、多克隆抗体、抗血清、嵌合抗体、人源化抗体和人抗体；优选的，所述抗体选自多特异性抗体、单链Fv(scFv)、单链抗体、抗独特型(抗-Id)抗体、双抗体、微型抗体、纳米抗体、单结构域抗体、Fab片段、F(ab’)片段、二硫化物连接的双特异性Fv(sdFv)和胞内抗体。The antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that the antibody or antigen-binding fragment is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antisera, chimeric antibodies, humanized antibodies and human antibodies; preferably, the antibody is selected from the group consisting of multispecific antibodies, single-chain Fv (scFv), single-chain antibodies, anti-idiotypic (anti-Id) antibodies, diabodies, minibodies, nanobodies, single domain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fv (sdFv) and intracellular antibodies.
6. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码权利要求1-5任一所述的抗体或抗原结合片段。An isolated polynucleotide, characterized in that the polynucleotide encodes the antibody or antigen-binding fragment according to any one of claims 1-5.
7. 一种重组载体，其包含权利要求6所述的多核苷酸，以及任选的调控序列；A recombinant vector comprising the polynucleotide of claim 6 and an optional regulatory sequence;

   优选的，所述重组载体为克隆载体或表达载体；Preferably, the recombinant vector is a cloning vector or an expression vector;

   更优选的，所述调控序列选自前导序列、多聚腺苷酸化序列、前肽序列、启动子、信号序列、转录终止子，或其任何组合。 More preferably, the regulatory sequence is selected from a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, a transcription terminator, or any combination thereof.
8. 一种产品，其特征在于，包含如权利要求1-7任一所述的抗体或抗原结合片段、多核苷酸或重组载体中的一种或多种，并容纳于合适的容器中。A product, characterized in that it comprises one or more of the antibodies or antigen-binding fragments, polynucleotides or recombinant vectors as described in any one of claims 1 to 7, and is contained in a suitable container.
9. 根据权利要求8所述的产品，其特征在于，所述产品为试剂盒形式；The product according to claim 8, characterized in that the product is in the form of a kit;

   优选的，所述试剂盒包括但不限于：ELISA试剂盒、流式分选试剂盒或IHC试剂盒。Preferably, the kit includes but is not limited to: an ELISA kit, a flow cytometry kit or an IHC kit.
10. 权利要求1-5任一所述的抗体或抗原结合片段的如下任一应用：Any of the following uses of the antibody or antigen-binding fragment of any one of claims 1 to 5:

    (1)在Claudin-18.2蛋白的检测、鉴定或筛选中的应用，或在制备Claudin-18.2的检测、鉴定或筛选产品中的应用；(1) Application in the detection, identification or screening of Claudin-18.2 protein, or in the preparation of products for the detection, identification or screening of Claudin-18.2;

    优选的，所述检测包括定量检测或定性检测；Preferably, the detection includes quantitative detection or qualitative detection;

    更优选的，所述检测包括免疫原性检测；More preferably, the detection comprises immunogenicity detection;

    进一步优选的，所述检测的方式包括但不限于流式检测、ELISA检测或IHC检测；Further preferably, the detection method includes but is not limited to flow cytometry, ELISA detection or IHC detection;

    (2)在肿瘤的诊断或伴随诊断中的应用，或在制备肿瘤的诊断或伴随诊断试剂盒中的应用。 (2) Use in the diagnosis or companion diagnosis of tumors, or in the preparation of diagnostic or companion diagnosis kits for tumors.