Antibodies, antigen-binding fragments, and medical uses thereof

Abstract

The present application provides an antibody or antigen-binding fragment thereof specifically binding to a claudin family protein and medical uses thereof. The antibody or antigen-binding fragment thereof of the present application has high specificity and affinity to the claudin family protein. In addition, the present application also provides a preparation method and application of the antibody.

Description

Technical Field

[0001] This invention relates to the field of therapeutic monoclonal antibodies, particularly antibodies specific to tight junction protein-6, and their use in treating diseases. Background Technology

[0002] Tight junction protein-6 (CLDN6) is a type 2 membrane protein containing 220 amino acids. It belongs to the tight junction protein (CLDN) family, which comprises 27 different proteins characterized by four transmembrane domains and two extracellular loops. Various CLDN family members are located at tight junctions between epithelial cells. Recently, CLDN6 has also been associated with several key intracellular signaling pathways and the promotion of tumor progression in cancer. CLDN6 expression is elevated in a variety of human cancers, including ovarian cancer and endometrial malignancies, while its expression is rarely or not detected in the corresponding tissues of normal adults. In patients with ovarian and endometrial cancer, high levels of CLDN6 expression in malignant tissues are independent prognostic markers of poor progression-free survival and overall survival. Combined data show that high expression in both cancerous and normal tissues in adults makes CLDN6 an attractive target for developing anticancer therapies.

[0003] Anti-CLDN6 antibodies are described in WO2022096700A1, WO2021006328A1, WO2020168059A1, and WO2023285560A1. Several bispecific antibodies and antibody-drug conjugates targeting CLDN6 have entered clinical trials, such as AMG-794, SAIL66, TORL-1-23, and DS9606a.

[0004] CLDN6 shares high homology with its family members such as CLDN9, CLDN4, and CLDN3. Therefore, developing CLDN6-specific antibodies is challenging. Due to the limited number of available CLDN6-specific monoclonal antibodies, there is a need in the art for better anti-CLDN6 antibodies with effective internalization or other functional properties not possessed by known antibodies. Additional diagnostic tools are also needed to detect CLDN6 expression in CLDN6-related disease conditions. This invention aims to address these and other needs. Summary of the Invention

[0005] One object of the present invention is to provide a novel antibody or antigen-binding fragment thereof that binds to CLDN6 (hereinafter also referred to as an antibody, etc.), a molecule containing the antibody, and a pharmaceutical composition having cytotoxic activity, etc., containing the antibody or the molecule as an active ingredient.

[0006] To achieve this objective, the inventors conducted extensive research and developed the present invention by developing novel anti-CLDN6 antibodies and molecules containing such antibodies.

[0007] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CLDN6, wherein the antibody comprises: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 12, 14, 15, or 16, and The light chain variable region comprises the amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 13; CDRs are defined according to the numbering systems of Kabat, IMGT, Chothia, AbM, or Contact.

[0008] In some embodiments, the antibody that specifically binds to CLDN6 or its antigen-binding fragment comprises: a heavy chain variable region containing HCDR1, HCDR2, and HCDR3 and a light chain variable region containing LCDR1, LCDR2, and LCDR3, wherein: (a) A heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 12, and The light chain variable region comprises the amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 13; (b) A heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 14, and The light chain variable region comprises the amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 13; (c) Heavy chain variable region, said heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 15, and Light chain variable region, wherein the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 13; or (d) Heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 16, and The light chain variable region comprises the amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 13; In one aspect, the present invention provides a novel antibody or antigen-binding fragment thereof that specifically binds to CLDN6, comprising: a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 regions and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 regions, wherein: a) HCDR1 has an amino acid sequence as shown in SEQ ID NO: 01 or SEQ ID NO: 07; b) HCDR2 has an amino acid sequence as shown in SEQ ID NO: 02, SEQ ID NO: 08, or SEQ ID NO: 11; c) HCDR3 has an amino acid sequence as shown in SEQ ID NO: 03, SEQ ID NO: 09, or SEQ ID NO: 10; d) LCDR1 has an amino acid sequence as shown in SEQ ID NO: 04; e) LCDR2 has an amino acid sequence as shown in SEQ ID NO: 05; f) LCDR3 has an amino acid sequence as shown in SEQ ID NO: 06.

[0009] In some embodiments, the heavy chain variable region sequence comprises HCDR1 having the amino acid sequence shown in SEQ ID NO: 01, HCDR2 having the amino acid sequence shown in SEQ ID NO: 02, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 03; or HCDR1 having the amino acid sequence shown in SEQ ID NO: 07, HCDR2 having the amino acid sequence shown in SEQ ID NO: 08, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 09; or HCDR1 having the amino acid sequence shown in SEQ ID NO: 07, HCDR2 having the amino acid sequence shown in SEQ ID NO: 08, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 10; or HCDR1 having the amino acid sequence shown in SEQ ID NO: 01, HCDR2 having the amino acid sequence shown in SEQ ID NO: 11, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 03.

[0010] In some embodiments, the light chain variable region sequence comprises LCDR1 having the amino acid sequence shown in SEQ ID NO: 04, LCDR2 having the amino acid sequence shown in SEQ ID NO: 05, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 06.

[0011] In some embodiments, the antibody or its antigen-binding fragment comprises: a) a heavy chain variable region sequence comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 02, and SEQ ID NO: 03, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 05, and SEQ ID NO: 06, respectively; or b) a heavy chain variable region sequence comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 07, SEQ ID NO: 08, and SEQ ID NO: 09, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 05, and SEQ ID NO: 06, respectively; or c) a heavy chain variable region sequence comprising HCDR1, HCDR2, and LCDR3 as shown in SEQ ID NO: 07, SEQ ID NO: 08, and SEQ ID NO: 09, respectively. 10. HCDR1, HCDR2, and HCDR3 are shown; and the light chain variable region sequence contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 05, and SEQ ID NO: 06, respectively; or d) the heavy chain variable region sequence contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 11, and SEQ ID NO: 03, respectively; and the light chain variable region sequence contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 05, and SEQ ID NO: 06, respectively.

[0012] In some embodiments, the antibody or its antigen-binding fragment is selected from mouse antibodies, chimeric antibodies, humanized antibodies, human antibodies or their antigen-binding fragments.

[0013] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 12, 14-16, or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence; and / or a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 13, or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence.

[0014] In some embodiments, the antibody or its antigen-binding fragment comprises: a) a heavy chain variable region as shown in SEQ ID NO: 12 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; and / or a light chain variable region as shown in SEQ ID NO: 13 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; or b) a heavy chain variable region as shown in SEQ ID NO: 14 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; and / or a light chain variable region as shown in SEQ ID NO: 13 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; or c) a heavy chain variable region as shown in SEQ ID NO: 15 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; and / or a light chain variable region as shown in SEQ ID NO: 14 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; and / or a light ... The light chain variable region shown in SEQ ID NO: 13 or having at least 80%, 85%, 90%, 95% or 99% sequence identity with it; or d) the heavy chain variable region shown in SEQ ID NO: 16 or having at least 80%, 85%, 90%, 95% or 99% sequence identity with it; and / or the light chain variable region shown in SEQ ID NO: 13 or having at least 80%, 85%, 90%, 95% or 99% sequence identity with it.

[0015] In some embodiments, the antibody or its antigen-binding fragment comprises: a) a heavy chain variable region as shown in SEQ ID NO: 12 and / or a light chain variable region as shown in SEQ ID NO: 13; or b) a heavy chain variable region as shown in SEQ ID NO: 14 and / or a light chain variable region as shown in SEQ ID NO: 13; or c) a heavy chain variable region as shown in SEQ ID NO: 15 and / or a light chain variable region as shown in SEQ ID NO: 13; or d) a heavy chain variable region as shown in SEQ ID NO: 16 and / or a light chain variable region as shown in SEQ ID NO: 13.

[0016] In some embodiments, the antibody or its antigen-binding fragment comprises: a) a heavy chain variable region as shown in SEQ ID NO: 12 and a light chain variable region as shown in SEQ ID NO: 13; or b) a heavy chain variable region as shown in SEQ ID NO: 14 and a light chain variable region as shown in SEQ ID NO: 13; or c) a heavy chain variable region as shown in SEQ ID NO: 15 and a light chain variable region as shown in SEQ ID NO: 13; or d) a heavy chain variable region as shown in SEQ ID NO: 16 and a light chain variable region as shown in SEQ ID NO: 13.

[0017] In some embodiments, the antibody further comprises a human antibody constant region; preferably, the heavy chain constant region of the human antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their conventional variants, and the light chain constant region of the human antibody constant region is selected from the κ and λ chain constant regions of human antibodies and their conventional variants; more preferably, the antibody comprises a heavy chain constant region as shown in SEQ ID NO: 17 and a human light chain constant region as shown in SEQ ID NO: 18.

[0018] In some embodiments, the antibody that specifically binds to CLDN6 or its antigen-binding fragment comprises: a) a heavy chain having the amino acid sequence shown in SEQ ID NO: 19 and a light chain having the amino acid sequence shown in SEQ ID NO: 20; or b) a heavy chain having the amino acid sequence shown in SEQ ID NO: 21 and a light chain having the amino acid sequence shown in SEQ ID NO: 20; or c) a heavy chain having the amino acid sequence shown in SEQ ID NO: 22 and a light chain having the amino acid sequence shown in SEQ ID NO: 20; or d) a heavy chain having the amino acid sequence shown in SEQ ID NO: 23 and a light chain having the amino acid sequence shown in SEQ ID NO: 20.

[0019] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), sdAb, sdFv, dimerized V region (biantibody), disulfide-stabilized Fv (dsFv), and CDR-containing peptides.

[0020] The present invention also provides a multispecific antibody comprising the above-mentioned antibody or its antigen-binding fragment, preferably, the multispecific antibody being a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

[0021] The present invention also provides an immunoconjugate comprising the above-described antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is conjugated to one or more therapeutic agents; preferably, the therapeutic agent is a cytotoxic agent.

[0022] The present invention also provides a chimeric antigen receptor comprising: (a) an antigen-binding site, said antigen-binding site being the above-mentioned antibody or its antigen-binding fragment or the above-mentioned multispecific antibody; (b) a transmembrane domain; and (c) an intracellular signal transduction domain.

[0023] The present invention also provides an isolated nucleic acid molecule that encodes the above-mentioned antibody or its antigen-binding fragment or the above-mentioned multispecific antibody.

[0024] The present invention also provides an expression vector comprising the isolated nucleic acid molecules described above.

[0025] The present invention also provides a host cell comprising the above-mentioned nucleic acid molecule or the above-mentioned carrier, wherein the host cell is selected from the group consisting of prokaryotic cells and eukaryotic cells; preferably eukaryotic cells, more preferably mammalian cells.

[0026] The present invention also provides a pharmaceutical composition comprising the above-described antibody or antigen-binding fragment or the above-described multispecific antibody, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0027] The present invention also provides a therapeutic conjugate comprising the above-described antibody or its antigen-binding fragment or the above-described multispecific antibody, wherein the antibody or its antigen-binding fragment or the multispecific antibody is conjugated to one or more therapeutic agents; preferably, the therapeutic agent is a cytotoxic agent.

[0028] The present invention also provides a diagnostic conjugate comprising the above-mentioned antibody or its antigen-binding fragment or the above-mentioned multispecific antibody, wherein the antibody or its antigen-binding fragment or the multispecific antibody is conjugated to one or more diagnostic agents; preferably, the diagnostic agent is selected from the group consisting of detectable markers, enzyme- or metal-containing substances, fluorescent substances, luminescent substances and colored substances.

[0029] The present invention also provides a method for immunological detection or measurement of CLDN6, wherein the method comprises detecting or measuring CLDN6 by contacting the antibody described above or its antigen-binding fragment or the multispecific antibody described above.

[0030] The present invention also provides a method for diagnosing diseases associated with human CLDN6 positive cells, wherein the method comprises detecting or measuring CLDN6 positive cells by contacting the antibody or its antigen-binding fragment, the multispecific antibody or the diagnostic conjugate described above.

[0031] The present invention also provides a method for treating or preventing cancers expressing CLDN6, comprising the steps of administering a therapeutically effective amount of the above-mentioned antibody or antigen-binding fragment, the above-mentioned multispecific antibody, the above-mentioned pharmaceutical composition, or the above-mentioned chimeric antigen receptor to a subject who requires treatment or prevention of cancer expressing CLDN6.

[0032] In some implementations, the cancers expressing CLDN6 are selected from ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, bladder cancer, kidney cancer, colon cancer, small bowel cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, germ cell tumors and their metastatic forms.

[0033] Advantages of this invention: Compared with the prior art, the present invention has the following beneficial effects: (1) The antibody of the present invention can specifically recognize / bind to CLDN6 with high affinity (single digits nM) and specificity.

[0034] (2) The antibodies of the present invention can be effectively internalized through CLDN6-mediated endocytosis.

[0035] (3) Some of the antibodies of the present invention are fully human antibodies, and therefore can be safely administered to subjects without causing immunogenic reactions. Therefore, the antibodies of the present invention have great clinical value. Attached Figure Description

[0036] Figure 1 In vitro binding characterization of recombinant anti-CLDN6 antibody to CLDN6-CHO (A), CLDN9-CHO (B), CLDN4-CHO (C), CLDN3-CHO (D), PA-1 (E) and CHO K1 (F) cells.

[0037] Figure 2 The dose-response curve of recombinant anti-CLDN6 antibody internalization in PA-1 cells was determined by indirect killing assay. Detailed Implementation

[0038] In this invention, unless otherwise defined, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures used herein for cell culture, molecular genetics, nucleic acid chemistry, and immunology are standard procedures widely used in their respective fields. Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0039] Terminology Definition

[0040] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as they may vary. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0041] To interpret this specification, the following definitions will apply, and where appropriate, terms used in the singular may also include plural forms, and vice versa. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0042] The terms “CLDN6” and “CLDN6 antigen” are used interchangeably herein and include any variant, isotype, and species homolog of human CLDN6 that is expressed naturally in cells or on cells transfected with the human CLDN6 gene.

[0043] As used in this article, the term "antibody" includes complete antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or their single chains.

[0044] An antibody is a glycoprotein or its antigen-binding portion comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. Each light chain contains a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and scattered with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0045] As used herein, the term “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD3). It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CHI domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CHI domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of an antibody; (v) dAb fragments consisting of VH domains (Ward et al., (1989) Nature 341: 544-546); (vi) separate complementarity-determining regions (CDRs); and (vii) combinations of two or more separate CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked using a recombination approach via a synthetic linker that allows them to be made into a single protein chain, where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242 : 423-426 ; and Huston et al. (1988) Proc. Natl. Acad. Sci. USÅ85 : 5879- 5883). Such single-chain antibodies are also intended to be covered within the term "antigen-binding portion" of antibody.

[0046] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence.

[0047] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated in a recombinant manner, such as (a) antibodies isolated from transgenic or transchromosomally modified animals (e.g., mice) of the human immunoglobulin gene or hybridomas prepared therefrom (further described in Part I below), (b) antibodies isolated from host cells transformed to express antibodies, such as antibodies isolated from transfected tumors, (c) antibodies isolated from a recombinant human antibody library, and (d) antibodies prepared, expressed, generated, or isolated by any other method involving the splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may be mutagenized in vitro (or, when using animals with transgenic human Ig sequences, in vivo somatic cell mutagenization), so that the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, while derived from and associated with human germline VH and VL sequences, may not be naturally present in a human antibody germline library in vivo.

[0048] The term "CDR" refers to one of the six hypervariable regions within the antibody variable domain that primarily facilitate antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (LCDR1, LCDR2, LCDR3 or L1, L2, L3) of the light chain variable domain and CDR1, CDR2, and CDR3 (HCDR1, HCDR2, HCDR3 or H1, H2, H3) of the heavy chain variable domain. Furthermore, CDRs can be defined according to various other numbering systems known in the art, such as the Chothia numbering system (Chothia & Lesk (1987) J.Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or the definition from Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). The correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77). (2003). Those skilled in the art can readily identify each CDR defined by the numbering system.

[0049] Methods and techniques for identifying CDRs within amino acid sequences of HCVR and LCVR are well known in the art and can be used to identify CDRs within the specific HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, Chothia (Chothia et al. (1989) Nature 342:877-883) based on the three-dimensional structure of antibodies and CDR loop topology, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, and AbM (University of Bath), Contact (University College London), the International Immunogenetics Database (IMGT) (imgt.cines.fr / on the World Wide Web), and North CDR definitions based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art can readily identify CDRs defined by each numbering system.

[0050] The following are useful comparisons of CDR numbers:

[0051] Note 1: Some of these definitions (particularly for Chothia rings) vary depending on the individual publication consulted; Note 2: Any numbering scheme may be used for these CDR definitions, except for the Contact definition using Chothia or Martin (enhanced Chothia); Note 3: When using the Kabat numbering convention, the end of the Chothia HCDR1 ring varies between H32 and H34 depending on the ring length. This is because the Kabat numbering scheme places the indentation at H35A and H35B.

[0052] The term "conservative modification" or "conservative substitution" refers to replacing an amino acid in a protein with another amino acid that has similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, skeletal structure, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will understand that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., Page 224, (4thedition)). Furthermore, substitution with structurally or functionally similar amino acids is unlikely to impair biological activity.

[0053] In some embodiments, the anti-CLDN6 antibody or fragment thereof of the present invention encompasses antibodies or fragments thereof having post-translational modifications on the light chain variable region, the heavy chain variable region, or the light or heavy chain.

[0054] In some implementations, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. Glycosylation sites on the antibody can be conveniently added or deleted by altering the amino acid sequence, thereby creating or removing one or more glycosylation sites. In some applications, modifications to remove unwanted glycosylation sites may be useful, for example, the removal of the fucose module to enhance antibody-dependent cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277: 26733). In other applications, galactosylation modifications can be performed to improve complement-dependent cytotoxicity (CDC).

[0055] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to create Fc region variants in order to enhance the efficiency of the antibody, for example, in the treatment of cancer or cell proliferation diseases. Fc region variants may comprise human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0056] As used herein, the term "nucleic acid molecule" refers to both DNA and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but double-stranded DNA is preferred. A nucleic acid is "effectively linked" when it has a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.

[0057] The preparation method for nucleic acids is a conventional method in the art. Preferably, the preparation method includes the following steps: obtaining a nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned protein by artificial full-length sequence synthesis.

[0058] Those skilled in the art will recognize that base sequences encoding amino acid sequences of proteins can be appropriately substituted, deleted, altered, inserted, or added to provide polynucleotide homologs. The polynucleotide homologs of the present invention can be prepared by substituting, deleting, or adding one or more bases of a gene encoding a protein sequence within a range that maintains antibody activity.

[0059] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which another DNA fragment can be attached. In another embodiment, the vector is a viral vector into which another DNA fragment can be attached to a viral genome. The vectors disclosed herein are capable of self-replication in host cells where they have been introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors), or they can integrate into the host cell's genome after introduction into the host cell, thereby replicating together with the host genome (e.g., non-free mammalian vectors).

[0060] Recombinant expression vectors can be obtained using conventional methods in the art, i.e., by linking the nucleic acid molecules of the present invention to various expression vectors. The expression vector is one of many conventional vectors in the art, as long as it can carry the aforementioned nucleic acid molecules. Preferred vectors include various plasmids, granules, bacteriophages, or viral vectors, etc.

[0061] As used herein, the term “transfected tumor” includes recombinant eukaryotic host cells expressing antibodies, such as CHO cells, NS / O cells, HEK293 cells, plant cells, or fungi, including yeast cells.

[0062] The DNA molecule sequence of the antibody or fragment thereof according to the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening. Furthermore, sequences encoding the light and heavy chains can be fused together to form a single-chain antibody.

[0063] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This is typically done by cloning the sequence into a vector, transforming cells with that vector, and then isolating the relevant sequence from the proliferating host cells using conventional methods.

[0064] In addition, relevant sequences can be synthesized artificially, especially when the fragment length is short. Typically, several small fragments are synthesized first, and then linked together to obtain a long sequence fragment.

[0065] Currently, the DNA sequence encoding the antibody (or a fragment thereof or a derivative thereof) of this invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or, for example, vectors) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequence of this invention through chemical synthesis.

[0066] Generally, the obtained host cells are cultured under conditions suitable for expressing the antibodies of the present invention. Then, the antibodies of the present invention are purified using conventional immunoglobulin purification steps, such as conventional separation and purification methods well known to those skilled in the art, such as protein A-agarose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography.

[0067] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980)).

[0068] The antibodies according to the invention can be expressed in cells or on cell membranes, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods based on its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional refolding treatment, protein precipitant (e.g., salt precipitation) treatment, centrifugation, permeation lysis of cells, sonication, ultracentrifugation, molecular sieve chromatography (gel chromatography), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), other liquid chromatography methods, and combinations thereof.

[0069] As used in this article, the term "k" d (sec) -1 This refers to the dissociation rate constant of a specific antibody-antigen interaction. The value is also known as the koff value.

[0070] As used in this article, the term "k" a (M) -1 x sec -1 This refers to the binding rate constant of a specific antibody-antigen interaction.

[0071] As used in this article, the term "K" D "(M)" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. "KD" refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka), expressed as molar concentration (M). In view of this disclosure, the KD value of an antibody can be determined using methods in the art. For example, the KD of an antibody can be determined by using surface plasmon resonance, for example by using a biosensor system, such as the system, or by using biolayer interferometry techniques, such as the Octet RED96 system.

[0072] The term "epitope" or "antigenic determinant" refers to a site on an antigen that an immunoglobulin or antibody specifically binds to (e.g., a specific site on the CLDN6 molecule). Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996).

[0073] The terms "specific binding," "selective binding," "specific binding," and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, antibodies bind at a concentration of less than approximately 10-1. -7 M, for example, less than approximately 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D (Combined)

[0074] As used herein, the term "pharmaceutical composition" is intended to refer to a mixture containing one or more of the compounds described herein, or their physiologically / pharmaceutical acceptable salts or prodrugs, along with other chemical components (e.g., physiologically / pharmaceutical acceptable carriers and excipients). The purpose of a pharmaceutical composition is to facilitate administration to a living organism, to promote the absorption of the active ingredient, and to allow it to exert its biological activity.

[0075] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, "application" and "treatment" refer to the contact between an exogenous drug, therapeutic agent, diagnostic reagent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid. "Application" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, investigational, and experimental methods. Cell handling includes contacting cells with reagents, as well as contacting fluids with reagents, wherein the fluid is in contact with cells. "Application" and "treatment" also mean, for example, in vitro and ex vivo treatment of cells by means of reagents, diagnostics, conjugate compositions, or by means of another cell. When applied to humans, veterinarians, or research subjects, "treatment" refers to therapeutic treatment, preventative measures, research, and diagnostic applications.

[0076] In addition, this disclosure includes a medicament for treating diseases associated with CLDN6, comprising an antibody of this disclosure or an antigen-binding fragment thereof as an active ingredient.

[0077] There are no limitations on diseases related to CLDN6, as long as they are CLDN6-related. For example, the therapeutic response induced by the molecules disclosed in this disclosure can be reduced by binding to human CLDN6. Therefore, when the molecules of this disclosure are present in formulations and formulations suitable for therapeutic applications, they are very useful for people with tumors, cancers, or infectious diseases.

[0078] Furthermore, this disclosure relates to a method for immunological detection or measurement of CLDN6, a reagent for immunological detection or measurement of CLDN6, a method for immunological detection or measurement of cells expressing CLDN6, and a diagnostic reagent for diagnosing diseases associated with CLDN6-positive cells, comprising, as an active ingredient, an antibody or antigen-binding fragment that specifically recognizes human CLDN6 as described in this disclosure.

[0079] In this disclosure, the method for detecting or measuring the amount of CLDN6 can be any known method. For example, it includes immunoassay or assay.

[0080] Immunological detection or assay is a method that detects or measures the amount of antibody or antigen by using labeled antigens or antibodies. Examples of immunological detection or assays include radiolabeled antibody assays (RIA), enzyme immunoassays (EIA or ELISA), fluorescence immunoassays (FIA), luminescent immunoassays, Western blotting, and physicochemical methods.

[0081] The aforementioned diseases associated with CLDN6-positive cells can be diagnosed by detecting or measuring cells expressing CLDN6 using the antibody or antibody fragment of the present invention.

[0082] To detect cells expressing peptides, known immunoassay methods can be used, preferably immunoprecipitation, fluorescent cell staining, or immunohistochemical staining. Alternatively, fluorescent antibody staining using the FMAT8100HTS (Applied Biosystem) can be employed.

[0083] Example

[0084] The invention is further illustrated by the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods without detailed conditions in the following examples generally follow the conditions described in Conventional Conditions, such as those in Sambrook. J et al. "Guide to Molecular Cloning Laboratory" (translated by Huang Peitang et al., Beijing: Science Press, 2002), or the manufacturer's recommended conditions (such as the product instructions). Unless otherwise stated, percentages and parts are by weight. Unless otherwise stated, the experimental materials and reagents used in the following examples are commercially available.

[0085] The room temperature described in the examples is a conventional room temperature in the art, typically 10-30°C.

[0086] Example 1: Immunization and Antibody Screening

[0087] immunity

[0088] Twenty B-CLDN6 KO mice (RenLite) were immunized three times on days 0, 14, and 28 with 60 μL of the full-length human CLDN6 plasmid pDNA-h-CLDN6-V5-PADRE (150 μg) and CpG adjuvant. Serum titers were examined by flow cytometry using CLDN6-overexpressing cells KOP293-hCLDN6(T)-V5(P). On day 42, full-length human CLDN6 plasmid pDNA-h-CLDN6-V5-PADRE (150 μg) and KOP293-hCLDN6(T)-V5(P) cells (1 × 10⁻⁶) were used. 7 Mice were given booster immunizations.

[0089] Sequencing and screening

[0090] Plasma B cells secreting CLDN6-specific antibodies were screened using the Beacon platform. Reverse transcription and PCR amplification were performed to obtain the VH and VL sequences. The VH and VL regions were cloned into human IgG1 and Kappa constant region expression vectors, respectively. The vectors were transfected into CHO-S cells in 24-well plates using the ExpiCHO-STM cell expression system. After incubation for 3–5 days, the supernatant was collected. The specificity of the antibodies in the supernatant was characterized by FACS. Four antibodies specifically binding to human CLDN6 were identified.

[0091] The amino acid sequences and CDR sequences of the heavy chain variable region and light chain variable region of each antibody are shown in the following tables (Tables 1 to 3). The amino acid residues of the CDR in VH / VL were numbered and annotated according to the Kabat numbering system.

[0092] Table 1. CDR sequences of the heavy chain variable region from clones sequenced from B cells.

[0093] Table 2. CDR sequences of the light chain variable region from clones derived from B cell sequencing

[0094] Table 3. Sequences of the heavy and light chain variable regions from clones sequenced from B cells.

[0095] Example 2: Expression, purification, and binding affinity detection of recombinant antibodies

[0096] The cDNA sequences encoding the VH and VL regions of the four selected clones were directly synthesized into DNA fragments with a 5' in-frame leader sequence (MGWSCIILFLVATATGVHS). These DNA fragments were cloned into selected vectors using the NEBuilder DNA Assembly Cloning Kit (New England Biolabs). The VH region was cloned into the pFUSE-CHIg_hG1 vector (InvivoGen #pfuse-hchg1), in the same frame as the hIgG1 heavy chain constant region in the vector. The VL region was cloned into the pFUSE2-CLIg_hk vector (InvivoGen, #pfuse2-hclk), in the same frame as the hIg kappa light chain constant region in the vector. The amino acid sequences of the hIgG1 heavy chain constant region and the hIg kappa light chain constant region are as follows: Heavy chain constant region (SEQ ID NO: 17): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light chain constant region (SEQ ID NO: 18): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Table 4. Heavy and light chain sequences from clones sequenced from B cells

[0097] Heavy chain expression plasmids and light chain plasmids were co-transfected into Expi293F cells (ThermoFisher, #A14527) using the ExpiFectamine 293 Transfection Kit (ThermoFisher, A29129), or co-transfected into ExpiCHO-S cells (ThermoFisher, #A29127) using the ExpiFectamine CHO Transfection Kit (ThermoFisher, A29129). The plasmid DNA concentration was 1.0 μg / mL of suspension cells, and the LC:HC vector ratio was 1:1, according to the manufacturer's instructions. Transfected cells were cultured at 37°C on an orbital shaker with 8% CO2 for 5 to 7 days. Conditioned culture medium was collected, and antibodies were purified using a HiTrap MabSelect SuRe column (Cytiva, #17549112) on an AKTA Pure 25 instrument (Cytiva). The eluted antibody was neutralized with Tris buffer (pH 9.0) and then exchanged with PBS buffer. Product concentration was measured by UV absorption, and mass was determined by SDS-PAGE and HPLC.

[0098] Using CHO-overexpressing cells (CLDN6-CHO, CLDN9-CHO, CLDN4-CHO, and CLDN3-CHO), PA-1 ovarian cancer cells, and non-CHO-overexpressing K1 cells, the binding of hIgG1 mAb to CLDN6 on the cell surface was determined by FACS analysis.

[0099] CHO-overexpressing cells were cultured in F12K medium supplemented with 10% FBS, 1% penicillin and streptomycin, and a selective antibiotic. PA-1 cells were cultured in EMEM medium supplemented with 10% FBS and 1% penicillin and streptomycin. All cell lines were cultured in a humid environment at 37°C and 5% CO2.

[0100] To determine the binding of hIgG1 mAb to the CLDN receptor on the cell surface, cells were first harvested and then... 6Cells were resuspended at 100 cells / mL in cell staining buffer (BioLegend). Then, the cells were treated on ice for 10 minutes with human Fc receptor blocking reagent (BioLegend). The resulting cell suspension was aliquoted into 50 μL aliquots. 50 μL of hIgG1 mAb at different concentrations were mixed with each aliquot, with the final hIgG1 mAb concentration ranging from 0.4 pM to 22.2 nM, or 10 pM to 600 nM. The cells were incubated on ice for 1 hour, followed by two washes with cell staining buffer. 50 μL of secondary antibody (PE-conjugated goat anti-human Fc, eBioscience™, 1:400 dilution) was added to each sample to resuspend the cells. The cells were then incubated on ice for another 30 minutes. The cells were then washed twice with cell staining buffer and resuspended in 4% PFA for fixation. The samples were analyzed using iQue3 to measure median fluorescence intensity using the corresponding channel. TORL-3-7 (TORL-1-23 mAb, see WO2020191342) is a positive control. The VH and VL values ​​for TORL-3-7 are shown below. Results are as follows... Figure 1 As shown in AF and Table 5, the results confirm that the anti-CLDN6 antibody of the present invention specifically binds to CLDN6 initially expressed in cancer cells or overexpressed in CHO cells in a concentration-dependent manner. Simultaneously, the antibody was observed to bind least to CLDN9, CLDN4, and CLDN3.

[0101] >VH of TORL-3-7 (SEQ ID NO: 24)

[0102] EVQLLESGGGLVQPGGSMRLSCAASGFTFSNYWMNWVRQAPGKGLEWVAQIRLKSDNYATHYADSVKGRFTISRDDSKNTVYLQMNSLRAEDTGVYYCNDGPPSGSWGQGTLLTVSS

[0103] >VL of TORL-3-7 (SEQ ID NO: 25)

[0104] DIQMTQSPSSSLSASVGDRVTITCRISENIYSYLAWYQQKPGKAPKLLVYNAKILVEGVPSRFSGSGSGTDFTLTISSLQPEDFGTYYCQHHYTVPWTFGQGTKLEIK

[0105] Table 5. K+ binding of anti-CLDN6 recombinant antibody to CLDN6-positive PA-1 cells and CLDN6-CHO cells D value

[0106] Example 3: Characterization of the internalization of the anti-CLDN6 recombinant antibody in PA-1 cells by indirect killing assay.

[0107] To assess antibody endocytosis induced by anti-CLDN6 antibody binding, an indirect killing assay was used to characterize antibody internalization. A Fab anti-human IgG Fc-MMAF conjugate with a cleavable linker (Moradec, AH-202AF) was incubated with and conjugated to recombinant anti-CLDN6 antibody. TORL-3-7 was used as a positive control. The resulting complex was then incubated with CLDN6-expressing cells. Upon binding to the CLDN6 receptor on the cell surface, the complex was internalized, and the bound MMAF was released after lysosomal cleavage of the linker. The released MMAF subsequently inhibited cell division by blocking tubulin polymerization. In a separate assay, CLDN6-expressing PA-1 cells were seeded at 2500 cells / well in 96-well plates and incubated overnight. The recombinant anti-CLDN6 antibody was mixed with the Fab anti-human IgG Fc-MMAF conjugate with a cleavable linker at a 1:6 ratio (mol / mol) and incubated for 10 minutes to form the complex. Then, serial dilutions of the complex from 1.5 pM to 10 nM were added to each well and incubated for 72 hours. Cell viability was measured using Cell Titer Glo 2.0 (Promega, G9243). Dose-response curves were plotted and fitted using GraphPad Prism9. Figure 2 As shown in Table 6, all the anti-CLDN6 antibodies of this invention exhibit strong cytotoxicity, indicating that MMAF is effectively delivered into cells. These anti-CLDN6 antibodies also demonstrate sufficient internalization activity.

[0108] Table 6. IC50 of indirect lethality test 50 To evaluate the internalization of recombinant anti-CLDN6 antibody in PA-1 cells

[0109] Note: "NA" indicates IC 50 It cannot be measured.

[0110] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the disclosed teachings, and these modifications and variations are also included within the scope of the invention. The scope of protection of the invention is defined by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CLDN6, wherein, The antibody comprises: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 12, 14, 15, or 16, and The light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:

13.

2. An antibody or antigen-binding fragment thereof that specifically binds to CLDN6, comprising: a heavy chain variable region including HCDR1, HCDR2, and HCDR3 regions and a light chain variable region including LCDR1, LCDR2, and LCDR3 regions, wherein: a) HCDR1 has the amino acid sequence shown in SEQ ID NO: 01 or SEQ ID NO: 07; b) HCDR2 has the amino acid sequence shown in SEQ ID NO: 02, SEQ ID NO: 08 or SEQ ID NO: 11; c) HCDR3 has the amino acid sequence shown in SEQ ID NO: 03, SEQ ID NO: 09 or SEQ ID NO: 10; d) LCDR1 has the amino acid sequence shown in SEQ ID NO: 04; e) LCDR2 has the amino acid sequence shown in SEQ ID NO: 05; f) LCDR3 has the amino acid sequence shown in SEQ ID NO:

06.

3. The antibody or antigen-binding fragment thereof that specifically binds to CLDN6 according to any one of claims 1-2, wherein the heavy chain variable region sequence comprises HCDR1, each having the amino acid sequence shown in SEQ ID NO: 01, HCDR2, having the amino acid sequence shown in SEQ ID NO: 02, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 03; or HCDR1, each having the amino acid sequence shown in SEQ ID NO: 07, HCDR2, having the amino acid sequence shown in SEQ ID NO: 08, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 09; or HCDR1, each having the amino acid sequence shown in SEQ ID NO: 07, HCDR2, having the amino acid sequence shown in SEQ ID NO: 08, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 10; or HCDR1, each having the amino acid sequence shown in SEQ ID NO: 01, HCDR2, having the amino acid sequence shown in SEQ ID NO: 11, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 03; and / or The light chain variable region sequence includes LCDR1, each having the amino acid sequence shown in SEQ ID NO: 04, LCDR2, having the amino acid sequence shown in SEQ ID NO: 05, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

06.

4. The antibody or antigen-binding fragment thereof that specifically binds to CLDN6 according to any one of claims 1-3, wherein: a) The heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 02 and SEQ ID NO: 03 respectively; and the light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 05 and SEQ ID NO: 06 respectively; or b) The heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 07, SEQ ID NO: 08 and SEQ ID NO: 09 respectively; and the light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 05 and SEQ ID NO: 06 respectively; or c) The heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 07, SEQ ID NO: 08 and SEQ ID NO: 10 respectively; and the light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 05 and SEQ ID NO: 06 respectively; or d) The heavy chain variable region sequence includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 11 and SEQ ID NO: 03 respectively; and the light chain variable region sequence includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 05 and SEQ ID NO: 06 respectively.

5. The antibody or antigen-binding fragment thereof that specifically binds to CLDN6 according to any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof is selected from mouse antibodies, chimeric antibodies, humanized antibodies, human antibodies or antigen-binding fragments thereof.

6. An antibody or antigen-binding fragment thereof that specifically binds to CLDN6 according to any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 12, 14-16, or a sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith; and / or a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 13, or a sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith.

7. The antibody or antigen-binding fragment thereof that specifically binds to CLDN6 according to any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof comprises: a) The heavy chain variable region as shown in SEQ ID NO: 12 or having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith; and / or the light chain variable region as shown in SEQ ID NO: 13 or having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith; or b) The heavy chain variable region as shown in SEQ ID NO: 14 or having at least 80%, 85%, 90%, 95% or 99% sequence identity with it; and / or the light chain variable region as shown in SEQ ID NO: 13 or having at least 80%, 85%, 90%, 95% or 99% sequence identity with it. or c) The heavy chain variable region as shown in SEQ ID NO: 15 or having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith; and / or the light chain variable region as shown in SEQ ID NO: 13 or having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith. or d) Heavy chain variable regions as shown in SEQ ID NO: 16 or having at least 80%, 85%, 90%, 95% or 99% sequence identity with it; and / or light chain variable regions as shown in SEQ ID NO: 13 or having at least 80%, 85%, 90%, 95% or 99% sequence identity with it.

8. An antibody or antigen-binding fragment thereof that specifically binds to CLDN6 according to any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof comprises: a) The heavy chain variable region as shown in SEQ ID NO: 12, and / or the light chain variable region as shown in SEQ ID NO: 13; or b) The heavy chain variable region as shown in SEQ ID NO: 14, and / or the light chain variable region as shown in SEQ ID NO: 13; or c) The heavy chain variable region as shown in SEQ ID NO: 15, and / or the light chain variable region as shown in SEQ ID NO: 13; or d) Heavy chain variable region as shown in SEQ ID NO: 16, and / or light chain variable region as shown in SEQ ID NO:

13.

9. An antibody or antigen-binding fragment thereof that specifically binds to CLDN6 according to any one of claims 1-8, wherein the antibody or antigen-binding fragment thereof comprises: a) The heavy chain variable region as shown in SEQ ID NO: 12, and the light chain variable region as shown in SEQ ID NO: 13; or b) The heavy chain variable region as shown in SEQ ID NO: 14, and the light chain variable region as shown in SEQ ID NO: 13; or c) The heavy chain variable region as shown in SEQ ID NO: 15, and the light chain variable region as shown in SEQ ID NO: 13; or d) The heavy chain variable region as shown in SEQ ID NO: 16, and the light chain variable region as shown in SEQ ID NO:

13.

10. An antibody or antigen-binding fragment thereof that specifically binds to CLDN6 according to any one of claims 1-9, wherein the antibody further comprises a human antibody constant region; preferably, the heavy chain constant region of the human antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their conventional variants, and the light chain constant region of the human antibody constant region is selected from the κ and λ chain constant regions of human antibodies and their conventional variants; more preferably, the antibody comprises a heavy chain constant region as shown in SEQ ID NO: 17 and a human light chain constant region as shown in SEQ ID NO:

18.

11. The antibody or antigen-binding fragment thereof that specifically binds to CLDN6 according to any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof comprises: a) A heavy chain having the amino acid sequence shown in SEQ ID NO: 19 and a light chain having the amino acid sequence shown in SEQ ID NO: 20; or b) A heavy chain having the amino acid sequence shown in SEQ ID NO: 21 and a light chain having the amino acid sequence shown in SEQ ID NO: 20; or c) A heavy chain having the amino acid sequence shown in SEQ ID NO: 22 and a light chain having the amino acid sequence shown in SEQ ID NO: 20; or d) A heavy chain having the amino acid sequence shown in SEQ ID NO: 23 and a light chain having the amino acid sequence shown in SEQ ID NO:

20.

12. The antibody that specifically binds to CLDN6 according to any one of claims 1-11, or the antigen-binding fragment thereof, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), sdAb, sdFv, dimerized V region (biantibody), disulfide bond-stabilized Fv (dsFv), and CDR-containing peptides.

13. A multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-12; preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

14. An immunoconjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-12 or a multispecific antibody according to claim 13, wherein the antibody or antigen-binding fragment thereof or the multispecific antibody is conjugated to one or more therapeutic agents; preferably, the therapeutic agent is a cytotoxic agent.

15. A chimeric antigen receptor comprising: (a) an antigen-binding site, said antigen-binding site being an antibody or an antigen-binding fragment thereof according to any one of claims 1-12 or a multispecific antibody according to claim 13; (b) Transmembrane domains; and (c) Intracellular signal transduction domains.

16. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-12 or a multispecific antibody according to claim 13.

17. An expression vector comprising the isolated nucleic acid molecule according to claim 16.

18. A host cell comprising a nucleic acid molecule according to claim 16 or a vector according to claim 17, wherein the host cell is selected from the group consisting of prokaryotic cells and eukaryotic cells; preferably eukaryotic cells, more preferably mammalian cells.

19. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1-12 or a multispecific antibody according to claim 14, and one or more pharmaceutically acceptable carriers, diluents or excipients.

20. A therapeutic conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-12 or a multispecific antibody according to claim 13, wherein the antibody or antigen-binding fragment thereof or the multispecific antibody is conjugated to one or more therapeutic agents; preferably, the therapeutic agent is a cytotoxic agent.

21. A diagnostic conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-12 or a multispecific antibody according to claim 13, wherein the antibody or antigen-binding fragment thereof or the multispecific antibody is conjugated to one or more diagnostic agents; preferably, the diagnostic agent is selected from the group consisting of detectable markers, enzyme- or metal-containing substances, fluorescent substances, luminescent substances and colored substances.

22. A method for immunological detection or measurement of CLDN6, wherein the method comprises detecting or measuring CLDN6 by contacting an antibody or an antigen-binding fragment thereof according to any one of claims 1-12 or a multispecific antibody according to claim 13.

23. A method for diagnosing a disease associated with human CLDN6 positive cells, wherein the method comprises detecting or measuring the CLDN6 positive cells by contacting an antibody or antigen-binding fragment thereof according to any one of claims 1-12, a multispecific antibody according to claim 13, or a diagnostic conjugate according to claim 21.

24. A method of treating or preventing cancer expressing CLDN6, comprising administering to a subject requiring treatment or prevention of cancer expressing CLDN6 a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-12, or a multispecific antibody according to claim 13, or a pharmaceutical composition according to claim 19, or a chimeric antigen receptor according to claim 15.

25. The method for treating or preventing cancers expressing CLDN6 according to claim 24, wherein the cancers expressing CLDN6 are selected from ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, bladder cancer, kidney cancer, colon cancer, small intestine cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, germ cell tumors and their metastatic forms.

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