Antibody targeting claudin-1, ADC thereof and use thereof

Abstract

Provided in the present invention are a mouse anti-CLDN1 antibody, a chimeric antibody and a humanized antibody thereof. Compared with existing CLDN1 antibodies, the antibody provided in the present invention has a higher specificity and affinity for the CLDN1 protein, and shows a stronger antibody-dependent cellular cytotoxicity. In addition, compared with existing CLDN1 antibodies, the antibody provided in the present invention also has a stronger ability for internalization into CLDN1-expressing cells, and shows an improved cell-killing activity and anti-tumor effect after conjugation with a cytotoxic compound. Further provided in the present invention is an antibody-drug conjugate containing the antibody.

Description

An antibody and ADC targeting tight junction protein 1 and their applications

[0001] Cross-references to related applications

[0002] This patent application claims priority to Chinese invention patent application No. CN202510355383.2, filed on March 25, 2025, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This invention belongs to the field of antibody drugs. Specifically, this invention relates to antibodies against tight junction protein 1 (Claudin-1; CLDN1) and antibody-drug conjugates targeting CLDN1, as well as their uses. Background Technology

[0004] Human tight junction protein 1 (Claudin-1; CLDN1) belongs to the tight junction protein family. Under physiological conditions, the Claudin protein family plays an important role in maintaining cell polarity in epithelial and endothelial cells, cell adhesion and fixation, and ion transport via bypass pathways. In various solid tumors, aberrant expression of the Claudin protein family is involved in tumor proliferation, invasion, and epidermal-mesenchymal transition (EMT). In normal tissues, Claudin proteins are distributed at tight junctions, and their epitopes are masked; however, in malignant tumors, tight junctions are disrupted, exposing Claudins epitopes on the surface of tumor cells. This difference in distribution makes Claudin family proteins potential therapeutic targets for various tumors.

[0005] CLDN1 is overexpressed in various tumors, such as head and neck squamous cell carcinoma, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, ovarian cancer, and thyroid cancer. Studies have shown that CLDN1 is significantly upregulated in head and neck squamous cell carcinoma, and its overexpression is associated with poor prognosis of head and neck squamous cell carcinoma (Oncology. 2025, 103(2): 107-111.); abnormal expression of CLDN1 can disrupt the differentiation program of normal colon cells and affect the number of goblet cells in the intestine (Gut. 2014, 63(4), 622-34.); CLDN1 is significantly upregulated in hepatocellular carcinoma (J Hepatol. 2023, 78(2): 343-355.); with the progression of cervical cancer, the copy number and protein expression of the CLDN1 gene both increase significantly (Oncotarget. 2016, 7(52), 87449-87461). Furthermore, it was found that CLDN1 overexpression promotes the reconstruction of the tumor extracellular matrix, forming a collagen barrier outside the tumor, preventing immune surveillance and leading to immune escape (J Hepatol. 2023, 78(2):343-355.). Therefore, CLDN1 can serve as a novel therapeutic target for various tumors, and there is an urgent need in this field for antibodies and related drugs with higher specificity and bioactivity for human CLDN1 for the treatment of cancer patients. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a novel anti-CLDN1 antibody that has lower non-specific binding to general cells but can bind to CLDN1 protein with high specificity and high affinity, thereby having a high tumor cell selectivity. At the same time, it mediates the direct killing of tumor cells by killer cells through efficient binding to the CLDN1 protein expressed by tumor cells. In addition, the antibody should also have a strong ability to internalize into CLDN1-expressing cells, thus making it suitable for preparation into antibody-drug conjugates. Therefore, an antibody-drug conjugate with better targeting and killing effect on CLDN1-expressing tumor cells is provided.

[0007] Therefore, one object of the present invention is to provide an antibody or fragment thereof that specifically binds to CLDN1, particularly CLDN1 expressed on tumor cells. Another object of the present invention is to provide an antibody-drug conjugate or a salt thereof that targets CLDN1.

[0008] The technical solution of the present invention is as follows.

[0009] First aspect

[0010] The present invention provides an antibody or antigen-binding fragment thereof against tight junction protein 1 (CLDN1), wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to CLDN1, particularly human CLDN1.

[0011] In the context of this invention, unless otherwise stated, the term "CLDN1" covers any form or structural region of CLDN1.

[0012] In the context of this invention, the term "antigen-binding fragment" encompasses various functional fragments of the antibody that specifically binds to CLDN1, which retain the antibody's ability to bind to the antigen and the corresponding biological activity. It is known in the art that the antibody's ability to bind to the antigen and the corresponding biological activity can be achieved from fragments of the intact antibody, which can be obtained using conventional techniques known to those skilled in the art and screened for functionality in the same manner as for the intact antibody. For example, antigen-binding fragments of the antibody can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody.

[0013] Specifically, the anti-CLDN1 antibody or its antigen-binding fragment provided by the present invention comprises complementarity-determining regions (CDRs) of the heavy chain, namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and complementarity-determining regions (CDRs) of the light chain, namely light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3). According to a specific embodiment of the present invention, the heavy chain CDRs contained in the anti-CLDN1 antibody or its antigen-binding fragment are derived from the amino acid sequence shown in SEQ ID NO. 5 or SEQ ID NO. 7; and / or, the light chain CDRs contained in the anti-CLDN1 antibody or its antigen-binding fragment are derived from the amino acid sequence shown in SEQ ID NO. 6 or SEQ ID NO. 8.

[0014] The amino acid sequences shown in any one of SEQ ID NO. 5 to SEQ ID NO. 8 provided above are the amino acid sequences of the heavy chain variable region (VH) or light chain variable region (VL) of the exemplary antibody provided in the "Best Mode for Carrying the Invention" section of this application. Using any one or a combination of antibody heavy chain or light chain complementarity-determining regions (CDRs) known in the art (e.g., Chothia, Kabat, IMGT, Contact, AbM, CCG, etc.), those skilled in the art can readily determine the heavy chain CDRs and light chain CDRs contained therein. Combinations of heavy chain CDRs and light chain CDRs can be obtained according to the definition tools known or conventional in the art, and antibodies or fragments thereof containing each of these combinations of heavy chain CDRs and light chain CDRs are within the scope of protection of this invention.

[0015] Preferably, the anti-CLDN1 antibody or its antigen-binding fragment provided by the present invention comprises heavy chain CDRs and light chain CDRs from the heavy chain variable region and light chain variable region shown in the following amino acid sequence pairings:

[0016] (1) SEQ ID NO.5 + SEQ ID NO.6; or

[0017] (2)SEQ ID NO.7+SEQ ID NO.8.

[0018] As described above, for example, the CCG definition can be used to classify the CDRs in the above amino acid sequence pairings, as shown in the embodiments of the present invention.

[0019] Accordingly, in the anti-CLDN1 antibody or its antigen-binding fragment provided by the present invention, the heavy chain CDRs and light chain CDRs are as follows:

[0020] (1) HCDR1, HCDR2, and HCDR3, sequentially comprising the amino acid sequences shown in SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11; and LCDR1, LCDR2, and LCDR3, sequentially comprising the amino acid sequences shown in SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14; or

[0021] (2) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.9, SEQ ID NO.15, and SEQ ID NO.16; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.17, SEQ ID NO.13, and SEQ ID NO.14.

[0022] As described above, the anti-CLDN1 antibody or its antigen-binding fragment provided by the present invention specifically binds to tight junction protein 1 (CLDN1), preferably primate or rodent CLDN1, such as human, monkey, or mouse CLDN1. Optionally, the antibody or its antigen-binding fragment provided by the present invention may or may not have species cross-binding activity with human, monkey, or mouse CLDN1.

[0023] Preferably, the anti-CLDN1 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), both of which include the aforementioned CDRs and the framework region (FR) therebetween. The arrangement of each region from the N-terminus to the C-terminus is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0024] More preferably, in the anti-CLDN1 antibody or its antigen-binding fragment provided by the present invention, the heavy chain variable region may contain the amino acid sequence shown in SEQ ID NO.5 or SEQ ID NO.7, or contain an amino acid sequence having at least 75% identity with the amino acid sequence; and / or, the light chain variable region contains the amino acid sequence shown in SEQ ID NO.6 or SEQ ID NO.8, or contains an amino acid sequence having at least 75% identity with the amino acid sequence.

[0025] In the context of this invention, the term "at least 75% identity" refers to a maximum of 25% difference in the amino acid sequence that may exist in any frame region within the heavy chain variable region or the light chain variable region, or in any domain or sequence other than the heavy chain variable region and the light chain variable region in the antibody or its antigen-binding fragment of this invention. This difference may result from amino acid deletions, additions, or substitutions at any position, wherein the substitutions may be conservative or non-conservative. The term "at least 75% identity" encompasses any percentage of identity between at least 75% and 100% identity, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity.

[0026] According to a specific embodiment of the present invention, the anti-CLDN1 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise a combination of the following amino acid sequences:

[0027] (1) The amino acid sequence shown in SEQ ID NO. 5, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 5; and the amino acid sequence shown in SEQ ID NO. 6, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 6; or

[0028] (2) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.7; and the amino acid sequence shown in SEQ ID NO.8, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.8.

[0029] Preferably, the anti-CLDN1 antibody provided by the present invention can be a mouse antibody, rabbit antibody, or human antibody, or it can be a mouse antibody, a chimeric antibody, or a fully or partially humanized antibody. The CLDN1 antibody can also be a derivatized antibody, such as an antibody obtained by CDR transplantation, affinity maturation, point mutation modification, or chemical modification based on an initial mouse monoclonal antibody. The chemical modification includes glycosylation, acetylation, polyethylene glycol modification, phosphorylation, amidation, protease cleavage, linkage with cellular ligands or effector molecules, protection of active reactive groups, and / or blocking. Preferably, the antigen-binding fragment of the antibody can be a single-chain variable fragment (scFv), a bivalent single-chain variable fragment (BsFv), a disulfide-stabilized variable fragment (dsFv), (dsFv)2, an antigen-binding fragment (Fab), Fab' fragment (Fab'), (Fab' fragment)2 (F(ab')2), or a variable fragment (Fv), etc. Regarding the antigen-binding fragment of the antibody described in this invention, it can be any fragment of the antibody capable of specifically binding to CLDN1.

[0030] In addition to the heavy chain and / or light chain variable regions, the anti-CLDN1 antibody or its antigen-binding fragment provided by the present invention further comprises a heavy chain constant region (CH) and / or a light chain constant region (CL), preferably comprising a human or mouse heavy chain constant region and / or a light chain constant region. Preferably, the anti-CLDN1 antibody or its fragment comprises a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE and / or a κ or λ type light chain constant region.

[0031] According to a specific embodiment of the present invention, the anti-CLDN1 antibody is a monoclonal antibody, preferably a mouse, chimeric, or humanized monoclonal antibody. According to a specific embodiment of the present invention, the monoclonal antibody comprises a heavy chain constant region sequence of IgG1, such as the human IgG1 heavy chain constant region shown in SEQ ID NO.3; and / or comprises a kappa light chain constant region, such as the human kappa light chain constant region shown in SEQ ID NO.4.

[0032] According to a specific embodiment of the present invention, the anti-CLDN1 antibody of the present invention is a monoclonal antibody. Preferably, the anti-CLDN1 antibody provided by the present invention is an immunoglobulin, for example, the type of the immunoglobulin is human IgA, IgD, IgE, IgG or IgM. More preferably, the antibody is a human IgG1 subtype.

[0033] Second aspect

[0034] The present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the anti-CLDN1 antibody or its antigen-binding fragment described herein.

[0035] The term "nucleotide sequence encoding the anti-CLDN1 antibody or its antigen-binding fragment according to the present invention" refers to a nucleotide sequence encoding the heavy chain CDRs, light chain CDRs, light chain variable regions, heavy chain variable regions, heavy chains, and / or light chains contained in the antibody or its antigen-binding fragment. For example, the nucleic acid molecule provided by the present invention contains nucleotide sequences encoding each of the heavy chain CDRs and light chain CDRs contained in the aforementioned antibody or its antigen-binding fragment; contains nucleotide sequences encoding the heavy chain variable regions and light chain variable regions contained in the aforementioned antibody or its antigen-binding fragment; or contains nucleotide sequences encoding the heavy chains and light chains contained in the aforementioned antibody or its antigen-binding fragment.

[0036] Third aspect

[0037] The nucleic acid molecules of this invention can be cloned into a vector, and then transformed or transfected into host cells. Therefore, in a third aspect, this invention also provides a vector containing the nucleic acid molecules of this invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a phage vector, etc. The vectors or nucleic acid molecules of this invention can be used to transform or transfect host cells, for purposes such as preserving or expressing antibodies.

[0038] Fourth aspect

[0039] The present invention also provides a host cell comprising the nucleic acid molecules and / or vectors of the present invention, or the host cell being transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells.

[0040] Fifth aspect

[0041] The anti-CLDN1 antibody or its antigen-binding fragment provided by this invention can be obtained using any method known in the art. For example, this invention also provides a method for preparing the anti-CLDN1 antibody or its antigen-binding fragment, the method comprising culturing the host cells provided by this invention while allowing the host cells to express the heavy and light chains of the antibody. Optionally, the method further includes a step of recovering the generated anti-CLDN1 antibody.

[0042] Sixth aspect

[0043] The anti-CLDN1 antibody or its antigen-binding fragment provided by the present invention can also be directly or indirectly linked to other parts, such as heavy chain CDRs, light chain CDRs, heavy chain variable regions, light chain variable regions, heavy chains, and light chains of other antibodies; or, such as small molecule compounds, such as cytotoxic compounds used to prepare antibody-drug conjugates; or, such as cell surface receptors, sugars, polymers, etc., that modify the antibody or its antigen-binding fragment.

[0044] Accordingly, in a sixth aspect, the present invention also provides the use of the said anti-CLDN1 antibody or its antigen-binding fragment, nucleic acid molecule, vector, or host cell in the preparation of antibody-drug conjugates (ADCs).

[0045] Antibody-drug conjugates (ADCs) generally consist of three parts: an antibody or antibody-like ligand; a small molecule drug; and a linker that conjugates the antibody or antibody-like ligand to the drug. ADCs utilize the specific recognition ability of antibodies or antibody-like ligands on specific antigens on the surface of cells (such as tumor cells) and leverage the internalization activity of the antibodies or antibody-like ligands to deliver the small molecule drug (such as an anti-tumor drug) into the cell, thereby causing intracellular release of the small molecule drug to exert a killing effect. The anti-CLDN1 antibody or its antigen-binding fragment, nucleic acid molecule, vector, or host cell of the present invention can be used to prepare such drugs, for example, by directly or indirectly conjugating an anti-CLDN1 antibody or its antigen-binding fragment encoded by an anti-CLDN1 antibody or its antigen-binding fragment or a nucleic acid molecule to a small molecule drug via a linker.

[0046] Seventh aspect

[0047] This invention provides an antibody-drug conjugate targeting CLDN1 or a salt thereof, comprising the anti-CLDN1 antibody or its antigen-binding fragment provided by this invention. The salt can be a pharmaceutically acceptable salt.

[0048] The antibody-drug conjugate can be formed by conjugating the anti-CLDN1 antibody or its antigen-binding fragment provided by this invention with a cytotoxic compound. The cytotoxic compound can be a tubulin inhibitor, a topoisomerase inhibitor, a DNA binder, etc. For example, the tubulin inhibitor can be maytansine compounds such as DM1 and DM4, sarsaparilla toxin compounds such as Monomethyl Dolastatin 10, MMAE, and MMAF, tubulolysin compounds, Cryptophycin derivatives, Taltobulin, muscarine, chalcogenide, eribulin, and derivatives of the aforementioned drugs; the topoisomerase inhibitor can be camptothecin compounds such as Dxd, exatecan, and their derivatives, doxorubicin metabolite PNU-159682 derivative, and irinotecan and its metabolite SN38, etc.; the DNA binder can be PBD derivatives and Duocarmycin and its derivatives, etc.

[0049] Furthermore, the present invention also provides the following preferred embodiments for the antibody-drug conjugate.

[0050] First, this invention provides an antibody-drug conjugate or a salt thereof targeting CLDN1, wherein the antibody-drug conjugate or the salt thereof has the general formula The structure is shown. In the context of this invention, unless otherwise specified, the various groups or structures in this general formula are as follows:

[0051] Ab represents the anti-CLDN1 antibody or its antigen-binding fragment provided by this invention.

[0052] E L Selected from the following groups ( (This indicates that Ab is linked to the sulfhydryl group of cysteine):

[0053] E L -1a and / or E L -1b: and / or E L -2: E L -3: E L -4: E L -5: E L -6:

[0054] M is a phenylene or a phenylene substituted with one or more substituents, or a chemical bond; in the substituted phenylene, the substituent is selected from alkyl (e.g., C1-6 alkyl, preferably C1-4 alkyl), haloalkyl (e.g., haloC1-6 alkyl, preferably haloC1-4 alkyl, such as trifluoromethyl), alkoxy (e.g., C1-6 alkoxy, preferably C1-4 alkoxy, preferably methoxy), halogen, ester, amide, and cyano.

[0055] SP1 is selected from C1-8 alkylene, C1-8 cycloalkylene, or C1-21 (preferably C1-16, more preferably C1-11, more preferably C5-9) straight-chain heteroalkylene, wherein the C1-21 straight-chain heteroalkylene comprises 1-11 (preferably 1-6, more preferably 3-5) heteroatoms selected from N, O, or S, wherein each of the C1-8 alkylene, C1-8 cycloalkylene, and C1-21 straight-chain heteroalkylene is independently and optionally substituted by one or more substituents selected from hydroxyl, amino, sulfonic acid, and cyano groups.

[0056] SP2 is selected from -NH(CH2CH2O) a CH2CH2CO-、-NH(CH2CH2O) a CH2CO-、-S(CH2) a CO- or chemical bond, where a is an integer from 1 to 20.

[0057] A represents a short peptide structure consisting of 2-4 amino acids or a combination of a short peptide structure consisting of 2-4 amino acids and a self-releasing fragment. The short peptide structure consisting of 2 amino acids can be NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO, NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO, or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO, or NH-Val-Cit-CO; 3 The short peptide structure formed by 1 amino acid can be NH-Glu-Val-Ala-CO, NH-Glu-Val-Cit-CO, or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO; the short peptide structure formed by 4 amino acids can be NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-Gly-CO, preferably NH-Gly-Gly-Phe-Gly-CO. Preferably, A is NH-Val-Ala-CO, NH-Val-Cit-CO, NH-Gly-Gly-Phe-Gly-CO, or NH-Ala-Ala-Ala-CO, or A is a combination of NH-Val-Ala-CO, NH-Val-Cit-CO, NH-Gly-Gly-Phe-Gly-CO, or NH-Ala-Ala-Ala-CO with a self-releasing structural fragment, where NH represents the amino terminus of the short peptide structure and CO represents the carboxyl terminus of the short peptide structure. Group A can be linked to SP2 through the amino group at the amino terminus of its short peptide structure. In some embodiments, the self-releasing structural fragment is selected from -NH-CH2- or PABC. In a preferred embodiment, A is selected from NH-Val-Ala-CO or NH-Val-Cit-CO-PABC.

[0058] m can be 1 to 10, and m can be an integer or a non-integer.

[0059] D indicates a cytotoxic compound, which, as mentioned above, can be a tubulin inhibitor, a topoisomerase inhibitor, a DNA binder, etc.

[0060] In the general formula In this formula, M can preferably be a halogen-substituted phenylene, particularly a fluorine-substituted phenylene. In this general formula, SP1 can preferably be a C1-11, preferably C5-9, more preferably C7 straight-chain heteroalkylene, containing 1-6, preferably 3-5, more preferably 4 heteroatoms selected from N, O, or S. In this general formula, a in SP2 can further preferably be an integer from 1 to 10, more preferably an integer from 1 to 6. In particular, SP2 can preferably be a chemical bond. In this general formula, m can further preferably be an integer or non-integer of 1 to 8 (e.g., 1 to 5 or 1 to 6), more preferably an integer or non-integer of 3 to 8. For example, m can further preferably be an integer or non-integer of 3 to 5, such as 4.

[0061] In this general formula, further, the group The following structures can be selected, where the wavy line indicates a connection to cysteine ​​in the Ab or to cytotoxic compound D:

[0062] (1)

[0063] (2)

[0064] (3)

[0065] (4)

[0066] (5)

[0067] (6)

[0068] (7)

[0069] (8)

[0070] Furthermore, the antibody-drug conjugate or its salt provided by the present invention preferably has a structure as shown in structural formula Ia (general formula for bridged site-directed conjugation structure) and / or Ib (general formula for bridged site-directed open-ring conjugation structure):

[0071] and / or

[0072] In structural formulas Ia and / or Ib, Ab, m, groups M, SP1, SP2, A, and D are consistent with the general formula above. The definitions of Ab, m, group M, SP1, SP2, A, and D are the same.

[0073] Alternatively, the antibody-drug conjugate or its salt provided by the present invention may have a structure as shown in structural formulas Ic and / or Id:

[0074] and / or

[0075] In structural formulas Ic and / or Id, Ab, m, groups A and D are consistent with the general formula above. The definitions of Ab, m, and groups A and D are the same.

[0076] According to a specific embodiment of the present invention, the antibody-drug conjugate targeting CLDN1 or its salt provided by the present invention has the following structure:

[0077] (1)

[0078] (2)

[0079] (3)

[0080] (4)

[0081] (5)

[0082] (6)

[0083] (7)

[0084] (8)

[0085] (9)

[0086] (10)

[0087] (11)

[0088] (12)

[0089] (13)

[0090] (14)

[0091] Eighth aspect

[0092] The anti-CLDN1 antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, or antibody-drug conjugate or its salt provided by the present invention can be included in a composition, more particularly in a pharmaceutical composition, such as a pharmaceutical formulation, for use in various purposes as needed.

[0093] Therefore, the present invention also provides a composition comprising the anti-CLDN1 antibody or its antigen-binding fragment provided by the present invention, a nucleic acid molecule, a carrier, a host cell, or an antibody-drug conjugate or its salt. Preferably, the composition is a pharmaceutical composition, which optionally further comprises pharmaceutically acceptable excipients. The pharmaceutical compositions provided by the present invention can be formulated into various dosage forms known in the medical or pharmaceutical fields and administered in an applicable manner.

[0094] Ninth aspect

[0095] This invention also provides the use of the anti-CLDN1 antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, antibody-drug conjugate or its salt or composition in the preparation of a medicament for the prevention, treatment and / or improvement of a disease associated with CLDN1 expression (including CLDN1 positivity, high or overexpression). Further, the disease can be a solid tumor, such as a CLDN1-positive solid tumor. The anti-CLDN1 antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, antibody-drug conjugate or its salt or composition may exert their effects by binding to CLDN1 to exert an ADCC effect or by the cytotoxic toxicity of cytotoxic compounds in the antibody-drug conjugate, but is not limited thereto. For example, the diseases mentioned can be liver cancer, head and neck squamous cell carcinoma (HNSCC), squamous cell lung cancer (SQCLC), thyroid cancer, ovarian cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, cervical cancer, etc.

[0096] Tenth aspect

[0097] This invention also provides a method for preventing, treating, and / or improving a disease, the method comprising administering to a subject in need an anti-CLDN1 antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, antibody-drug conjugate or its salt or combination thereof, wherein the disease is associated with CLDN1 expression (including CLDN1 positivity, high or overexpression). Further, the disease can be a solid tumor, such as a CLDN1-positive solid tumor. For example, the disease can be liver cancer, head and neck squamous cell carcinoma (HNSCC), squamous cell lung cancer (SQCLC), thyroid cancer, ovarian cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, cervical cancer, etc. The subject can be a mammal, preferably a primate or rodent, such as a human.

[0098] The methods for preventing, treating and / or improving diseases provided by the present invention depend on a variety of factors when applied, including the specific active ingredient of the pharmaceutical composition applied, the patient's age, weight, sex or physical and medical condition, the severity of the disease to be treated, the route of administration, etc.

[0099] The method provided by this invention can also be used in combination with other drugs or methods. These other drugs or methods refer to those that can be administered in combination with the anti-CLDN1 antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, antibody-drug conjugate or its salt or composition described in this invention, such as small molecule chemical drugs, targeted drugs, recombinant protein drugs such as antibodies, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapeutic drugs, and radiotherapy. The combined administration of these two methods can be carried out in any form, such as simultaneously, continuously, or at intervals.

[0100] Eleventh aspect

[0101] This invention also provides the use of the aforementioned anti-CLDN1 antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, antibody-drug conjugate or its salt or composition in the preparation of reagents for diagnosing diseases associated with CLDN1 expression (including CLDN1 positivity, high or overexpression). Further, the disease can be a solid tumor, such as a CLDN1-positive solid tumor. For example, the disease can be liver cancer, head and neck squamous cell carcinoma (HNSCC), squamous cell lung cancer (SQCLC), thyroid cancer, ovarian cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, cervical cancer, etc.

[0102] Twelfth aspect

[0103] This invention also provides a method for diagnosing a disease, the method comprising contacting an anti-CLDN1 antibody or a fragment thereof, a nucleic acid molecule, a carrier, a host cell, an antibody-drug conjugate or a salt thereof, or a combination thereof, with a sample from a subject, wherein the disease is associated with CLDN1 expression (including CLDN1 positivity, high or overexpression). Further, the disease can be a solid tumor, such as a CLDN1-positive solid tumor. For example, the disease can be liver cancer, head and neck squamous cell carcinoma (HNSCC), squamous cell lung cancer (SQCLC), thyroid cancer, ovarian cancer, colorectal cancer, breast cancer, gastric cancer, pancreatic cancer, cervical cancer, etc. The subject can be a mammal, preferably a primate or rodent, such as a human.

[0104] Thirteenth aspect

[0105] This invention provides a kit comprising the anti-CLDN1 antibody of the present invention or its antigen-binding fragment, a nucleic acid molecule, a vector, a host cell, an antibody-drug conjugate or its salt or combination thereof. The kit can be used for the aforementioned prevention, treatment and / or improvement, or for the aforementioned diagnosis. Depending on the intended application, the kit may also contain other reagents, such as buffers. For example, the kit is for detecting CLDN1 expression (including overexpression) in any biological sample using an ELISA.

[0106] This invention provides anti-CLDN1 mouse antibodies, chimeric antibodies, and humanized antibodies against CLDN1. Compared to existing CLDN1 antibodies, the antibodies provided by this invention exhibit lower non-specific binding to CLDN1-negative cells but can bind to the CLDN1 protein with higher affinity, such as recombinant cells overexpressing CLDN1 protein or tumor cells, thus possessing higher tumor selectivity and better safety. Regarding antibody-mediated tumor-killing activity, the antibodies provided by this invention exhibit relatively stronger antibody-mediated cytotoxicity and a more significant tumor-suppressing effect. Furthermore, experiments have demonstrated that the antibodies provided by this invention have a strong ability to internalize into CLDN1-expressing cells, and their internalization activity is higher than that of existing CLDN1 antibodies. Taking topoisomerase inhibitors and microtubule inhibitors as examples, the antibodies provided by this invention, after conjugation with cytotoxic compounds, show more significant CLDN1-positive cell-killing activity and tumor-suppressing effects.

[0107] Therefore, the anti-CLDN1 mouse antibody, chimeric antibody and its humanized antibody provided by this invention will have important application potential in tumor targeted therapy, targeted killing ADC drug development, and targeted and immunotherapy combination therapy. Attached Figure Description

[0108] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0109] Figure 1 shows the distribution of CLDN1 expression levels in different tumor tissues;

[0110] Figures 2A and 2B illustrate the internalization activity of the anti-CLDN1 chimeric antibody provided by the present invention in different tumor cells;

[0111] Figure 3 shows the ADCC activity of the anti-CLDN1 chimeric antibody provided by the present invention;

[0112] Figures 4A to 4C illustrate the binding activity of the anti-CLDN1 humanized antibody provided by the present invention with CLDN1-negative cells;

[0113] Figures 5A to 5D illustrate the internalization activity of the anti-CLDN1 humanized antibody provided by the present invention in different tumor cells;

[0114] Figure 6 shows the in vivo efficacy of the anti-CLDN1 humanized antibody provided by the present invention in the Huh7 CDX mouse model;

[0115] Figure 7 shows the in vivo efficacy of the antibody-drug conjugate targeting CLDN1 provided by the present invention in the Huh7 CDX mouse model;

[0116] Figure 8 shows the in vivo efficacy of the antibody-drug conjugate targeting CLDN1 provided by the present invention in the Cal27 CDX mouse model;

[0117] Figure 9 shows the in vivo efficacy of the antibody-drug conjugate targeting CLDN1 provided by the present invention in the SCC4 CDX mouse model;

[0118] Figure 10 shows the in vivo efficacy of the antibody-drug conjugate targeting CLDN1 provided by the present invention in the Cal27 CDX mouse model;

[0119] Figure 11 shows the in vivo efficacy of the antibody-drug conjugate targeting CLDN1 provided by the present invention in the HCC1954CDX mouse model.

[0120] The best way to implement an invention

[0121] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0122] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products.

[0123] The following examples demonstrate the construction of recombinant expression cells or recombinant expression plasmids using different antigen proteins, including:

[0124] Human CLDN1 (hCLDN1): UniProtKB-O95832;

[0125] Monkey CLDN1 (cynoCLDN1): UniProtKB-F6TZW7;

[0126] Mouse CLDN1 (mCLDN1): UniProtKB-O88551.

[0127] In the following examples, antibody OM7D3B3-H3L3 (derived from WO2017162678) was used as a positive control antibody. This antibody was developed by Alentis Theraputics and is currently in Phase I clinical trials for the indication of solid tumors. Its variable region sequence is as follows:

[0128] >OM7D3B3-H3L3_VH

[0129] >OM7D3B3-H3L3_VL

[0130] The following examples use the human IgG1 heavy chain constant region and the human kappa light chain constant region to construct chimeric antibodies, humanized antibodies, or positive control antibodies, with the following sequences:

[0131] Human IgG1 heavy chain constant region:

[0132] Human kappa light chain constant region:

[0133] Example 1: Expression of CLDN1 in different tumor tissues

[0134] CLDN1 has been reported to be significantly expressed in various tumors, such as liver cancer, head and neck squamous cell carcinoma (HNSCC), squamous cell lung cancer (SQCLC), thyroid cancer, ovarian cancer, and colorectal cancer. Its overexpression is associated with poor prognosis in patients with these tumors. The inventors of this application detected CLDN1 expression in relevant tumor tissues using tumor microarray (TMA) IHC staining.

[0135] The antigen retrieval and staining steps in the tumor tissue microarray were performed using a Leica Bond III staining system. CLDN1 antibody (Abcam, catalog number: ab211737) was diluted 1:2000 and incubated with the microarray at room temperature for 1 hour. After washing, the microarray was stained with the secondary antibody Goat Anti-Human IgG (H+L)-HRP (1:300) using a Leica DS9800 staining system and incubated at room temperature for 30 minutes. The microarray was then washed and developed with DAB working solution for 5 minutes, followed by hematoxylin staining for 15 minutes. The microarray was dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. The microarray was scanned using a fluorescence imaging scanner, analyzed using Halo analysis software, and the results (- / + / ++ / +++) were interpreted by a pathologist, who also calculated the H-score.

[0136] As shown in Figure 1, CLDN1 is expressed in more than 90% of liver cancer tissues, more than 85% of head and neck squamous cell carcinoma (HNSCC) tissues, 70-80% of cervical cancer tissues, and 50-60% of lung squamous cell carcinoma (SQCLC) tissues.

[0137] Example 2: Animal Immunization and Hybridoma Screening

[0138] Six- to eight-week-old female Balb / c mice were immunized with human and monkey CLDN1 overexpressing cell lines HEK293-hCLDN1, Huh7-hCLDN1, and pcDNA3.1-hCLDN1 plasmids, respectively. Immunization was performed three times at a frequency of once every two weeks, followed by intraocular blood collection. Serum titers were analyzed by flow cytometry using human, monkey, and mouse CLDN1 overexpressing cell lines NIH3T3-hCLDN1, NIH3T3-cynoCLDN1, and NIH3T3-mCLDN1. Mice with high binding activity were selected for subsequent hybridoma fusion.

[0139] After booster immunization of selected mice, their spleens were harvested, ground, and the resulting cells were electrofused with mouse myeloma cells SP20. The cells were cultured in HAT and HT media for 10-14 days, and the hybridoma supernatant was collected. Cellular binding screening was performed on the obtained hybridoma supernatant using the FACS method. The initial screening cell lines were HEK293-hCLDN1 and HEK293-vector (transfected with a blank vector), and the secondary screening cell lines were NIH3T3-hCLDN1, NIH3T3-cynoCLDN1, and NIH3T3. FACS-positive clones were selected for further subcloning. After 5-7 days of culture, the cell-level binding activity of the supernatant was detected again. Finally, multiple monoclonal hybridomas with cross-binding activity between human and monkey CLDN1 were obtained.

[0140] Example 3: Production and Identification of Human-Mouse Chimeric Antibodies

[0141] (i) Gene extraction of candidate clones, construction of chimeric antibody expression vectors, expression and purification

[0142] After culturing monoclonal hybridoma cells to the logarithmic growth phase, cells (approximately 1E6 cells / clone) were collected, RNA was extracted, and cDNA was obtained through reverse transcription. Amplification and sequencing were then performed to obtain the VH and VL sequences of the antibody. The VH and VL sequences of the antibody were constructed into pTT5 expression vectors containing the constant regions of the human IgG1 heavy chain and the human kappa light chain, respectively, to obtain recombinant expression plasmids of the antibody heavy and light chains. The heavy and light chain plasmids were mixed in a specific ratio and transiently transferred into CHO cells. After culturing, the supernatant was collected and purified to obtain the expressed chimeric antibody.

[0143] (II) Activity Characterization of Chimeric Antibodies

[0144] 1) Binding of chimeric antibodies to human and monkey CLDN1 overexpressing cells and CLDN1 endogenous expressing cells

[0145] The cellular-level binding activity of the obtained chimeric antibodies was detected using human and monkey CLDN1 overexpressing cell lines NIH3T3-hCLDN1 and NIH3T3-cynoCLDN1, as well as endogenously CLDN1-expressing hepatocellular carcinoma cells Huh7 and cervical squamous cell carcinoma cells Siha.

[0146] Specifically, CLDN1-expressing cells in logarithmic growth phase were adjusted to 2E6 cells / mL and seeded into 96-well plates at 50 μL / well. Serially diluted test antibody (starting concentration 30 μg / mL, 4-fold dilution, 8 concentration points) was added at 50 μL / well and mixed with the cells. After incubation at 4°C for 1 h, the cells were centrifuged, the supernatant was discarded, and the cells were washed twice with PBS. A 1:1000 dilution of goat anti-human Fcγ fragment-specific APC antibody (Jackson ImmunoResearch, catalog number: 109-135-098) was added to each well, and the cells were incubated at 4°C in the dark for 30 min. After washing twice with PBS, the cells were resuspended in 30 μL of PBS and analyzed by flow cytometry using an iQue Screener PLUS (Saorius). The results were analyzed using GraphPad Prism.

[0147] As shown in Table 1, the chimeric antibody ch8H1 can specifically bind to human and monkey CLDN1 overexpressing cells and tumor cells. Moreover, the EC50 value of ch8H1 binding to tumor cells with different levels of endogenous CLDN1 expression is significantly better than that of the positive control antibody OM7D3B3-H3L3.

[0148] Table 1. Binding activity of the anti-CLDN1 chimeric antibody provided in this invention with different CLDN1-expressing cells. --: Not detected

[0149] 2) Internalization of chimeric antibodies on CLDN1 endogenously expressed cells

[0150] The internalization activity of the obtained chimeric antibodies was detected using an antibody internalization kit (Sartorius, catalog number: 90565), Huh7 liver cancer cells endogenously expressing CLDN1, and Siha cervical squamous cell carcinoma cells.

[0151] Specifically, the antibody and internalization reagent were first adjusted to 100 μg / mL. Antibody labeling was performed using a ratio of 1 μL antibody + 1 μL internalization reagent + 48 μL total culture medium, followed by incubation at 37°C and 5% CO2 for 15 min. After incubation, the mixture was serially diluted 2-fold to create 8 concentration points. Simultaneously, CLDN1-expressing cells in logarithmic growth phase were adjusted to 2E6 cells / mL and seeded at 20 μL / well in 96-well plates. The diluted mixture was then mixed with the cells at 20 μL / well and incubated at 37°C and 5% CO2 for 2 h. Cells were collected and analyzed by flow cytometry using iQue Screener PLUS (Saorius). The results were analyzed using GraphPad Prism.

[0152] As shown in Figures 2A and 2B, the chimeric antibody ch8H1 can be effectively internalized, and its internalization activity is superior to that of the positive control antibody OM7D3B3-H3L3.

[0153] 3) Antibody-dependent cytotoxic (ADCC) activity of chimeric antibodies

[0154] Engineered Jurkat cells were used as effector cells, stably expressing firefly luciferase driven by the Human FcγRIIIa receptor and NFAT response element. The bioactivity of the antibody in the ADCC mechanism of action was quantified via the luciferase produced by NFAT pathway activation.

[0155] Specifically, target cells, namely human liver cancer cells Huh7 (5E5 cells / mL), were seeded at 40 μL / well into 96-well blank microplates (Coster, catalog number: 3917). The test antibody (initial concentration 30 μg / mL, 4-fold dilution, 9 concentration points) and blank control PBS were added at 20 μL / well, mixed well, and incubated at 37°C and 5% CO2 for 1 h. Then, ADCC effector cells (3.75E6 cells / mL) were added at 40 μL / well (effector / target cell ratio 7.5:1), and incubated at 37°C and 5% CO2 for 6 h. After removing the 96-well blank microplates and allowing them to return to room temperature, 100 μL of Bio-Lite Luciferase Assay System reagent (Vazyme, catalog number: DD1201-03) was added to each well, and the plates were shaken for 5 min. The relative fluorescence value was then detected using a SpectraMax M5e microplate reader. The results were analyzed using GraphPad Prism (RLU unit).

[0156] As shown in Figure 3, the chimeric antibody ch8H1 exhibits significant ADCC activity against target-expressing cells, and its activity is higher than that of the positive control antibody OM7D3B3-H3L3.

[0157] Example 4: Humanization of Antibodies and Identification of Humanized Antibodies

[0158] (I) Antibody humanization and the production and activity evaluation of humanized antibodies

[0159] Antibody humanization was performed using CDR region transplantation, with CDRs defined according to CCG rules.

[0160] Specifically, a variable region structure model of the antibody was established based on the chimeric antibody VH and VL sequences (MOE software). The human antibody with the highest homology was selected to provide the antibody framework. The CDR regions from the mouse anti-VH and VL antibodies were transplanted into the human antibody framework, forming the humanized antibody variable region sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Simultaneously, based on the established antibody variable region structure model, amino acid residue reversion mutations were performed to ensure the activity of the humanized antibody.

[0161] Following the method described in Example 3(a), the expression vector for the humanized antibody was constructed, expressed, and purified. The binding and internalization activities of the humanized antibodies containing different mutations with CLDN1-expressing cells (including human and monkey CLDN1-overexpressing cells and tumor cells) were evaluated, and humanized antibodies with activity comparable to or better than the original antibodies were selected. The light and heavy chain variable region sequences of the chimeric antibody ch8H1 and the finally screened humanized antibody hz8H1 are shown in Table 2 (underlined and bolded sequences are CDR sequences).

[0162] Table 2. Variable region sequences of anti-CLDN1 chimeric antibodies and humanized antibodies

[0163] (II) Activity Characterization of Humanized Antibodies

[0164] 1) Binding of humanized antibodies to human and monkey CLDN1 overexpressing cells and CLDN1 endogenous expressing cells

[0165] Following the method described in Example 3(II) 1), the humanized antibodies were subjected to cellular-level binding detection using human and monkey CLDN1 overexpressing cells HEK293-hCLDN1 and HEK293-cynoCLDN1, as well as human head and neck squamous cell carcinoma cells Cal27, SCC4, liver cancer cells Huh7, and lung squamous cell carcinoma cells NCI-H1703 that endogenously express CLDN1.

[0166] As shown in Table 3, the humanized antibody hz8H1 can specifically bind to human and monkey CLDN1 overexpressing cells and tumor cells. Moreover, the EC50 value of hz8H1 binding to overexpressing cells and tumor cells with different levels of endogenous CLDN1 expression is significantly better than that of the positive control antibody OM7D3B3-H3L3.

[0167] Table 3. Binding activity of the anti-CLDN1 humanized antibody provided in this invention with different CLDN1-expressing cells.

[0168] 2) Binding of humanized antibodies to CLDN1-negative cells

[0169] Following the method described in Example 3(II)1), the non-specific binding activity of the obtained humanized antibody was detected at the cellular level using CLDN1-negative cells (HEK293-Vector, CHOK1) and small cell lung cancer cells SHP77.

[0170] As shown in Figures 4A to 4C, the humanized antibody hz8H1 showed virtually no nonspecific binding activity with these cells, and its binding activity was significantly lower than that of the positive control antibody OM7D3B3-H3L3.

[0171] 3) Internalization of humanized antibodies on CLDN1 endogenously expressed cells

[0172] Following the method described in Example 3(II)2), the internalization activity of the obtained humanized antibody was detected using human head and neck squamous cell carcinoma cells Cal27 and SCC4, liver cancer cells Huh7, and lung squamous cell carcinoma cells NCI-H1703 with different levels of endogenous CLDN1 expression.

[0173] As shown in Figures 5A to 5D, the humanized antibody hz8H1 can be effectively internalized in tumor cells with different levels of endogenous CLDN1 expression, and its internalization activity is superior to that of the positive control antibody OM7D3B3-H3L3.

[0174] Example 5: In vivo efficacy evaluation of anti-CLDN1 humanized antibody in tumor-bearing mouse model

[0175] Human hepatocellular carcinoma cells (Huh7) were inoculated into the right posterior dorsal region of female BALB / c nude mice (6-8 weeks old). Three days after inoculation, the average tumor volume was approximately 80 mm². 3 Dosing was administered in groups, and the specific grouping and dosing details are shown in Table 4.

[0176] As shown in Table 4, subcutaneous injection was used for drug administration. The day of administration was designated as D0, and the drug was administered once each on D0, D7, D14, and D21. After grouping, the tumor volume and body weight of the mice were measured three times a week, and the relationship between changes in body weight and tumor volume and drug administration time was recorded. At the end of the experiment, the tumor-bearing mice were euthanized. The tumor growth inhibition rate (TGI) was calculated (%) and statistical analysis was performed.

[0177] Table 4. Grouped dosing regimens for in vivo efficacy evaluation

[0178] As shown in Figure 6 and Table 5, at the end of the experiment (D24), the mean tumor volume of mice in the G1 (Isotype Control), G2 (OM7D3B3-H3L3, 25 mg / kg), and G3 (hz8H1, 25 mg / kg) groups was 1306.91 mm. 3 913.04mm 3 830.31mm 3 The TGI (%) of groups G2 and G3 were 31.82% and 38.74%, respectively. Therefore, compared with the Isotype Control, both OM7D3B3-H3L3 (p<0.05) and hz8H1 (p<0.01) showed significant antitumor effects, with hz8H1 exhibiting superior antitumor activity compared to OM7D3B3-H3L3.

[0179] Table 5. In vivo efficacy of anti-CLDN1 humanized antibody in Huh7 CDX mouse model

[0180] Note (the same applies below):

[0181] a. Mean ± SEM.

[0182] b. Tumor growth inhibition rate is calculated as TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment) / (mean tumor volume at the end of treatment in solvent control group - mean tumor volume at the start of treatment in solvent control group)] × 100%.

[0183] c. Compared with the control group, the p-value is calculated based on the endpoint tumor volume.

[0184] d. “*” indicates that the p-value is less than or equal to 0.05, “**” indicates that the p-value is less than or equal to 0.01, “***” indicates that the p-value is less than or equal to 0.001, and “ns” indicates that the p-value is not significant.

[0185] Example 6: Preparation of antibody-drug conjugates targeting CLDN1

[0186] (I) Synthesis of drug-containing linkers

[0187] The following drug-containing linker payload was synthesized according to the method described in patent application publications WO2023 / 109965A1 or WO2018 / 095422A1.

[0188] MWD-L1:

[0189] Mass spectrometry: [M+2H] 2+ =840.9

[0190] MWF-L6 (also known as MF-16):

[0191] Mass spectrometry: [M+1] + =1450

[0192] MWF-L7:

[0193] Mass spectrometry: [M+1] + =1493.5

[0194] MWF-L8:

[0195] Mass spectrometry: [M+1] + =1598.4

[0196] MWD-L7:

[0197] Mass spectrometry: [M+1] + =1682

[0198] MWD-L8:

[0199] Mass spectrometry: [M+1] + =1645.7

[0200] MWD-L9:

[0201] Mass spectrometry: [M+1] + =1713.7

[0202] C-1:

[0203] Mass spectrometry: [M+1] + =1927

[0204] C-2:

[0205] Mass spectrometry: [M+1] + =1987

[0206] C-3:

[0207] Mass spectrometry: [M+1] + =1963

[0208] C-4:

[0209] Mass spectrometry: [M+1] + =1995

[0210] (II) Preparation of antibody-drug conjugates targeting CLDN1

[0211] 1. Preparation of antibody-C-3ADC

[0212] The antibody or irrelevant control hIgG1 was diluted to 10 mg / mL using 10 mmol / L histidine-histidine hydrochloride buffer. A 10 mg / mL TCEP aqueous solution was added to the 10 mg / mL antibody dilution at a 10:1 (TCEP:antibody) molar ratio. Reduction was performed by incubation at 25°C for 120 min. The reactants were then transferred to conjugation buffer (50 mmol / L PB + 50 mmol / L NaCl + 2 mmol / L EDTA·Na2), followed by the addition of 20 mg / mL C-3. Conjugation was then performed with the reduced antibody at a 4.8:1 (C-3:antibody) molar ratio at 25°C for 60 min. After the reactants were transferred to hydrolysis buffer (50 mmol / L PB), the conjugated antibody was hydrolyzed at 35°C for 120 min. Finally, impurities were removed using hydrophobic chromatography packing material Butyl Sepharose 4FastFlow, and the collected chromatography buffer was ultrafiltered and replaced with 10 mmol / L Pb, pH 7.4 buffer. The obtained ADC molecule was named "antibody name-C-3".

[0213] 2. Preparation of antibody-MF-L6 ADC

[0214] The antibody or irrelevant control hIgG1 was diluted to 10 mg / mL using 10 mmol / L histidine-histidine hydrochloride buffer. A 10 mg / mL TCEP aqueous solution was added to the 10 mg / mL antibody dilution at a 10:1 (TCEP:antibody) molar ratio. Reduction was performed by incubation at 25°C for 120 min. The reactants were then transferred to conjugation buffer (50 mmol / L PB + 50 mmol / L NaCl + 2 mmol / L EDTA·Na2), followed by the addition of 20 mg / mL MF-L6. Conjugation was then performed with the reduced antibody at a 4.8:1 (MF-L6:antibody) molar ratio at 25°C for 60 min. Subsequently, the reactants were transferred to hydrophobic chromatography buffer (50 mmol / L PB). Impurities were then removed using Toyopearl Butyl 650M hydrophobic chromatography material, and the collected chromatography solution was ultrafiltered and transferred to 50 mmol / L PB buffer. Hydrolyze at 35°C for 120 min, then replace the buffer with 10 mmol / L PB at pH 7.4 by ultrafiltration. The obtained ADC molecule is named "antibody name-MF-L6".

[0215] (III) Purity and DAR value analysis of antibody-drug conjugates targeting CLDN1

[0216] 1. Purity of size exclusion high-performance liquid chromatography (SEC-HPLC)

[0217] Instrument: High-performance liquid chromatograph (HPLC);

[0218] Chromatographic column: TSKgel G3000SWXL (5μm, 7.8mm×30cm) column;

[0219] Mobile phase: 0.1 mol / L phosphate buffer (PB) + 0.2 mol / L L-arginine hydrochloride (pH 6.8);

[0220] Chromatographic conditions: column temperature: 30℃; flow rate: 0.6 ml / min; detection wavelength: 280 nm; injection volume: 20 μl; isocratic elution.

[0221] 2. Antibody-C-3ADC: Hydrophobic Interaction High Performance Liquid Chromatography (HIC-HPLC) DAR Value

[0222] Instrument: High-performance liquid chromatograph (HPLC);

[0223] Chromatographic column: Proteomix HIC Butyl-NP5 (4×35mm) column;

[0224] Mobile phases: Phase A: 0.025 mol / L sodium phosphate + 1.2 mol / L ammonium sulfate (pH 7.0), Phase B: 0.025 mol / L sodium phosphate (pH 7.0), Phase C: 100% isopropanol (IPA);

[0225] Chromatographic conditions: column temperature: 25℃; flow rate: 0.8 ml / min; detection wavelength: 280 nm; injection volume: 30 μl; gradient elution.

[0226] Washing conditions:

[0227] DAR calculation formula:

[0228] DAR = ∑(weighted peak area) / 100, that is, DAR = (D0 peak area ratio × 0 + D1 peak area ratio × 1 + D2 peak area ratio × 2 + D3 peak area ratio × 3 + D4 peak area ratio × 4 + D5 peak area ratio × 5 + D6 peak area ratio × 6 + D7 peak area ratio × 7 + D8 peak area ratio × 8) / 100.

[0229] 3. Antibody-MF-L6 ADC: Hydrophobic interaction high performance liquid chromatography (HIC-HPLC) DAR value instrument: high performance liquid chromatograph (HPLC);

[0230] Chromatographic column: SHIMSEN Ankylo HIC-Ph column (5μm, 4.6mm×100mm);

[0231] Mobile phases: Phase A: 25 mmol / L sodium phosphate + 1.2 mol / L ammonium sulfate + 20 mmol / L tetrabutylammonium bromide (pH 7.4), Phase B: 25 mmol / L sodium phosphate + 20 mmol / L tetrabutylammonium bromide (pH 7.4), Phase C: 100% isopropanol (IPA);

[0232] Chromatographic conditions: column temperature: 30℃; flow rate: 0.6 ml / min; detection wavelength: 280 nm; injection volume: 30 μl; gradient elution.

[0233] Washing conditions:

[0234] DAR calculation formula:

[0235] DAR = ∑(weighted peak area) / 100, that is, DAR = (D0 peak area ratio × 0 + D1 peak area ratio × 1 + D2 peak area ratio × 2 + D3 peak area ratio × 3 + D4 peak area ratio × 4 + D5 peak area ratio × 5 + D6 peak area ratio × 6 + D7 peak area ratio × 7 + D8 peak area ratio × 8) / 100.

[0236] The purity of the prepared ADC molecules and the results of DAR value detection are shown in Table 6.

[0237] Table 6. Purity and DAR value of antibody-drug conjugates targeting CLDN1

[0238] Example 7 Cell-killing evaluation of antibody-drug conjugates targeting CLDN1

[0239] The cytotoxic activity of the antibody-drug conjugates targeting CLDN1 was detected using HEK293-hCLDN1 cells overexpressing human CLDN1, as well as human hepatocellular carcinoma cells Huh7, human ovarian carcinoma cells OVCAR3, and human head and neck squamous cell carcinoma cells SCC4.

[0240] Specifically, the cell count was adjusted to 4E4-6E4 cells / mL according to the growth rate, and 50 μL / well was seeded into 96-well blank plates (Coster, catalog number: 3917). Simultaneously, 50 μL / well of the test antibody (initial concentration 30 μg / mL, 4-fold dilution, 9 concentration points) and blank control PBS were added. The plates were incubated at 37°C in CO2 for 6 days. After removing the 96-well blank plates and allowing them to return to room temperature, 100 μL of Cell Titer Glo luminescent cell viability assay reagent (Promega, catalog number: G7573) was added to each well, and the plates were shaken for 5 min. The relative fluorescence unit (RLU) was then measured using a SpectraMax M5e microplate reader.

[0241] As shown in Table 7, the antibody-drug conjugates hz8H1-MF-L6 and hz8H1-C-3 targeting CLDN1 showed good cell-killing activity in various cell types, and their activity was higher than that of the positive control antibody-drug conjugates OM7D3B3-H3L3-MF-L6 and OM7D3B3-H3L3-C-3.

[0242] Table 7. Cell-killing activity of the antibody-drug conjugate targeting CLDN1 provided in this invention in different CLDN1-expressing cells. --: Not detected

[0243] Example 8: In vivo efficacy evaluation of antibody-drug conjugates targeting CLDN1 in tumor-bearing mouse models.

[0244] (I) In vivo efficacy evaluation of antibody-drug conjugates obtained by conjugation with MF-L6

[0245] Human hepatocellular carcinoma Huh7 cells were inoculated into the right posterior dorsal region of female BALB / c nude mice (6-8 weeks old). Six days after inoculation, the average tumor volume was approximately 150 mm. 3 The drugs were administered in time-groups; human head and neck squamous cell carcinoma Cal27 cells were inoculated into the right posterior dorsal region of NOD SCID female mice (6-8 weeks old). Eleven days after inoculation, the average tumor volume was approximately 130 mmHg. 3 Dosing was administered in time-grouped groups; specific groupings and dosing details are shown in Table 8.

[0246] As shown in Table 8, intravenous injection was used for drug administration. The day of administration for each group was recorded as D0, and all mice were given the drug once on D0. After grouping, the tumor volume and body weight of the mice were measured three times a week, and the relationship between changes in body weight and tumor volume and drug administration time was recorded. At the end of the experiment, the tumor-bearing mice were euthanized. The tumor growth inhibition rate (TGI) was calculated (%) and statistical analysis was performed.

[0247] Table 8. Grouped dosing regimens for in vivo efficacy evaluation

[0248] 1) In vivo efficacy in the human liver cancer Huh7 model

[0249] As shown in Figure 7 and Table 9, at the end of the experiment (D28), the mean tumor volume of mice in the G1 (Vehicle), G2 (hz8H1-MF-L6, 10 mg / kg), and G3 (hz8H1-MF-L6, 5 mg / kg) groups was 1514.07 mm. 3 67.69mm 3 102.55mm 3 The TGI (%) of groups G2 and G3 were 106.00% and 103.48%, respectively. Therefore, compared with the Vehicle, hz8H1-MF-L6 (10 mg / kg, p<0.001; 5 mg / kg, p<0.001) significantly and completely inhibited tumor proliferation.

[0250] Table 9. In vivo efficacy of antibody-drug conjugates targeting CLDN1 in Huh7 CDX mouse model

[0251] 2) In vivo efficacy in the human head and neck squamous cell carcinoma Cal27 model

[0252] As shown in Figure 8 and Table 10, at the end of the experiment (D41), the mean tumor volume of mice in the G1 (Vehicle) group, G2 (hz8H1-MF-L6, 10 mg / kg) group, G3 (hz8H1-MF-L6, 3 mg / kg) group, G4 (OM7D3B3-H3L3-MF-L6, 10 mg / kg) group, and G5 (OM7D3B3-H3L3-MF-L6, 3 mg / kg) group was 1400.12 mm. 3 232.26mm 3 844.56mm 3 400.84mm 3 1287.36mm 3 The TGI (%) of each treatment group were 83.35%, 39.59%, 71.24%, and 7.81%, respectively. Therefore, at day 41, compared with the Vehicle, hz8H1-MF-L6 (10 mg / kg, p<0.001) and OM7D3B3-H3L3-MF-L6 (10 mg / kg, p<0.001) still significantly inhibited tumor proliferation, with the former showing better tumor-suppressing effect than the latter; hz8H1-MF-L6 (3 mg / kg, p<0.01) also showed better tumor-suppressing effect than OM7D3B3-H3L3-MF-L6 (3 mg / kg, p>0.05).

[0253] Table 10. In vivo efficacy of antibody-drug conjugates targeting CLDN1 in Cal27 CDX mouse model

[0254] (II) In vivo efficacy evaluation of antibody-drug conjugates obtained by conjugation with C-3

[0255] Human head and neck squamous cell carcinoma SCC4 cells were subcutaneously injected into the right anterior rib area of ​​female NCG mice (6-8 weeks old). Fifteen days after inoculation, the average tumor volume was approximately 150 mm. 3 Drug administration was divided into groups based on time; human head and neck squamous cell carcinoma Cal27 cells were subcutaneously inoculated into the right forelimb of NCG female mice (6-8 weeks old). 25 days after inoculation, when the average tumor volume was approximately 150 mm³, drug administration was divided into groups based on time; human breast cancer HCC1954 cells were inoculated into the right posterior region of CB-17SCID female mice (6-8 weeks old). 6 days after inoculation, when the average tumor volume was approximately 140 mm³, drug administration was divided into groups based on time. 3 Dosing was administered in time-grouped groups; specific groupings and dosing details are shown in Table 11.

[0256] As shown in Table 11, intravenous injection was used for drug administration. The day of administration was designated as D0. Drug was administered once on D0 or twice, on D0 and D7. Tumor volume and body weight were measured two or three times per week after grouping, and the relationship between changes in body weight and tumor volume and drug administration time was recorded. At the end of the experiment, the tumor-bearing mice were euthanized. The tumor growth inhibition rate (TGI) was calculated (%) and statistical analysis was performed.

[0257] Table 11. Grouped dosing regimens for in vivo efficacy evaluation

[0258] 1) In vivo efficacy in a human head and neck squamous cell carcinoma SCC4 model

[0259] As shown in Figure 9 and Table 12, at the end of the experiment (D54), the mean tumor volume of mice in the G1 (Vehicle), G2 (OM7D3B3-H3L3-C-3, 3 mg / kg), and G3 (hz8H1-C-3, 3 mg / kg) groups was 1665.24 mm. 3 0mm 3 0mm 3 The TGI (%) in each treatment group was 100%. Therefore, at day 54, compared with the Vehicle, OM7D3B3-H3L3-C-3 (3 mg / kg, p<0.001) and hz8H1-C-3 (3 mg / kg, p<0.001) still significantly and completely inhibited tumor proliferation, and their tumor-suppressing effects were comparable.

[0260] Table 12. In vivo efficacy of antibody-drug conjugates targeting CLDN1 in SCC4 CDX mouse model

[0261] 2) In vivo efficacy in the human head and neck squamous cell carcinoma Cal27 model

[0262] As shown in Figure 10 and Table 13, at the end of the experiment (D21), the mean tumor volume of mice in the G1 (Vehicle), G2 (OM7D3B3-H3L3-C-3, 3 mg / kg), and G3 (hz8H1-C-3, 3 mg / kg) groups was 974.67 mm. 3 728.61mm 3 500.28mm 3 The TGI (%) of each treatment group was 26.36% and 47.92%, respectively. Therefore, at day 21, compared with the Vehicle, OM7D3B3-H3L3-C-3 (3 mg / kg, p<0.05) and hz8H1-C-3 (3 mg / kg, p<0.001) showed significant antitumor effects, with hz8H1-C-3 exhibiting superior antitumor efficacy compared to OM7D3B3-H3L3-C-3.

[0263] Table 13. In vivo efficacy of antibody-drug conjugates targeting CLDN1 in Cal27 CDX mouse model

[0264] 3) In vivo efficacy in the human breast cancer HCC1954 model

[0265] As shown in Figure 11 and Table 14, at the end of the experiment (D39), the mean tumor volume of mice in the G1 (Vehicle), G2 (OM7D3B3-H3L3-C-3, 3 mg / kg), and G3 (hz8H1-C-3, 3 mg / kg) groups was 1368.29 mm. 3 37.04mm 3 29.73mm 3 The TGI (%) of each treatment group was 108.22% and 108.86%, respectively. Therefore, at day 39, compared with the Vehicle, both OM7D3B3-H3L3-C-3 (3 mg / kg, p<0.001) and hz8H1-C-3 (3 mg / kg, p<0.001) significantly and completely inhibited tumor proliferation.

[0266] Table 14. In vivo efficacy of antibody-drug conjugates targeting CLDN1 in the HCC1954 CDX mouse model

[0267] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

Claims

1. An antibody against tight junction protein 1 (CLDN1) or an antigen-binding fragment thereof, said anti-CLDN1 antibody or antigen-binding fragment comprising heavy chain complementarity-determining regions (CDRs), namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and light chain complementarity-determining regions (CDRs), namely light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), wherein the heavy chain CDRs and light chain CDRs are as follows: (1) HCDR1, HCDR2, and HCDR3, sequentially comprising the amino acid sequences shown in SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11; and LCDR1, LCDR2, and LCDR3, sequentially comprising the amino acid sequences shown in SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14; or (2) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.9, SEQ ID NO.15, and SEQ ID NO.16; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.17, SEQ ID NO.13, and SEQ ID NO.

14.

2. The anti-CLDN1 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-CLDN1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise a combination of the following amino acid sequences: (1) The amino acid sequence shown in SEQ ID NO. 5, or an amino acid sequence having at least 75% identity with said amino acid sequence; and the amino acid sequence shown in SEQ ID NO. 6, or an amino acid sequence having at least 75% identity with said amino acid sequence; or (2) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.8, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.

8.

3. The anti-CLDN1 antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antigen-binding fragment of the antibody can be a single-chain variable fragment (scFv), a bivalent single-chain variable fragment (BsFv), a disulfide-stabilized variable fragment (dsFv), (dsFv)2, an antigen-binding fragment (Fab), Fab' fragment (Fab'), (Fab' fragment)2 (F(ab')2), or a variable fragment (Fv).

4. The anti-CLDN1 antibody or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The anti-CLDN1 antibody or its antigen-binding fragment further comprises a heavy chain constant region (CH) and / or a light chain constant region (CL), preferably comprising a human or mouse heavy chain constant region and / or a light chain constant region; Preferably, the anti-CLDN1 antibody or its fragment comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

5. The anti-CLDN1 antibody or its antigen-binding fragment according to any one of claims 1 to 4, characterized in that, The anti-CLDN1 antibody is a monoclonal antibody, preferably a mouse, chimeric, or humanized monoclonal antibody. Preferably, the monoclonal antibody comprises a heavy chain constant region sequence of IgG1, such as the human IgG1 heavy chain constant region shown in SEQ ID NO.3; and / or comprises a kappa light chain constant region, such as the human kappa light chain constant region shown in SEQ ID NO.

4.

6. The anti-CLDN1 antibody or its antigen-binding fragment according to any one of claims 1 to 5, characterized in that, The anti-CLDN1 antibody is a monoclonal antibody; Preferably, the anti-CLDN1 antibody is an immunoglobulin, for example, the type of the immunoglobulin is human IgA, IgD, IgE, IgG or IgM, and more preferably, the antibody is human IgG1 subtype.

7. A nucleic acid molecule comprising a nucleotide sequence encoding an anti-CLDN1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 6.

8. A vector comprising the nucleic acid molecule of claim 7.

9. A host cell comprising the nucleic acid molecule of claim 7 or the vector of claim 8, or transformed or transfected by the nucleic acid molecule of claim 7 or the vector of claim 8.

10. Use of the anti-CLDN1 antibody or its antigen-binding fragment as described in any one of claims 1 to 6, the nucleic acid molecule as described in claim 7, the vector as described in claim 8, or the host cell as described in claim 9 in the preparation of antibody-drug conjugates (ADCs).

11. An antibody-drug conjugate targeting CLDN1 or a salt thereof, comprising the anti-CLDN1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 6.

12. The antibody-drug conjugate targeting CLDN1 or a salt thereof according to claim 11, characterized in that, The antibody-drug conjugate is formed by conjugating the anti-CLDN1 antibody or its antigen-binding fragment with a cytotoxic compound. Preferably, the cytotoxic compound is a microtubule inhibitor, a topoisomerase inhibitor, or a DNA binder.

13. The antibody-drug conjugate targeting CLDN1 or a salt thereof according to claim 11 or 12, characterized in that, The antibody-drug conjugate or its salt has the general formula The structure shown is as follows, wherein: Ab represents the anti-CLDN1 antibody or its antigen-binding fragment; E L Selected from the following groups ( (This indicates that Ab is linked to the sulfhydryl group of cysteine): E L -1a and / or E L -1b: and / or E L -2： E L -3： E L -4： E L -5： E L -6： M is a phenylene or a phenylene substituted with one or more substituents, or a chemical bond; in the substituted phenylene, the substituent is selected from alkyl, haloalkyl, alkoxy, halogen, ester, amide, and cyano groups; SP1 is selected from C1-8 alkylene, C1-8 cycloalkylene, or C1-21 linear heteroalkylene, wherein the C1-21 linear heteroalkylene comprises 1-11 heteroatoms selected from N, O, or S, wherein each of the C1-8 alkylene, C1-8 cycloalkylene, and C1-21 linear heteroalkylene is independently and optionally substituted by one or more substituents selected from hydroxyl, amino, sulfonic acid, and cyano groups; SP2 is selected from -NH(CH2CH2O) a CH2CH2CO-、-NH(CH2CH2O) a CH2CO-、-S(CH2) a CO- or chemical bond, where a is an integer from 1 to 20; A represents a short peptide structure consisting of 2-4 amino acids or a combination of a short peptide structure consisting of 2-4 amino acids and a self-releasing structural fragment; m is between 1 and 10, and m can be an integer or a non-integer; D represents the cytotoxic compound.

14. The antibody-drug conjugate targeting CLDN1 or a salt thereof according to any one of claims 11 to 13, characterized in that, Group Selected from the following structures, where the wavy line indicates a connection to cysteine ​​in the Ab or to cytotoxic compound D: (1) (2) (3) (4) (5) (6) (7) (8) 15. The antibody-drug conjugate targeting CLDN1 or a salt thereof according to any one of claims 11 to 14, characterized in that, The antibody-drug conjugate or its salt has a structure as shown in structural formulas Ia and / or Ib: and / or In structural formulas Ia and / or Ib, Ab, m, group M, SP1, SP2, A, and D are consistent with the general formula in claim 13. The definitions of Ab, m, group M, SP1, SP2, A, and D are the same.

16. The antibody-drug conjugate targeting CLDN1 or a salt thereof according to any one of claims 11 to 15, characterized in that, The antibody-drug conjugate or its salt has a structure as shown in structural formulas Ic and / or Id: and / or In structural formulas Ic and / or Id, Ab, m, groups A and D correspond to the general formula in claim 13. The definitions of Ab, m, and groups A and D are the same.

17. The antibody-drug conjugate targeting CLDN1 or a salt thereof according to any one of claims 11 to 16, characterized in that, The antibody-drug conjugate or its salt has the following structure: (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) 18. A composition comprising an anti-CLDN1 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 6, a nucleic acid molecule as claimed in claim 7, a vector as claimed in claim 8, a host cell as claimed in claim 9, or an antibody-drug conjugate targeting CLDN1 or a salt thereof as claimed in any one of claims 11 to 17, and optionally pharmaceutically acceptable excipients.

19. Use of the anti-CLDN1 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6, the nucleic acid molecule of claim 7, the vector of claim 8, the host cell of claim 9, the antibody-drug conjugate targeting CLDN1 as described in any one of claims 11 to 17 or a salt thereof, or the composition of claim 18 in the preparation of a medicament for the prevention, treatment and / or improvement of a disease; Preferably, the disease is associated with CLDN1 expression (including CLDN1 positivity, high or overexpression); Preferably, the disease is a solid tumor, such as a solid tumor that expresses CLDN1 positively; Preferably, the disease is liver cancer, head and neck squamous cell carcinoma, lung squamous cell carcinoma, thyroid cancer, ovarian cancer, colorectal cancer, breast cancer, stomach cancer, pancreatic cancer, or cervical cancer.

20. Use of the anti-CLDN1 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6, the nucleic acid molecule of claim 7, the vector of claim 8, the host cell of claim 9, the antibody-drug conjugate targeting CLDN1 of any one of claims 11 to 17 or a salt thereof, or the composition of claim 18 in the preparation of a reagent for diagnosing a disease; Preferably, the disease is associated with CLDN1 expression (including CLDN1 positivity, high or overexpression); Preferably, the disease is a solid tumor, such as a solid tumor that expresses CLDN1 positively; Preferably, the disease is liver cancer, head and neck squamous cell carcinoma, lung squamous cell carcinoma, thyroid cancer, ovarian cancer, colorectal cancer, breast cancer, stomach cancer, pancreatic cancer, or cervical cancer.

21. A kit comprising an anti-CLDN1 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 6, a nucleic acid molecule as claimed in claim 7, a vector as claimed in claim 8, a host cell as claimed in claim 9, an antibody-drug conjugate targeting CLDN1 as claimed in any one of claims 11 to 17, or a salt thereof, or a composition as claimed in claim 18.

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