Antibody or antigen-binding fragment thereof targeting claudin 1 and use thereof

Abstract

Disclosed in the present invention are an antibody or an antigen-binding fragment thereof targeting Claudin 1 and use thereof. The antibody or the antigen-binding fragment thereof targeting Claudin 1 exhibits good affinity for Claudin 1, has good endocytic activity in cells expressing Claudin 1, and thus has potential application value in the diagnosis and treatment of diseases mediated by Claudin 1.

Description

An antibody targeting Claudin 1 or its antigen-binding fragment and its applications

[0001] This application claims priority to Chinese patent application 2025103035458, filed on March 14, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to an antibody or its antigen-binding fragment targeting Claudin 1 and its applications. Background Technology

[0003] Claudin1 (CLDN1) is a four-transmembrane protein belonging to the Claudin protein family, encoded by the multi-gene family CLDN. Claudin proteins are expressed in all epithelial and endothelial cells. As crucial components of tight junctions, they are located at the apical and lateral membranes of epithelial cells, forming a continuous seal around the cell through self-aggregation and intercellular interactions, acting as a physical barrier to prevent solutes and water from freely passing through the extracellular space. These tight junctions prevent molecules from crossing between cells and maintain cell polarity; therefore, Claudin proteins play a vital role in regulating intercellular permeability and maintaining intercellular adhesion between epithelial and endothelial cell layers. In addition, CLDN1 also mediates cell adhesion, signal transduction, and epithelial-mesenchymal differentiation (EMT).

[0004] Claudin1 plays a double-edged sword role in various cancers, acting as both a tumor promoter and a tumor suppressor. In some cancers, decreased CLDN1 expression is associated with cancer progression and invasion, while in others, its absence may improve patient survival. Furthermore, the localization of CLDN1 in disease states (nuclear or cytoplasmic junctions) is a key focus, as its localization may be related to tumor invasiveness. In liver and digestive system cancers, upregulated CLDN1 exhibits non-connecting functions and may drive extracellular matrix remodeling, forming a dense collagen barrier around the tumor. This barrier helps protect the tumor from the immune system and immunotherapy, leading to poorer patient prognosis. Antibodies targeting CLDN1 exposed on tumors can coordinate cancer cell death and improve the contact between immune cells and immunotherapy through antibody-dependent cell-mediated cytotoxicity and by promoting collagen barrier disruption.

[0005] The relationship between Claudin1 and fibrotic diseases is a hot topic in medical research. Studies have shown that non-connected Claudin1 (njCLDN1) plays a crucial role in fibrosis. Using monoclonal antibodies targeting exposed njCLDN1, researchers found that Claudin1 is a mediator and target of liver fibrosis. Targeting CLDN1 can restore inflammation-induced pro-fibrotic signaling and cell fate in hepatocytes and inhibit myofibroblast differentiation in hepatic stellate cells. Further research indicates that Claudin1 has anti-fibrotic effects in lung and kidney fibrosis models, suggesting its potential role as a therapeutic target for fibrosis across organs and tissues.

[0006] Although some anti-Claudin1 monoclonal antibodies are already in clinical use, the development of more anti-Claudin1 monoclonal antibodies is still urgently needed to enhance their specificity and function. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides an antibody targeting Claudin 1 or its antigen-binding fragment and its applications. The antibody targeting Claudin 1 or its antigen-binding fragment provided by this invention exhibits strong specific binding ability to Claudin 1, while showing weak non-specific binding ability to Claudin 7 or Claudin 19. The antibody targeting Claudin 1 or its antigen-binding fragment possesses good specific binding and internalization capabilities, characteristics that make it potentially valuable in the diagnosis and treatment of Claudin 1-mediated diseases.

[0008] Specifically, the first aspect of the present invention provides an antibody or antigen-binding fragment thereof targeting Claudin 1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3.

[0009] The HCDR1 contains the amino acid sequence shown in SEQ ID NO:47.

[0010] The HCDR2 contains the amino acid sequence shown in SEQ ID NO:48.

[0011] The HCDR3 contains the amino acid sequence EKY;

[0012] The LCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0013] The LCDR2 comprises the amino acid sequence shown in SEQ ID NO:49, and,

[0014] The LCDR3 contains an amino acid sequence as shown in SEQ ID NO:3.

[0015] In some embodiments of the present invention, the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and EKY, respectively, and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; or,

[0016] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:7, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:8, and SEQ ID NO:6, respectively; or...

[0017] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:9, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:6, respectively; or...

[0018] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:9, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:11, and SEQ ID NO:6, respectively; or...

[0019] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:12, and SEQ ID NO:6, respectively; or...

[0020] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:13, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:14, and SEQ ID NO:6, respectively; or...

[0021] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:15, SEQ ID NO:2, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:16, and SEQ ID NO:6, respectively; or...

[0022] The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:17, and SEQ ID NO:6, respectively; or...

[0023] The heavy chain variable region contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and EKY, respectively, and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:18, respectively.

[0024] In some preferred embodiments of the present invention, the heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:7, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:8, and SEQ ID NO:6, respectively; or,

[0025] The heavy chain variable region contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:9, and EKY, respectively, and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:6, respectively.

[0026] In some embodiments, LCDR1, LCDR2, and LCDR3 are LCDR1, LCDR2, and LCDR3 in the light chain variable regions as shown in any of SEQ ID NO:24-32, wherein the CDR is defined according to the Kabat, IMGT, Combined, Chothia, or AbM numbering system.

[0027] In some embodiments, HCDR1, HCDR2 and HCDR3 are respectively HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in any of SEQ ID NO:19,20,21,22 or23, wherein the CDR is defined according to the Kabat, IMGT, Combined, Chothia or AbM numbering system.

[0028] In some embodiments of the present invention, the antibody or its antigen-binding fragment is selected from at least one of the following:

[0029] (1) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:19; and / or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:24; or,

[0030] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:20; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:25; or,

[0031] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:21; and / or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:26; or,

[0032] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:21; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:27; or,

[0033] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:19; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:28; or,

[0034] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:22; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:29; or,

[0035] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:23; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:30; or,

[0036] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:19; and / or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:31; or,

[0037] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:19; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:32; or,

[0038] (2) The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:19; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:24; or,

[0039] The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:20; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:25; or,

[0040] The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:21; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:26; or,

[0041] The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:21; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:27; or,

[0042] The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:19; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:28; or,

[0043] The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:22; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:29; or,

[0044] The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:23; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:30; or,

[0045] The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:19; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:31; or,

[0046] The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:19; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:32.

[0047] In some preferred embodiments of the present invention, the amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity does not involve changes to the CDR sequence.

[0048] In some embodiments of the present invention, the antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0049] In some embodiments of the present invention, the antibody or its antigen-binding fragment is selected from one or more of the following:

[0050] (1) Fab, Fab', F(ab')2, Fv, scFv or sdAb;

[0051] (2) Monoclonal or polyclonal antibodies;

[0052] (3) Monospecific antibodies, bispecific antibodies, or multispecific antibodies;

[0053] (4) Fully human antibodies, humanized antibodies, or chimeric antibodies;

[0054] (5) Antibody prodrug.

[0055] In some preferred embodiments of the present invention, the antibody or its antigen-binding fragment is a full-length antibody.

[0056] In some preferred embodiments of the present invention, the antibody or its antigen-binding fragment includes at least one constant region selected from human IgG1, IgG2, IgG3, IgG4, IgA1, IgA, IgM, IgD and IgE and variants thereof.

[0057] In some embodiments of the present invention, the antibody or its antigen-binding fragment is selected from any of the following:

[0058] (I) The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:38; or,

[0059] The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:34; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:39; or,

[0060] The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:35; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:40; or,

[0061] The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:35; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:41; or,

[0062] The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:42; or,

[0063] The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:36; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:43; or,

[0064] The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:37; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:44; or,

[0065] The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:45; or,

[0066] The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:46; or,

[0067] (II) The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:38; or,

[0068] The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:34; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:39; or,

[0069] The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:35; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:40; or,

[0070] The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:35; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:41; or,

[0071] The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:42; or,

[0072] The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:36; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:43; or,

[0073] The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:37; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:44; or,

[0074] The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:45; or,

[0075] The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:46.

[0076] In some preferred embodiments of the present invention, the amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity does not involve changes to the CDR sequence.

[0077] A second aspect of the present invention provides an isolated nucleic acid that encodes an antibody or an antigen-binding fragment thereof as described in the first aspect.

[0078] A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the second aspect.

[0079] In some embodiments of the present invention, the recombinant expression vector is selected from viral vectors and non-viral vectors.

[0080] In some preferred embodiments of the present invention, the non-viral vector is selected from plasmids, linear DNA fragments, and RNA.

[0081] In some preferred embodiments of the present invention, the recombinant expression vector is a plasmid.

[0082] In some specific embodiments of the present invention, the backbone plasmid of the recombinant expression vector is the pLVX-puro plasmid.

[0083] A fourth aspect of the present invention provides a transformant comprising a nucleic acid as described in the second aspect or a recombinant expression vector as described in the third aspect, wherein the transformant is a non-animal or non-plant variety.

[0084] In some embodiments of the present invention, the transformant is a eukaryotic cell.

[0085] In some preferred embodiments of the present invention, the eukaryotic cells are mammalian cells.

[0086] In some specific embodiments of the present invention, the mammalian cells are CHO cells, 293T cells, or derived cells thereof.

[0087] The fifth aspect of the present invention provides a method for preparing an antibody against Claudin1 or an antigen-binding fragment thereof, the method comprising culturing a transformant as described in the fourth aspect and obtaining the antibody or an antigen-binding fragment thereof from the culture.

[0088] A sixth aspect of the present invention provides a chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof as described in the first aspect.

[0089] In some embodiments of the present invention, the chimeric antigen receptor further includes a co-stimulatory domain and a signal transduction domain.

[0090] A seventh aspect of the present invention provides a genetically modified cell that expresses the chimeric antigen receptor as described in the sixth aspect.

[0091] In some embodiments of the present invention, the genetically modified cells are T cells or NK cells.

[0092] The eighth aspect of the present invention provides an antibody-drug conjugate comprising a cytotoxic agent or tag, and an antibody or an antigen-binding fragment thereof as described in the first aspect.

[0093] A ninth aspect of the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in the first aspect, a nucleic acid as described in the second aspect, a recombinant expression vector as described in the third aspect, a transformant as described in the fourth aspect, a genetically modified cell as described in the seventh aspect, or an antibody-drug conjugate as described in the eighth aspect, and a pharmaceutically acceptable carrier and / or excipients.

[0094] The tenth aspect of the present invention provides a method for detecting Claudin1, which includes the step of contacting a sample to be tested with an antibody or an antigen-binding fragment thereof as described in the first aspect.

[0095] In some embodiments of the present invention, the detection is for non-diagnostic and / or therapeutic purposes.

[0096] The eleventh aspect of the present invention provides the use of an antibody or antigen-binding fragment thereof as described in the first aspect, a genetically modified cell as described in the seventh aspect, an antibody-drug conjugate as described in the eighth aspect, or a pharmaceutical composition as described in the ninth aspect in the preparation of a medicament for treating and / or preventing Claudin1-mediated diseases.

[0097] In some embodiments of the present invention, the disease is cancer.

[0098] In some specific embodiments of the present invention, the cancer is selected from one or more of liver cancer, breast cancer, colorectal cancer, and ovarian cancer.

[0099] The twelfth aspect of the present invention provides a method for treating and / or preventing Claudin1-mediated diseases or conditions, the method comprising administering to a subject in need a therapeutic or preventative amount of an antibody or antigen-binding fragment thereof as described in the first aspect, a genetically modified cell as described in the seventh aspect, an antibody-drug conjugate as described in the eighth aspect, or a pharmaceutical composition as described in the ninth aspect.

[0100] In some embodiments of the present invention, the disease is cancer.

[0101] In some specific embodiments of the present invention, the cancer is selected from one or more of liver cancer, breast cancer, colorectal cancer, and ovarian cancer.

[0102] The thirteenth aspect of the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described in the first aspect, a nucleic acid as described in the second aspect, a recombinant expression vector as described in the third aspect, a transformant as described in the fourth aspect, a gene-modified cell as described in the seventh aspect, an antibody-drug conjugate as described in the eighth aspect, or a pharmaceutical composition as described in the ninth aspect.

[0103] The fourteenth aspect of the present invention provides an antibody or antigen-binding fragment thereof as described in the first aspect, a genetically modified cell as described in the seventh aspect, an antibody-drug conjugate as described in the eighth aspect, or a pharmaceutical composition as described in the ninth aspect, for the treatment and / or prevention of Claudin1-mediated diseases.

[0104] In some embodiments of the present invention, the disease is cancer.

[0105] In some specific embodiments of the present invention, the cancer is selected from one or more of liver cancer, breast cancer, colorectal cancer, and ovarian cancer.

[0106] Unless otherwise stated, the present invention will be implemented using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, all of which are within the scope of the art.

[0107] To facilitate a better understanding of this invention, certain technical terms are specifically defined as follows. Unless otherwise expressly defined elsewhere in this document, the technical terms or expressions used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0108] For definitions and terminology in this field, those skilled in the art may refer at least in part to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues follow the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids. Singular forms used herein (including the claims) include their corresponding plural forms unless otherwise expressly specified herein.

[0109] The term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as these variations are suitable for carrying out the disclosed methods.

[0110] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0111] As used herein, the term “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted inclusively, that is, including at least one in the quantity or element list, but also including more than one, and optionally, additional unlisted items. Only when explicitly indicated by the opposite terms, such as “only one” or “exactly one” or when “consisting of” is used in the claims, will it refer to only one number or one element of the list.

[0112] Unless the context clearly indicates otherwise, the words “one” and “a” should be understood as “at least one” as used in this article.

[0113] The term "antibody" refers to any form of antibody that has the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camel-derived single-domain antibodies.

[0114] The term "isolated antibody" refers to the purified state of a binding compound, and in this context, it means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth media. The term "isolated" does not mean the complete absence of such substances or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic application of the binding compound described herein.

[0115] The term "monoclonal antibody" refers to an antibody derived from a basic homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations, which may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise a large number of antibodies targeting different epitopes (or specific to different epitopes). The modifier "monoclonal" indicates the characteristic of antibodies derived from a basic homogeneous group of antibodies and should not be construed as requiring the production of antibodies through any particular method.

[0116] The term "full-length antibody" refers to an immunoglobulin molecule that, in its natural state, contains at least four peptide chains: two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region (abbreviated as CH in this document). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this document) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into highly variable complementarity-determining regions (CDRs) and regions separated by more conserved regions called framework regions (FRs). Each VH or VL region consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region 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.

[0117] The term "antigen-binding fragment" of an antibody ("parental antibody") includes a fragment or derivative of an antibody, typically comprising at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments well known in the art; biantibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments; and multispecific antibodies. In some preferred embodiments of the invention, the antigen-binding fragments of the invention are selected from Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments; and multispecific antibodies. When antigen-binding activity is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative maintains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or higher of the antigen-binding affinity of the parent antibody. It is also anticipated that the antigen-binding fragment of the antibody may include conserved or non-conserved amino acid substitutions that do not significantly alter its biological activity (referred to as “conserved variants” or “functionally conserved variants” of the antibody). The term “binding compound” refers to both the antibody and its binding fragment.

[0118] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment containing both VH and VL domains, which are located within a single polypeptide chain. Fv polypeptides typically also include a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired structure for antigen binding.

[0119] The term "domain antibody" refers to an immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0120] The term "fully human antibody" refers to an antibody that contains only the sequence of human immunoglobulin proteins. If produced in mice, in mouse cells, or in hybridomas derived from mouse cells, a fully human antibody may contain mouse glycans. Similarly, a "mouse antibody" refers to an antibody that contains only the sequence of mouse immunoglobulins. Alternatively, if produced in rats, in rat cells, or in hybridomas derived from rat cells, a fully human antibody may contain rat glycans. Likewise, a "rat antibody" refers to an antibody that contains only the sequence of rat immunoglobulins.

[0121] The term "Fc region" is used in this document to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is based on the EU numbering system, also known as the EU index, e.g., Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0122] The term "antibody probody" refers to a conditionally activated antibody that has undergone reversible modification to its antigen-binding region through genetic engineering. Antibody probody retains the core structure of the antibody but adds a masking peptide to conceal the target active region, allowing the antibody to remain inactive in normal tissues and only be activated by specific enzymes or chemical signals in the disease microenvironment (such as tumors). The structure of an antibody probody typically includes the antibody, a masking peptide linked to the N-terminus of the antibody light chain, and a linker that can be hydrolyzed by enzymes.

[0123] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes are typically composed of various chemically active surface molecules such as amino acid or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational and non-conformational epitopes lies in the fact that, in the presence of denaturing solvents, the former loses binding, rather than the latter.

[0124] The term "affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. In some embodiments of the invention, affinity is measured using surface plasmon resonance (SPR) techniques, such as the affinity between the antibody and antigen of the present invention. In some preferred embodiments of the invention, a specific method for measuring affinity is the BIAcore method described herein.

[0125] The terms “nucleic acid,” “polynucleotide,” “nucleic acid molecule,” and “polynucleotide molecule” are used interchangeably herein (unless the context otherwise indicates) and refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0126] The term "construct" refers to any recombinant polynucleotide molecule (such as plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecules) derived from any source, capable of integration into the genome or autonomous replication, containing one or more polynucleotide molecules that are functionally linked (i.e., operably linked). In some preferred embodiments of the invention, the recombinant construct comprises polynucleotides of the invention operably linked to transcription initiation regulatory sequences that drive and / or direct transcription of the polynucleotides of the invention in a host cell. Expression of the polynucleotides of the invention can be driven and / or directed using both heterologous and non-heterologous (i.e., endogenous) promoters.

[0127] The term "vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introducing heterologous DNA into host cells). One type of vector is the "plasmid," which is a circular double-stranded DNA loop into which an additional DNA segment can be ligated. Another type of vector is the viral vector, in which an additional DNA segment can be ligated into the viral genome. Some vectors can replicate autonomously in the host cells they are introduced into (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian vectors). After introduction into the host cell, other vectors (e.g., non-free-living mammalian vectors) integrate into the host cell's genome and thus replicate along with the host genome. Furthermore, some vectors can guide the expression of operatively linked genes. These types of vectors are referred to herein as "expression vectors."

[0128] As used herein, the term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a target gene upon transformation, transfection, or transduction into host cells. An expression vector contains one or more phenotypic selection markers and origins of replication to ensure vector maintenance and to provide amplification within the host when needed. In some preferred embodiments of the invention, the expression vector comprises the constructs of the invention and / or the polynucleotides of the invention.

[0129] As used herein, the term "CLAUDIN 1-mediated disease" refers to a disease in which CLAUDIN 1 is involved in the onset, progression, or prolongation of the disease, such as involving the activation (e.g., aberrant or hyperactivation) of CLAUDIN 1, and where the involvement of CLAUDIN 1 directly or indirectly produces at least one of the following effects: disease initiation / occurrence; increased / deeper pathological changes in the disease; expanded site / range of disease impact; aggravated bodily damage caused by the disease; increased pain caused by the disease; insensitivity / resistance to treatment; decreased tendency for spontaneous remission; and worsened prognosis. In some embodiments of the invention, the CLAUDIN 1-mediated disease is an inflammatory disease; in some specific embodiments of the invention, the CLAUDIN 1-mediated disease is liver cancer, breast cancer, colorectal cancer, and ovarian cancer.

[0130] As used herein, the term “treatment” or “cure” for any disease or condition, in one embodiment, means improving the disease or condition (i.e., slowing or halting or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, “treatment” or “cure” means alleviating or improving at least one bodily parameter, including those physical parameters that may not be identifiable by the patient. In yet another embodiment, “treatment” or “cure” means regulating the disease or condition physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of bodily parameters), or in both ways. Unless explicitly described herein, methods for assessing the treatment and / or prevention of disease are generally known in the art.

[0131] The term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. As used herein, the term “cyno” or “cyno-eating macaque” refers to or is derived from the cyno-eating macaque.

[0132] The term "combination" refers to the administration of one or more other therapeutic agents, including simultaneous (co-) administration and sequential administration in any order.

[0133] The terms "therapeutic effective amount," "therapeutic effective dose," and "effective amount" refer to the amount of the CLAUDIN 1 antibody or its antigen-binding fragment thereof, when administered alone or in combination with other therapeutic agents to cells, tissues, or subjects, that effectively prevents or improves the symptoms of one or more diseases or conditions, or the development of such diseases or conditions. Therapeutic effective dose also refers to an amount of antibody or its antigen-binding fragment sufficient to cause symptom improvement, such as the amount that treats, cures, prevents, or improves the associated medical condition, or accelerates the treatment, cure, prevention, or improvement of such condition. When administered to an individual as a single active ingredient, the therapeutic effective dose refers only to that ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that cause the therapeutic effect, whether administered in combination, sequentially, or simultaneously. The effective amount of the therapeutic agent will result in an increase of at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% in diagnostic criteria or parameters.

[0134] The term "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation or composition that is non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0135] Anti-CLAUDIN 1 antibody

[0136] In one aspect, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to CLAUDIN 1. The terms "anti-CLAUDIN 1 antibody," "anti-CLAUDIN 1," "CLAUDIN 1 antibody," "antibody targeting Claudin 1," or "antibody binding to CLAUDIN 1" refer to antibodies capable of binding to the CLAUDIN 1 protein or fragments thereof with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting CLAUDIN 1. Unless otherwise specified, "CLDN1," "CLAUDIN 1," and "Claudin 1" are used interchangeably, and capitalization is not important.

[0137] The antibodies of the present invention can be generated using any suitable method for antibody production. Any suitable form of CLAUDIN 1 can be used as an immunogen (antigen) for antibody production. By way of example and not limitation, any variant of CLAUDIN 1 or a fragment thereof can be used as an immunogen.

[0138] In some implementations, the generated anti-CLAUDIN 1 antibody can be produced by screening a phage display library.

[0139] In some embodiments, the amino acid sequences of the light and heavy chain variable regions and CDRs of the anti-CLAUDIN 1 antibody of the present invention are shown in Table 6. The light and heavy chain variable regions of the above antibody are formed by combining the human clone heavy chain constant region Fc and light chain constant region κ with each heavy chain variable region and light chain variable region, respectively, to form complete antibody heavy and light chains.

[0140] The precise amino acid sequence boundary of the variable region CDR of the antibody of the present invention can be determined using any of many well-known schemes, including those based on the three-dimensional structure of the antibody and the topology of the CDR loop, such as Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), and those based on antibody sequence variability, such as Kabat et al. (Sequences of Proteins of Immunological Interest, 4th edition, USDapartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), and the International ImMunoGeneTics database (IMGT) (1999 Nucleic Acids). Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering of a large number of crystal structures. Example methods for defining antibody CDRs in this invention are shown in Table 1.

[0141] Table 1: Example Method for Defining Antibody CDRs in this Invention

[0142] Here, Haa-Hbb can refer to the amino acid sequence from position aa (Kabat coding rule) to position bb (Chothia coding rule) starting from the N-terminus of the antibody heavy chain. For example, H31-H35 can refer to the amino acid sequence from position 31 to position 35 starting from the N-terminus of the antibody heavy chain, according to the Kabat coding rule.

[0143] Unless otherwise stated, the CDR of the antibodies of the present invention can be determined by those skilled in the art according to any scheme in the art (e.g., different assignment systems or combinations).

[0144] It should be noted that the boundaries of the CDRs of the variable region of the same antibody may differ based on different assignment systems. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems may differ. Therefore, when referring to antibodies defined with the specific CDR sequence of this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries defined by this invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0145] Antibodies with different specificities (i.e., different binding sites against different antigens) have different core binding receptors (CDRs). However, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Minimal overlapping regions can be determined using at least two of the Kabat, Chothia, AbM, Contact, and North methods, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining CDR sequence can be determined through the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be substituted with conserved amino acid residues.

[0146] In some implementations, anti-CLAUDIN1 antibodies are obtained by engineering using standard molecular biology techniques.

[0147] In some embodiments, amino acid changes include amino acid deletions, insertions, or substitutions. In some embodiments, the anti-CLAUDIN 1 antibody or its antigen-binding fragment of the present invention comprises those antibodies having amino acid sequences that have been mutated by amino acid deletions, insertions, or substitutions, but still possess at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the antibodies described above (particularly in the CDR region depicted in the above sequence). In some embodiments, when the antibody of the present invention is compared with the CDR region depicted in the specific sequence, the number of amino acid mutations in the CDR region that have been mutated by amino acid deletions, insertions, or substitutions does not exceed 1, 2, 3, 4, or 5. In some embodiments, when the antibody of the present invention is compared with the frame region in the specific sequence, the number of amino acid mutations in the frame region that have been mutated by amino acid deletions, insertions, or substitutions does not exceed 1, 2, 3, 4, or 5.

[0148] The term "percentage (%) amino acid sequence identity," or simply "identity," is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0149] In some embodiments, the full-length antibody disclosed in this invention includes a light chain variable region and a heavy chain variable region. Those skilled in the art can also select light chain constant regions and heavy chain constant regions from different antibody sources according to actual needs, such as light chain constant regions (or their conventional variants) and heavy chain constant regions (or their conventional variants) derived from human antibodies. Furthermore, the combinations of light chain variable regions and heavy chain variable regions in Table 6 can form single-chain antibodies (scFv), Fab, or other antigen-binding fragments containing scFv or Fab.

[0150] In some embodiments, the CLAUDIN 1 antibody is an IgG antibody, such as IgG1, IgG2, IgG3 or IgG4 antibody or a modified form thereof, as described in the following sections.

[0151] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to produce an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0152] In some implementations, it may be necessary to produce cysteine-engineered antibodies, such as "thioMAb", in which one or more residues of the antibody are replaced with cysteine ​​residues.

[0153] In some embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0154] antibody expression

[0155] In another aspect, the present invention provides a polynucleotide molecule encoding the anti-CLAUDIN 1 antibody or its antigen-binding fragment described herein. The polynucleotide molecule may comprise a polynucleotide molecule containing an amino acid sequence encoding the light chain variable region and / or the heavy chain variable region of the antibody, or a polynucleotide molecule containing an amino acid sequence encoding the light chain and / or the heavy chain of the antibody.

[0156] In some embodiments, the polynucleotide molecule encoding the antibody of the present invention includes a polynucleotide molecule that has been mutated by nucleotide deletion, insertion or substitution, but still has at least about 60%, 70%, 80%, 90%, 95% or 100% identity with the coding region corresponding to the CDR depicted in the sequence described above.

[0157] In another aspect, the present invention provides an expression vector comprising a polynucleotide molecule as described herein, preferably a eukaryotic expression vector. In some embodiments, the polynucleotide molecule as described herein is contained in one or more expression vectors.

[0158] In another aspect, the present invention provides a host cell comprising a polynucleotide molecule as described herein or an expression vector as described herein, preferably a eukaryotic cell, more preferably a mammalian cell.

[0159] In another aspect, the present invention provides a method for preparing an anti-CLAUDIN 1 antibody or an antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or the antigen-binding fragment thereof in a host cell as described herein under conditions suitable for expression of the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the host cell.

[0160] This invention provides mammalian host cells for expressing the recombinant antibodies of this invention, including a variety of immortalized cell lines available from the American Type Culture Collection (ATCC). These particularly include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells, and many other cell lines. Mammal host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines exhibit high expression levels.

[0161] In one embodiment, the present invention provides a method for preparing an anti-CLAUDIN 1 antibody, wherein the method includes, when an expression vector is introduced into a mammalian host cell, producing the antibody by culturing the host cell for a sufficient period of time to allow the antibody to be expressed in the host cell, or more preferably by secreting the antibody into a culture medium in which the host cell grows.

[0162] Antibodies can be recovered from the culture medium using standard protein purification methods. The antibody molecules prepared as described herein can be purified using known existing techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the antibody molecules of this invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and HPLC.

[0163] Antibodies expressed in different cell lines or in transgenic animals may have different glycosylations. However, all antibodies encoded by the nucleic acid molecules provided herein or containing the amino acid sequences provided herein are part of the invention, regardless of their glycosylation. Similarly, in some embodiments, non-fucosylated antibodies are advantageous because they generally have stronger efficacy in vitro and in vivo than their fucosylated counterparts and are unlikely to be immunogenic because their sugar structure is a normal component of native human serum IgG.

[0164] Pharmaceutical compositions and pharmaceutical preparations

[0165] In another aspect, the present invention provides a pharmaceutical composition comprising an anti-CLAUDIN 1 antibody or an antigen-binding fragment thereof as described in the present invention, a polynucleotide molecule as described in the present invention, an expression vector as described in the present invention, a transformant as described in the present invention, a genetically modified cell as described in the present invention, or an antibody-drug conjugate as described in the present invention, and a pharmaceutically acceptable carrier or excipient. It should be understood that the anti-CLAUDIN 1 antibody or pharmaceutical composition thereof provided by the present invention can integrate suitable carriers, excipients, and other reagents in a formulation for co-administration, thereby providing improved transfer, delivery, and tolerability, etc.

[0166] The term "pharmaceutical composition" refers to a formulation that allows the biologically effective form of the active ingredient contained therein to be present, and does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the formulation.

[0167] Pharmaceutical formulations containing the anti-CLAUDIN 1 antibody described herein can be prepared by mixing the anti-CLAUDIN 1 antibody of the present invention, having the desired purity, with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. editor (1980)). Preferably, the formulation is in the form of an aqueous solution or a lyophilized preparation.

[0168] The pharmaceutical compositions or formulations of the present invention may further comprise one or more other active ingredients required for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. In some embodiments, the other active ingredients are immune checkpoint inhibitors or growth inhibitors, which are suitably combined in amounts effective for the intended use. In some embodiments, the pharmaceutical compositions of the present invention further comprise a composition encoding a polynucleotide molecule of an anti-CLAUDIN 1 antibody.

[0169] In another aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0170] In another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein.

[0171] Medical uses and treatment methods

[0172] Any anti-CLAUDIN 1 antibody or corresponding immunoconjugate provided herein may be used for therapeutic purposes. It should also be understood that, when discussing "antibody," compositions containing antibodies are also included. The anti-CLAUDIN 1 antibody of the present invention may be used in therapeutic or preventative amounts in any embodiment of the therapeutic or preventative method described herein.

[0173] In another aspect, the present invention provides the use of the antibodies or antigen-binding fragments thereof described herein, the pharmaceutical compositions described herein, or the pharmaceutical combinations described herein in the preparation of medicaments for treating and / or preventing CLAUDIN 1-mediated diseases, preferably cancer or tumors; more preferably, the diseases are selected from liver cancer, breast cancer, colorectal cancer, and ovarian cancer.

[0174] In another aspect, the present invention provides antibodies or antigen-binding fragments thereof described herein, pharmaceutical compositions or combinations thereof described herein, for the treatment and / or prevention of CLAUDIN 1-mediated diseases, preferably cancer or tumors; more preferably, the diseases are selected from liver cancer, breast cancer, colorectal cancer and ovarian cancer.

[0175] In another aspect, the present invention provides a method for treating and / or preventing CLAUDIN 1-mediated diseases or conditions, comprising administering to a subject in need a therapeutically or preventively effective amount of an antibody or antigen-binding fragment thereof described herein, a pharmaceutical composition described herein, or a combination of pharmaceuticals described herein, preferably the disease being cancer or a tumor; more preferably, the disease being selected from liver cancer, breast cancer, colorectal cancer, and ovarian cancer.

[0176] In some embodiments, the administration methods of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intra-articular, intra-articular (e.g., in arthritic joints), inhalation, aerosol delivery, or local administration to the lesion.

[0177] The term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual undergoing treatment. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any adverse feelings or direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. For the antibodies of this invention, the ability to reduce the severity of the disease in one or more ways, i.e., to exert a therapeutic effect, is manifested in one or more of the following: increased patient lifespan (survival); delayed disease progression; and reduced need for medical care.

[0178] The present invention also provides for the combined administration of a therapeutically effective amount of one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to a subject. The antibodies of the present invention can be used alone or in combination with other therapeutic agents in a therapy. In some embodiments, the antibodies of the present invention are co-administered with at least one additional therapeutic agent.

[0179] Methods for diagnosis and detection

[0180] In another aspect, the present invention provides a method for detecting the presence of CLAUDIN 1 in a sample using an antibody or antigen-binding fragment thereof described herein. The term "detection" as used herein includes quantitative or qualitative detection. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In some embodiments, the biological sample comprises cells or tissue. In some embodiments, CLAUDIN 1 is human CLAUDIN 1 or cynomolgus monkey CLAUDIN 1. The method includes the steps of contacting the sample with an antibody or antigen-binding fragment thereof described herein or a detection composition containing the antibody or antigen-binding fragment thereof, and detecting the presence of a conjugate or binding signal formed by the binding of the antibody or antigen-binding fragment thereof to CLAUDIN 1. When used for detection purposes, the antibody or antigen-binding fragment thereof described herein may be labeled to indicate whether the conjugate has been formed. In some embodiments, the method may be an in vitro or in vivo method.

[0181] In some embodiments, CLAUDIN 1 is detected before treatment, for example, before initiating treatment or before a treatment interval. In one embodiment, an anti-CLAUDIN 1 antibody or an antigen-binding fragment thereof is provided for diagnostic or detection methods.

[0182] This invention includes all combinations of the specific embodiments described. Further embodiments of the invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the invention, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. For all purposes, all disclosures, patents, and patent applications cited herein, including in citations, are incorporated herein by reference in their entirety. The compounds of this invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other methods, and equivalent substitutions well known to those skilled in the art, with preferred embodiments including but not limited to the embodiments of this invention.

[0183] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0184] The reagents and raw materials used in this invention are all commercially available.

[0185] The positive and progressive effects of this invention are as follows: the antibody or its antigen-binding fragment targeting Claudin 1 provided by this invention has good specific binding ability and internalization ability. Its internalization ability is comparable to or better than that of the control molecule, making it potentially valuable in the diagnosis and treatment of Claudin 1-mediated diseases. Attached Figure Description

[0186] Figure 1 shows the FASC flow cytometry identification of human CLDN1-HEK293 cells.

[0187] Figure 2 shows the FASC flow cytometry identification of monkey CLDN1-HEK293 cells.

[0188] Figure 3 shows the FASC flow cytometry identification of human CLDN7-HEK293 cells.

[0189] Figure 4 shows the FASC flow cytometry identification of human CLDN19-HEK293 cells.

[0190] Figure 5 shows the endocytic activity of the non-specifically modified antibody in huCLDN1-HEK293, huCLDN7-HEK293, huCLDN19-HEK293 and Huh-7 cells. Detailed Implementation

[0191] Other aspects and advantages of this application will readily become apparent to those skilled in the art from the detailed description below. The detailed description below shows and describes only exemplary embodiments of this application. As those skilled in the art will recognize, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the concept and scope of the invention to which this application pertains. Accordingly, the descriptions in this application are merely exemplary and not restrictive.

[0192] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0193] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0194] The control antibody ALE-F02 used in this invention has a sequence derived from WO2017162678A1, with the light chain sequence being SEQ ID NO:50 and the heavy chain sequence being SEQ ID NO:51. It was produced using conventional recombinant antibody production processes.

[0195] Light chain:

[0196] Heavy chain:

[0197] The control antibody 6F6C3 used in this invention has a sequence derived from US9931416B2, with the light chain sequence being SEQ ID NO:52 and the heavy chain sequence being SEQ ID NO:53. It was produced using conventional recombinant antibody production processes.

[0198] Light chain:

[0199] Heavy chain:

[0200] Note: The underlined and bolded parts of the above sequence represent the CDR area, numbered according to the Kabat rule.

[0201] Example 1: Construction of Claudin1, Claudin7, and Claudin19 overexpression cell lines

[0202] 1.1 Construction of HEK293 overexpression cell lines (hereinafter referred to as huCLDN1-HEK293, cynoCLDN1-HEK293, huCLDN7-HEK293, and huCLDN19-HEK293) expressing human Claudin1, monkey Claudin1, and their related human Claudin7 and human Claudin19:

[0203] The coding nucleic acid sequences of full-length human Claudin1 (Uniprot ID: O95832), monkey Claudin1 (Uniprot ID: A0A2K5WWF3), human Claudin7 (Uniprot ID: O95471), and human Claudin19 (Uniprot ID: Q8N6F1) were constructed into the pLVX-puro plasmid (purchased from Clontech, catalog number: Cat#632164). The resulting plasmids were then electroporated into HEK293 cells using an electroporator (purchased from Invitrogen, Neon™ Transfection System, catalog number: MP922947). CRL-1573 TMAfter electroporation, the resulting cells were transferred to DMEM medium (Gibco, catalog number 11995065) containing 10% FBS (purchased from Gibco, catalog number 15140-141) and free of antibiotics. The cells were then cultured in 10×10 cm cell culture dishes for 48 hours, followed by incubation at an average rate of 10... 4 Cells were aliquoted into 96-well cell culture plates at a density of 1 cell / well, and puromycin was added to a final concentration of 2 μg / mL as a selection pressure. Cell lines that formed clones were picked for identification after about 2 weeks.

[0204] 1.2 Flow cytometry identification of huCLDN1-HEK293 and cynoCLDN1-HEK293 cells:

[0205] The cells in logarithmic growth phase were digested and plated into 96-well plates. After washing with FACS buffer (1×PBS buffer containing 2% FBS), the cells were incubated at 4°C for 30 min with serially diluted primary antibodies (positive control antibodies ALE-F02 and 6F6C3) in PBS. After washing, the prepared fluorescent secondary antibody anti-human IgG Fc (purchased from Abcam, catalog number: 98596) was added and incubated at 4°C for 30 min. Finally, the cells were detected by flow cytometry (purchased from Beckman, catalog number: CytoFLEXAOO-1-1102). The experimental results are shown in Figures 1 and 2. The results showed that the huCLDN1-HEK293 cell line with high expression of human CLDN1 and the cynoCLDN1-HEK293 cell line with monkey CLDN1 were obtained.

[0206] 1.3 Flow cytometry identification of huCLDN7-HEK293 and huCLDN19-HEK293 cells from the same family:

[0207] The cells in logarithmic growth phase were digested and plated into 96-well plates. After washing with FACS buffer (1×PBS buffer containing 2% FBS), the cells were fixed and permeabilized according to the instructions of the cell permeabilization kit (eBioscience, catalog number: 88-8824-00). Then, APC anti-DYKDDDDK Tag (Biolegend, catalog number: 637308) with serially diluted PBS was added, and the cells were incubated at 4°C for 30 min. Finally, the cells were analyzed by flow cytometry (Beckman, catalog number: CytoFLEXAOO-1-1102). The experimental results are shown in Figures 3 and 4. The results showed that the huCLDN7-HEK293 cell line with high expression of human CLDN7 and the huCLDN19-HEK293 cell line with high expression of human CLDN19 were obtained.

[0208] Example 2: Obtaining Anti-Claudin 1 Antibody

[0209] Immunotube assay and cell screening were used to select phage display antibody gene libraries. Both immunotube assay and cell screening aim to enrich specific antibodies against the antigens, serving as two complementary and validation methods. A library of hundreds of billions of human antibodies was used, employing a positive cross-selection process using both immunotube solid-phase assay and cell screening. Negative selection (using Claudin7 / 19 cells overexpressing HEK293) was incorporated during the screening process. After multiple rounds of selection, positive clones specifically binding to human and monkey Claudin1 were obtained. The specific steps are as follows:

[0210] Immunotube solid-phase screening: The antigen protein hu-Claudin 1-VLP (purchased from ACRO, catalog number: CL1-H52P6) was coated on the surface of an immunotube with high adsorption capacity. A phage display antibody library was added to the immunotube, and the antigen protein adsorbed on the immunotube surface was incubated, washed, and eluted. This process involved four rounds of panning to ultimately enrich Fab monoclonal antibodies specific to the antigen. In this example, after four rounds of panning, Fab monoclonal antibodies against human Claudin 1 were enriched. The specific method is described below. In the first round of screening, 1 mL of 100 μg / mL hu-Claudin 1-VLP protein was added to the immunotherapy tube and incubated overnight at 4°C. The coating solution was discarded the next day, and the tube was blocked with 5% milk PBS for 2 hours. After washing twice with PBS, a phage display library of hundreds of billions of cells was added and incubated for 2 hours. The tube was then washed 6 times with PBST, followed by 2 times with PBS to remove non-specifically bound phages. Finally, 0.8 mL of 0.05% phage protein was added to the immunotherapy tube. EDTA trypsin digestion solution was used to elute phages that specifically bound the target antigen. These phages were then used to infect logarithmic-phase SS320 cells (purchased from Lucigen, catalog number: 60512-1), incubated at 37°C for 30 min, and then cultured at 220 rpm for 1 h. VSCM13 helper phages were then added, incubated for 30 min, and cultured again at 220 rpm for 1 h. The culture was then centrifuged and transferred to yeast extract-tryptone medium (Sigma-Aldrich, catalog number: Y2377) containing kanamycin and chloramphenicol, and cultured overnight at 30°C and 220 rpm. The phages were precipitated the next day for subsequent 2–4 rounds of screening. The antigens used for phage screening in the second, third, and fourth rounds were hu-Claudin 1-VLP protein (starting from the second round, phages were incubated with empty VLP for 2 hours before being added to the hu-Claudin 1-VLP protein-coated immunosorbent assay tubes to remove non-specific binding). The antigen concentrations decreased sequentially to 20 μg / mL, 4 μg / mL, and 1 μg / mL, respectively. In addition, the PBST washing intensity gradually increased, with 10, 14, and 14 elutions, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the enrichment effect of the phage pools eluted from each round, coated with hu-Claudin 1-VLP protein. Clones were randomly selected from each round of screening for sequence analysis, and positive clones were selected for ELISA screening. Simultaneously, non-specific detection of empty VLP was performed to exclude non-specific binding.

[0211] Cell screening: Using the huCLDN1-HEK293 overexpressing cell line as the screening antigen, antibodies against human CLDN1 were screened from the phage display library. The specific method is as follows: The huCLDN1-HEK293 overexpressing cell line or the negative screening family huCLDN7-HEK293 and huCLDN19-HEK293 overexpressing cell lines were cultured in T25 culture flasks. When the cell line reached approximately 90% confluence, the optimal growth state was achieved. The culture supernatant was removed, and the cells were washed once with PBS (manufacturer: Yuanpei, catalog number: B310KJ). Then, 5 mL of 4% paraformaldehyde (purchased from: Sangon Biotech, catalog number: E672002-0500) was added for fixation for 1 hour. Finally, the cells were washed twice with PBS, and the resulting material was ready for use as antigen material for phage cell screening. During screening, the phages were first incubated with fixed huCLDN7-HEK293 and huCLDN19-HEK293 cell culture flasks at room temperature for 1 hour. Then, the supernatant phages after adsorption were aspirated and incubated with fixed human CLDN1-HEK293 cell culture flasks for 2 hours. After washing twice with PBS, 3 mL of glycine-HCl (pH 2.0) was added and gently mixed for 10 min to elute phages that specifically bind to the target membrane protein CLDN1. Then, the eluted supernatant was used to infect SS320 cells in the logarithmic growth phase (purchased from Lucigen, catalog number: 60512-1), incubated for 30 min, and then cultured at 220 rpm for 1 h. Next, VSCM13 helper phage (manufacturer: HonorGene, catalog number: HG-VSW0964) was added, incubated for 30 min, and then cultured at 220 rpm for another 1 h. After centrifugation, the medium was transferred to yeast extract-tryptone medium (manufacturer: Sigma-Aldrich, catalog number: Y2377) containing kanamycin and chloramphenicol. The resulting phages were then used for the second round of screening. This process was repeated, and sequence analysis was performed on the clones randomly selected in each round. The results showed that after three rounds of screening, the fourth round of screening resulted in significant sequence enrichment.

[0212] The monoclonal antibodies obtained from the panning process were sequenced and full-length antibodies were constructed. Further cellular-level FACS binding validation (using engineered HEK293 cells expressing human Claudin1 protein, huCLDN1-HEK293, experimental method as in Example 1) and non-binding validation with Claudin7 and Claudin19 (huCLDN7-HEK293 cell lines and huCLDN19-HEK293 cell lines) were performed (experimental method as in Example 1). Finally, candidate antibodies that specifically bind to human Claudin1 and monkey Claudin1 at the cellular level were obtained. The names and sequence numbers of some of the constructed antibodies are shown below.

[0213] Antibody 1 (AbM regimen):

[0214] HCDR1: SEQ ID NO:1; HCDR2: SEQ ID NO:2; HCDR3: EKY;

[0215] LCDR1: SEQ ID NO:4; LCDR2: SEQ ID NO:5; LCDR3: SEQ ID NO:6;

[0216] VH: SEQ ID NO: 19; VL: SEQ ID NO: 24;

[0217] Heavy chain: SEQ ID NO:33; Light chain: SEQ ID NO:38.

[0218] Example 3: Detection of in vitro binding activity of anti-Claudin 1 antibody

[0219] 3.1 FACS detection of the binding activity of anti-Claudin 1 antibody on huCLDN1-HEK293 and cynoCLDN1-HEK293 cells.

[0220] huCLDN1-HEK293 and cynoCLDN1-HEK293 cells were prepared into cell suspensions and the cell density was adjusted to 1×10⁻⁶. 6 Cells / mL. Using a 96-well round-bottom plate, add 100 μL of cell suspension to each well using a 100 μL pipette. Centrifuge at 300 g / min for 5 min. Discard the supernatant. Dilute the antibody with FACS buffer to create a concentration gradient of 20.000, 5.000, 1.250, 0.313, 0.078, 0.020, 0.005, and 0.001 μg / mL. Using a 100 μL pipette, add 100 μL of the antibody dilution to each well. Incubate at 4°C for 60 min, then wash the cells twice with FACS buffer. Dilute the fluorescent secondary antibody anti-human IgG Fc (purchased from Abcam, catalog number: 98596) 1:200 with FACS buffer, adding 100 μL to each well. Incubate at 4°C for 30 min, then wash the cells twice with FACS buffer. The mean fluorescence intensity (MFI) of cells incubated with each antibody concentration was detected using flow cytometry, and concentration-curve analysis was performed to assess the antibody-bound EC. 50 The concentrations and results are shown in Tables 2 and 3. FACS combined experiments showed that antibody 1 had good affinity for human and monkey Claudin 1 antigen, and its affinity for human and monkey Claudin 1 antigen was superior to that of control antibodies ALE-F02 and 6F6C3.

[0221] Table 2: Affinity of antibodies to human Claudin 1 antigen as detected by FACS (EC50) 50 value)

[0222] Table 3: Affinity of antibodies to monkey Claudin 1 antigen as detected by FACS (EC50) 50 value)

[0223] 3.2 FACS detection of the binding activity of anti-Claudin1 antibody in Huh-7 cells.

[0224] Huh-7 cells were prepared into a cell suspension and the cell density was adjusted to 1×10⁻⁶. 6 Cells / mL. Using a 96-well round-bottom plate, add 100 μL of cell suspension to each well using a 100 μL pipette. Centrifuge at 300 g / min for 5 min. Discard the supernatant. Dilute the antibody with FACS buffer to create a concentration gradient of 20.000, 5.000, 1.250, 0.313, 0.078, 0.020, 0.005, and 0.001 μg / mL. Using a 100 μL pipette, add 100 μL of the antibody dilution to each well. Incubate at 4°C for 60 min, then wash the cells twice with FACS buffer. Dilute the fluorescent secondary antibody anti-human IgG Fc (purchased from Abcam, catalog number: 98596) 1:200 with FACS buffer, adding 100 μL to each well. Incubate at 4°C for 30 min, then wash the cells twice with FACS buffer. The mean fluorescence intensity (MFI) of cells incubated with each antibody concentration was detected using flow cytometry, and concentration-curve analysis was performed to assess the antibody-bound EC. 50 The concentrations and results are shown in Table 4. FACS combined experiments showed that antibody 1 had good affinity for Huh-7 cells, and its affinity for Huh-7 cells was superior to that of control antibodies ALE-F02 and 6F6C3.

[0225] Table 4: Affinity of antibodies to Huh-7 cells as detected by FACS (EC50) 50 value)

[0226] 3.3 FACS detection of the binding activity of anti-Claudin1 antibody in Claudin7, Claudin19 HEK293 cells.

[0227] huCLDN7-HEK293 cells and huCLDN19-HEK293 cells were respectively prepared into cell suspensions, and the cell density was adjusted to 1×10⁻⁶. 6Cells / mL. Using a 96-well round-bottom plate, add 100 μL of cell suspension to each well using a 100 μL pipette. Centrifuge at 300 g / min for 5 min. Discard the supernatant. Dilute the antibody with FACS buffer to create a concentration gradient of 20.000, 5.000, 1.250, 0.313, 0.078, 0.020, 0.005, and 0.001 μg / mL. Using a 100 μL pipette, add 100 μL of the antibody dilution to each well. Incubate at 4°C for 60 min, then wash the cells twice with FACS buffer. Dilute the fluorescent secondary antibody anti-human IgG Fc (purchased from Abcam, catalog number: 98596) 1:200 with FACS buffer, adding 100 μL to each well. Incubate at 4°C for 30 min, then wash the cells twice with FACS buffer. The mean fluorescence intensity (MFI) of cells incubated with each antibody concentration was detected using flow cytometry, and concentration-curve analysis was performed to assess the antibody-bound EC. 50 The concentrations and results are shown in Table 5. FACS binding assays showed that antibody 1 had less nonspecific binding to Claudin7 and Claudin19 than the control antibody ALE-F02.

[0228] Table 5: Affinity of antibodies to human Claudin 7 and Claudin 19 antigens as detected by FACS (EC5) 50 value) “N / A” indicates that no test was performed.

[0229] Example 4 Non-specific modification of antibody affinity maturation

[0230] The antibody molecule obtained from the fully human library screening still exhibits non-specific binding with cells overexpressing the CLDN7 / 19 family protein. Antibody engineering was used to reduce this non-specific binding and simultaneously improve the binding affinity to the target CLDN1 cells.

[0231] 4.1 Design and Construction of Affinity Maturation Non-Specific Modification Library

[0232] Nonspecific modification of antibody 1 was performed to improve affinity and reduce nonspecific binding. This nonspecific modification was based on M13 phage display technology, using codon-based primers (in primer synthesis, each codon is composed of NNK) to introduce mutations in the CDR region, constructing four phage display libraries: Libraries 1 and 2 were single-point combination mutations, with Library 1 containing a CDRL1+CDRL3+CDRH3 combination mutation and Library 2 containing a CDRL2+CDRH1+CDRH2 combination mutation; Libraries 3 and 4 were double-point saturation mutations, with Library 3 containing a double-point saturation mutation of CDRL3 and Library 4 containing a double-point saturation mutation of CDRH3. The specific library construction method is as follows: First, primers containing point mutations are synthesized; second, using the antibody to be modified 1 as a PCR amplification template, sequences containing mutations in the CDR region are amplified. Fragments containing different CDR mutations are combined using bridging PCR. Then, the point mutation antibody is ligated into the phage display vector via double enzyme digestion (HindIII and NotI) and double-end ligation. Finally, the antibody sequence with the mutation site is transformed into *E. coli* SS320 via electroporation. For details on library capacity calculation and phage library preparation, please refer to Example 2.

[0233] 4.2 Screening of Non-specific Modified Libraries Based on Affinity Maturation

[0234] For specific procedures on library screening, please refer to Example 2. After initial screening, affinity sequencing, and sequence analysis of the libraries, a total of 72 candidate molecules were selected for construction, expression, and functional screening.

[0235] 4.3 Validation of candidate molecules for non-specific modification based on affinity maturation

[0236] Based on the physicochemical properties, affinity activity, and non-specific binding activity of the antibodies, eight candidate molecules were selected. The CDR sequence was determined using the AbM-defined CDR method. The sequence information of the affinity-matured anti-Claudin 1 antibodies is shown in Tables 6 and 7.

[0237] Table 6: CDR and variable region amino acid sequences of affinity-matured anti-Claudin 1 antibodies

[0238] Table 7: Full-length amino acid sequence of heavy and light chains of anti-Claudin 1 antibody with affinity maturation

[0239] Example 5: Detection of in vitro binding activity of nonspecifically modified antibodies

[0240] Seventy-two unique lead molecules obtained after antibody engineering were expressed in 0.5 mL of CHO cells in eukaryotic cells. Titer expression levels in the supernatant were measured, followed by ELISA affinity verification using hu-Claudin 1-VLP coating. FACS non-specific binding verification was performed on the overexpressing cell lines huCLDN7-HEK293 and huCLDN19-HEK293 at two concentration points (2 μg / mL and 20 μg / mL). Based on affinity and non-specificity data, eight lead molecules were obtained and purified by 10 mL of CHO cell expression in eukaryotic cells. Subsequent FACS non-specific curve analysis was performed on the overexpressing cell lines huCLDN7-HEK293 and huCLDN19-HEK293 (results are shown in Table 8). The results showed that the non-specifically modified antibody of this invention has good affinity for human Claudin 1, but weak affinity for human Claudin 7 or Claudin 19.

[0241] Table 8: Affinity of antibodies to human Claudin 1, Claudin 7 and Claudin 19 antigens as detected by FACS (EC50) 50 value) “N / A” indicates that no test was performed.

[0242] Example 6: Detection of endocytic activity of nonspecifically modified antibodies

[0243] The endocytic activity of anti-Claudin1 antibody in huCLDN1-HEK293, huCLDN7-HEK293, huCLDN19-HEK293, and Huh-7 cells was detected using pHrodo (purchased from Invitrogen, Zenon pHrodo iFL IgG Labeling Reagents, catalog number: Z25611) to trace live cell endocytosis.

[0244] Cells in the logarithmic growth phase were collected, suspended, and their density adjusted to 1 × 10⁻⁶ cells. 5Cells / mL. A 96-well plate was used, and 200 μL of cell suspension was added to each well using a 200 μL pipette. The plate was incubated overnight at 37°C. The antibody concentration was diluted to 80 nM with PBS, and the pHrodo reagent concentration to 240 nM. The antibody and pHrodo reagent were mixed at a 1:1 volume ratio and incubated at room temperature for 10 minutes to ensure thorough mixing with the test sample. 50 μL of pHrodo-bound antibody was added to each well, and the plate was incubated at 37°C for 48 hours. Cells were collected at each time point and washed twice with FACS buffer. The mean fluorescence intensity (MFI) of cells incubated at each antibody concentration was detected using flow cytometry. The results are shown in Figure 5. Antibodies 2 and 3 showed similar endocytic activity to antibody 1 in Huh-7 and huCLDN1-HEK293 cells. FACS analysis showed that the endocytic activity of antibodies 2 and 3 in huCLDN1-HEK293 cells after 48 hours was superior to the control antibody ALE-F02. On huCLDN7-HEK293 and huCLDN19-HEK293 cells, antibody 2 showed weaker non-specific endocytosis than antibody 1 and ALE-F02 antibody.

[0245] The sequences involved in this invention are shown in Table 9.

[0246] Table 9: Sequence List (all are amino acid sequences; for sequence definitions / descriptions, please refer to the preceding sections of this article).

[0247] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof targeting Claudin 1, characterized in that, The antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region includes light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3. The HCDR1 contains the amino acid sequence shown in SEQ ID NO:

47. The HCDR2 contains the amino acid sequence shown in SEQ ID NO:

48. The HCDR3 contains the amino acid sequence EKY; The LCDR1 contains the amino acid sequence shown in SEQ ID NO:

4. The LCDR2 comprises the amino acid sequence shown in SEQ ID NO:49, and, The LCDR3 contains an amino acid sequence as shown in SEQ ID NO:

3.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:7, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:8, and SEQ ID NO:6, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:9, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:6, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:9, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:11, and SEQ ID NO:6, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:12, and SEQ ID NO:6, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:13, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:14, and SEQ ID NO:6, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:15, SEQ ID NO:2, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:16, and SEQ ID NO:6, respectively; or... The heavy chain variable region comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and EKY, respectively, and the light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:17, and SEQ ID NO:6, respectively; or... The heavy chain variable region contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and EKY, respectively, and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:18, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody or its antigen-binding fragment is selected from at least one of the following: (1) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:20; and / or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:25; or, The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:21; and / or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:26; or, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:19; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:24; or, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:21; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:27; or, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:19; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:28; or, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:22; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:29; or, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:23; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:30; or, The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:19; and / or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:31; or, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:19; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:32; or, (2) The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:20; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:25; or, The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:21; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:26; or, The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:19; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:24; or, The heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:21; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:27; or, the heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 19; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 28; or, the heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 22; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 29; or, the heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 23; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 30; or, the heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 19; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 31; or, the heavy chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 19; and / or, the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO:

32.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein, wherein the antibody or antigen-binding fragment thereof is selected from one or more of: (1) a Fab, Fab', F(ab')2, Fv, scFv, or sdAb; (2) a monoclonal antibody or a polyclonal antibody; (3) a mono-specific antibody, a bi-specific antibody, or a multi-specific antibody; (4) a murine antibody, a fully human antibody, a humanized antibody, or a chimeric antibody; (5) an antibody prodrug; Preferably, the antibody or antigen-binding fragment thereof is a full-length antibody, preferably comprising at least one constant region selected from human IgGl, IgG2, IgG3, IgG4, IgAl, IgA, IgM, IgD, and IgE, and variants thereof.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein, the antibody or antigen-binding fragment thereof is selected from any one of: (I) The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:34; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:39; or, The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:35; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:40; or, The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:38; or, The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:35; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:41; or, The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:42; or, The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:36; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:43; or, The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:37; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:44; or, The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:45; or, The heavy chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:46; or, (II) The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:34; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:39; or, The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:35; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:40; or, The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:38; or, The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:35; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:41; or, The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:42; or, The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:36; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:43; or, The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:37; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:44; or, The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:45; or, The heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:33; and / or, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

46.

6. An isolated nucleic acid, comprising, The isolated nucleic acid encodes an antibody or antigen-binding fragment thereof as described in any one of claims 1-5.

7. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 6; Preferably, the recombinant expression vector is selected from: viral vectors and non-viral vectors; More preferably, the non-viral vector is selected from: plasmids, linear DNA fragments, and RNA; More preferably, the recombinant expression vector is a plasmid, and the backbone plasmid of the recombinant expression vector is the pLVX-puro plasmid.

8. A transformant characterized in that, The transformant comprises the isolated nucleic acid as described in claim 6 or the recombinant expression vector as described in claim 7, wherein the transformant is a non-animal or non-plant variety; preferably, the transformant is a eukaryotic cell, more preferably, the eukaryotic cell is a mammalian cell, such as CHO cells, 293T cells or their derivatives.

9. A method of preparing an antibody or antigen-binding fragment thereof against Claudin 1, characterized in that, The method includes culturing the transformant as described in claim 8 and obtaining the antibody or its antigen-binding fragment from the culture.

10. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises an antibody or an antigen-binding fragment thereof as described in any one of claims 1-5; Preferably, the chimeric antigen receptor further includes a co-stimulatory domain and a signal transduction domain.

11. A genetically modified cell, wherein, The cell expresses the chimeric antigen receptor as described in claim 10; preferably, the gene-modified cell is a T cell or an NK cell.

12. An antibody drug conjugate, characterized in that, The antibody-drug conjugate comprises a cytotoxic agent or tag, and an antibody or antigen-binding fragment thereof as described in any one of claims 1-5.

13. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises an antibody or antigen-binding fragment thereof as described in any one of claims 1-5, an isolated nucleic acid as described in claim 6, a recombinant expression vector as described in claim 7, a transformant as described in claim 8, a genetically modified cell as described in claim 11, or an antibody-drug conjugate as described in claim 12, and a pharmaceutically acceptable carrier and / or excipients.

14. A method of detecting Claudin 1, characterized by, It includes the step of contacting the sample to be tested with an antibody or antigen-binding fragment thereof as described in any one of claims 1-5; preferably, the detection is for non-diagnostic and / or therapeutic purposes.

15. A method of treating and / or preventing a Claudin 1 -mediated disease or disorder, characterized in that, The method includes administering to a subject in need a therapeutic or preventative effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-5, a genetically modified cell as described in claim 11, an antibody-drug conjugate as described in claim 12, or a pharmaceutical composition as described in claim 13; Preferably, the disease is cancer; More preferably, the cancer is selected from one or more of liver cancer, breast cancer, colorectal cancer, and ovarian cancer.

16. A kit comprising, The kit comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-5, the isolated nucleic acid as described in claim 6, the recombinant expression vector as described in claim 7, the transformant as described in claim 8, the gene-modified cell as described in claim 11, the antibody-drug conjugate as described in claim 12, or the pharmaceutical composition as described in claim 13.

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