CLDN18.2 antigen-binding protein and its use

Abstract

The present invention provides a CLDN18.2 antigen-binding protein and uses thereof. The present invention provides isolated antigen binding proteins capable of binding to CLDN18.2, as well as chimeric antigen receptors comprising the isolated antigen binding proteins, and methods for preparing and using the antigen binding proteins and chimeric antigen receptors.

Description

[Technical Field]

[0001] This application relates to the technical field of biopharmaceuticals, and more particularly to antigen-binding proteins capable of binding to CLDN18.2. [Background technology]

[0002] Gastric cancer is the second most deadly malignant tumor in the world. According to 2015 data from the World Health Organization, 754,000 people worldwide die from gastric cancer each year. Pancreatic cancer is one of the most malignant tumors, and according to data from Non-Patent Literature 1, more than 200,000 people worldwide die from pancreatic cancer each year. The standard first-line treatment for advanced or recurrent gastric cancer is chemotherapy. Adding trastuzumab to chemotherapy has provided some degree of survival benefit to patients with HER2-positive tumors, but HER2-positive gastric cancer accounts for only 15% of all cases. The development of safe and effective treatments is urgently needed.

[0003] CLDN18.2 (Claudin 18.2) is expressed only in differentiated parietal cells and not in normal tissues. Recent studies have shown that CLDN18.2 is overexpressed in over 77% of gastric cancer patients and over 80% of pancreatic cancer patients, as well as in solid tumors such as lung cancer, esophageal cancer, and ovarian cancer. CLDN18.2 belongs to the family of tight junction proteins that regulate the flow of molecules between laminar cells. However, in tumors, tight junctions are disrupted, and CLDN proteins lose their major role. Because CLDN18.2 is abundant in gastric tumors, researchers estimate that half of patients with advanced gastric cancer could be candidates for new therapies targeting CLDN18.2 antibodies. Furthermore, because this unique target is not present in healthy tissues other than the gastric wall, treatment side effects can be minimized. These characteristics indicate that CLDN18.2 is an ideal target for the development of therapeutic monoclonal antibodies.

[0004] Because CLDN18.2 is a membrane protein, it has been difficult to obtain high-quality antibodies that target the native protein using conventional antibody screening methods. Currently, in vivo immunization methods (e.g., DNA injection) are widely used, but these require multiple immunizations of animals. CLDN18 has two splice variants (CLDN18.1 and CLDN18.2), both located in the first extracellular loop. These variants differ only in the N-terminal 69th amino acid sequence, with the rest of the sequence being identical. Since CLDN18.1 is expressed in normal lung tissue, it is necessary to screen for antibodies that bind to CLDN18.2 but not to CLDN18.2. On the other hand, the CLDN18.2 sequence exhibits extremely high homology between humans and mice. Therefore, obtaining antibodies that specifically bind to CLDN18.2 is a matter of urgency. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Application No. WO 06 / 030220 [Patent Document 2] International Application No. WO 06 / 003388 [Patent Document 3] U.S. Publication No. 6,150,584 [Patent Document 4] U.S. Publication No. 6,458,592 [Patent Document 5] U.S. Publication No. 6,420,140 [Patent Document 6] International Application No. WO2015 / 036583 [Patent Document 7] Chinese Patent Application No. CN103509114A [Non-patent literature]

[0006] [Non-Patent Document 1] The Lancet, 2016 (medical journal) [Non-Patent Document 2] Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121 - 129 (2003) [Non - Patent Document 3] Roque et al., Biotechnol. Prog. 20:639 - 654 (2004) [Non - Patent Document 4] Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991) [Non - Patent Document 5] Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6 [Non - Patent Document 6] Hamers - Casterman et al., Nature 363:446 - 448 (1993); Sheriff et al, Nature Struct. Biol. 3:733 - 736 (1996) [Non - Patent Document 7] Ward et al. (Nature, 1989 Oct 12; 341(6242):544 - 6) [Non - Patent Document 8] Holt et al., Trends Biotechnol., 2003, 21(11):484 - 490 [Non - Patent Document 9] "Improved Tools for Biological Sequence Comparison" by W.R. Pearson and D.J. Lipman [Non - Patent Document 10] Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci.), 85:2444 - 2448, 1988 [Non - Patent Document 11] DJ Lipman and WRPearson, "Fast and highly sensitive protein similarity search," Science, 227:1435-1441, 1989. [Non-Patent Document 12] S. Altschul, W. Gish, W. Miller, EWMyers, and D. Lipman, "Basic Local Alignment Search Tools," Journal of Molecular Biology, 215:403-410, 1990. [Non-Patent Document 13] Brummell et al.,(1993)Biochem 32:1180-8 [Non-Patent Document 14] de Wildt et al.,(1997)Prot.Eng.10:835-41 [Non-Patent Document 15] Komissarov et al.,(1997)J.Biol.Chem.272:26864-26870 [Non-Patent Document 16] Hall et al.,(1992)J.Immunol.149:1605-12 [Non-Patent Document 17] Kelley and O'Connell (1993)Biochem.32:6862-35 [Non-Patent Document 18] Adib-Conquy et al.,(1998)Int.Immunol.10:341-6 and Beers et al.,(2000)Clin.Can.Res.6:2835-43 [Non-Patent Document 19] Kufer et al,cited supra;Cao and Suresh,Bioconjugate Chemistry,9 (6), 635-644 (1998) [Non-Patent Document 20] van Spriel et al., Immunology Today, 21 (8), 391-397 (2000) [Non-Patent Document 21] PROTOCOL published by Lotta von Boehmer (doi:10.1038 / nprot.2016.102) [Overview of the project] [Problems that the invention aims to solve]

[0007] This application provides an antigen-binding protein capable of binding to human CLDN18.2, exhibiting one or more desired functional properties, such as being specifically able to bind to human CLDN18.2, not inherently binding to human CLDN18.1, and potentially possessing CDC activity and / or antitumor activity. This application also provides nucleic acid molecules encoding the isolated antigen-binding protein, expression vectors, host cells, and methods for preparing the isolated antigen-binding protein. The isolated antigen-binding protein of this application can be used to prevent and / or treat diseases and / or disorders such as tumors and / or cancer. [Means for solving the problem]

[0008] In one embodiment, the present application provides an isolated antigen-binding protein comprising HCDR3 having the amino acid sequence shown in SEQ ID NO: 67.

[0009] In some embodiments, the isolated antigen-binding protein HCDR3 comprises an amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 12, and SEQ ID NO: 18.

[0010] In some embodiments, the isolated antigen-binding protein comprises HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 68 or SEQ ID NO: 11.

[0011] In some embodiments, the isolated antigen-binding protein HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 17.

[0012] In some embodiments, the isolated antigen-binding protein comprises HCDR1, wherein HCDR1 comprises an amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 16.

[0013] In some embodiments, the isolated antigen-binding protein comprises H-FR1, the C-terminus of H-FR1 directly or indirectly ligated to the N-terminus of HCDR1, and H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 72.

[0014] In some embodiments, the isolated antigen-binding protein H-FR1 comprises an amino acid sequence represented by SEQ ID NO: 34, SEQ ID NO: 43, and SEQ ID NO: 49.

[0015] In some embodiments, the isolated antigen-binding protein comprises H-FR2, which is located between HCDR1 and HCDR2 and comprises the amino acid sequence shown in SEQ ID NO: 73.

[0016] In some embodiments, the H-FR2 of the isolated antigen-binding protein contains the amino acid sequence shown in SEQ ID NO: 35 or SEQ ID NO: 44.

[0017] In some embodiments, the isolated antigen-binding protein comprises H-FR3, which is located between HCDR2 and HCDR3, and includes an amino acid sequence indicated by SEQ ID NO: 80 or SEQ ID NO: 45.

[0018] In some embodiments, the isolated antigen-binding protein H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 36 or SEQ ID NO: 42.

[0019] In some embodiments, the isolated antigen-binding protein comprises H-FR4, the N-terminus of which is ligated to the C-terminus of which is it is which is it

[0020] In some embodiments, the isolated antigen-binding protein H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 46 or SEQ ID NO: 37.

[0021] In some embodiments, the isolated antigen-binding protein comprises VH, the VH comprising the amino acid sequence shown in SEQ ID NO: 78.

[0022] In some embodiments, the VH of the isolated antigen-binding protein comprises an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 15, and SEQ ID NO: 19.

[0023] In some embodiments, the isolated antigen-binding protein comprises LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 69.

[0024] In some embodiments, the isolated antigen-binding protein LCDR3 comprises an amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 23, and SEQ ID NO: 25.

[0025] In some embodiments, the isolated antigen-binding protein comprises LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 70.

[0026] In some embodiments, the isolated antigen-binding protein LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 22.

[0027] In some embodiments, the isolated antigen-binding protein comprises LCDR1, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 71.

[0028] In some embodiments, the isolated antigen-binding protein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 21.

[0029] In some embodiments, the isolated antigen-binding protein comprises L-FR1, the C-terminus of which is directly or indirectly ligated to the N-terminus of which is which is which is which is which has an amino acid sequence represented by SEQ ID NO: 75.

[0030] In some embodiments, the isolated antigen-binding protein L-FR1 comprises an amino acid sequence represented by SEQ ID NO: 38, SEQ ID NO: 47, SEQ ID NO: 50, and SEQ ID NO: 53.

[0031] In some embodiments, the isolated antigen-binding protein comprises L-FR2, which is located between LCDR1 and LCDR2, and includes the amino acid sequence shown in SEQ ID NO: 39.

[0032] In some embodiments, the isolated antigen-binding protein comprises L-FR3, which is located between LCDR2 and LCDR3 and comprises the amino acid sequence shown in SEQ ID NO: 76.

[0033] In some embodiments, the isolated antigen-binding protein L-FR3 comprises an amino acid sequence represented by SEQ ID NO: 40, SEQ ID NO: 48, and SEQ ID NO: 51.

[0034] In some embodiments, the isolated antigen-binding protein comprises L-FR4, the N-terminus of which is ligated to the C-terminus of which is

[0035] In some embodiments, the isolated antigen-binding protein L-FR4 comprises an amino acid sequence represented by SEQ ID NO: 41, SEQ ID NO: 52, and SEQ ID NO: 54.

[0036] In some embodiments, the isolated antigen-binding protein comprises a VL, the VL comprising the amino acid sequence shown in SEQ ID NO: 79.

[0037] In some embodiments, the VL of the isolated antigen-binding protein comprises an amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 20, and SEQ ID NO: 24.

[0038] In some embodiments, the isolated antigen-binding protein comprises VH and VL selected from any group below: 1) VH containing the amino acid sequence shown in SEQ ID NO: 1, VL containing the amino acid sequence shown in SEQ ID NO: 5, 2) VH containing the amino acid sequence shown in SEQ ID NO: 15, VL containing the amino acid sequence shown in SEQ ID NO: 5, 3) VH containing the amino acid sequence shown in SEQ ID NO: 9, VL containing the amino acid sequence shown in SEQ ID NO: 13, 4) VH containing the amino acid sequence shown in SEQ ID NO: 19, VL containing the amino acid sequence shown in SEQ ID NO: 20, and 5) VH containing the amino acid sequence shown in SEQ ID NO: 19, and VL containing the amino acid sequence shown in SEQ ID NO: 24.

[0039] In some embodiments, the isolated antigen-binding protein includes a constant region of the antibody heavy chain.

[0040] In some embodiments, the antibody heavy chain constant region is derived from the human IgG heavy chain constant region.

[0041] In some embodiments, the antibody heavy chain constant region is derived from the human IgG1 heavy chain constant region.

[0042] In some embodiments, the isolated antigen-binding protein includes a constant region of the antibody light chain.

[0043] In some embodiments, the antibody light chain constant region is derived from the human Igκ constant region.

[0044] In some embodiments, the isolated antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

[0045] In some embodiments, the antigen-binding fragment includes Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv and / or dAb.

[0046] In some embodiments, the antibody is selected from one or more of the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0047] In some embodiments, as verified by FACS, the isolated antigen-binding protein does not substantially compete with the reference antibody for binding to CLDN18.2, and the reference antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH of the reference antibody comprises the amino acid sequence shown in SEQ ID NO: 55, and the VL of the reference antibody comprises the amino acid sequence shown in SEQ ID NO: 56.

[0048] In some embodiments, the isolated antigen-binding protein can specifically bind to CLDN18.2 but not substantially to CLDN18.1.

[0049] In some embodiments, the CLDN18.2 to which the isolated antigen-binding protein can specifically bind includes mouse CLDN18.2, cynomolgus monkey CLDN18.2, and / or human CLDN18.2.

[0050] In some embodiments, the isolated antigen-binding protein has CDC activity.

[0051] In some embodiments, the isolated antigen-binding protein can inhibit tumor growth and / or tumor cell proliferation.

[0052] In another embodiment, the application also provides a chimeric antigen receptor comprising a target moiety comprising the antigen-binding protein described herein.

[0053] In some embodiments, the chimeric antigen receptor comprises a costimulatory domain, the costimulatory domain comprising a costimulatory domain derived from one or more proteins selected from the group consisting of the ligands CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83, CD40, and MyD88.

[0054] In some embodiments, the co-stimulatory domain of the chimeric antigen receptor is an intracellular co-stimulatory signaling region derived from 4-1BB.

[0055] In some embodiments, the co-stimulatory domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 85.

[0056] In some embodiments, the chimeric antigen receptor comprises an intracellular signaling domain, the intracellular signaling domain comprising a domain containing CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma-associated herpesvirus (HSKV), DAP10, DAP-12, and at least one ITAM.

[0057] In some embodiments, the intracellular signaling domain of the chimeric antigen receptor is a signaling domain derived from CD3ζ.

[0058] In some embodiments, the intracellular signaling domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 86.

[0059] In some embodiments, the chimeric antigen receptor comprises a transmembrane domain, the transmembrane domain comprising one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0060] In some embodiments, the transmembrane domain of the chimeric antigen receptor is a transmembrane domain derived from CD8.

[0061] In some embodiments, the transmembrane domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 84.

[0062] In some embodiments, the chimeric antigen receptor includes a hinge region between the target portion and the transmembrane domain, wherein the hinge region comprises one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0063] In some embodiments, the hinge region of the chimeric antigen receptor is a hinge region derived from CD8.

[0064] In some embodiments, the hinge region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 83.

[0065] In some embodiments, the chimeric antigen receptor further comprises a signal peptide.

[0066] In some embodiments, the signal peptide of the chimeric antigen receptor is a signal peptide derived from the CD8 protein.

[0067] In some embodiments, the signal peptide of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 88.

[0068] In some embodiments, the chimeric antigen receptor further comprises a low-density lipoprotein receptor-associated protein or a fragment thereof.

[0069] In some embodiments, the low-density lipoprotein receptor-related protein or its fragments comprises one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1 to 12 and their functional fragments.

[0070] In some embodiments, the low-density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 91.

[0071] In another embodiment, the application also provides polypeptide molecules comprising the isolated antigen-binding protein or the chimeric antigen receptor.

[0072] In some embodiments, the polypeptide molecule includes a fusion protein.

[0073] In another embodiment, the application also provides an immune complex comprising the isolated antigen-binding protein.

[0074] In another embodiment, the application also provides one or more isolated nucleic acid molecules encoding the isolated antigen-binding protein, the chimeric antigen receptor, or the polypeptide molecule.

[0075] In some embodiments, the nucleic acid molecule includes a nucleotide sequence represented by any of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.

[0076] In some embodiments, the nucleic acid molecule comprises a base sequence selected from any of the following groups: 1) The base sequence shown in Sequence ID No. 26, and the base sequence shown in Sequence ID No. 27, 2) The base sequence shown in Sequence ID No. 30, and the base sequence shown in Sequence ID No. 27, 3) The nucleotide sequence shown in Sequence ID No. 28, and the nucleotide sequence shown in Sequence ID No. 29, 4) The base sequence shown in Sequence ID No. 31, and the base sequence shown in Sequence ID No. 32, and 5) The nucleotide sequence shown in SEQ ID NO: 31, and the nucleotide sequence shown in SEQ ID NO: 33.

[0077] In another embodiment, the application also provides a vector comprising the nucleic acid molecule.

[0078] In another embodiment, the application also provides cells comprising the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, or the vector.

[0079] In some embodiments, the cells are immune effector cells.

[0080] In some embodiments, the cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphocyte progenitor cells, and / or pluripotent stem cells.

[0081] In some embodiments, the cells are T cells.

[0082] In some embodiments, the cells further comprise and / or express low-density lipoprotein receptor-related proteins or fragments thereof.

[0083] In some embodiments, the low-density lipoprotein receptor-related protein or its fragments comprises one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1 to 12 and their functional fragments.

[0084] In some embodiments, the low-density lipoprotein receptor-related protein or fragment thereof is low-density lipoprotein receptor-related protein 5 and / or 6 or fragment thereof.

[0085] In some embodiments, the low-density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 91.

[0086] In another embodiment, the application also provides a pharmaceutical composition comprising the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0087] In another embodiment, the application also provides a method for preparing the isolated antigen-binding protein, which includes culturing the cells under conditions in which the antigen-binding protein is expressed.

[0088] In another embodiment, the application also provides the use of the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition in the preparation of pharmaceuticals for preventing, alleviating, and / or treating diseases and / or disorders.

[0089] In some embodiments, the disease and / or disorder includes cancer.

[0090] In some embodiments, the cancer includes solid tumors and / or hematological malignancies.

[0091] In some embodiments, the cancers include gastric cancer and / or colon cancer.

[0092] In another embodiment, the application also provides a method for detecting CLDN18.2 in a sample, comprising administering the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cells, and / or the pharmaceutical composition.

[0093] In another embodiment, the application also provides a reagent or kit for detecting CLDN18.2 in a sample, comprising the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cells, and / or the pharmaceutical composition.

[0094] In another embodiment, the application also provides the use of the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cells, and / or the pharmaceutical composition in the preparation of a kit for detecting the presence and / or content of CLDN18.2 in a sample.

[0095] Those skilled in the art will readily understand other aspects and advantages of this application from the detailed description below. The detailed description below illustrates only exemplary embodiments of this application. As those skilled in the art will understand, the content of this application allows them to modify the specific embodiments disclosed without departing from the spirit and scope of the invention. Furthermore, the drawings and descriptions accompanying this application should be considered in all respects to be illustrative and not restrictive. [Brief explanation of the drawing]

[0096] The specific features of the invention described in this application are described in the attached claims. Further understanding of the features and advantages of the invention can be gained by referring to the exemplary embodiments and drawings described in detail below.

[0097] [Figure 1] This graph shows the binding activity assay of the antigen-binding protein of this application and high-expression human CLDN18.2 cells by flow cytometry. [Figure 2] This graph shows the binding activity assay of the antigen-binding protein of this application and high-expression human CLDN18.1 cells by flow cytometry. [Figure 3] This graph shows the binding activity assay of the antigen-binding protein of this application and tumor cell lines, as determined by flow cytometry. [Figure 4] This graph shows the species cross-binding activity analysis of the antigen-binding protein of this application. [Figures 5a-5b] This graph shows the CDC activity assay of the antigen-binding protein of this application. [Figure 6] This graph shows the competitive binding activity analysis of the antigen-binding protein of this application. [Figure 7] This graph shows the change in body weight of mice after administration. [Figure 8] This graph shows the relative change (%) in body weight of mice after administration. [Figure 9] This graph shows the change in tumor volume in mice after administration. [Figure 10] This graph shows the tumor suppression rate in mice after administration. [Figure 11] This graph shows the survival curve of mice after administration. [Figure 12A-B] This graph shows the identification of the antigen-binding activity of the CLDN18.2 single-chain antibody. [Figure 13] This graph shows the identification of the binding activity of the CLDN18.2 single-chain antibody to non-target cells. [Figure 14] This graph shows the antitumor activity of the CLDN18.2 antigen-binding protein of this application in a mouse model of human gastric cancer. [Figure 15A-D] This graph shows the expression status of CLDN18.2-specific CARs in T cells. [Figure 16] This graph shows the in vitro killing activity assay of CAR-T cells. [Figure 17A-B] This graph shows the detection of factor secretion by CLDN18.2-specific CARs. [Figure 18A-D] This graph shows the antitumor activity of CLDN18.2-specific CAR-T cells in a mouse colon cancer CDX model. [Modes for carrying out the invention]

[0098] The embodiments of the invention of this application will be described below with reference to specific examples, and those skilled in the art will be able to easily understand the invention and other advantages and effects of this application from the contents disclosed herein.

[0099] (Term definition) In this application, the terms “CLDN18.2” or “Claudin18.2” are used interchangeably and generally refer to isoform 2 of the cell-binding claudin Claudin18. This term includes “full-length” unprocessed CLDN18.2 and all forms of CLDN18.2 arising from cell processing. CLDN18.2 may include complete CLDN18.2 and fragments, functional variants, isoforms, species homologs, derivatives, analogs, and analogs having at least one epitope in common with CLDN18.2. The amino acid sequence of CLDN18.2 (e.g., human CLDN18.2) may be known in the art. For example, the human CLDN18.2 sequence can be shown by GeneBank accession number NM_001002026.3. For example, the mouse CLDN18.2 sequence can be shown by GeneBank accession number NM_001194921.1. For example, the cynomolgus monkey CLDN18.2 nucleotide sequence can be represented by the GeneBank accession number XM_001114708.4.

[0100] In this application, the terms “CLDN18.1” or “Claudin18.1” are used interchangeably and generally refer to isoform 1 of the cell-binding claudin Claudin18. This term includes “full-length” unprocessed CLDN18.1 and any form of CLDN18.2 arising from cell processing. CLDN18.2 may include complete CLDN18.1 and fragments, functional variants, isoforms, species homologs, derivatives, analogs, and analogs having at least one epitope in common with CLDN18.1. The amino acid sequence of CLDN18.1 (e.g., human CLDN18.1) may be known in the art. For example, the human CLDN18.1 sequence can be shown by GeneBank accession number NM_016369.4. For example, the mouse CLDN18.1 sequence can be shown by GeneBank accession number NM_019815.3. For example, the cynomolgus monkey CLDN18.1 nucleotide sequence can be represented by the GeneBank accession number XM_005545863.2.

[0101] In this application, the term “isolated” generally means obtained from its natural state by artificial means. If “isolated” substances or components exist in nature, it is possible that the natural environment in which the “isolated” substance or component is located has been altered, or that the substance has been isolated from its natural environment, or both. For example, living animals naturally contain polynucleotides or polypeptides that have not been isolated, and isolation refers to a high-purity version of the same polynucleotide or polypeptide isolated from this natural state. The term “isolated” does not exclude the presence of mixtures of artificial or synthetic substances, or other impurities that do not affect the activity of the substance.

[0102] In this application, the term “isolated antigen-binding protein” typically means a protein obtained from nature by artificial means and possessing antigen-binding ability. The “isolated antigen-binding protein” may include an antigen-binding moiety and a framework or framework portion that allows the antigen-binding moiety to adopt a conformation that facilitates the binding of the antigen-binding moiety to an antigen, of optional choice. The antigen-binding protein may include, for example, an antibody-derived protein framework region (FR) or an alternative or artificial framework region into which a CDR or CDR derivative has been implanted. Such frameworks include, but are not limited to, antibody-derived framework regions containing mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein, and fully synthesized framework regions containing, for example, biocompatible polymers. See, for example, Non-Patent Documents 2 and 3. Examples of antigen-binding proteins include human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, single-chain antibodies, diabodies, triabodies, tetrabodies, Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv, dAb, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies and their fragments.

[0103] In this application, the terms “variable domain” and “variable region” are used interchangeably and generally refer to a portion of the heavy and / or light chain of an antibody. The variable domains of the heavy and light chains are respectively referred to as “V H " and "V L These domains can be called (or "VH" and "VL" respectively). These domains are usually the most variable parts of the antibody (compared to other antibodies of the same class) and contain the antigen-binding site.

[0104] In this application, the term "variable" generally means that a portion of the variable domain is highly different in sequence between antibodies. The variable domain mediates antigen binding and determines the specificity of a particular antibody to a particular antigen. However, variability is not evenly distributed throughout the variable domain. It is usually concentrated in three portions called hypervariable regions (CDRs or HVRs) within the variable domains of both the light and heavy chains. More highly conserved portions within the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains primarily have a β-sheet structure and contain four FR regions linked by CDRs that form three loop-like links and, in some cases, portions of the β-sheet structure. The CDRs in each chain are held very close to the CDRs of the other chain by the FR regions and play a role in the formation of the antigen-binding site of the antibody (see Non-Patent Literature 4).

[0105] In this application, the term “antibody” generally refers to immunoglobulins or their fragments or derivatives, encompassing any polypeptide containing an antigen-binding site, whether produced in vitro or in vivo. This term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid, mutant, and transplanted antibodies. For the purposes of this invention, the term “antibody” includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function (e.g., specifically binding to human CLDN18.2). Such fragments contain an antigen-binding domain. A basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units and another polypeptide called the J chain, containing 10 antigen-binding sites, while IgA antibodies contain 2 to 5 basic quad units that bind and polymerize with the J chain to form polyvalent combinations. In the case of IgG, the quad unit is generally about 150,000 daltons. Each light chain is linked to a heavy chain by a covalent disulfide bond, but two heavy chains are linked to each other by one or more disulfide bonds depending on the isotype of the heavy chain. Each heavy and light chain also has regularly spaced intrachain disulfide crosslinks. Each heavy chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) related to the α and γ chains respectively, as well as four CH domains related to the μ and ε isotypes. Each light chain has a variable domain (VL) at its N-terminus, followed by a constant domain at the other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain. The pairing of VH and VL forms a single antigen-binding site together. For information on the structure and properties of various classes of antibodies, see, for example, Non-Patent Document 5.The light chain (L) derived from any vertebrate species can be assigned to one of two distinct types, called κ and λ, based on the amino acid sequence of its constant domain. Depending on the amino acid sequence of the constant domain of the heavy chain (CH), immunoglobulins can be assigned to various classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain named α, δ, ε, γ, and μ, respectively. Based on relatively small differences in CH sequence and function, γ and α are further classified into subclasses; for example, humans express the subclasses IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgK1.

[0106] In this application, the term "CDR" is also called "complementarity-determining region" and generally refers to a region within the variable domain of an antibody whose sequence is hypervariable and / or forms a structural loop. Generally, antibodies contain six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). In some embodiments, naturally occurring camelid antibodies consisting only of heavy chains can also function normally and be stabilized in the absence of light chains. See, for example, Non-Patent Document 6.

[0107] In this application, the term "FR" generally refers to a more highly conserved portion within the antibody variable domain, known as the framework region. Typically, the natural heavy and light chain variable domains each contain four FR regions, namely four in the VH (H-FR1, H-FR2, H-FR3, and H-FR4) and four in the VL (L-FR1, L-FR2, L-FR3, and L-FR4). For example, the VL of the isolated antigen-binding protein in this application may contain framework regions L-FR1, L-FR2, L-FR3, and L-FR4. The VH of the isolated antigen-binding protein in this application may contain framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0108] In this application, the term “antigen-binding fragment” generally refers to one or more fragments that have the ability to specifically bind to an antigen (e.g., CLDN18.2). In this application, antigen-binding fragments may include Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv and / or dAb.

[0109] In this application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As described above, an intact antibody can be digested with papain. The antibody, upon papain digestion, produces two identical antigen-binding fragments, namely the "Fab" fragment and the remaining "Fc" fragment (i.e., the Fc region, same as above). The Fab fragment may consist of one complete light chain and a variable region of the heavy chain and a first constant region (CH1) of the heavy chain (VH).

[0110] In this application, the term "Fab' fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which is a fragment slightly larger than a Fab fragment. For example, a Fab' fragment may include all of the light chain, all of the variable domains of the heavy chain, and all or part of the first and second constant regions of the heavy chain. For example, a Fab' fragment may also include some or all of the 220-330 amino acid residues of the heavy chain.

[0111] In this application, the term "F(ab')2" generally refers to an antibody fragment produced by digesting an intact antibody with pepsin. The F(ab')2 fragment includes two disulfide-bonded Fab fragments and a portion of a hinge region. The F(ab')2 fragment has divalent antigen-binding activity and can crosslink antigens.

[0112] In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody that includes all or part of the heavy chain variable region and the light chain variable region, but lacks the heavy chain constant region and the light chain constant region. The heavy chain variable region and the light chain variable region include, for example, the CDR. For example, an Fv fragment includes all or part of the amino-terminal variable regions of the heavy chain and the light chain, which consist of about 110 amino acids.

[0113] In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain and heavy chain variable regions are adjacent (via a synthetic linker, e.g., a short, flexible polypeptide linker) and can be expressed in the form of a single-chain polypeptide, retaining the specificity of the intact antibody from which the scFv is derived. Unless otherwise noted, as used in this application, an scFv may have the VL and VH variable regions in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), and an scFv may comprise a VL linker-VH or a VH-linker-VL.

[0114] In this application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having either a VH domain or a VL domain. See, for example, Non-Patent Documents 7 and 8, and, for example, Patent Documents 1 and 2 and other published patent applications of Domantis Ltd.

[0115] In this application, the term "monoclonal antibody" generally refers to a preparation of an antibody molecule with a single molecular composition. Monoclonal antibodies have high specificity for a single antigenic site. In contrast to conventional (polyclonal) antibody preparations, which contain different antibodies against antigenic determinants (epitopes), each monoclonal antibody targets a single antigenic determinant on an antigen. In addition to its specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma cultures that are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates a characteristic of antibodies obtained from a substantially homogeneous antibody population and is not interpreted as requiring antibody production by any particular method. For example, the monoclonal antibodies used in this application can be produced in hybridoma cells or by recombinant DNA methods.

[0116] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region originates from one species and the constant region originates from another species. Typically, the variable region originates from antibodies of experimental animals such as rodents ("parental antibodies"), and the constant region originates from human antibodies. Therefore, the resulting chimeric antibody is less likely to cause a harmful immune response in human individuals compared to parental (e.g., mouse-derived) antibodies.

[0117] In this application, the term “humanized antibody” generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulin. Small additions, deletions, insertions, substitutions, or modifications of amino acids in the CDR region are also permissible, as long as they retain the ability of the antibody to bind to a particular antigen. A humanized antibody optionally includes at least a portion of the constant region of a human immunoglobulin. A “humanized antibody” retains antigen specificity similar to that of the original antibody. A “humanized” form of a non-human (e.g., mouse) antibody may, at a minimum, include a chimeric antibody of a sequence derived from a non-human immunoglobulin. In some cases, CDR region residues of a human immunoglobulin (receptor antibody) may be replaced with residues in the CDR region of a non-human species (donor antibody) (e.g., mouse, rat, or non-human primate) having the desired properties, affinity, and / or capabilities. In some cases, FR region residues of a human immunoglobulin may be replaced with corresponding non-human residues. Humanized antibodies may contain amino acid modifications not present in receptor antibodies or donor antibodies. These modifications can be made to further improve antibody properties such as binding affinity.

[0118] The term "fully human antibody" generally refers to an antibody containing only human immunoglobulin protein sequences. Fully human antibodies may contain mouse glycans when produced in mice, mouse cells, or hybridomas derived from mouse cells. Similarly, "mouse antibody" or "rat antibody" refers to an antibody containing only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans, transgenic animals having human immunoglobulin germline sequences, by phage display or other molecular biological methods. Exemplary techniques that can be used to produce antibodies are described in Patent Documents 3, 4, and 5. Other techniques, such as the use of libraries, are known in the art.

[0119] In this application, the term "directly linked" is used in contrast to the term "indirectly linked," and generally refers to direct linking. For example, direct linking may occur when substances are directly linked to each other without the use of a spacer. The spacer may be a linker. For example, the linker may be a peptide linker. The term "indirectly linked" generally refers to cases where substances are not directly linked to each other. For example, indirect linking may occur via a spacer. For example, in the isolated antigen-binding protein of this application, the C-terminus of L-FR1 and the N-terminus of LCDR1 may be linked directly or indirectly.

[0120] In this application, the term “isolated nucleic acid molecule” generally refers to a nucleotide, deoxyribonucleotide, or ribonucleotide of any length in an isolated form, or an analogue thereof isolated from the natural environment or artificially synthesized.

[0121] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle capable of inserting protein-coding polynucleotides and expressing proteins. Vectors can express the genetic material elements carried by the vector in host cells by transforming, transfecting, or transfecting them. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages such as lambda phages or M13 phages, and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). Vectors may contain various elements that control expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. Vectors may also contain origins of replication. Vectors may contain components that facilitate entry into cells, such as viral particles, liposomes, or capsids, but they are not limited to these.

[0122] In this application, the term “cell” generally refers to a single cell, cell line, or cell culture that is or was a recipient of a plasmid or vector of a subject, comprising the nucleic acid molecules or vectors described in this invention. A cell may include the offspring of a single cell. Due to natural, accidental, or intentional mutation, the offspring may not necessarily be identical (in terms of total DNA complement morphology or genome) to the original parent cell. A cell may include a cell transfected in vitro with the vector described in this application. A cell may be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cells such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, or stem cells (e.g., ES cells, iPS cells, mesenchymal stem cells) or immune cells (e.g., T cells, NK cells, NKT cells, macrophages). In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell.

[0123] In this application, the term “pharmaceutical composition” generally refers to a composition for preventing / treating a disease or disorder. The pharmaceutical composition may include an isolated antigen-binding protein as described in this application, a nucleic acid molecule as described in this application, a carrier and / or a cell as described in this application, and optionally a pharmaceutically acceptable adjuvant. The pharmaceutical composition may also include one or more suitable formulations of a (pharmaceutically effective) carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier and / or preservative. The acceptable components of the composition are preferably nontoxic to the recipient at the dose and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0124] In this application, the term “pharmaceutically acceptable carrier” includes pharmaceutically acceptable vectors, excipients, or stabilizers that are nontoxic to cells or mammals to which they are exposed at commonly used doses and concentrations. Examples of physiologically acceptable carriers include buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming alleles (e.g., sodium), and / or nonionic surfactants.

[0125] In this application, the terms “specifically binding” or “specific” generally refer to measurable and reproducible interactions that can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules), such as binding between a target and an antibody. For example, an antibody that specifically binds to a target (which may be an epitope) may be an antibody that binds to that target with higher affinity, binding strength, ease, and / or longer duration than an antibody that binds to other targets. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved across different species of proteins. In some embodiments, specific binding may include, but is not required, exclusive binding.

[0126] In this application, the term "reference antibody" generally refers to an antibody capable of binding to an antigen (e.g., CLDN18.2). In some cases, the antigen-binding protein described in this application does not have competitive binding activity compared to the reference antibody. In some cases, the reference antibody described in this application may be zolbetuximab.

[0127] In this application, the term “substantially non-binding” generally means not binding at all, or binding with very weak binding activity. Here, very weak binding activity means, for example, in a flow cytometry binding activity assay, the average fluorescence intensity of an antibody detected to bind to CLDN18.1 is at least 50%, 55%, 60%, 70%, 80%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% lower than that of a CLDN18.1 antibody-positive control.

[0128] In this application, the term “subject” generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cattle, sheep, rabbits, mice, rats, or monkeys.

[0129] In this application, the term “tumor” generally refers to a neoplasm or solid lesion formed by abnormal cell proliferation. In this application, a tumor may be a solid tumor or a hematological tumor. For example, in this application, a tumor may be a CLDN18.2-positive tumor.

[0130] The term “cancer” generally refers to a disease characterized by the rapid and uncontrolled proliferation of abnormal cells. Cancer cells can spread locally through the bloodstream or lymphatic system, or to other parts of the body. Cancer as used in this application includes, but is not limited to, gastric cancer and / or colon cancer. The terms “tumor” and “cancer” are used interchangeably herein, and both terms, for example, encompass solid tumors and hematological malignancies, such as diffuse tumors or circulating tumors. As used herein, the terms “cancer” or “tumor” may include precancerous and malignant cancers and tumors.

[0131] In this application, such protein, polypeptide and / or amino acid sequence should be understood to include at least a variant or homolog having the same or similar function as the protein or polypeptide.

[0132] In this application, the variant may be, for example, a protein or polypeptide in which one or more amino acids in the amino acid sequence of the protein and / or polypeptide (e.g., an antibody or fragment thereof that specifically binds to the CLDN18.2 protein) are substituted, deleted, or added. For example, a functional variant may include a protein or polypeptide having amino acid modifications by substituting, deleting, or adding at least one amino acid, such as 1 to 30, 1 to 20, or 1 to 10, and further, for example, one, two, three, four, or five amino acids. The functional variant may substantially maintain the biological properties of the protein or polypeptide before modification (e.g., substitution, deletion, or addition). For example, the functional variant may maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding ability) of the protein or polypeptide before modification. For example, the substitution may be a conservative substitution.

[0133] In this application, the homolog may be a protein or polypeptide having at least about 85% (for example, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the protein and / or polypeptide (for example, an antibody or fragment thereof that specifically binds to the CLDN18.2 protein).

[0134] In this application, homology generally refers to the approximation, similarity, or association between two or more sequences. The "percentage of sequence homology" can be calculated by the following method: Compare two sequences aligned in a comparison window, determine the number of positions in the two sequences that contain the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met), obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., the size of the window), and multiply the result by 100 to obtain the percentage of sequence homology. Alignment to determine the percentage of sequence homology can be performed using various methods known in the art, 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 aligning sequences, including any algorithm necessary to achieve maximum alignment across the entire length of the sequences being compared or across the region of the target sequence. Such homology can also be measured by the FASTA and BLAST methods. For a description of the FASTA algorithm, see Non-Patent Documents 9, 10, and 11. For a description of the BLAST algorithm, see Non-Patent Document 12.

[0135] In this application, the term "includes" generally means to include, summarize, contain, or encompass. In some cases, it may also mean "for" or "consisting of."

[0136] In this application, the term "approximately" generally means fluctuating within a range of ±0.5% to 10% of a given value, for example, fluctuating within a range of ±0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of a given value.

[0137] (Detailed description of the invention) The CDR of an antibody, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region can come into contact with an antigen or antigenic epitope. The CDR of an antibody can be determined by several numbering systems, including CCG, Kabat, Chothia, IMGT, and Kabat / Chothia. These numbering systems are known in the art, and specific examples can be found at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different numbering systems based on the antibody sequence and structure. Differences in the CDR region may occur when using different numbering systems. In this application, the CDR may encompass CDR sequences obtained by any CDR assignment method, and also encompass variants thereof. The variants include amino acid sequences of the CDR in which one or more amino acids are substituted, deleted, and / or added. For example, 1 to 30, 1 to 20, or 1 to 10 amino acids may be substituted, deleted, and / or inserted, and these homologs may also be included, and such homologs may be amino acid sequences having at least about 85% (for example, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the CDR.

[0138] In one embodiment, the present application provides an isolated antigen-binding protein that may contain HCDR3. In the present application, the HCDR3 may contain the amino acid sequence shown in SEQ ID NO: 67. For example, the HCDR3 may contain the amino acid sequence shown in SEQ ID NO: 4. For example, the HCDR3 may contain the amino acid sequence shown in SEQ ID NO: 12. For example, the HCDR3 may contain the amino acid sequence shown in SEQ ID NO: 18.

[0139] In this application, the isolated antigen-binding protein may include HCDR2. In this application, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 68. In this application, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 11. For example, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 3. For example, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 17.

[0140] In this application, the isolated antigen-binding protein may include HCDR1. In this application, HCDR1 may include the amino acid sequence shown in SEQ ID NO: 2. In this application, HCDR1 may include the amino acid sequence shown in SEQ ID NO: 10. In this application, HCDR1 may include the amino acid sequence shown in SEQ ID NO: 16.

[0141] In this application, the isolated antigen-binding protein may include HCDR1, HCDR2, and HCDR3. In this application, HCDR1 may include the amino acid sequence shown in any of SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 16, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 68 or SEQ ID NO: 11, and HCDR3 may include the amino acid sequence shown in SEQ ID NO: 67.

[0142] In this application, the isolated antigen-binding protein HCDR1 may include an amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 16; HCDR2 may include an amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 17, and SEQ ID NO: 11; and HCDR3 may include an amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 12, and SEQ ID NO: 18.

[0143] In this application, the isolated antigen-binding proteins HCDR1, HCDR2, and HCDR3 may contain amino acid sequences selected from any of the following groups: 1) The HCDR1 may include the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 3, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 4. 2) The HCDR1 may include the amino acid sequence shown in SEQ ID NO: 10, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 11, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 12. 3) The HCDR1 may include the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 18.

[0144] In this application, the isolated antigen-binding protein may include H-FR1, the C-terminus of H-FR1 being directly or indirectly ligated to the N-terminus of HCDR1, and H-FR1 may include the amino acid sequence shown in SEQ ID NO: 72. For example, H-FR1 may include the amino acid sequence shown in SEQ ID NO: 34. For example, H-FR1 may include the amino acid sequence shown in SEQ ID NO: 43. For example, H-FR1 may include the amino acid sequence shown in SEQ ID NO: 49.

[0145] In this application, the isolated antigen-binding protein may include H-FR2, which is located between HCDR1 and HCDR2 and may include the amino acid sequence shown in SEQ ID NO: 73. For example, H-FR2 may include the amino acid sequence shown in SEQ ID NO: 35. For example, H-FR2 may include the amino acid sequence shown in SEQ ID NO: 44.

[0146] In this application, the isolated antigen-binding protein may include H-FR3, which is located between HCDR2 and HCDR3 and may include the amino acid sequence shown in SEQ ID NO: 80. In this application, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 45. For example, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 36. For example, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 42.

[0147] In this application, the isolated antigen-binding protein may include H-FR4, the N-terminus of H-FR4 being ligated to the C-terminus of HCDR3, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 74. For example, H-FR4 may include the amino acid sequence shown in SEQ ID NO: 46. For example, H-FR4 may include the amino acid sequence shown in SEQ ID NO: 37.

[0148] In this application, the isolated antigen-binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4. In this application, H-FR1 may include the amino acid sequence shown in SEQ ID NO: 72, H-FR2 may include the amino acid sequence shown in SEQ ID NO: 73, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 80 or SEQ ID NO: 45, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 74.

[0149] In this application, H-FR1 may include an amino acid sequence indicated by any of SEQ ID NO: 34, SEQ ID NO: 43, and SEQ ID NO: 49; H-FR2 may include an amino acid sequence indicated by SEQ ID NO: 35 or SEQ ID NO: 44; H-FR3 may include an amino acid sequence indicated by SEQ ID NO: 36 or SEQ ID NO: 42; and H-FR4 may include an amino acid sequence indicated by SEQ ID NO: 46 or SEQ ID NO: 37.

[0150] In this application, the isolated antigen-binding proteins H-FR1, H-FR2, H-FR3, and H-FR4 may contain amino acid sequences selected from any of the following groups: 1) H-FR1 may include the amino acid sequence shown in SEQ ID NO: 34, H-FR2 may include the amino acid sequence shown in SEQ ID NO: 35, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 36, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 37. 2) H-FR1 may include the amino acid sequence shown in SEQ ID NO: 43, H-FR2 may include the amino acid sequence shown in SEQ ID NO: 44, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 45, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 46. 3) H-FR1 may include the amino acid sequence shown in SEQ ID NO: 34, H-FR2 may include the amino acid sequence shown in SEQ ID NO: 35, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 42, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 37. 4) H-FR1 may include the amino acid sequence shown in SEQ ID NO: 49, H-FR2 may include the amino acid sequence shown in SEQ ID NO: 35, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 42, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 37.

[0151] In this application, the isolated antigen-binding protein may include HCDR1, HCDR2, HCDR3, H-FR1, HFR2, HFR3, and H-FR4. For example, HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3, and H-FR4 may each sequentially include the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 42, and SEQ ID NO: 37, or the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 49, SEQ ID NO: 35, SEQ ID NO: 42, and SEQ ID NO: 37.

[0152] In this application, the isolated antigen-binding protein may include VH. In this application, VH may include the amino acid sequence shown in SEQ ID NO: 78. For example, VH may include the amino acid sequence shown in SEQ ID NO: 1. For example, VH may include the amino acid sequence shown in SEQ ID NO: 9. For example, VH may include the amino acid sequence shown in SEQ ID NO: 15. For example, VH may include the amino acid sequence shown in SEQ ID NO: 19.

[0153] In this application, the isolated antigen-binding protein may include LCDR3. In this application, LCDR3 may include the amino acid sequence shown in SEQ ID NO: 69. For example, LCDR3 may include the amino acid sequence shown in SEQ ID NO: 8. For example, LCDR3 may include the amino acid sequence shown in SEQ ID NO: 14. For example, LCDR3 may include the amino acid sequence shown in SEQ ID NO: 23. For example, LCDR3 may include the amino acid sequence shown in SEQ ID NO: 25.

[0154] In this application, the isolated antigen-binding protein may include LCDR2. In this application, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 70. For example, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 7. For example, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 22.

[0155] In this application, the isolated antigen-binding protein may include LCDR1. In this application, LCDR1 may include the amino acid sequence shown in SEQ ID NO: 71. For example, LCDR1 may include the amino acid sequence shown in SEQ ID NO: 6. For example, LCDR1 may include the amino acid sequence shown in SEQ ID NO: 21.

[0156] In this application, the isolated antigen-binding protein may include LCDR1, LCDR2, and LCDR3. For example, LCDR1 may include the amino acid sequence shown in SEQ ID NO: 71, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 70, and LCDR3 may include the amino acid sequence shown in SEQ ID NO: 69.

[0157] In this application, the isolated antigen-binding protein LCDR1 may include the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 21, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 22, and the LCDR3 may include the amino acid sequence shown in any of SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 23, and SEQ ID NO: 25.

[0158] In this application, the isolated antigen-binding proteins LCDR1, LCDR2, and LCDR3 may contain amino acid sequences selected from any of the following groups: 1) LCDR1 may include the amino acid sequence shown in SEQ ID NO: 6, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 7, and LCDR3 may include the amino acid sequence shown in SEQ ID NO: 8. 2) LCDR1 may include the amino acid sequence shown in SEQ ID NO: 6, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 7, and LCDR3 may include the amino acid sequence shown in SEQ ID NO: 14. 3) LCDR1 may include the amino acid sequence shown in SEQ ID NO: 21, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 22, and LCDR3 may include the amino acid sequence shown in SEQ ID NO: 23. 4) LCDR1 may include the amino acid sequence shown in SEQ ID NO: 6, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 7, and LCDR3 may include the amino acid sequence shown in SEQ ID NO: 25.

[0159] In this application, the isolated antigen-binding protein may include L-FR1, the C-terminus of L-FR1 being directly or indirectly ligated to the N-terminus of LCDR1. In this application, L-FR1 may include the amino acid sequence shown in SEQ ID NO: 75. For example, L-FR1 may include the amino acid sequence shown in SEQ ID NO: 38. For example, L-FR1 may include the amino acid sequence shown in SEQ ID NO: 47. For example, L-FR1 may include the amino acid sequence shown in SEQ ID NO: 50. For example, L-FR1 may include the amino acid sequence shown in SEQ ID NO: 53.

[0160] In this application, the isolated antigen-binding protein may include L-FR2, which is located between LCDR1 and LCDR2. In this application, L-FR2 may include the amino acid sequence shown in Sequence ID No. 39.

[0161] In this application, the isolated antigen-binding protein may include L-FR3, which is located between LCDR2 and LCDR3. In this application, L-FR3 may include the amino acid sequence shown in SEQ ID NO: 76. For example, L-FR3 may include the amino acid sequence shown in SEQ ID NO: 40. For example, L-FR3 may include the amino acid sequence shown in SEQ ID NO: 48. For example, L-FR3 may include the amino acid sequence shown in SEQ ID NO: 51.

[0162] In this application, the isolated antigen-binding protein may include L-FR4, the N-terminus of which is ligated to the C-terminus of which is which is which. In this application, the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 77. For example, the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 41. For example, the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 52. For example, the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 54.

[0163] In this application, the isolated antigen-binding protein may include L-FR1, L-FR2, L-FR3, and L-FR4. For example, L-FR1 may include the amino acid sequence shown in SEQ ID NO: 75, L-FR2 may include the amino acid sequence shown in SEQ ID NO: 39, L-FR3 may include the amino acid sequence shown in SEQ ID NO: 76, and L-FR4 may include the amino acid sequence shown in SEQ ID NO: 77.

[0164] In this application, the isolated antigen-binding protein L-FR1 may include an amino acid sequence represented by any of SEQ ID NO: 38, SEQ ID NO: 47, SEQ ID NO: 50, and SEQ ID NO: 53; L-FR2 may include an amino acid sequence represented by SEQ ID NO: 39; L-FR3 may include an amino acid sequence represented by any of SEQ ID NO: 40, SEQ ID NO: 48, and SEQ ID NO: 51; and L-FR4 may include an amino acid sequence represented by any of SEQ ID NO: 41, SEQ ID NO: 52, and SEQ ID NO: 54.

[0165] In this application, the isolated antigen-binding proteins L-FR1, L-FR2, L-FR3, and L-FR4 may contain any of the following amino acid sequences: 1) L-FR1 may include the amino acid sequence shown in SEQ ID NO: 38, L-FR2 may include the amino acid sequence shown in SEQ ID NO: 39, L-FR3 may include the amino acid sequence shown in SEQ ID NO: 40, and L-FR4 may include the amino acid sequence shown in SEQ ID NO: 41. 2) L-FR1 may include the amino acid sequence shown in SEQ ID NO: 47, L-FR2 may include the amino acid sequence shown in SEQ ID NO: 39, L-FR3 may include the amino acid sequence shown in SEQ ID NO: 48, and L-FR4 may include the amino acid sequence shown in SEQ ID NO: 41. 3) L-FR1 may include the amino acid sequence shown in SEQ ID NO: 50, L-FR2 may include the amino acid sequence shown in SEQ ID NO: 39, L-FR3 may include the amino acid sequence shown in SEQ ID NO: 51, and L-FR4 may include the amino acid sequence shown in SEQ ID NO: 52. 4) L-FR1 may include the amino acid sequence shown in SEQ ID NO: 53, L-FR2 may include the amino acid sequence shown in SEQ ID NO: 39, L-FR3 may include the amino acid sequence shown in SEQ ID NO: 40, and L-FR4 may include the amino acid sequence shown in SEQ ID NO: 54.

[0166] In this application, the isolated antigen-binding protein comprises LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3, and L-FR4. For example, LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3, and L-FR4 may each sequentially include the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 47, SEQ ID NO: 39, SEQ ID NO: 48, and SEQ ID NO: 41, the amino acid sequences shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 50, SEQ ID NO: 39, SEQ ID NO: 51, and SEQ ID NO: 52, or the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 53, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 54.

[0167] In this application, the isolated antigen-binding protein comprises VL. In this application, VL may comprise the amino acid sequence shown in SEQ ID NO: 79. For example, VL may comprise the amino acid sequence shown in SEQ ID NO: 5. For example, VL may comprise the amino acid sequence shown in SEQ ID NO: 13. For example, VL may comprise the amino acid sequence shown in SEQ ID NO: 20. For example, VL may comprise the amino acid sequence shown in SEQ ID NO: 24.

[0168] In this application, the isolated antigen-binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. In this application, HCDR1 may include an amino acid sequence shown in any of SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 16; HCDR2 may include an amino acid sequence shown in SEQ ID NO: 68 or SEQ ID NO: 11; HCDR3 may include an amino acid sequence shown in SEQ ID NO: 67; LCDR1 may include an amino acid sequence shown in SEQ ID NO: 71; LCDR2 may include an amino acid sequence shown in SEQ ID NO: 70; and LCDR3 may include an amino acid sequence shown in SEQ ID NO: 69.

[0169] In this application, HCDR1 may include an amino acid sequence indicated by SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 16; HCDR2 may include an amino acid sequence indicated by SEQ ID NO: 3, SEQ ID NO: 17, and SEQ ID NO: 11; HCDR3 may include an amino acid sequence indicated by SEQ ID NO: 4, SEQ ID NO: 12, and SEQ ID NO: 18; LCDR1 may include an amino acid sequence indicated by SEQ ID NO: 6 or SEQ ID NO: 21; LCDR2 may include an amino acid sequence indicated by SEQ ID NO: 7 or SEQ ID NO: 22; and LCDR3 may include an amino acid sequence indicated by SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 23, and SEQ ID NO: 25.

[0170] In this application, the isolated antigen-binding proteins HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 may contain any of the following amino acid sequences: 1) The HCDR1 may include the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 3, the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 4, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 6, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 8. 2) The HCDR1 may include the amino acid sequence shown in SEQ ID NO: 10, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 11, the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 12, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 6, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 14. 3) The HCDR1 may include the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 17, the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 18, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 6, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 8. 4) The HCDR1 may include the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 17, the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 18, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 21, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 22, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 23. 5) The HCDR1 may include the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 17, the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 18, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 6, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 25.

[0171] In this application, the isolated antigen-binding protein may comprise VH and VL. In this application, VH may comprise the amino acid sequence shown in SEQ ID NO: 78, and VL may comprise the amino acid sequence shown in SEQ ID NO: 79.

[0172] In this application, the VH of the isolated antigen-binding protein may include an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 15, and SEQ ID NO: 19, and the VL may include an amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 20, and SEQ ID NO: 24.

[0173] In this application, the isolated antigen-binding protein may comprise VH and VL selected from any of the following groups: 1) VH may contain the amino acid sequence shown in SEQ ID NO: 1, VL may contain the amino acid sequence shown in SEQ ID NO: 5, 2) VH which may contain the amino acid sequence shown in SEQ ID NO: 15, VL which may contain the amino acid sequence shown in SEQ ID NO: 5, 3) VH may contain the amino acid sequence shown in SEQ ID NO: 9, VL may contain the amino acid sequence shown in SEQ ID NO: 13, 4) VH which may contain the amino acid sequence shown in SEQ ID NO: 19, VL which may contain the amino acid sequence shown in SEQ ID NO: 20, and 5) VH may contain the amino acid sequence shown in SEQ ID NO: 19, and VL may contain the amino acid sequence shown in SEQ ID NO: 24.

[0174] In this application, the isolated antigen-binding protein may include an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from the human IgG heavy chain constant region. In some embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from the human IgG1 heavy chain constant region.

[0175] In this application, the isolated antigen-binding protein may include an antibody light chain constant region. The antibody light chain constant region may be derived from a human Igκ constant region.

[0176] It should be noted that the isolated antigen-binding proteins of this application may contain heavy chain and / or light chain sequences with one or more conserved sequence modifications. So-called "conserved sequence modifications" refer to amino acid modifications that do not significantly affect or alter the antibody binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding proteins described herein through standard techniques known in the art, such as site mutations and PCR-mediated mutations. Conservative amino acid substitutions are substitutions of amino acid residues with similar side chains. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR region of the isolated antigen-binding proteins of this application can be substituted with other amino acid residues from the same side chain group. Those skilled in the art will know that some conserved sequence modifications do not result in loss of antigen-binding ability, specifically see, for example, Non-Patent Documents 13, 14, 15, 16, 17, and 18.

[0177] Various assays known in the art can be used to identify, screen, or characterize the physical / chemical properties and / or biological activity of the CLDN18.2 antigen-binding protein of this application.

[0178] In some embodiments, the antigen-binding activity of the antigen-binding protein or fusion protein of this application can be measured by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blotting), flow cytometry (e.g., FACS), immunohistochemistry, and immunofluorescence.

[0179] In this application, the isolated antigen-binding protein can specifically bind to the CLDN18.2 antigen. Antigen-binding proteins that "specifically bind" to the CLDN18.2 antigen typically bind to CLDN18.2 but do not, or substantially, bind to other proteins that lack the CLDN18.2 sequence. Whether an antigen-binding protein (e.g., an antibody) binds to the antigen CLDN18.2 can be confirmed using any assay known in the art. For example, fluorescence-activated cell sorting (FACS) can be used to measure the specific binding activity of the isolated antigen-binding protein to CLDN18.2. In some embodiments, the specific binding may be concentration-dependent. For example, in FACS binding activity experiments, the mean fluorescence intensity of CLDN18.2 increases with increasing CLDN18.2 antibody concentration.

[0180] In this application, the antigen-binding protein can bind to the human CLDN18.2 protein. The antigen-binding protein of this application may also cross-react with mouse (e.g., mouse) and / or monkey (e.g., cynomolgus monkey) CLDN18.2, which can be detected, for example, by FACS. In this application, “cross-reaction” means the ability of an antibody to react with homologous proteins of other species.

[0181] In this application, the antigen-binding protein can induce a CDC effect. For example, the CLDN18.2 antigen-binding protein of this application can induce a potent CDC effect in SP2 / 0-human CLDN18.2 cells. For example, the CLDN18.2 antigen-binding protein of this application can induce a potent CDC effect in MC38-human CLDN18.2 cells. For example, the induction method may be dose-dependent.

[0182] In this application, the isolated antigen-binding protein, as verified by FACS, does not substantially compete with the reference antibody for binding to CLDN18.2. The reference antibody may include a heavy chain variable region (VH) and a light chain variable region (VL), the VH of the reference antibody may include the amino acid sequence shown in SEQ ID NO: 55, and the VL of the reference antibody may include the amino acid sequence shown in SEQ ID NO: 56.

[0183] In this application, the antigen-binding protein can suppress tumor growth and / or proliferation of tumor cells. The tumor may be a CLDN18.2-expressing tumor (e.g., colon cancer). The colon cancer cells may be MC38-human CLDN18.2 cells.

[0184] Chimeric antigen receptor In another embodiment, the application also provides a chimeric antigen receptor (CAR) which may include a target moiety that binds to the CLDN18.2 protein, for example, the target moiety that binds to the CLDN18.2 protein may be the antigen-binding protein of the application. For example, the target moiety may exist in the form of an scFv. For example, the scFv may include the CDR, VH, and / or VL of the isolated antigen-binding protein of the application. For example, the VH and VL of the scFv may be linked by SEQ ID NO: 89. For example, the scFv may include the amino acid sequence shown in SEQ ID NO: 90.

[0185] In this application, the CAR may include other domains in addition to the extracellular target portion that binds to the CLDN18.2 protein.

[0186] In this application, the CAR may include a co-stimulatory signaling region capable of providing a stimulatory signal. For example, the co-stimulatory signaling region may include an intracellular co-stimulatory signaling region derived from one or more proteins selected from the group consisting of ligands for CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83, CD40, and MyD88.

[0187] For example, the co-stimulatory signaling region may be an intracellular co-stimulatory signaling region derived from 4-1BB. For example, the co-stimulatory signaling region may include the amino acid sequence shown in SEQ ID NO: 85.

[0188] In some cases, the CAR may include an intracellular signaling domain that may contain a domain having at least one ITAM motif. The intracellular signaling domain can transmit an activation signal into the cell. For example, the intracellular signaling domain may include an intracellular signaling domain derived from one or more proteins selected from the group consisting of domains containing CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma-associated herpesvirus (HSKV), DAP10, DAP-12, and other ITAMs.

[0189] For example, the intracellular signaling region may be a signaling domain derived from CD3ζ. For example, the intracellular signaling region may include the amino acid sequence shown in SEQ ID NO: 86.

[0190] In some cases, the CAR may include a transmembrane domain, which is a sequence within a cell surface protein that spans the cell membrane and may include a hydrophobic α-helix. The transmembrane domain can be derived from any type I transmembrane protein. The transmembrane domain may be a synthetic sequence that is predicted to form a hydrophobic helix. For example, the transmembrane domain may be derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0191] For example, the transmembrane domain may be a transmembrane domain derived from CD8. For example, the transmembrane domain may include the amino acid sequence shown in Sequence ID No. 84.

[0192] In some cases, the CAR may include a hinge region that may be located between the extracellular target portion and the transmembrane domain. For example, the hinge region may be derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0193] For example, the hinge region may be a hinge region derived from CD8. For example, the hinge region may include the amino acid sequence shown in Sequence ID No. 83.

[0194] In this application, the CAR may further include a signal peptide at the N-terminus of the target portion that binds to the binding CLDN18.2 protein. For example, the signal peptide may be a signal peptide derived from the CD8 protein. For example, the signal peptide may include the amino acid sequence shown in SEQ ID NO: 88.

[0195] In this application, the CAR may also include low-density lipoprotein receptor-related proteins or fragments thereof. For example, the low-density lipoprotein receptor-related proteins or fragments thereof may be located at the C-terminus of the CAR. For example, the low-density lipoprotein receptor-related proteins or fragments thereof may include low-density lipoprotein receptor-related proteins 1 to 12 and their functional fragments. For example, the low-density lipoprotein receptor-related proteins or fragments thereof may be low-density lipoprotein receptor-related proteins 5 and / or 6 or fragments thereof. For example, the low-density lipoprotein receptor-related proteins or fragments thereof may include the amino acid sequence shown in SEQ ID NO: 91.

[0196] In this application, the sequence of the low-density lipoprotein receptor-related protein or fragment within the CAR can be ligated to the C-terminal sequence of the CAR via a self-cleaving peptide (e.g., 2A peptides such as T2A, P2A, and E2A). For example, the low-density lipoprotein receptor-related protein or fragment can be ligated to the C-terminal of an intracellular signaling region via T2A. For example, the cleaving peptide may include the amino acid sequence shown in Sequence ID No. 87.

[0197] In this application, the CAR may sequentially include a target region (e.g., an antigen-binding protein) that binds to the CLDN18.2 protein from the N-terminus to the C-terminus, the hinge region, the transmembrane domain, the costimulatory signaling region, and the intracellular signaling region. For example, the CAR may sequentially include the scFv, a hinge region derived from CD8, a transmembrane domain derived from CD8, a costimulatory signaling region derived from 4-1BB, and an intracellular signaling region derived from CD3ζ, from the N-terminus to the C-terminus.

[0198] The present application also includes a vector capable of expressing the CAR or immune effector cells, the vector may sequentially include a nucleic acid molecule encoding a target portion (e.g., the antigen-binding protein) that binds to the CLDN18.2 protein, a nucleic acid molecule encoding the hinge region, a nucleic acid molecule encoding the transmembrane domain, a nucleic acid molecule encoding the costimulatory signaling region, a nucleic acid molecule encoding the intracellular signaling region, and a nucleic acid molecule encoding the low-density lipoprotein receptor-related protein or a fragment thereof.

[0199] The present application also includes a vector capable of expressing the CAR or immune effector cells, the vector may sequentially include a nucleic acid molecule encoding a target portion (e.g., the antigen-binding protein) that binds to the CLDN18.2 protein, a nucleic acid molecule encoding the hinge region, a nucleic acid molecule encoding the transmembrane domain, a nucleic acid molecule encoding the costimulatory signaling region, a nucleic acid molecule encoding the intracellular signaling region, a nucleic acid molecule encoding the cleaved peptide, and a nucleic acid molecule encoding the low-density lipoprotein receptor-related protein or a fragment thereof.

[0200] Polypeptide molecules, immune complexes, nucleic acid molecules, vectors, cells, and pharmaceutical compositions In another embodiment, the present application provides polypeptide molecules that may comprise the isolated antigen-binding protein or the chimeric antigen receptor of the present application.

[0201] In this application, the polypeptide molecule may include a fusion protein. For example, the isolated antigen-binding protein of this application can fuse with other functional molecules (e.g., antibodies or receptor ligands) to form a bispecific or multispecific molecule. The bispecific or multispecific molecule can specifically bind to at least two different binding sites or target molecules. The bispecific or multispecific molecule can be prepared by genetic modification, somatic hybridization, or chemical methods. For further details, see, for example, Non-Patent Documents 19 and 20.

[0202] In another embodiment, the application also provides an immune complex which may contain the isolated antigen-binding protein of the application.

[0203] In this application, the isolated antigen-binding protein or fragment thereof can be linked to another reagent such as a chemotherapeutic agent, toxin, immunotherapy agent, imaging probe, or spectroscopic probe. Such linkage may be by one or more covalent or non-covalent interactions and may include chelating activity. Multiple types of linkers (known in the art) can be used to form an immune complex. The immune complex may be provided in the form of a fusion protein that can be expressed from a polynucleotide encoding the immune complex. The immune complex may include, for example, an antibody-drug conjugate (ADC). In an ADC, the antibody and therapeutic agent can be crosslinked via a cleavable linker such as a peptide linker, disulfide linker, or hydrazone linker.

[0204] In another embodiment, the present application provides one or more nucleic acid molecules capable of encoding the isolated antigen-binding protein or the chimeric antigen receptor of the present application. For example, the nucleic acid molecules can be produced or synthesized by (i) in vitro amplification, for example by polymerase chain reaction (PCR) amplification; (ii) recombinant cloning; (iii) purification, for example by enzymatic cleavage and gel electrophoresis fractionation; or (iv) synthesis, for example by chemical synthesis.

[0205] In this application, the nucleic acid molecule may include any of the nucleotide sequences represented by SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.

[0206] In this application, the nucleic acid molecule may include a base sequence selected from any of the following groups: 1) The base sequence shown in Sequence ID No. 26, and the base sequence shown in Sequence ID No. 27, 2) The base sequence shown in Sequence ID No. 30, and the base sequence shown in Sequence ID No. 27, 3) The nucleotide sequence shown in Sequence ID No. 28, and the nucleotide sequence shown in Sequence ID No. 29, 4) The base sequence shown in Sequence ID No. 31, and the base sequence shown in Sequence ID No. 32, 5) The nucleotide sequence shown in SEQ ID NO: 31, and the nucleotide sequence shown in SEQ ID NO: 33.

[0207] In another embodiment, the present application provides a vector which may contain nucleic acid molecules of the present application. The vector may also contain other genes, such as marker genes, that enable selection of the vector in a suitable host cell and under suitable conditions. The vector may also contain expression regulatory elements that enable appropriate expression of coding regions in a suitable host. Such regulatory elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other regulatory elements that regulate gene transcription or mRNA translation. The vector can be used to express the genetic material elements carried by the vector in host cells by transforming, transducing, or transfecting the host cells. The vector may be a plasmid, cosmid, virus, phage, or other vector commonly used in genetic engineering. For example, the vector is an expression vector. The vector may also contain components that facilitate entry into cells, such as, but not limited to, viral particles, liposomes, or capsids.

[0208] In another embodiment, the present application provides cells that may contain the isolated antigen-binding protein of the present application, the chimeric antigen receptor of the present application, the polypeptide molecule of the present application, the nucleic acid molecule of the present application, or the vector of the present application. In some embodiments, each of the host cells may contain one or one nucleic acid molecule or vector of the present application. In some embodiments, each of the host cells may contain multiple (e.g., two or more) or multiple types (e.g., two or more) nucleic acid molecules or vectors of the present application. For example, the vector of the present application can be introduced into the host cell (e.g., a plant cell, a fungal or yeast cell, or other eukaryotic cell). In some embodiments, the cell may be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cell. The vector of the present application can be introduced into the host cell by methods known in the art.

[0209] In some embodiments, the cells may be immune effector cells. In some embodiments, the cells may be T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphocyte progenitor cells, and / or pluripotent stem cells.

[0210] In some embodiments, the cells may be T cells.

[0211] In this application, the cells may contain and / or express the CAR. In this application, the cells may contain and / or express the CAR and the low-density lipoprotein receptor-related protein or a fragment thereof.

[0212] In another embodiment, the application also provides a pharmaceutical composition which may comprise the isolated antigen-binding protein of the application, the chimeric antigen receptor of the application, the polypeptide molecule of the application, the immune complex of the application, the nucleic acid molecule of the application, the vector and / or cell of the application, and optionally a pharmaceutically acceptable carrier.

[0213] In some embodiments, the pharmaceutical composition may comprise one or more suitable formulations of pharmaceutically effective adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably nontoxic to the recipient at the doses and concentrations used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0214] In some embodiments, the pharmaceutical composition may contain one or more active compounds, for example, active compounds having complementary activities that generally do not adversely affect each other. The type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.

[0215] In some embodiments, the pharmaceutically acceptable carrier may include any solvent, dispersion medium, coating, isotonic agent, and absorption retarder suitable for drug administration, and is generally safe and non-toxic.

[0216] In some embodiments, the pharmaceutical composition may be administered parenterally, transdermally, intracavitaryly, intra-arterially, subarachnoidally and / or intranasally, or by direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject via infusion or injection. In some embodiments, the administration of the pharmaceutical composition may be carried out by different means such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the pharmaceutical composition may be administered without interruption. As described in Patent Document 6, uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the amount of therapeutic drug flowing into the patient's body.

[0217] Preparation method In another embodiment, the present application provides a method for preparing the antigen-binding protein. The method comprises culturing the cells described in the present application under conditions in which the isolated antigen-binding protein is expressed. This can be carried out, for example, by using a suitable culture medium, a suitable temperature and culture time, and such methods are understood by those skilled in the art.

[0218] Any suitable method for producing monoclonal antibodies can produce the antigen-binding protein of this application. For example, animals can be immunized with the bound or naturally occurring CLDN18.2 protein or a fragment thereof. Appropriate immunization methods, including adjuvants, immunostimulants, and repeated booster immunizations, can be used, and one or more routes can be used.

[0219] Any suitable form of CLDN18.2 can be used as an immunogen (antigen) to produce CLDN18.2-specific non-human antibodies and to screen the biological activity of said antibodies. The priming immunogen may be full-length mature human CLDN18.2 containing natural homodimers, or single / multiple epitope-containing peptides. The immunogen may be used alone or in combination with one or more immunoenhancing agents known in the art.

[0220] Chimeric human antibodies can be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. In this application, the antibody may be an IgG antibody, and the IgG1 subtype may be used. By screening the antibody using the biological assays described in the following examples, the necessary constant domain sequence can be optimized to produce the desired biological activity. Similarly, any type of light chain can be used in the compounds and methods of this application. For example, a κ chain or a variant thereof can be used in the compounds and methods of this application.

[0221] Method and Use In another embodiment, the application also provides the use of the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition in the preparation of pharmaceuticals for preventing, alleviating, and / or treating diseases and / or disorders.

[0222] In another embodiment, the application also provides methods for preventing, alleviating and / or treating diseases and / or disorders, the methods of which may include administering the isolated antigen-binding proteins, the chimeric antigen receptors, the polypeptide molecules, the immune complexes, the nucleic acid molecules, the vectors, the cells and / or the pharmaceutical compositions of the application to a subject of interest. In the application, such administration may be carried out by different means, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0223] In another embodiment, the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cells, and / or the pharmaceutical composition of this application can be used to prevent, alleviate, and / or treat diseases and / or disorders.

[0224] In this application, the aforementioned diseases and / or disorders may include cancer.

[0225] In this application, the term "cancer" may include solid tumors and / or hematological malignancies.

[0226] In this application, the term "cancer" may include CLDN18.2-positive tumors.

[0227] In this application, the term "cancer" may include gastric cancer and / or colon cancer.

[0228] In this application, the isolated antigen-binding protein may be administered together with one or more other antibodies to effectively suppress tumor growth in the subject. The isolated antigen-binding protein may also be administered together with a chemotherapeutic agent.

[0229] In another embodiment, the application also provides a method for detecting CLDN18.2 in a sample. The method comprises administering the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cells, and / or the pharmaceutical composition of the application. The method may be ex vivo and / or in vitro.

[0230] In another embodiment, the application also provides a reagent or kit for detecting CLDN18.2 in a sample, which may include the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cells, and / or the pharmaceutical composition.

[0231] In another aspect, the present application also provides the use of the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a reagent or kit for detecting CLDN18.2.

[0232] In the present application, the reagent or kit can be used to detect the presence and / or content of CLDN18.2 in a sample.

[0233] Without being bound by any theory, the following examples are for explaining the antigen-binding protein, preparation method and use of the present application, and are not intended to limit the scope of the invention of the present application.

Examples

[0234] (Example 1 Preparation of CLDN18.1 and CLDN18.2 Positive Control Antibodies) The CLDN18.1 positive control antibody is a commercially available anti-CLDN18 rabbit monoclonal antibody (abcam, Cat#ab203563), which can simultaneously recognize CLDN18.1 and CLDN18.2, and the antigen-binding site is intracellular. In the present application, this antibody is used as the CLDN18.1 positive control antibody and named Anti-Claudin18 antibody.

[0235] According to Patent Document 7, we commissioned Suzhou Jinweizhi Biotechnology Co., Ltd. to perform conventional gene synthesis based on the full-length amino acid sequences of the 175D10 clone (IMAB362) antibody heavy and light chains (for example, the amino acid sequences shown in SEQ ID NO: 81 and SEQ ID NO: 82). The genes were then cloned into our eukaryotic expression vectors pCMV-k and pCMV-IgG1 (NDL), and plasmids containing pCMV-IMAB362-VL and pCMV-IMAB362-VH were constructed. These two vector plasmids were simultaneously transfected into Expi293 cells in a 3:2 (VL:VH) ratio for transient expression. After 5 days, the cell culture supernatant was collected, purified by Protein A affinity chromatography, and BCA was quantified to obtain a CLDN18.2-positive control antibody, which was named zolbetuximab.

[0236] (Example 2: Construction of cell lines that stably and highly express CLDN18.1 and CLDN18.2) Following the nucleotide sequences of human CLDN18.1, human CLDN18.2, mouse CLDN18.1, mouse CLDN18.2, cynomolgus monkey CLDN18.1, and cynomolgus monkey CLDN18.2 shown in Table 1, conventional gene synthesis was commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd., and the genes were cloned into the lentivirus expression vector pHAGE-full EF1a-IzsGreen provided by our company. After activating puncture bacteria containing the obtained plasmids and performing plasmid extraction, lentiviruses were used to infect 293T cells, CHO, and SP2 / 0 cells using our lentivirus packaging and infection system. After detecting the infection efficiency of the cells obtained after infection using FACS, the cells were monoclonalized using gradient dilution and verified by FACS sorting to obtain monoclonal cell lines that stably and highly express CLDN18.1 and CLDN18.2.

[0237] [Table 1]

[0238] To validate cell lines that stably and highly express CLDN18.2 using FACS, 30,000 transfected cells were seeded in a V-shaped 96-well plate, the positive antibody solbetuximab was added, a PBS-negative control well was provided, and the cells were incubated at 4°C for 1 hour. The 96-well plate was then washed once with FACS buffer, and the secondary antibody (goat F(ab')2 anti-human IgG-Fc (DyLight® 650) (abcam, Cat#ab98593)) was added. After incubation at 4°C for 30 minutes, the 96-well plate was washed twice with FACS buffer. Subsequently, the fluorescence intensity of the cells was monitored using an iQue Screener flow cytometer (purchased from IntelliCyt), and monoclonal cells with the highest MFI value for proliferation were selected. These cells were then cultured and cryopreserved.

[0239] To validate cell lines that stably and highly express CLDN18.1 using FACS, cells were divided into two processing methods. One method involved counting the cells, then directly resuspending them in FACS buffer and plating them into well plates. The primary antibody used was solbetuximab, a CLDN18.2-positive antibody provided in Example 1. The other method involved immobilization and membrane disruption (using fixative FXP008 and membrane disruptor FXP009, manufactured by Shizheng Bai Biological Co., Ltd.) after cell counting, enabling the antibody to bind to antigen sites within the cells. The primary antibody used was the commercially available CLDN18 antibody provided in Example 1. These cells were simultaneously set in the instrument and measured. Monoclonal cells that strongly bound to the commercially available CLDN18 antibody but not to solbetuximab were selected, cultured, and cryopreserved.

[0240] The cell lines that stably and highly express the constructed CLDN18 are 293T-human CLDN18.1 (293T-human CLDN18.1), 293T-human CLDN18.2 (293T-human CLDN18.2), CHO-human CLDN18.1 (CHO-human CLDN18.1), CHO-human CLDN18.2 (CHO-human CLDN18.2), SP2 / 0-human CLDN18.1 (SP2 / 0-human CLDN18.1), and SP2 / 0-human CLDN18.2 (SP2 / 0-human They were labeled as CLDN18.2), 293T-mouse CLDN18.1 (293T-mouseCLDN18.1), 293T-mouse CLDN18.2 (293T-mouseCLDN18.2), 293T-cynomolgus monkey CLDN18.1 (293T-macacaCLDN18.1), and 293T-cynomolgus monkey CLDN18.2 (293T-macacaCLDN18.2), and all are monoclonal stable cell lines.

[0241] (Example 3: Generation of CLDN18.2 mouse antigen-binding protein) 1. Mouse Immunology Using the full-length nucleotide sequence of the first extracellular segment of human CLDN18.2, conventional gene synthesis was commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd., and the resulting plasmid was cloned into a eukaryotic expression vector provided by our company. The constructed plasmid was pCMV-CLDN18.2-D1, and a DNA immunogen was obtained by plasmid extraction from the resulting aspirated bacterial fluid. The cellular immunogen was SP2 / 0-human CLDN18.2 cells constructed in Example 2. In this experiment, a total of nine Balb / c mice were immunized. Six of these mice were immunized with a combination of cellular and DNA antigens, and the other three mice were immunized with whole-cell immunization. A classical mouse immunization schedule was used, and after two immunizations, mouse serum was collected. Serum titers were measured by FACS using 293T human CLDN18.2 cells, and mice meeting the titer requirements were selected for the final booster immunization. If the titer does not meet the requirements, it is necessary to increase the immunization by 1-2 times until the serum titer meets the requirements. Three days after the last immunization, mouse spleen tissue is collected under sterile conditions, the spleen cells are pulverized and made into a suspension, the spleen cells are subjected to erythrocyte hemolysis to lyse the spleen cells, and then divided into smaller portions and frozen in a refrigerator at -80°C.

[0242] 2. Hybridoma cell fusion Mouse myeloma cells SP2 / 0 were resuscitated beforehand and cultured and subcultured in DMEM medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions. On the day of fusion, PEG 1450 solution (Sigma Aldrich, Cat#25322-68-3) and high-glucose DMEM medium were preheated in a 37°C water bath. The SP2 / 0 cells were observed under a microscope, and it was determined that the cells were optimally in the logarithmic growth phase. The SP2 / 0 cells were collected by centrifugation at 400g for 5 minutes, resuspended in an appropriate amount of preheated DMEM medium, and counted with trypan blue. The cell viability needed to be 90% or higher, and then prepared by placing in a 37°C water bath. Mouse splenocytes were resuscitated in a 37°C water bath, washed with 20 ml of preheated DMEM, centrifuged at 400 g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 25 ml of preheated DMEM. The cells were diluted to a specific ratio and counted using a hemocytometer. The cells were mixed according to the cell number ratio (spleen cells: SP2 / 0 = 4:1), centrifuged at 400 g for 5 minutes, and the supernatant was discarded. Cell fusion was performed by slowly adding 1 ml of preheated PEG1450 solution. Total volume 1 × 10⁶ 7 Individual spleen cells were plated into 96-well plates, and the culture medium consisted of 10% FBS, 1% biantibody, 2% HAT supplement, and 10% ClonaCell. TM The medium used was HY Medium C (STEMCELL, CAT#:03803), a high-glucose DMEM medium. The medium was changed the day before measurement. The replacement medium was high-glucose DMEM medium containing 10% FBS, 1% biantibody, and 1% HT supplement. After 24 hours, the cell culture supernatant from a 96-well plate was taken and detected by flow cytometry.

[0243] 3. Flow cytometry screening of hybridoma cells Selected 293T-human CLDN18.2 cells and CHO-human CLDN18.2 cells constructed in Example 2 were fused, and the resulting parental clonal hybridoma cells were subjected to a first-round flow cytometry initial screening. That is, the cells were digested and counted separately, resuspended in flow cytometry staining buffer (PBS containing 0.1% BSA), and 1 × 10⁶ cells were used for the final screening.6 The cells were adjusted to a cell density of 1 / ml, 30 μl / well was added to a 96-well V-bottom plate, 50 μl / well of hybridoma cell culture supernatant was added to the 96-well V-bottom plate plated with cell antigens, and a positive control (5 μg / ml solbetuximab antibody, 30 μl / well) and a negative control (30 μl / well of PBS buffer) were added simultaneously. The cells were co-incubated at 4°C for 1 hour, washed once with flow cytometry staining buffer, the corresponding secondary antibody was added at 30 μl / well, the cells were co-incubated at 4°C for 30 minutes, washed twice with flow cytometry staining buffer, the cells were shaken to disperse, 25 μl of FACS buffer was added to each well, and the cells were set in the instrument for detection (iQue Screener flow cytometer, IntelliCyt). In the initial screening, parent clones showing CLDN18.2 positivity were selected and the cultures were expanded into 24-well cell culture plates. After 3 days, the cell supernatant was collected and re-screened with FACS to select four types of cells: CHO-human CLDN18.2, CHO-human CLDN18.1, 293T-human CLDN18.2, and 293T-human CLDN18.1. These were plated separately, and CLDN18.2-positive and CLDN18.1-negative hybridoma parent clones were screened from among them. Positive hybridoma parent clones were monocloned using gradient dilution, and after 2-3 rounds of subcloning and FACS selection, hybridoma monoclonal cells secreting CLDN18.2-specific antibodies were obtained. These cells could produce positive binding with 293T-human CLDN18.2 cells and CHO-human CLDN18.2 cells, but negative binding with 293T-human CLDN18.1 and CHO-human CLDN18.1 cells.

[0244] The results are shown in Table 2. The subclones that tested positive for binding to CLDN18.2 in the hybridoma cell culture supernatant included 3A6, 7E3, 5E6, 14E12, and 17B10, a total of five monoclonal cell lines.

[0245] [Table 2]

[0246] (Example 4: Sequencing of the variable region of the CLDN18.2 mouse antigen-binding protein) The sequences of the heavy chain variable region and light chain variable region of the CLDN18.2 mouse antigen-binding protein were obtained by sequencing using the degenerate primer amplification method. The primers used were synthesized by Suzhou Jinweizhi Co., Ltd., as referred to Non-Patent Literature 21. Specifically, five CLDN18.2-positive hybridoma monoclonal cells from Example 3 were collected, total cellular RNA was extracted using the QIAGEN RNeasy Plus Mini Kit, RNA integrity was detected by 1% agarose gel electrophoresis, and RNA concentration was measured using a NanoDrop nucleic acid quantitative analyzer. 1 µg of RNA was reverse transcribed into cDNA using an RNA reverse transcription kit and stored at -20°C. Using 5 μl of cDNA as a template, the PCR reaction system was constructed according to the operating instructions for the PCR high-fidelity enzyme (Beijing Quan Shi Jin Co., Ltd., AP231), the PCR procedure was set, the annealing temperature was set using gradient descent, and the second round of PCR was performed using 1 µl of the first round PCR product as a template. The PCR reaction system and procedure were the same as described above. All second-round PCR products were loaded onto 1% agarose gels for electrophoresis, and bands of appropriate size and suitability for specific purposes were excised. Gel recovery was performed, blunt-end cloning vectors (Beijing Quanshi Jin Co., Ltd., CB501) were ligated, and Trans1-T1 competent cells were transformed. These were then spread onto 2YT plates and incubated inverted in a 37°C incubator for 12-16 hours. Single clone colonies were selected using pipette tips on an ultra-clean workbench, and after the colonies were activated, sequencing was sent to Suzhou Jinweizhi Co., Ltd. Following sequencing, the VH and VL gene sequences of five hybridoma clones were obtained, as shown in Table 3.

[0247] [Table 3]

[0248] (Preparation of CLDN18.2 Antigen-Binding Protein, Example 5) Using the five heavy-chain and light-chain variable region sequences obtained by sequencing in Example 4 as templates, primers were designed respectively, and primer synthesis was commissioned to Suzhou Genewiz Biotechnology Co., Ltd. PCR amplification, agarose gel electrophoresis and gel recovery were performed using a high-fidelity enzyme. The recovered plasmid DNA was subjected to homologous recombination (Vazyme, C112) with eukaryotic expression vectors (pCMV-IgG1NDL and pCMV-κ) containing the human IgG1 constant region and cleaved with an enzyme. Suzhou Genewiz Biotechnology Co., Ltd. was requested to sequence and identify the correct positive recombinant vector. After correct sequencing, the plasmid was extracted, the heavy chain and the light chain were co-transfected into Expi293 cells, and after 5 days, centrifuged to recover the cell culture supernatant, which was purified by Protein A affinity chromatography to obtain complete human and mouse IgG1 and Igκ antigen-binding proteins. These five antigen-binding proteins were named c5E6, c7E3, c3A6, c14E12, and c17B10, respectively.

[0249] (Measurement of Specific Binding Activity of CLDN18.2 Antigen-Binding Protein and CLDN18.2, Example 6) By fluorescence-activated cell sorting technology (FACS), using an iQue Screener flow cytometer (purchased from IntelliCyt), the specific binding activity of the chimeric antibody to target cells was detected using PBS containing 0.1% BSA as a buffer. Three target cells, a stable transformed cell line expressing human CLDN18.2, a stable transformed cell line expressing human CLDN18.1, and a tumor cell line, were selected and the binding activities were measured respectively.

[0250] 1. Detection of Binding Activity of the Antigen-Binding Protein of Example 5 to High-Expression Human CLDN18.2 Cells by Flow Cytometry The cells were 293T-human CLDN18.2, CHO-human CLDN18.2, and SP2 / 0-human CLDN18.2 cells constructed in Example 2. The cells were digested and counted, and then resuspended in flow cytometry staining buffer. 1 × 10⁶ cells were used. 6 Cell density was adjusted to 30 μl / ml, and 30 μl / well was added to a 96-well V-bottom plate. Primary antibody was added at 30 μl / well, and seven concentration gradients were created by diluting the antibody from an initial concentration of 30 μg / ml with flow cytometry staining buffer in a 2-fold or 3-fold gradient. A PBS negative control was prepared for each antibody, and the positive control antibody was solbetuximab obtained by purification in Example 1. The cells were incubated at 4°C for 1 hour, washed once with flow cytometry staining buffer, and 30 μl / well of secondary antibody (abcam, Cat#ab98593) was added. The cells were incubated at 4°C for 30 minutes, washed twice with flow cytometry staining buffer, and the cells were shaken to disperse. 25 μl of flow cytometry staining buffer was added to each well, and the cells were allowed to stand until set up in the instrument. The original data was substituted into GraphPad 8.0 software, and the results of the graphing and calculations are shown in Figure 1.

[0251] 2. Detection of binding activity of the antigen-binding protein from Example 5 and high-expression human CLDN18.1 cells by flow cytometry. The positive control antibody was a commercially available anti-CLDN18 antibody (abcam, Cat#ab203563). The antigen-binding site is located in the intracellular portion of the CLDN18.2 4-transmembrane protein, requiring intracellular staining analysis by flow cytometry. Specifically, the cells used were 293T-human CLDN18.1 cells and SP2 / 0-human CLDN18.1 cells constructed in Example 1. After cell digestion and counting, the cells were immobilized and subjected to membrane disruption treatment. The treated cells were then stained 1 × 10⁶ cells in flow cytometry staining buffer. 6The cells were resuspended to a concentration of / ml and added to a 96-well V-bottom plate at a rate of 30 μl / well. Primary antibody was added at a rate of 30 μg / well, and seven concentration gradients were created by diluting the antibody from an initial concentration of 30 μg / ml with flow cytometry staining buffer in a 3-fold gradient. A PBS negative control was prepared for each antibody, and the dilution conditions for the positive control antibody were the same as above. The cells were incubated at 4°C for 1 hour, washed once with flow cytometry staining buffer, and 30 μl / well of secondary antibodies (abcam, Cat#ab98593 and Cat#ab150079) were added. The cells were incubated at 4°C for 30 minutes, washed twice with flow cytometry staining buffer, and the cells were shaken to disperse. 25 μl of flow cytometry staining buffer was added to each well, and the cells were allowed to stand until set up in the instrument. The original data was substituted into GraphPad 8.0 software, and the graphed and calculated results are shown in Figure 2.

[0252] 3. Detection of antigen-binding activity of antigen-binding proteins and tumor cell systems by flow cytometry in Example 5 Human CLDN18.2, constructed according to the method described in Example 2, was constructed, and MC38-human CLDN18.2 tumor cells that stably and highly express it were selected as target cells. The cells were digested and counted, resuspended in flow cytometry staining buffer, and 1 × 10⁶ samples were taken. 6 The solution was adjusted to / ml and 30 μl / well was added to a 96-well V-bottom plate. 30 μl / well of primary antibody was added, and seven concentration gradients were created by diluting the antibody from an initial concentration of 30 μg / ml with flow cytometry staining buffer in a 3-fold gradient. A PBS negative control was prepared for each antibody, and the positive control antibody was zolbetuximab purified in Example 1, with the same dilution conditions as above. The cells were incubated at 4°C for 1 hour, washed once with flow cytometry staining buffer, 30 μl / well of secondary antibody (abcam, Cat#ab98593) was added, incubated at 4°C for 30 minutes, washed twice with flow cytometry staining buffer, the cells were shaken to disperse, 25 μl of flow cytometry staining buffer was added to each well, and the cells were allowed to stand until set up in the instrument. The original data was substituted into GraphPad 8.0 software, and the graphed and calculated results are shown in Figure 3.

[0253] By the above procedures, five types of human and mouse antigen-binding proteins were expressed and purified, and their antigen-binding activities were verified by flow cytometry. As shown in Figure 1, all five types of antigen-binding proteins showed concentration-dependent binding activities to human CLDN18.2, and most of them were more potent than the solubilized zolbetuximab-positive antibody, and the detection results of the three types of cells were consistent. As shown in Figure 2, all five types of antigen-binding proteins could specifically bind to human CLDN18.2 but did not bind to human CLDN18.1. As shown in Figure 3, all five types of antigen-binding proteins strongly bound to mouse colon cancer MC38 cells that stably and highly expressed human CLDN18.2, and the strength of the binding activity showed concentration dependence, and some were more potent than the solubilized zolbetuximab-positive antibody.

[0254] (Example 7 Analysis of cross-species binding activity of CLDN18.2 antigen-binding protein) By fluorescence-activated cell sorting technology (FACS), using a CytoFLEX flow cytometer (purchased from BECKMAN COULTER), and using PBS containing 0.1% BSA as a buffer, the cross-species binding activities of the above antigen-binding proteins with mouse (mouseCLDN18.2) and cynomolgus monkey (macacaCLDN18.2) were detected. Specifically, 293T mouse CLDN18.2 cells and 293T cynomolgus monkey CLDN18.2 cells are stable transformed cell lines constructed in Example 2. The cells were digested and counted, and 1×10 6The cells were resuspended in flow cytometry staining buffer to a cell density of 1 / ml and added to a 96-well V-bottom plate at a rate of 30 μl / well. The primary antibody was gradient diluted 3-fold with flow cytometry staining buffer from an initial concentration of 10 μg / ml to create six concentration gradients. A PBS negative control was prepared for each antibody, and the positive control was zolbetuximab, with the same dilution conditions as above. The cells were incubated at 4°C for 1 hour, washed once with flow cytometry staining buffer, 30 μl / well of the secondary antibody (abcam, Cat#ab98593) was added, incubated at 4°C for 30 minutes, washed twice with flow cytometry staining buffer, the cells were shaken to disperse, 30 μl of flow cytometry staining buffer was added to each well, and the cells were set in the instrument for measurement. The original data was substituted into GraphPad 8.0 software, and the results of the graphs and calculations are shown in Figure 4. Five different CLDN18.2 chimeric antigen-binding proteins and the positive control antibody zolbetuximab all bind to mouse (mouseCLDN18.2) and cynomolgus monkey (macacaCLDN18.2), and the binding activity shows a gradient-dependent relationship. This indicates that these five chimeric antigen-binding proteins and zolbetuximab not only specifically bind to human CLDN18.2 (humanCLDN18.2), but also possess cross-species binding activity in humans, mice, and cynomolgus monkeys.

[0255] (Example 8: CDC activity of CLDN18.2 antigen-binding protein) The ability of CLDN18.2 antigen-binding proteins to induce CDC effects on SP2 / 0-human CLDN18.2 cells was detected using the cytotoxicity detection kit (Promega, Cat#G1780), and the specific process was as follows: (1) Prepare the culture medium (A: DMEM + 2% FBS + 1% biantibody, B: DMEM + 2% FBS + 1% biantibody + 10% rabbit complement), (2) Target cells SP2 / 0-human CLDN18.2 were centrifuged at 400g for 5 minutes, and then the target cell density was 4 × 10 5 Resuspend the cells in the above medium A to a concentration of cells / ml, and add 100 μl / well to a 96-well cell culture plate. (3) Dilute the antibody in medium B to three concentrations of 10 ug / ml, 2 ug / ml, and 0.4 ug / ml, add 100 μl / well to the detection well, create two overlapping wells at each concentration point, use solbetuximab as the positive control antibody and human IgG-Fc as the negative control. (4) The complement was Rabbit Complent 3-4 weeks (Cat#31061-3) purchased from PelFreez Bio, and the final complement concentration used in the experiment was 5%. (5) A control well was prepared according to the kit requirements, and the resulting mixture was co-incubated in a 37°C incubator for 4 hours. The absorbance value of 490 nM was then recorded using a microplate reader, the lysis rate of the target cells was calculated using the formula provided in the kit, and the data was analyzed and processed using GraphPad prism 8.

[0256] The results are shown in Figure 5a. When SP2 / 0-human CLDN18.2 cells were used as the target cells for killing, all detected antigen-binding proteins were able to induce a dose-dependent potent CDC effect, meaning they possessed significant CDC activity. In particular, c5E6, c7E3, and c17B10 showed higher CDC activity than zolbetuximab.

[0257] We further investigated the ability of the c5E6 antigen-binding protein to induce a CDC effect on CHO cells that stably overexpress human CLDN18.2. In short, CHO-human CLDN18.2 cells were constructed and obtained using our lentiviral transfection system as shown in Example 1, with the same procedure as described above, 1.2 × 10⁶ cells per well. 4Cells were plated at a cell density of 1, with a final rabbit complement concentration of 5%. Antigen-binding protein was diluted at a 5-fold gradient, i.e., 10 ug / ml, 2 ug / ml, 0.4 ug / ml, 0.08 ug / ml, 0.016 ug / ml, 0.0032 ug / ml, and 0.64 ng / ml. Two overlapping wells were provided for each concentration. Zolbetuximab was used as the positive control antibody, and human IgG-Fc as the isotype control antibody. After 4 hours of co-incubation, absorbance values ​​were measured using a microplate reader, and the lysis rate of target cells was calculated. The data were analyzed and processed using GraphPad prism 8.

[0258] The results are shown in Figure 5b, where the c5E6 antigen-binding protein was able to induce a dose-dependent potent CDC effect in CHO-human CLDN18.2 cells, exhibiting higher CDC activity than zolbetuximab.

[0259] (Example 9: Analysis of competitive binding activity of CLDN18.2 antigen-binding protein) c5E6 and zolbetuximab were labeled with biotin (EZ-Link Sulfo-NHS-LC-Biotin, Thermo, A39257), with 500 ug of each label. The labeling effect of the biotinylated antigen-binding protein molecules was measured using the OctetRED384 (sartorius) molecular interaction analyzer. An SA sensor was selected to immobilize the antibody, with an antibody concentration of 100 nM and a labeling time of 180 seconds. The labeled biotinylated antigen-binding proteins were named Biotinylated-c5E6, Biotinylated-c7E3, Biotinylated-c17B10, and Biotinylated-zolbetuximab.

[0260] Using fluorescence-activated cell sequencing (FACS), an iQue Screener flow cytometer (purchased from IntelliCyt) was used to detect the epitope competitive binding activity of the above antigen-binding protein and CHO-human CLDN18.2 cells (obtained in Example 2) in PBS containing 0.1% BSA as buffer. The specific process is as follows: (1) Using buffer, 1 × 10⁶ CHO-human CLDN18.2 cells 6 Prepare the solution at a concentration of cells / ml, and add 30 μl to each well of a 96-well V-bottom plate (corning 3894). (2) Prepare each antigen-binding protein to be tested in buffer so that the final concentration of the biotinylated-c5E6 antigen-binding protein is 0.5 μg / ml, and add 30 μl / well to a 96-well V-bottom plate. The final concentration of the biotinylated-zolbetuximab antigen-binding protein is 5 μg / ml, and add 30 μl / well to a 96-well V-bottom plate. (3) Prepare competitive antigen-binding proteins (c5E6, c7E3, c17B10 and zolbetuximab) in buffer, starting with a final antibody concentration of 333 ug / ml. Dilute the antibody in a 4-fold gradient to create 10 concentration gradients. PBS is provided as a negative control well. Add 30 μl / well of the prepared antibody at different concentrations to a 96-well V-bottom plate plated with target cells and the antibody to be tested, and mix thoroughly. (4) Incubate in a refrigerator at 4°C for 1 hour. (5) Add 110 μl of buffer to each well, centrifuge at 500 g for 5 minutes, discard the supernatant, and shake to disperse the cells. (6) Repeat step (5), (7) Prepare the fluorescent secondary antibody (BD phamingen APC Streptavidin, Cat#554067) in buffer in a 1:500 ratio, add 30 μl per well to the cells, mix well, and incubate in a refrigerator at 4°C for 30 minutes. (8) Add 170 μl of buffer to each well, centrifuge at 500 g for 5 minutes, discard the supernatant, and shake to disperse the cells. (9) Repeat step (8), (10) 25 μl of buffer was added to each well, mixed thoroughly, and then detected using a flow cytometer.

[0261] Figure 6 shows the results of flow cytometry epitope competitive binding activity detection. Both the isolated antigen-binding proteins c17B10 / c7E3 and biotinylated-c5E6 of this application were competitive, and the c17B10 / c7E3 antigen-binding protein completely competed with biotinylated-c5E6 for cell antigen binding at high concentrations (333 ug / ml), suggesting that they are the same antigen-binding epitope. In contrast, there was weak competition between zolbetuximab and biotinylated-c5E6. As the zolbetuximab antibody concentration increased, the median signal decreased, showing a certain concentration dependence. However, even when the zolbetuximab antibody concentration was increased to 333 ug / ml, the competitive activity and intensity were far weaker compared to other antigen-binding proteins. It was not possible to completely compete with the biotinylated-c5E6 antigen-binding protein, suggesting that the antigen-binding epitopes of zolbetuximab and c5E6 are not completely identical, but rather exhibit a cross-relationship or inclusion relationship (the epitope of zolbetuximab antibody is included in c5E6).

[0262] (Example 10: In vivo antitumor activity of CLDN18.2 antigen-binding protein) The antigen-binding proteins (c5E6, c7E3, c17B10) from Example 5, the positive control antibody zolbetuximab, and the isotype control antibody human IgG-Fc were selected, and their in vivo antitumor activity was measured in C57BL / 6 mice inoculated with mouse colon cancer cells. The above six antigen-binding proteins were obtained by purification using our Expi293 transient transfection system, and the endotoxin levels of the antigen-binding proteins were controlled to less than 4 EU / mg. The specific implementation scheme is as follows.

[0263] MC38 human CLDN18.2 cells were subcutaneously inoculated into the right ventral region of 115 female C57BL / 6 mice, with an inoculation volume of 1.1 × 10⁶ cells. 6 Seven days after vaccination, the tumor size was 31.33-116.31 mm. 3 (Average tumor size was 71.59 mm) 3 Eighty mice were selected and divided into 10 groups of eight mice each, according to tumor volume. The day the mice were randomly assigned to the groups was defined as Day 0. After group assignment, the mice were administered via the tail vein once a week for four consecutive weeks, divided into two groups: 30 mg / kg and 7.5 mg / kg. The mouse group assignments and administration plan are shown in Table 4. During the administration period, body weight and tumor size were measured three times a week. Tumor volume was calculated using the formula below, and tumor growth inhibition rate (TGI) and T / C were calculated based on tumor size. 3 When this was reached, the experiment was stopped and the mice were euthanized.

[0264] [Mathematics 1] TV = (length × width) 2 ) / 2

[0265] [Table 4]

[0266] The antitumor effects of zolbetuximab, c17B10, c5E6, and c7E3 administered via the tail vein through a subcutaneous MC38-human CLDN18.2 mouse colon cancer model were evaluated in female C57BL / 6 mice. The results showed that all administered mice were well-tolerated in the MC38-human CLDN18.2-bearing C57BL / 6 mice, and body weight remained stable without side effects. Changes in mouse body weight and relative changes (%) are shown in Figures 7 and 8, respectively. Analysis of tumor volume change curves and tumor growth inhibition curves prior to day 17 showed that the c5E6 and high-dose zolbetuximab groups exhibited better antitumor effects compared to the PBS control group, with a statistically significant difference in antitumor effect (****, P<0.0001), as shown in Figures 9 and 10. Analysis of Kaplan-Meier survival curves prior to day 40 revealed that, compared to the PBS control group, the low-dose c5E6 group, the high-dose c5E6 group, and the high-dose solbetuximab group all showed higher mouse survival rates, with a statistically significant difference (*, P<0.05). Among these, the c5E6 antigen-binding protein exhibited the strongest antitumor activity, as shown in Figure 11.

[0267] (Example 11: Production of CLDN18.2 single-chain antibody and identification of antigen-binding activity) In this example, cells stably expressing human CLDN18.2, i.e., SP2 / 0-hCLDN18.2 cells, were obtained using lentiviral infection and fluorescence-activated cell sorting. Cellular immunization was performed in Balb / c mice, with SP2 / 0-hCLDN18.2 cells as the immunogen. CLDN18.2-positive monoclonal cells were obtained using mouse hybridoma technology, flow cytometry screening, and subcloning, and IgG-type CLDN18.2 mouse monoclonal antibodies were obtained in vitro. Sequencing was performed using degenerate primer amplification to obtain the heavy chain variable region (V H ) and light chain variable region (V L The sequence of the antibody heavy chain variable region (V) is obtained via the synthetic linker peptide (Linker) gene. H ) and light chain variable region (V LThe recombinant gene is bound to the recombinant gene, and the antibody expressed by this recombinant gene is the CLDN18.2 single-chain antibody, namely 5E6-scFv (SEQ ID NO: 90). Lentivirus infection and fluorescence-activated cell sorting were performed simultaneously to obtain SP2 / 0-hCLDN18.1 cells that stably express human CLDN18.1.

[0268] Identification of specific binding activity of 5E6-scFv single-chain antibody and standard antibody (VH contains the amino acid sequence shown in SEQ ID NO: 92, and VL contains the amino acid sequence shown in SEQ ID NO: 93) by flow cytometry: SP2 / 0-hCLDN18.2 cells were counted, and the cells were resuspended in flow cytometry staining buffer. 6 The solution was adjusted to / ml and 30 μl / well was added to a 96-well V-bottom plate. Primary antibody was added at 30 μl / well, and seven concentration gradients were created by diluting the primary antibody from the working concentration of 80 μg / ml in a 4-fold gradient using flow cytometry staining buffer, with a PBS negative control prepared for each antibody. The cells were incubated at 4°C for 1 hour, washed once with flow cytometry staining buffer, and secondary antibody: goat F(ab')2 anti-human IgG-Fc (DyLight® 650) (abcam, Cat#ab98593) was added at 30 μl / well. The cells were incubated at 4°C for 30 minutes, washed twice with flow cytometry staining buffer, shaken to disperse the cells, and 25 μl / well of flow cytometry staining buffer was added. The cells were then awaited to be loaded into the machine. Simultaneously, SP2 / 0-hCLDN18.1 cells were counted and plated, and the primary antibody was diluted in a 3-fold gradient from the usage concentration of 10 ug / ml to create five concentration gradients. The secondary antibody was goat F(ab')2 anti-human IgG-Fc (DyLight® 650) (abcam, Cat#ab98593). The original data was input into GraphPad8.0 software for graphing and calculation, and Figure 12 shows that, similar to the standard antibody, the 5E6-scFv single-chain antibody can specifically bind to human CLDN18.2.

[0269] (Example 12: Identification of the binding activity of CLDN18.2 single-chain antibody against non-target cells) This example identified the binding activity of a 5E6-scFv single-chain antibody to various non-target cells in human tissue using fluorescence-activated cell sorting (FACS). The cells used were human precipitous fibroblasts (HFF), human immortalized epidermal keratinocytes (HaCaT), human normal hepatocytes (LO2), mesenchymal stem cells (MSC), normal human epidermal keratinocytes (KERA), human alveolar basal epithelial adenocarcinoma cells (A549), human breast cancer cells (MCF-7), and human dermal fibroblasts (BJ). Seven concentration gradients were created by diluting the antibody from a starting concentration of 30 ug / ml in a 3-fold gradient. The secondary antibody was goat F(ab')2 anti-human IgG-Fc (DyLight® 650) (abcam, Cat#ab98593). After incubation, the cells were centrifuged, resuspended in flow cytometry staining buffer, and set in the instrument. The results were then input into GraphPad 8.0 software and graphed. Figure 13 shows that the 5E6-scFv single-chain antibody did not exhibit nonspecific binding to various non-target cells in human tissue, and its specificity to tumor targets was initially verified to be the same as that of the standard antibody, meaning that there was no "off-target effect."

[0270] (Example 13: Antitumor activity of CLDN18.2 antigen-binding protein in a human gastric cancer mouse model) As in Example 10, human CLDN18.2 antigen-binding protein (c5E6), the positive control antibody zolbetuximab, and the isotype control antibody human IgG-Fc were selected to measure the in vivo antitumor activity in female nude mice inoculated with human gastric cancer cells (NUGC-4). The specific implementation scheme is as follows.

[0271] 28 female nude mice were subcutaneously inoculated with NUGC-4 cells, with an inoculation volume of 3 × 10⁶ cells. 6 Then, 7 days after vaccination, the tumor size was 80 mm. 3Twenty mice were selected and divided into four groups of five mice each, according to their tumor volume. The day the mice were randomly assigned to the groups was defined as Day 0. After group assignment, the mice were administered intraperitoneally twice a week for three consecutive weeks. The mouse group assignments and administration plan are shown in Table 5. During the administration period, body weight and tumor size were measured three times a week, and tumor volume was calculated using the following formula. A tumor volume of 2000 mm³ 3 When this was reached, the experiment was stopped and the mice were euthanized.

[0272] [Math 2] TV = (length × width) 2 ) / 2

[0273] [Table 5]

[0274] Using a subcutaneous NUGC-4 human gastric cancer tumor model, the antitumor effect of intraperitoneal administration of zolbetuximab and c5E6 antibody to nude mice was evaluated. The experiment was observed for 25 days after administration. The results showed that all administered mice, female nude mice with NUGC-4 tumors, were well-tolerated, and their body weight remained stable without side effects. The change in mouse body weight is shown in Figure 14A. The curve of change in tumor volume in mice during the observation period is shown in Figure 14B. Mice in the G3: zolbetuximab, 10 mg / kg group and the G4: c5E6, 10 mg / kg group showed consistent antitumor activity compared to the G2 isotype control group, with a statistically significant difference (p<0.05). There was no significant difference in antitumor activity between G3 and G4, indicating similar antitumor levels.

[0275] (Example 14: Stable expression of CLDN18.2-specific CAR in T cells) As shown in Figure 15A, the CLDN18.2-specific CAR structure in this embodiment consists of a human CD8 signal peptide (SEQ ID NO: 88), an anti-human CLDN18.2 single-chain antibody (SEQ ID NO: 90), a human CD8 hinge region (SEQ ID NO: 83), a human CD8 transmembrane domain (SEQ ID NO: 84), a human 4-1BB intracellular costimulatory domain (SEQ ID NO: 85), a human CD3ζ intracellular activation domain (SEQ ID NO: 86), and an added Ori novel element (SEQ ID NO: 91). First, this embodiment investigates the expression status of CLDN18.2-specific CARs in human T cells and the amplification factor of CART cells under conventional in vitro culture conditions. The specific method is as follows.

[0276] 1) Human PBMC cells frozen in liquid nitrogen were revived in a 37°C water bath, and centrifugation, resuspension, and rinsing (500g, 5 min, 400g, 5 min, 300g, 5 min) were performed three times in a system of 11ml PBS + 1ml PBMC. After rinsing, the human PBMC cells were mixed with CD3 MicroBeads, human (Miltenyi, 130-050-101), and CD3-positive selection was performed using a magnetic stand (i.e., CD3 + T cells were isolated and retained. CD3 + T cells were placed in a culture medium containing (4% FBS + X-VIVO (Lonza) + 20 ng / ml Factor I + 10 ng / ml Factor II) at a cell density of 1 × 10⁶ 6 The cells were resuspended to a concentration of cells / ml, and CD3 / CD28 magnetic beads (Thermo Fisher Scientific 40203D, washed twice with culture medium, aspirated with a magnetic stand, and allowed to stand for 1 minute) were added in a 1:3 ratio (cells:magnetic beads) to activate the T cells. The T cells were added to the magnetic beads and mixed well, and 700 μl of culture medium was added to each well of a 12-well plate, resulting in a cell count of 7 × 10⁶. 5 / well, density 1×10 6 The values ​​were given as cells / ml, and the sorting day was designated as day 0.

[0277] 2) Activated T cells were cultured in a 37°C CO2 incubator for 20 hours. Then, the corresponding viral supernatant was added at a ratio of 4 for the multiplicity of viral infection (MOI), and polybrene was added to a final concentration of 10 ug / ml. After mixing thoroughly by pipetting, the mixture was centrifuged at 1200 rpm for 1 hour using a horizontal centrifuge. The well plate was returned to the 37°C CO2 incubator and incubated for 24 hours, with the day of infection designated as day 1.

[0278] 3) After 24 hours, thoroughly mix the cells in each well of the 12-well plate by repeated pipetting, transfer to a 1.5 ml EP tube, centrifuge at 400 g for 5 minutes, remove the supernatant, and when the cell density is 7 × 10⁶ 5 Resuspend the cells in 1 ml of fresh X-VIVO complete medium until the cell density reaches 7 × 10⁶ cells / ml, and culture in a 37°C CO2 incubator. Replenish the medium when it turns yellow, count the cells every two days, and replenish with fresh X-VIVO complete medium until the cell density reaches 7 × 10⁶. 5 The cells were returned to their original cell / ml values, statistical counting results were recorded, graphed using GraphPad 8.0 software, and the amplification factor of CART cells under conventional culture conditions was calculated.

[0279] 4) Detection of cell positivity rate of CAR-T cells cultured for 9-14 days: Since the virus used to infect the cells is tagged with Myc, the Myc positivity rate was detected using a flow cytometer after the virus infected the cells, and the CAR expression positivity rate was obtained. The direct labeling and detection antibody used was Myc-Tag(9B11)Mouse mAb(Alexa Fluor(registered trademark)488 Conjugate)(Cell Signaling, 2279S).

[0280] Following an MOI of 4, cells were infected with human CLDN18.2 sequence viruses, including 5E6 sequence and standard sequence viruses. The results are shown in Figure 15B. The CAR positivity rate was detected by flow cytometry on day 12 of in vitro CART cell culture. The 5E6-CART positivity rate was 72.6%, while the Positive control CAR-T positivity rate was 65.4%. In this example, the CAR positivity rate was verified using a total of six different PBMC donors. Figure 15C shows that the 5E6-CART positivity rate in the six different PBMC donors remained at around 60%, the same as the Positive control CAR-T. Figure 15D shows that the cumulative amplification factor of 5E6-CART cells cultured in vitro for 12 days was approximately 240x, which is not significantly different from the amplification factor of Standard-CART.

[0281] (Example 15: CLDN18.2-specific CARs can specifically kill target cells in vitro.) In this example, CHO-hCLDN18.2 cells that stably and highly express human CLDN18.2 and CHO-hCLDN18.1 cells that stably and highly express human CLDN18.1 were first obtained via lentiviral infection and fluorescence-activated cell sorting. In this example, the specific killing ability of CAR-T cells in vitro was further evaluated using the LDH method with a cytotoxicity detection kit (Promega, Cat#G1780), and the procedure was as follows.

[0282] After centrifuging the CAR-T cells and Mock-T cells cultured for the usual 9 days in Example 14 above, they were resuspended in blank X-VIVO medium to obtain a cell density of 1 × 10⁶. 5 The cells were adjusted to a concentration of 5 × 10 / ml. Three types of target cells were used: CHO, CHO-hCLDN18.2, and CHO-hCLDN18.1. After digesting each of the three target cells, they were counted and then resuspended in blank X-VIVO medium to obtain a cell density of 5 × 10⁻¹⁴. 5The volume system was adjusted to 100 μl / ml, then mixed with 100 μl of target cell suspension and 100 μl of CAR-T / MockT cell suspension per well, and added to a sterile 96-well V-bottom plate. A control well was prepared according to the kit requirements, and the resulting mixture was co-incubated in a 37°C incubator for 24 hours. The absorbance value at 490 nM was recorded using a microplate reader, and the lysis rate of target cells was calculated using the formula provided in the kit. The data was analyzed and processed using Graphpad Prism 8. The results are shown in Figure 16. 5E6-CART specifically induces lysis of CLDN18.2-positive target cells in vitro, but does not lyse negative and CLDN18.1-positive cells. Similar to Standard-CART, this suggests that 5E6-CART cells possess highly specific target cell-killing activity against human CLDN18.2 in vitro.

[0283] (Example 16: Factor secretion status of CLDN18.2-specific CARs) This example analyzed the secretion of CAR-T receptors in response to IFN-γ and IL-2 during the process of killing target cells using the Human IFN-γ ELISA kit (R&D, DY285B) and the Human IL-2 ELISA kit (R&D, DY202). Specifically, 1 × 10⁶ wells were analyzed. 4Based on cell volume, target cells highly expressing CLDN18.2 (CHO-hCLDN18.2), control cells highly expressing CLDN18.1 (CHO-hCLDN18.1), and negative CHO cells were seeded into sterile 96-well plates. Effector cells, such as CAR-T cells (5E6-CART and Positive control CAR-T) and unmodified T cells (Mock T cells), were added to the target cells in an effector:target ratio of 5:1. After incubation for 24 hours, the supernatant was collected according to the kit instructions and its IL-2 and IFN-γ content was detected by enzyme immunosorbent assay (ELISA). The results are shown in Figures 17A and 17B. When 5E6-CART cells were co-incubated with CLDN18.2-positive cells, they secreted higher levels of the factor. However, when co-incubated with CLDN18.2-negative cells and CLDN18.1-positive cells, there was no significant factor secretion, and the results were the same as Standard-CART cells. Therefore, 5E6-CART cells have a specific cytokine-inducing effect on tumor cells that highly express CLDN18.2.

[0284] (Example 17: Antitumor activity of CLDN18.2-specific CAR-T cells in a mouse colon cancer CDX model) In this example, the human CLDN18.2 gene was first introduced into MC38 cells via lentivirus, and then mouse colon cancer cells that highly expressed human CLDN18.2, i.e., MC38-hCLDN18.2 cells, were selected using flow cytometry. A mouse colon cancer tumor model was constructed via subcutaneous injection into female B-NDG highly immunized mice, and the antitumor activity of CLDN18.2-specific CAR-T cells in the mouse body was verified. 1.5 × 10⁶ MC38-hCLDN18.2 cells were selected. 6 The mice were inoculated with the standard dose, and 8 days after inoculation, the tumor size was 41.68-120.6 mm. 3 (Average tumor size: 80.01 mm) 3Select mice from the specified group and randomly divide each group of 8 mice into 3 groups. CAR-T (or Mock T) cells cultured for 10 days were reinjected into the tail vein. The CAR-T reinjection volume for the 5E6-CART group and the Standard-CART group was 3 × 10⁻¹⁶. 6 The reinjection volume for cells / mouse and blank control mock T cell groups is 1 × 10⁻⁶. 7 The experiment involved cells / mice. As shown in Figure 18A, the tumor size and body weight of the mice were measured three times a week and observed for four weeks. After reinjection of CAR-T (or Mock T) cells into the tail vein of the mice, the mice in the 5E6-CART group lost more than 10% of their body weight on day 13, with a relative change of -34.51% in one mouse on day 15, and the first mouse died on day 13. The mice in the Standard-CART group also lost more than 10% of their body weight on day 16, with a relative change of -36.93% in one mouse on day 16, and the first mouse died on day 18. Due to the severe weight loss in the mice, the experiment was terminated with PG-D22, and no significant antitumor effect was observed at the end of the experiment, as shown in Figures 18B and 18C. After euthanizing and dissecting the mice, abnormal gastric injury and bleeding were found in both the 5E6-CART and Standard-CART groups, as shown in Figure 18D. In short, the 5E6-CART group mice exhibited low tolerability and clear side effects in this tumor model, particularly weight loss, hair standing on end, curling, and gastric bleeding, while the Mock T group mice were well-tolerated and had normal body weight.

Claims

1. It comprises HCDR3, HCDR2, and HCDR1, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, and An isolated antigen-binding protein that specifically binds to CLDN18.2, comprising LCDR3, LCDR2, and LCDR1, wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, and LCDR1 comprises the amino acid sequence shown in SEQ ID NO:

6.

2. The isolated antigen-binding protein according to claim 1, comprising VH and VL, wherein VH comprises the amino acid sequence shown in SEQ ID NO: 1 and VL comprises the amino acid sequence shown in SEQ ID NO:

5.

3. The isolated antigen-binding protein according to claim 1, comprising an antibody heavy chain constant region.

4. The isolated antigen-binding protein according to claim 3, wherein the antibody heavy chain constant region is derived from the human IgG1 heavy chain constant region.

5. The isolated antigen-binding protein according to claim 1, comprising an antibody light chain constant region.

6. The isolated antigen-binding protein according to claim 5, wherein the antibody light chain constant region is derived from a human Igκ constant region.

7. A chimeric antigen receptor comprising a target moiety containing the antigen-binding protein described in claim 1.

8. It comprises a co-stimulatory domain, an intracellular signaling domain, a transmembrane domain, a hinge region between the target region and the transmembrane domain, and a signal peptide. The aforementioned co-stimulatory domain includes a co-stimulatory domain derived from one or more proteins selected from the group consisting of the ligands CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83, CD40, and MyD88. The intracellular signaling domain includes an intracellular signaling region derived from one or more proteins selected from the group consisting of domains comprising CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma-associated herpesvirus (HSKV), DAP10, DAP-12, and at least one ITAM. The transmembrane domain comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. The chimeric antigen receptor according to claim 7, wherein the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

9. The aforementioned co-stimulatory domain is an intracellular co-stimulatory signaling region derived from 4-1BB, which includes the amino acid sequence shown in SEQ ID NO:

85. The intracellular signaling domain is a signaling domain derived from CD3ζ containing the amino acid sequence shown in Sequence ID No. 86, The aforementioned transmembrane domain is a transmembrane domain derived from CD8 containing the amino acid sequence shown in Sequence ID No. 84, The aforementioned hinge region is a hinge region derived from CD8 containing the amino acid sequence shown in Sequence ID No. 83, The aforementioned signal peptide is a signal peptide derived from the CD8 protein containing the amino acid sequence shown in SEQ ID NO:

88. The chimeric antigen receptor according to claim 8.

10. The chimeric antigen receptor according to claim 7, further comprising a low-density lipoprotein receptor-related protein or a fragment thereof.

11. The chimeric antigen receptor according to claim 10, wherein the low-density lipoprotein receptor-related protein or fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1 to 12 and their functional fragments.

12. The chimeric antigen receptor according to claim 10, wherein the low-density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:

91.

13. A polypeptide molecule comprising an isolated antigen-binding protein according to any one of claims 1 to 6 or a chimeric antigen receptor according to any one of claims 7 to 12.

14. An immune complex comprising an isolated antigen-binding protein according to any one of claims 1 to 6.

15. One or more isolated nucleic acid molecules encoding an isolated antigen-binding protein according to any one of claims 1 to 6 or a chimeric antigen receptor according to any one of claims 7 to 12.

16. A cell comprising an isolated antigen-binding protein according to any one of claims 1 to 6 or a chimeric antigen receptor according to any one of claims 7 to 12.

17. A pharmaceutical composition comprising an isolated antigen-binding protein according to any one of claims 1 to 6 and / or a chimeric antigen receptor according to any one of claims 7 to 12, and optionally a pharmaceutically acceptable carrier.

18. A pharmaceutical composition according to claim 17, used to prevent, alleviate and / or treat a disease and / or disorder. Here, the aforementioned diseases and / or disorders include cancer.

19. The pharmaceutical composition according to claim 18, wherein the cancer includes gastric cancer and / or colon cancer.

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