CN117098782A

CN117098782A - Antigen binding proteins targeting CLDN18.2 and uses thereof

Info

Publication number: CN117098782A
Application number: CN202280025144.5A
Authority: CN
Inventors: 杨沙沙; 何晓文; 吴凯; 王建涛; 王华菁; 杨焕凤
Original assignee: Oricell Therapeutics Co Ltd
Current assignee: Oricell Therapeutics Co Ltd
Priority date: 2021-04-02
Filing date: 2022-04-01
Publication date: 2023-11-21
Also published as: WO2022206976A1

Abstract

An isolated antigen binding protein capable of binding CLDN18.2. Chimeric antigen receptors comprising the isolated antigen binding proteins are also included. Also relates to a preparation method and application of the antigen binding protein or the chimeric antigen receptor.

Description

Antigen binding proteins targeting CLDN18.2 and uses thereof

Technical Field

The application relates to the field of biological medicine, in particular to an antigen binding protein capable of binding to CLDN18.2.

Background

Gastric cancer is a malignant tumor with the second global mortality rate, and 75.4 tens of thousands of gastric cancer patients die each year worldwide according to 2015 data of world health organization; pancreatic cancer is one of the most malignant tumors, and according to the data in journal 2016 of Lancet, more than 20 tens of thousands of pancreatic cancer patients die annually worldwide. The standard first-line treatment for advanced or recurrent gastric cancer is chemotherapy. Trastuzumab addition chemotherapy provides some survival benefit to HER 2-positive tumor patients, but only 15% of all gastric cancers are HER 2-positive. There is an urgent need to develop safe and effective treatments.

CLDN18.2 (Claudin 18.2) was expressed only in differentiated parietal cells and not in normal tissues. Recent studies have shown that CLDN18.2 is over-expressed in more than 77% of gastric cancer patients and more than 80% of pancreatic cancer patients, and in addition in solid tumors such as lung cancer, esophageal cancer and ovarian cancer. CLDN18.2 belongs to the family of tightly-linked proteins that can control molecular flow between lamina cells. However, in tumors, the tight junctions are broken and the CLDN protein loses its primary role. Because CLDN18.2 is present in large numbers in gastric tumors, researchers estimated that half of all advanced gastric cancer patients could be candidates for CLDN18.2 antibody target neotherapy. Furthermore, this unique target is not present in any healthy tissue other than the stomach wall, thereby minimizing the side effects of the treatment. These characteristics indicate that CLDN18.2 is an ideal target for therapeutic monoclonal antibody development.

Since CLDN18.2 is a membrane protein, it is difficult to obtain high quality antibodies against native proteins using conventional antibody screening methods. Most companies employ in vivo immunization, such as DNA injection, but require multiple rounds of immunization on animals. CLDN18 has two splice variants CLDN18.1 and CLDN18.2, which differ only in the N-terminal 69 amino acid sequence, present in the first extracellular loop, and the remaining sequences are identical. CLDN18.1 is expressed in normal lung tissue, and antibodies that bind to CLDN18.2 while not binding to CLDN18.1 need to be screened. On the other hand, the CLDN18.2 sequence has very high homology in humans and mice. Thus, there is a need to obtain antibodies that specifically bind CLDN 18.2.

Disclosure of Invention

The present application provides an antigen binding protein capable of binding to CLDN18.2 that exhibits one or more desired functional properties, e.g., that can specifically bind to human CLDN18.2 and that does not substantially bind to human CLDN18.1, that has CDC activity, and/or that has anti-tumor activity. The application also provides nucleic acid molecules encoding the isolated antigen binding proteins, expression vectors, host cells, and methods of making the isolated antigen binding proteins. The isolated antigen binding proteins of the application may be used for the prevention and/or treatment of diseases and/or disorders, such as tumors and/or cancers.

In one aspect, the application provides an isolated antigen binding protein comprising HCDR3, said HCDR3 comprising the amino acid sequence depicted in SEQ ID NO. 64.

In certain embodiments, the HCDR3 of the isolated antigen binding protein comprises an amino acid sequence set forth in any one of SEQ ID NO. 5, SEQ ID NO. 14, and SEQ ID NO. 22.

In certain embodiments, the isolated antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence depicted in SEQ ID NO. 63.

In certain embodiments, the HCDR2 of the isolated antigen binding protein comprises an amino acid sequence set forth in any one of SEQ ID NO. 4, SEQ ID NO. 13, and SEQ ID NO. 21.

In certain embodiments, the isolated antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 62.

In certain embodiments, the HCDR1 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO. 3 or SEQ ID NO. 20.

In certain embodiments, the isolated antigen binding protein comprises H-FR1, the C-terminus of said H-FR1 is directly or indirectly linked to the N-terminus of said HCDR1, and said H-FR1 comprises the amino acid sequence shown in SEQ ID NO. 68.

In certain embodiments, the H-FR1 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO. 37 or SEQ ID NO. 51.

In certain embodiments, the isolated antigen binding protein comprises H-FR2, said H-FR2 is located between said HCDR1 and said HCDR2, and said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 38.

In certain embodiments, the isolated antigen binding protein comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 69.

In certain embodiments, the H-FR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:39, SEQ ID NO:45, and SEQ ID NO: 48.

In certain embodiments, the isolated antigen binding protein comprises H-FR4, the N-terminus of H-FR4 is linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO. 40.

In certain embodiments, the isolated antigen binding protein comprises a VH comprising the amino acid sequence shown in SEQ ID No. 73.

In certain embodiments, the VH of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO. 2, SEQ ID NO. 12, SEQ ID NO. 19 and SEQ ID NO. 27.

In certain embodiments, the isolated antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence depicted in SEQ ID NO. 67.

In certain embodiments, the LCDR3 of the isolated antigen-binding protein comprises an amino acid sequence set forth in any one of SEQ ID NO. 10, SEQ ID NO. 17, SEQ ID NO. 25, and SEQ ID NO. 32.

In certain embodiments, the isolated antigen binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 66.

In certain embodiments, the LCDR2 of the isolated antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO. 9 or SEQ ID NO. 31.

In certain embodiments, the isolated antigen binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence depicted in SEQ ID NO. 65.

In certain embodiments, the LCDR1 of the isolated antigen-binding protein comprises the amino acid sequence set forth in SEQ ID NO. 8 or SEQ ID NO. 30.

In certain embodiments, the isolated antigen binding protein comprises L-FR1, the C-terminus of said L-FR1 is directly or indirectly linked to the N-terminus of said LCDR1, and said L-FR1 comprises the amino acid sequence shown in SEQ ID NO. 70.

In certain embodiments, the L-FR1 of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO. 41, SEQ ID NO. 46 and SEQ ID NO. 52.

In certain embodiments, the isolated antigen binding protein comprises L-FR2, said L-FR2 is located between said LCDR1 and said LCDR2, and said L-FR2 comprises the amino acid sequence set forth in SEQ ID NO. 42.

In certain embodiments, the isolated antigen binding protein comprises L-FR3, said L-FR3 is located between said LCDR2 and said LCDR3, and said L-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 71.

In certain embodiments, the L-FR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO. 43, SEQ ID NO. 49 and SEQ ID NO. 53.

In certain embodiments, the isolated antigen binding protein comprises L-FR4, the N-terminus of L-FR4 is linked to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO. 72.

In certain embodiments, the L-FR4 of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO. 44, SEQ ID NO. 47 and SEQ ID NO. 50.

In certain embodiments, the isolated antigen binding protein comprises a VL, and the VL comprises the amino acid sequence set forth in SEQ ID NO. 74.

In certain embodiments, the VL of the isolated antigen-binding protein comprises an amino acid sequence set forth in any one of SEQ ID NO. 7, SEQ ID NO. 16, SEQ ID NO. 24, and SEQ ID NO. 29.

In certain embodiments, the isolated antigen binding protein comprises any one of the group VH and VL selected from the group consisting of:

1) The VH comprises an amino acid sequence shown in SEQ ID NO. 2, and the VL comprises an amino acid sequence shown in SEQ ID NO. 7;

2) The VH comprises an amino acid sequence shown in SEQ ID NO. 12, and the VL comprises an amino acid sequence shown in SEQ ID NO. 16;

3) The VH comprises an amino acid sequence shown in SEQ ID NO. 19, and the VL comprises an amino acid sequence shown in SEQ ID NO. 24; and

4) The VH comprises the amino acid sequence shown in SEQ ID NO. 27, and the VL comprises the amino acid sequence shown in SEQ ID NO. 29.

In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region.

In certain embodiments, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

In certain embodiments, the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.

In certain embodiments, the isolated antigen binding protein comprises an antibody light chain constant region.

In certain embodiments, the antibody light chain constant region is derived from a human igκ constant region.

In certain embodiments, the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.

In certain embodiments, the antigen binding fragment comprises a Fab, fab ', fv fragment, F (ab') ₂ ，F(ab) ₂ scFv, di-scFv and/or dAb.

In certain embodiments, the antibody is selected from one or more of the following groups: monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

In certain embodiments, the isolated antigen binding protein is verified in FACS to substantially not compete with a reference antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:54 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:55 for binding to CLDN18.2.

In certain embodiments, the isolated antigen binding protein is capable of specifically binding CLDN18.2 and does not substantially bind CLDN18.1.

In certain embodiments, the CLDN18.2 comprises a mouse CLDN18.2, a cynomolgus CLDN18.2, and/or a human CLDN18.2.

In certain embodiments, the isolated antigen binding protein has CDC activity.

In certain embodiments, the isolated antigen binding protein is capable of inhibiting tumor growth and/or proliferation of tumor cells.

In another aspect, the application also provides a chimeric antigen receptor comprising a targeting moiety comprising an antigen binding protein of the application.

In certain embodiments, the chimeric antigen receptor comprises a co-stimulatory domain comprising a co-stimulatory domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B-H3, 2B4, fepsilon RI gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligand, CD40 and MyD88.

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

In certain embodiments, the co-stimulatory domain of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO. 79.

In certain embodiments, the chimeric antigen receptor comprises an intracellular signaling domain comprising an intracellular signaling domain derived from one or more proteins selected from the group consisting of: CD3 ζ, CD3 δ, CD3 γ, CD3 ε, CD79a, CD79b, fcεRIγ, fcεRIβ, fcγRIIa, bovine leukemia virus gp30, epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and domains comprising at least one ITAM.

In certain embodiments, the intracellular signaling domain of the chimeric antigen receptor is a signaling domain derived from cd3ζ.

In certain embodiments, the intracellular signaling domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO. 80.

In certain embodiments, the chimeric antigen receptor comprises a transmembrane region comprising 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, CD ε, CD3 ζ, 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.

In certain embodiments, the transmembrane region of the chimeric antigen receptor is a transmembrane region derived from CD 8.

In certain embodiments, the transmembrane region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO. 78.

In certain embodiments, the chimeric antigen receptor comprises a hinge region between the targeting moiety and the transmembrane region, the hinge region comprising 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, fc εRIgamma, BTLA, GITR, DAP, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

In certain embodiments, the hinge region of the chimeric antigen receptor is a CD 8-derived hinge region.

In certain embodiments, the hinge region of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO. 77.

In certain embodiments, the chimeric antigen receptor further comprises a signal peptide.

In certain embodiments, the chimeric antigen receptor the signal peptide is derived from a signal peptide of a CD8 protein.

In certain embodiments, the signal peptide of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO. 82.

In certain embodiments, the chimeric antigen receptor further comprises a low density lipoprotein receptor-related protein or fragment thereof.

In certain embodiments, 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 1-12 and functional fragments thereof.

In certain 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.

In certain embodiments, the low density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence shown in SEQ ID NO. 84.

In another aspect, the application also provides a polypeptide molecule comprising said isolated antigen binding protein or said chimeric antigen receptor.

In certain embodiments, the polypeptide molecule comprises a fusion protein.

In another aspect, the application also provides an immunoconjugate comprising the isolated antigen binding protein.

In another aspect, the application also provides an isolated nucleic acid molecule or molecules encoding the isolated antigen binding protein, the chimeric antigen receptor or the polypeptide molecule.

In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in any one of SEQ ID NO. 1, SEQ ID NO. 6, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 18, SEQ ID NO. 23, SEQ ID NO. 26, and SEQ ID NO. 28.

In certain embodiments, the nucleic acid molecule comprises any one of the nucleotide sequences selected from the group consisting of:

1) A nucleotide sequence shown in SEQ ID NO. 1 and a nucleotide sequence shown in SEQ ID NO. 6;

2) A nucleotide sequence shown as SEQ ID NO. 11 and a nucleotide sequence shown as SEQ ID NO. 15;

3) The nucleotide sequence shown as SEQ ID NO. 18 and the nucleotide sequence shown as SEQ ID NO. 23; and

4) The nucleotide sequence shown as SEQ ID NO. 26 and the nucleotide sequence shown as SEQ ID NO. 28.

In another aspect, the application also provides a vector comprising said nucleic acid molecule.

In another aspect, the application also provides a cell comprising the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule or the vector.

In certain embodiments, the cell is an immune effector cell.

In certain embodiments, the cells comprise 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, lymphoprogenitor cells, and/or pluripotent stem cells.

In certain embodiments, the cell is a T cell.

In certain embodiments, the cell further comprises and/or expresses a low density lipoprotein receptor-related protein or fragment thereof.

In another aspect, the application also provides a pharmaceutical composition comprising the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule and/or the cell, and optionally a pharmaceutically acceptable carrier.

In another aspect, the application also provides a method of preparing the isolated antigen-binding protein, the method comprising culturing the cell under conditions such that the antigen-binding protein is expressed.

In a further aspect, the application also provides the use of the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition for the preparation of a medicament for the prevention, alleviation and/or treatment of a disease and/or a disorder.

In certain embodiments, the disease and/or condition comprises cancer.

In certain embodiments, the cancer comprises a solid tumor and/or a hematological tumor.

In certain embodiments, the cancer comprises gastric cancer and/or colon cancer.

In another aspect, the application also provides a method of detecting CLDN18.2 in a sample comprising administering the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.

In another aspect, the application also provides a kit or kit for detecting CLDN18.2 in a sample comprising the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.

In a further aspect, the application also provides the use of the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition for the preparation of a kit for detecting the presence and/or amount of CLDN18.2 in a sample.

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the application as claimed. Accordingly, the drawings and descriptions of the present application are to be regarded as illustrative in nature and not as restrictive.

Drawings

The specific features of the application related to the application are shown in the appended claims. A better understanding of the features and advantages of the application in accordance with the present application will be obtained by reference to the exemplary embodiments and the accompanying drawings that are described in detail below. The drawings are briefly described as follows:

FIG. 1 shows the detection of binding activity of the isolated antigen binding proteins of the application to cells highly expressing human CLDN 18.2.

FIG. 2 shows the detection of binding activity of the isolated antigen binding proteins of the application to cells highly expressing human CLDN 18.1.

FIG. 3 shows the detection of binding activity of the isolated antigen binding proteins of the application to tumor cell lines.

FIG. 4 shows a cross-binding activity assay of the isolated antigen binding proteins of the application.

FIGS. 5a-5b show the detection of CDC activity of an isolated antigen binding protein of the application.

Figure 6 shows the change in body weight of mice after administration.

Fig. 7 shows the relative change in body weight (%) of mice after administration.

Fig. 8 shows tumor volume changes in mice after administration.

Fig. 9 shows tumor inhibition of mice after administration.

Figure 10 shows the survival curve of mice after dosing.

Figures 11A-D show the construction of CLDN 18.2-specific CARs and their expression in T cells.

Figure 12 shows the in vitro killing activity assay of CAR-T cells according to the application.

Figures 13A-B show the detection of factor secretion for CLDN 18.2-specific CARs.

Figures 14A-C show the tumor-inhibiting effect of CLDN 18.2-specific CAR-T cells in a mouse human gastric cancer model.

Detailed Description

Further advantages and effects of the present application will become readily apparent to those skilled in the art from the present disclosure, by describing embodiments of the present application with specific examples.

Definition of terms

In the present application, the terms "CLDN18.2", or "Claudin18.2" are used interchangeably and generally refer to subtype 2 of the cell attachment Claudin18. The term encompasses "full length", unprocessed CLDN18.2, and any form of CLDN18.2 resulting from cellular processing. CLDN18.2 can include intact CLDN18.2 and fragments thereof, functional variants, isoforms, species homologs, derivatives, analogs thereof, and analogs having at least one epitope in common with CLDN18.2. The amino acid sequence of CLDN18.2 (e.g., human CLDN 18.2) is known in the art. For example, the human CLDN18.2 nucleotide sequence can be shown under genbank accession No. nm_ 001002026.3. For example, the mouse CLDN18.2 nucleotide sequence can be shown under GeneBank accession No. nm_ 001194921.1. For example, the cynomolgus monkey CLDN18.2 nucleotide sequence may be shown under GeneBank accession No. xm_ 001114708.4.

In the present application, the terms "CLDN18.1", or "Claudin18.1" are used interchangeably and generally refer to subtype 1 of the cell attachment Claudin18. The term encompasses "full length", unprocessed CLDN18.1, and any form of CLDN18.2 resulting from cellular processing. CLDN18.2 can include intact CLDN18.1 and fragments thereof, functional variants, isoforms, species homologs, derivatives, analogs thereof, and analogs having at least one epitope in common with CLDN 18.1. The amino acid sequence of CLDN18.1 (e.g., human CLDN 18.1) is known in the art. For example, the human CLDN18.1 nucleotide sequence can be shown under genbank accession No. nm_ 016369.4. For example, the mouse CLDN18.1 nucleotide sequence can be shown under GeneBank accession No. nm_ 019815.3. For example, the cynomolgus monkey CLDN18.1 nucleotide sequence may be shown under GeneBank accession No. xm_ 005545863.2.

In the present application, the term "isolated" generally refers to those obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide that has been isolated from the natural state and is of high purity is said to be isolated. The term "isolated" does not exclude the incorporation of artificial or synthetic substances, nor the presence of other impure substances that do not affect the activity of the substance.

In the present application, the term "isolated antigen binding protein" generally refers to a protein having antigen binding ability obtained from a natural state by artificial means. The "isolated antigen bindingThe protein "may comprise an antigen-binding portion and optionally a framework or framework portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen by the antigen-binding portion. The antigen binding proteins may comprise, for example, an antibody-derived protein Framework Region (FR) or an alternative protein framework region or artificial framework region with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to, framework regions comprising antibody sources that are introduced, for example, to stabilize mutations in the three-dimensional structure of the antigen binding protein, as well as fully synthetic framework regions comprising, for example, biocompatible polymers. See, e.g., korndorfer et al, 2003, proteins: structure, function, andBioiInformatics, 53 (1): 121-129 (2003); roque et al, biotechnol. Prog.20:639-654 (2004). Examples of antigen binding proteins include, but are not limited to: human antibodies, humanized antibodies; a chimeric antibody; a recombinant antibody; a single chain antibody; a bifunctional antibody; a trifunctional antibody; a four-functional antibody; fab, fab ', fv fragment, F (ab') ₂ ，F(ab) ₂ scFv, di-scFv, dAb, igD antibody; igE antibodies; igM antibodies; an IgG1 antibody; an IgG2 antibody; an IgG3 antibody; or IgG4 antibodies and fragments thereof.

In the present application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain. The variable domains of the heavy and light chains, respectively, may be referred to as "V _H "and" V _L "(or" VH "and" VL ", respectively). These domains are typically the most variable portions of an antibody (relative to other antibodies of the same type) and comprise antigen binding sites.

In the present application, the term "variable" generally refers to the fact that certain segments of the variable domain may differ greatly in sequence between antibodies. The variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains. It is typically concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called Framework Regions (FR). The variable domains of the natural heavy and light chains each comprise four FR regions, mostly in a β -sheet configuration, connected by three CDRs, which form a circular connection and in some cases form part of a β -sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and the CDRs from the other chain together promote the formation of the antigen binding site of the antibody (see Kabat et al, sequences of Immunological Interest, fifth Edition, national Institute of Health, bethesda, md. (1991)).

In the present application, the term "antibody" generally refers to an immunoglobulin or fragment or derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. Unless otherwise modified by the term "intact", as in "intact antibodies", for the purposes of the present application the term "antibody" also 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 bind CLDN 18.2). Typically, such fragments should include an antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetramer units with another polypeptide called the J chain and contain 10 antigen binding sites, whereas IgA antibodies comprise 2-5 basic 4-chain units that can polymerize in conjunction with the J chain to form multivalent combinations. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the alpha and gamma chains, followed by four CH domains for the mu and epsilon isoforms. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to heavy chain Corresponds to the first constant domain (CH 1). Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The VH and VL pairs together form a single antigen binding site. For the structure and properties of antibodies of different classes, see e.g. Basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw (eds), appleton&Lange, norwalk, conn, 1994, pages 71 and chapter 6. L chains from any vertebrate species can be divided into one of two distinct types, termed kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to different classes or isotypes based on the amino acid sequence of the constant domain of the heavy Chain (CH). Currently there are five classes of immunoglobulins: igA, igD, igE, igG and IgM, have heavy chains named α, δ, ε, γ and μ, respectively. Based on the relatively small differences in CH sequence and function, the γ and α classes are further divided into subclasses, e.g., humans express the following subclasses: igG1, igG2A, igG2B, igG3, igG4, igA1 and IgK1.

In the present application, the term "CDR" is also referred to as "complementarity determining region", and generally refers to a region in the variable domain of an antibody whose sequence is highly variable and/or forms a structurally defined loop. Typically, an antibody comprises six CDRs; three in VH (HCDR 1, HCDR2, HCDR 3), and three in VL (LCDR 1, LCDR2, LCDR 3). In certain embodiments, naturally occurring camelid antibodies consisting of only heavy chains are also able to function normally and stably in the absence of light chains. See, e.g., hamers-Casterman et al, nature 363:446-448 (1993); sheiff et al, nature Structure. Biol.3:733-736 (1996).

In the present application, the term "FR" generally refers to the more highly conserved portion of the antibody variable domain, which is referred to as the framework region. Typically, the variable domains of the natural heavy and light chains each comprise four FR regions, namely four in VH (H-FR 1, H-FR2, H-FR3, and H-FR 4), and four in VL (L-FR 1, L-FR2, L-FR3, and L-FR 4). For example, VL of the isolated antigen binding proteins of the application may comprise framework regions L-FR1, L-FR2, L-FR3, and L-FR4. The VH of the isolated antigen binding proteins of the application may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

In the present application, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind an antigen (e.g., CLDN 18.2). In the present application, the antigen binding fragment may include Fab, fab', F (ab) ₂ Fv fragment, F (ab') ₂ scFv, di-scFv and/or dAb.

In the present application, the term "Fab" generally refers to an antigen-binding fragment of an antibody. As described above, papain can be used to digest intact antibodies. The antibodies, after digestion with papain, produce two identical antigen-binding fragments, a "Fab" fragment, and a residual "Fc" fragment (i.e., fc region, supra). Fab fragments can be composed of a complete L chain with a heavy chain variable region and the H chain (V _H ) Is a constant region (C) _H 1) Composition is prepared.

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

In the present application, the term "F (ab') 2" generally refers to an antibody fragment produced by pepsin digestion of an intact antibody. The F (ab') 2 fragment contains two Fab fragments held together by disulfide bonds and a partial hinge region. F (ab') 2 fragments have divalent antigen binding activity and are capable of cross-linking antigens.

In the present application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy and light chain variable regions, and lacking the heavy and light chain constant regions. The heavy chain variable region and the light chain variable region include, for example, CDRs. For example, fv fragments comprise all or part of the amino terminal variable region of about 110 amino acids of the heavy and light chains.

In the present application, the term "scFv" generally refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and are capable of expression as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specifically stated otherwise, as used herein, an scFv may have the VL and VH variable regions described in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), an scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

In the present application, the term "dAb" generally refers to an antigen binding fragment having a VH domain, a VL domain or having a VH domain or a VL domain, reference to, for example, ward et al (Nature, 1989Oct 12;341 (6242): 544-6), reference to Holt et al, trends Biotechnol.,2003,21 (11): 484-490; and other published patent applications such as WO 06/030220, WO 06/003388 and Domantis Ltd.

In the present application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules consisting of single molecules. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" refers to the characteristics of the antibody as obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in the present application may be prepared in hybridoma cells or may be prepared by recombinant DNA methods.

In the present application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody") and the constant region is derived from a human antibody such that the resulting chimeric antibody has a reduced likelihood of eliciting an adverse immune response in a human individual as compared to the parent (e.g., mouse-derived) antibody.

In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (e.g., a mouse antibody) are replaced with the corresponding amino acids derived from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may also be permissible, provided that they still retain the ability of the antibody to bind to a particular antigen. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. "humanized antibodies" retain antigen specificity similar to the original antibody. A "humanized" form of a non-human (e.g., murine) antibody may minimally comprise chimeric antibodies derived from sequences of non-human immunoglobulins. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired properties, affinity and/or capability. In some cases, the FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, the humanized antibody may comprise amino acid modifications that are not in the recipient antibody or in the donor antibody. These modifications may be made to further improve the properties of the antibody, such as binding affinity.

The term "fully human antibody" generally refers to an antibody comprising only human immunoglobulin protein sequences. If it is produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell, the fully human antibody may contain a murine sugar chain. Similarly, "mouse antibody" or "rat antibody" refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans by phage display or other molecular biological methods in transgenic animals with human immunoglobulin germline sequences. Exemplary techniques that can be used to make antibodies are in U.S. patents: 6,150,584, 6,458,592, 6,420,140. Other techniques, such as the use of libraries, are known in the art.

In the present application, the term "directly connected" is opposed to the term "indirectly connected," which generally refers to a direct connection. For example, the direct linkage may be where there is no spacer between the substances. The spacer may be a linker. For example, the linker may be a peptide linker. The term "indirect linkage" generally refers to the situation where the materials are not directly linked. For example, the indirect connection may be the case where the connection is through a spacer. For example, in the isolated antigen binding proteins of the application, the C-terminus of the L-FR1 and the N-terminus of the LCDR1 can be directly or indirectly linked.

In the present application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs thereof, isolated from the natural environment or synthesized.

In the present application, the term "vector" generally refers to a nucleic acid vector into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell. For example, the carrier may comprise: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal virus species used as vectors may include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus (e.g., SV 40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. It is also possible for the vector to include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not just these.

In the present application, the term "cell" generally refers to a single cell, cell line or cell culture that may or may not be the recipient of a subject plasmid or vector, which comprises a nucleic acid molecule according to the present application or a vector according to the present application. Cells may include progeny of a single cell. The offspring may not necessarily be identical to the original parent cell (either in the form of the total DNA complement or in the genome) due to natural, accidental or deliberate mutation. Cells may include cells transfected in vitro with the vectors of the application. The cells may be bacterial cells (e.g., E.coli), yeast cells, 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. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK293 cell.

In the present application, the term "pharmaceutical composition" generally refers to a composition for preventing/treating a disease or disorder. The pharmaceutical composition may comprise an isolated antigen binding protein of the application, a nucleic acid molecule of the application, a vector of the application and/or a cell of the application, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may further comprise one or more (pharmaceutically effective) suitable formulations of carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. Pharmaceutical compositions of the application include, but are not limited to, liquid, frozen and lyophilized compositions.

In the present application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers which are non-toxic to the cells or mammals to which they are exposed at the dosages and concentrations employed. Physiologically acceptable carriers can include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelators, sugar alcohols, salt-forming counter ions, such as sodium; and/or nonionic surfactants.

In the present application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds a target (which may be an epitope) may be an antibody that binds the target with greater affinity, avidity, more readily, and/or for a greater duration than it binds other targets. In certain embodiments, the antibodies specifically bind to epitopes on proteins that are conserved among proteins of different species. In certain embodiments, specific binding may include, but is not required to be, exclusively binding.

In the present application, the term "reference antibody" generally refers to an antibody that can bind to an antigen (e.g., CLDN 18.2). In some cases, the antigen binding proteins of the application do not have competitive binding activity compared to a reference antibody. In some cases, the reference antibody of the application may be zolbetuximab.

In the present application, the term "substantially unbound" generally means bound with little or no binding activity. Wherein a weak binding activity may refer to, for example, detecting that the average fluorescence intensity value of an antibody that binds to CLDN18.1 is at least about 50%, about 55%, about 60%, about 70%, about 80%, about 90%, about 91%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100% lower in comparison to a CLDN18.1 antibody positive control in a flow binding activity assay.

In the present application, the term "subject" generally refers to a human or non-human animal, including but not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

In the present application, the term "tumor" generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In the present application, the tumor may be a solid tumor or a hematological tumor. For example, in the present application, the tumor may be a CLDN18.2 positive tumor.

The term "cancer" generally refers to a disease characterized by rapid and uncontrolled growth of abnormal cells. Cancer cells may spread to other parts of the body locally or through the blood stream and lymphatic system. Cancers in the present application include, but are not limited to, stomach cancer, colon cancer, and the like. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid tumors and liquid tumors, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" may include premalignant as well as malignant cancers and tumors.

In the context of the present application, reference to protein, polypeptide and/or amino acid sequences is also to be understood as comprising at least the following ranges: variants or homologues having the same or similar function as the protein or polypeptide.

In the present application, the variant may be, for example, a protein or polypeptide having one or more amino acids substituted, deleted or added in the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or fragment thereof that specifically binds CLDN18.2 protein). For example, the functional variant may comprise a protein or polypeptide that has been altered in amino acids by at least 1, such as 1-30, 1-20, or 1-10, and yet another such as 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and/or insertions. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%,70%,80%,90%, or 100% of the biological activity (e.g., antigen binding capacity) of the protein or the polypeptide prior to alteration. For example, the substitution may be a conservative substitution.

In the present application, the homolog may be a protein or polypeptide having at least about 85% (e.g., 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 the polypeptide (e.g., an antibody or fragment thereof that specifically binds CLDN18.2 protein).

In the present application, the homology generally refers to similarity, similarity or association between two or more sequences. "percent sequence homology" can be calculated by: the two sequences to be aligned are compared in a comparison window, the number of positions in the two sequences where the same nucleobase (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) is present is determined to give the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., window size), and the result is multiplied by 100 to produce the percent sequence homology. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length sequence being compared or over the region of the target sequence. The homology can also be determined by the following method: FASTA and BLAST. For a description of FASTA algorithm, see w.r.pearson and d.j.lipman, "improved tools for biological sequence comparison", proc.Natl. Acad.Sci., U.S. Proc., 85:2444-2448, 1988; "quick sensitive protein similarity search" by d.j.lipman and w.r.pearson, science,227:1435-1441, 1989. For a description of the BLAST algorithm, see "a basic local contrast (alignment) search tool", journal of molecular biology, 215:403-410, 1990.

In the present application, the term "comprising" generally means containing, summarizing, containing or comprising. In some cases, the meaning of "as", "consisting of … …" is also indicated.

In the present application, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 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% above or below the specified value.

Detailed Description

The CDRs of an antibody, also known as complementarity determining regions, are part of the variable region. The amino acid residues of this region may be contacted with an antigen or epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, kabat, chothia, IMGT, a combination of Kabat/Chothia et al. These coding systems are known in the art and can be found in particular in http:// www.bioinf.org.uk/abs/index. Html # kabat num. The CDR regions can be determined by one skilled in the art using different coding systems depending on the sequence and structure of the antibody. Using different coding systems, CDR regions may differ. In the present application, the CDR encompasses CDR sequences partitioned according to any CDR partitioning scheme; variants thereof are also contemplated, including amino acid substitutions, deletions and/or additions to the amino acid sequence of the CDRs. Such as 1-30, 1-20 or 1-10, and further such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or insertions; homologues thereof are also contemplated, which may be amino acid sequences having at least about 85% (e.g., 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 sequences of the CDRs.

In one aspect, the application provides an isolated antigen binding protein that may comprise HCDR3. In the present application, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 64. For example, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 5. For example, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 14. For example, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 22.

In the present application, the isolated antigen binding protein may comprise HCDR2. In the present application, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 63. In the present application, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 4. For example, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 13. For example, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 21.

In the present application, the isolated antigen binding protein may comprise HCDR1. In the present application, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 62. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 3. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 20.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3. In the present application, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 62, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 63, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 64.

In the present application, the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 20, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO. 4, SEQ ID NO. 13 and SEQ ID NO. 21, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO. 5, SEQ ID NO. 14 and SEQ ID NO. 22.

In the present application, the HCDR1, HCDR2 and HCDR3 of the isolated antigen binding protein may comprise an amino acid sequence selected from any one of the following groups:

1) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 4, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 5;

2) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 13, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 14; and

3) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 20, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 21, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 22.

In the present application, the isolated antigen binding protein may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 68. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 37. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 51.

In the present application, the isolated antigen binding protein may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 38.

In the present application, the isolated antigen binding protein may comprise H-FR3, said H-FR3 being located between said HCDR2 and said HCDR3, and said H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 69. In the present application, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 39. For example, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 45. For example, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 48.

In the present application, the isolated antigen binding protein may comprise H-FR4, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 40.

In the present application, the isolated antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4. In the present application, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 68, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 38, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 69, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 40.

In the present application, the H-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 37 or SEQ ID NO. 51, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 38, the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO. 39, SEQ ID NO. 45 and SEQ ID NO. 48, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 40.

In the present application, the H-FR1, H-FR2, H-FR3 and H-FR4 of said isolated antigen binding protein may comprise an amino acid sequence selected from any one of the following groups:

1) The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 37, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 38, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 39, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 40;

2) The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 37, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 38, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 45, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 40;

3) The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 37, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 38, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 48, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 40; and

4) The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 51, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 38, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 39, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 40.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, HFR2, HFR3 and H-FR4. For example, the HCDR1, HCDR2, HCDR3, H-FR1, HFR2, HFR3 and H-FR4 may each comprise, in order: SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39 and SEQ ID NO. 40; SEQ ID NO. 3, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 45 and SEQ ID NO. 40; SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 48 and SEQ ID NO. 40; or the amino acid sequences shown in SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 51, SEQ ID NO. 38, SEQ ID NO. 39 and SEQ ID NO. 40.

In the present application, the isolated antigen binding protein may comprise VH. In the present application, the VH may comprise the amino acid sequence shown in SEQ ID NO. 73. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO. 2. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO. 12. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO. 19. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO 27.

In the present application, the isolated antigen binding protein may comprise LCDR3. In the present application, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 67. For example, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 10. For example, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 17. For example, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25. For example, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 32.

In the present application, the isolated antigen binding protein may comprise LCDR2. In the present application, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 66. For example, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 9. For example, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 31.

In the present application, the isolated antigen binding protein may comprise LCDR1. In the present application, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 65. For example, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 8. For example, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 30.

In the present application, the isolated antigen binding protein may comprise LCDR1, LCDR2 and LCDR3. For example, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 65, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 66, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 67.

In the present application, the LCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 8 or SEQ ID NO. 30, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 9 or SEQ ID NO. 31, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO. 10, SEQ ID NO. 17, SEQ ID NO. 25 and SEQ ID NO. 32.

In the present application, the LCDR1, LCDR2 and LCDR3 of the isolated antigen binding protein may comprise an amino acid sequence selected from any one of the group consisting of:

1) The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 8, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 9, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 10;

2) The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 8, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 9, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 17;

3) The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 8, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 9, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25; and

4) The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 30, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 31, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 32.

In the present application, the isolated antigen binding protein may comprise L-FR1, and the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR 1. In the present application, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 70. For example, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 41. For example, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 46. For example, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 52.

In the present application, the isolated antigen binding protein may comprise L-FR2, said L-FR2 being located between said LCDR1 and said LCDR 2. In the present application, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 42.

In the present application, the isolated antigen binding protein may comprise L-FR3, said L-FR3 being located between said LCDR2 and said LCDR 3. In the present application, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 71. For example, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 43. For example, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 49. For example, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 53.

In the present application, the isolated antigen binding protein may comprise L-FR4, the N-terminus of L-FR4 being linked to the C-terminus of LCDR 3. In the present application, the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 72. For example, the L-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 44. For example, the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 47. For example, the L-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 50.

In the present application, the isolated antigen binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4. For example, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 70, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 42, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 71, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 72.

In the present application, the L-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO. 41, SEQ ID NO. 46 and SEQ ID NO. 52, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 42, the L-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO. 43, SEQ ID NO. 49 and SEQ ID NO. 53, and the L-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NO. 44, SEQ ID NO. 47 and SEQ ID NO. 50.

In the present application, the L-FR1, L-FR2, L-FR3 and L-FR4 of said isolated antigen binding protein can comprise any one of the following amino acid sequences:

1) The L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 41, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 42, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 43, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 44;

2) The L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 46, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 42, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 43, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 47;

3) The L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 46, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 42, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 49, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 50; and

4) The L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 52, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 42, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 53, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 50.

In the present application, the isolated antigen binding proteins comprise LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3 and L-FR4. For example, the LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3 and L-FR4 may each comprise, in order: SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 and SEQ ID NO. 44; SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 17, SEQ ID NO. 46, SEQ ID NO. 42, SEQ ID NO. 43 and SEQ ID NO. 47; SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 25, SEQ ID NO. 46, SEQ ID NO. 42, SEQ ID NO. 49 and SEQ ID NO. 50; or SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 52, SEQ ID NO. 42, SEQ ID NO. 53 and SEQ ID NO. 50.

In the present application, the isolated antigen binding protein comprises VL. In the present application, the VL may comprise the amino acid sequence shown in SEQ ID NO. 74. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 7. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 16. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 24. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 29.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3. In the present application, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 62, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 63, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 64, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 65, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 66, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 67.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3. In the present application, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 20, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO. 4, SEQ ID NO. 13 and SEQ ID NO. 21, the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO. 5, SEQ ID NO. 14 and SEQ ID NO. 22, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 8 or SEQ ID NO. 30, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 9 or SEQ ID NO. 31, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO. 10, SEQ ID NO. 17, SEQ ID NO. 25 and SEQ ID NO. 32.

In the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the isolated antigen binding protein may comprise any one of the following sets of amino acid sequences:

1) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 4, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 5, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 8, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 9, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 10;

2) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 13, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 14, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 8, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 9, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 17;

3) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 20, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 21, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 22, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 8, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 9, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 25; and

4) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 4, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 5, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 30, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 31, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 32.

In the present application, the isolated antigen binding protein may comprise VH and VL. In the present application, the VH may comprise the amino acid sequence shown in SEQ ID NO. 73, and the VL may comprise the amino acid sequence shown in SEQ ID NO. 74.

In the present application, the VH of the isolated antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID No. 2, SEQ ID No. 12, SEQ ID No. 19 and SEQ ID No. 27, and the VL may comprise the amino acid sequence shown in any one of SEQ ID No. 7, SEQ ID No. 16, SEQ ID No. 24 and SEQ ID No. 29.

In the present application, the isolated antigen binding protein may comprise any one selected from the group consisting of VH and VL:

1) The VH may comprise the amino acid sequence shown in SEQ ID NO. 2, and the VL may comprise the amino acid sequence shown in SEQ ID NO. 7;

2) The VH may comprise the amino acid sequence shown in SEQ ID NO. 12 and the VL may comprise the amino acid sequence shown in SEQ ID NO. 16;

3) The VH may comprise the amino acid sequence shown in SEQ ID NO. 19 and the VL may comprise the amino acid sequence shown in SEQ ID NO. 24; and

4) The VH may comprise the amino acid sequence shown in SEQ ID NO. 27 and the VL may comprise the amino acid sequence shown in SEQ ID NO. 29.

In the present application, the isolated antigen binding protein may comprise an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In certain embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region.

In the present application, the isolated antigen binding protein may comprise an antibody light chain constant region. The antibody light chain constant region may be derived from a human igκ constant region.

Furthermore, it is noted that the isolated antigen binding proteins of the application may comprise heavy and/or light chain sequences to which one or more conservative sequence modifications exist. By "conservative sequence modifications" is meant amino acid modifications that do not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications may be introduced into the isolated antigen binding proteins of the application by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are substitutions of amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues having similar side chains are known in the art. These groups of amino acid residues include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in the CDR regions of the isolated antigen binding proteins of the application may be replaced with other amino acid residues of the same side chain set. Those skilled in the art know that some conservative sequence modifications do not result in the disappearance of antigen binding, see, for example, brummell et al, (1993) Biochem 32:1180-8; de Wildt et al, (1997) Prot.Eng.10:835-41; komissarov et al, (1997) J.biol. Chem.272:26864-26870; hall et al, (1992) J.Immunol.149:1605-12; kelley and O' Connell (1993) biochem.32:6862-35; adib-Conquy et al, (1998) int. Immunol.10:341-6 and beer et al, (2000) Clin. Can. Res.6:2835-43.

The physical/chemical properties and/or biological activity of CLDN18.2 antigen binding proteins of the application can be identified, screened or characterized by various assays known in the art.

In certain embodiments, the antigen binding activity of an antigen binding protein or fusion protein of the application can be tested, for example, by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., western blot), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, and the like.

In the present application, the isolated antigen binding protein is capable of specifically binding to CLDN18.2 antigen. An antigen binding protein that "specifically binds" to CLDN18.2 antigen can typically bind to CLDN18.2 but does not bind or substantially does not bind to other proteins lacking the CLDN18.2 sequence. Whether an antigen binding protein (e.g., an antibody) binds to the antigen CLDN18.2 can be determined using any assay known in the art. For example, the specific binding activity of the isolated antigen binding protein to CLDN18.2 can be determined using flow cytometry fluorescence sorting (FACS). In certain embodiments, the specific binding may be a concentration-dependent binding. For example, in FACS binding activity experiments, the mean fluorescence intensity of CLDN18.2 increased with increasing CLDN18.2 antibody concentration.

In the present application, the antigen binding protein may bind to human CLDN18.2 protein. In certain instances, the antigen binding proteins of the application can also cross-react with CLDN18.2 in mice (e.g., mice) and/or monkeys (e.g., cynomolgus monkeys). For example, by FACS. In the present application, "cross-reactive" generally refers to the ability of an antibody to react with homologous proteins from other species.

In the present application, the antigen binding protein is capable of eliciting a CDC effect. For example, the CLDN18.2 antigen binding proteins of the application are capable of inducing a strong CDC effect against SP2/0-humancldn18.2 cells. For example, the CLDN18.2 antigen binding proteins of the application are capable of inducing a strong CDC effect against MC38-humancldn18.2 cells. For example, the induction mode may be in a dose dependent manner.

In the present application, the isolated antigen binding protein was validated in FACS that it did not substantially compete with the reference antibody for binding to CLDN18.2. The reference antibody may comprise a heavy chain variable region (VH) and a light chain variable region (VL), e.g., the VH thereof may comprise the amino acid sequence shown in SEQ ID No. 54 and the VL thereof may comprise the amino acid sequence shown in SEQ ID No. 55.

In the present application, the antigen binding proteins are capable of inhibiting tumor growth and/or proliferation of tumor cells. The tumor may be a CLDN18.2 expressing tumor, e.g., colon cancer. The colon cancer cell may be an MC 38-humanDN18.2 cell.

Chimeric antigen receptor

In another aspect, the application also provides a Chimeric Antigen Receptor (CAR) that can comprise a targeting moiety that binds to CLDN18.2 protein, e.g., the targeting moiety that binds to CLDN18.2 protein can be an antigen binding protein of the application. For example, the targeting moiety may be in the form of an scFv. For example, the scFv may comprise CDRs, VH and/or VL of an isolated antigen binding protein of the application. For example, the VH and VL of the scFv may be linked by the amino acid sequence shown in SEQ ID NO. 83. For example, the scFv may comprise a VH of 5C10 and a VL of 5C 10. For example, the scFv may comprise VH of 5F3 and VL of 5F 3. For example, the scFv may comprise a VH of 14C3 and a VL of 14C 3. For example, the scFv may comprise a VH of 1C7 and a VL of 1C 7.

For example, the scFv may comprise the amino acid sequence shown as SEQ ID NO. 85.

In the present application, the CAR comprises an extracellular targeting moiety that binds to CLDN18.2 protein, and may also comprise other domains.

In the present application, the CAR may include a co-stimulatory signal region that may provide a stimulatory signal. For example, the costimulatory signal region may comprise an intracellular costimulatory signal region of one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B-H3, 2B4, fepsilon RI gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligand, CD40 and MyD88.

For example, the costimulatory signal region may be an intracellular costimulatory signal region derived from 4-1 BB. For example, the costimulatory signal region may comprise the amino acid sequence shown as SEQ ID NO. 79.

In certain cases, the CAR may comprise an intracellular signaling region, which may comprise a domain having at least one ITAM motif. The intracellular signaling domain may transmit an activation signal to the interior of the cell. For example, the intracellular signal region may comprise an intracellular signal region derived from one or more proteins selected from the group consisting of: CD3 ζ, CD3 δ, CD3 γ, CD3 ε, CD79a, CD79b, fcεRIγ, fcεRIβ, fcγRIIa, bovine leukemia virus gp30, epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and other domains comprising at least one ITAM.

For example, the intracellular signaling region may be a signaling domain derived from cd3ζ. For example, the intracellular signal region may comprise the amino acid sequence shown in SEQ ID NO. 80.

In some cases, the CAR can comprise a transmembrane domain, which is a sequence in a cell surface protein that spans the cell membrane, which can comprise a hydrophobic alpha helix. The transmembrane domain may be derived from any type I transmembrane protein. The transmembrane domain may be a synthetic sequence predicted to form a hydrophobic helix. For example, the transmembrane region may comprise 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, CD ε, CD3 ζ, 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.

For example, the transmembrane region may be a transmembrane region derived from CD 8. For example, the transmembrane region may comprise the amino acid sequence shown in SEQ ID NO. 78.

In some cases, the CAR can include a hinge region, which can be located between the extracellular targeting moiety and the transmembrane domain. For example, the hinge region may comprise a hinge region of 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, fc εRIgamma, BTLA, GITR, DAP, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

For example, the hinge region may be a CD8 derived hinge region. For example, the hinge region may comprise the amino acid sequence shown in SEQ ID NO. 77.

In the present application, the CAR may further comprise a signal peptide at the N-terminus of the targeting moiety that binds to CLDN18.2 protein. For example, the signal peptide may be a signal peptide derived from a CD8 protein. For example, the signal peptide may comprise the amino acid sequence shown in SEQ ID NO. 82.

In the present application, the CAR may further comprise a low density lipoprotein receptor-related protein or a fragment thereof. For example, the low density lipoprotein receptor-related protein or fragment thereof may be located at the C-terminus of the CAR. For example, the low density lipoprotein receptor-related protein or fragment thereof may comprise low density lipoprotein receptor-related proteins 1-12 and functional fragments thereof. For example, the low density lipoprotein receptor-related protein or fragment thereof may be low density lipoprotein receptor-related protein 6 or fragment thereof. For example, the low density lipoprotein receptor-related protein or fragment thereof may comprise the amino acid sequence shown in SEQ ID NO. 84.

In the present application, the sequence of the low density lipoprotein receptor-related protein or fragment thereof in the CAR may be linked to the C-terminal sequence of the CAR by a self-cleaving peptide (e.g., a 2A peptide such as T2A, P2A, E a). For example, the low density lipoprotein receptor-related protein or fragment thereof may be linked to the C-terminus of the intracellular signaling region by T2A. For example, the cleavage peptide may comprise the amino acid sequence shown in SEQ ID NO. 81.

In the present application, from the N-terminus to the C-terminus, the CAR may comprise a targeting moiety that binds to CLDN18.2 protein (e.g., the antigen binding protein), the hinge region, the transmembrane domain, the costimulatory signaling region, and the intracellular signaling region, in that order. For example, from the N-terminus to the C-terminus, the CAR may comprise the scFv, a hinge region derived from CD8, a transmembrane region derived from CD8, a costimulatory signaling region derived from 4-1BB, and an intracellular signaling region derived from cd3ζ in that order.

In the present application, from the N-terminus to the C-terminus, the CAR may comprise a targeting moiety that binds to CLDN18.2 protein (e.g., the antigen binding protein), the hinge region, the transmembrane domain, the costimulatory signaling region, and the intracellular signaling region, in that order. For example, from the N-terminus to the C-terminus, the CAR may comprise, in order, the scFv, the hinge region derived from CD8, the transmembrane region derived from CD8, the costimulatory signaling region derived from 4-1BB, the intracellular signaling region derived from cd3ζ, and a low density lipoprotein receptor-related protein or fragment thereof.

In the present application, a vector capable of expressing the CAR or immune effector cell is also included, in which vector a nucleic acid molecule encoding a targeting moiety that binds to CLDN18.2 protein (e.g., the antigen binding protein), encoding the hinge region, encoding the transmembrane domain, encoding the costimulatory signaling region, encoding the intracellular signaling region, and encoding the low density lipoprotein receptor-related protein or fragment thereof may be included in sequence.

In the present application, a vector capable of expressing the CAR or immune effector cell is also included, in which vector a nucleic acid molecule encoding a targeting moiety that binds to CLDN18.2 protein (e.g., the antigen binding protein), encoding the hinge region, encoding the transmembrane domain, encoding the costimulatory signaling region, encoding the intracellular signaling region, encoding the shear peptide, and encoding the low density lipoprotein receptor-related protein or fragment thereof may be included in sequence.

Polypeptide molecules, immunoconjugates, nucleic acid molecules, vectors, cells and pharmaceutical compositions

In another aspect, the application provides a polypeptide molecule, which may comprise an isolated antigen binding protein of the application or a chimeric antigen receptor of the application.

In the present application, the polypeptide molecule may comprise a fusion protein. For example, the isolated antigen binding proteins of the application may be fused to other functional molecules (e.g., antibodies or receptor ligands) to form bispecific molecules. The bispecific molecule can specifically bind to at least two different binding sites or targeting molecules. The bispecific molecule may be prepared by genetic engineering, somatic hybridization, or chemical methods. See, for example, kufer et al, computed supra; cao and Suresh, bioconjugate Chemistry,9 (6), 635-644 (1998); and van Spriel et al, immunology Today,21 (8), 391-397 (2000).

In another aspect, the application also provides immunoconjugates which may comprise the isolated antigen binding proteins of the application.

In the present application, the isolated antigen binding proteins or fragments thereof of the present application may be linked to another agent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, spectroscopic probe, or the like. The linkage may be through one or more covalent bonds, or non-covalent interactions, and may include chelation. A variety of linkers (which may be known in the art) may be used to form the immunoconjugate. Furthermore, the immunoconjugate may be provided in the form of a fusion protein, which may be expressed from a polynucleotide encoding the immunoconjugate. The immunoconjugate may further comprise, for example, an antibody-drug conjugate (ADC). In ADC, the antibody and therapeutic agent may be cross-linked by a linker that is cleavable, such as a peptide linker, disulfide linker, or hydrazone linker.

In another aspect, the application provides one or more nucleic acid molecules that may encode an isolated antigen binding protein of the application or a chimeric antigen receptor of the application. For example, it may be produced or synthesized by: (i) amplified in vitro, e.g. by Polymerase Chain Reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified, e.g. fractionated by cleavage and gel electrophoresis, or (iv) synthesized, e.g. by chemical synthesis.

In the present application, the nucleic acid molecule may comprise a nucleotide sequence shown in any one of SEQ ID NO. 1, SEQ ID NO. 6, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 18, SEQ ID NO. 23, SEQ ID NO. 26 and SEQ ID NO. 28.

In the present application, the nucleic acid molecule may comprise any one of the nucleotide sequences selected from the group consisting of:

In another aspect, the application provides a vector which may comprise a nucleic acid molecule according to the application. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation, and the like. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell. The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector may include components that assist it in entering the cell, such as viral particles, liposomes, or protein shells, but not exclusively.

In another aspect, the application provides a cell which may comprise an isolated antigen binding protein of the application, a chimeric antigen receptor of the application, a polypeptide molecule of the application, a nucleic acid molecule of the application or a vector of the application. In certain embodiments, each or each host cell may comprise one or more nucleic acid molecules or vectors of the application. In certain embodiments, each or each host cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors of the application. For example, the vectors of the application may be introduced into such host cells, e.g., eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like. In certain embodiments, the cell may be a bacterial cell (e.g., E.coli), a yeast cell, or other eukaryotic cell. The vectors of the application may be introduced into the host cell by methods known in the art.

In certain embodiments, the cell may be an immune effector cell. In certain embodiments, the cells may 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, lymphoprogenitor cells, and/or pluripotent stem cells.

In certain embodiments, the cell may be a T cell.

In the present application, the cell may comprise and/or express the CAR. In the present application, the cell may comprise and/or express the CAR and the low density lipoprotein receptor-related protein or fragment thereof.

In another aspect, the application also provides a pharmaceutical composition, which may comprise an isolated antigen binding protein of the application, a chimeric antigen receptor of the application, a polypeptide molecule of the application, an immunoconjugate of the application, a nucleic acid molecule of the application, a vector of the application and/or a cell of the application, and optionally a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition may further comprise one or more (pharmaceutically effective) suitable formulations of adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. Pharmaceutical compositions of the application include, but are not limited to, liquid, frozen and lyophilized compositions.

In certain embodiments, the pharmaceutical compositions may also contain more than one active compound, typically those active compounds having complementary activity that 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.

In certain embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents compatible with drug administration, generally safe, non-toxic.

In certain embodiments, the pharmaceutical composition may comprise parenteral, transdermal, endoluminal, intra-arterial, intrathecal and/or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition may be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition may be administered without interruption. The uninterrupted (or continuous) administration may be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient, as described in WO 2015/036583.

Preparation method

In another aspect, the application provides a method of preparing said antigen binding protein. The method may comprise culturing the host cell of the application under conditions such that the antigen binding protein is expressed. For example, such methods are known to those of ordinary skill in the art by using an appropriate medium, an appropriate temperature, an appropriate incubation time, and the like.

Any method suitable for producing monoclonal antibodies may be used to produce the antigen binding proteins of the application. For example, animals may be immunized with the conjugated or naturally occurring CLDN18.2 protein or fragments thereof. Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.

Any suitable form of CLDN18.2 can be used as an immunogen (antigen) for generating non-human antibodies specific for CLDN18.2 and screening the antibodies for biological activity. The priming immunogen may be full length mature human CLDN18.2, including natural homodimers, or a peptide containing single/multiple epitopes. The immunogens may be used alone or in combination with one or more immunogenicity enhancing agents known in the art.

The chimeric human antibody may be selected from any kind of immunoglobulin including IgM, igD, igG, igA and IgE. In the present application, the antibody may be an IgG antibody, and an IgG1 subtype may be used. Optimization of the necessary constant domain sequences to produce the desired biological activity can be achieved by screening antibodies using the biological assays described in the examples below. Also, any type of light chain may be used in the compounds and methods of the application. For example, kappa chains or variants thereof are useful in the compounds and methods of the present application.

Method and use

In a further aspect, the application provides the use of said isolated antigen binding protein, said chimeric antigen receptor, said polypeptide molecule, said immunoconjugate, said nucleic acid molecule, said vector, said cell and/or said pharmaceutical composition for the preparation of a medicament for the prevention, alleviation and/or treatment of a disease and/or a disorder.

In another aspect, the application also provides a method of preventing, alleviating or treating a disease and/or disorder, which method may comprise administering to a subject in need thereof the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition of the application. In the present application, the administration may be performed in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

In another aspect, the isolated antigen binding proteins, the chimeric antigen receptors, the polypeptide molecules, the immunoconjugates, the nucleic acid molecules, the vectors, the cells and/or the pharmaceutical compositions of the application may be used for preventing, alleviating or treating diseases and/or disorders.

In the present application, the disease and/or condition may include cancer.

In the present application, the cancer may include a solid tumor and/or a hematological tumor.

In the present application, the cancer may comprise CLDN18.2 positive tumors.

In the present application, the cancer may include stomach cancer and/or colon cancer.

In the present application, the isolated antigen binding protein may be administered with one or more other antibodies so as to effectively inhibit tumor growth in a subject. The isolated antigen binding proteins may also be administered with a chemotherapeutic agent.

In another aspect, the application also provides a method of detecting CLDN18.2 in a sample. The method comprises administering the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition of the application. The method may be an ex vivo and/or an in vitro method.

In another aspect, the application also provides a kit or a kit for detecting CLDN18.2 in a sample, which may comprise said isolated antigen binding protein, said chimeric antigen receptor, said polypeptide molecule, said immunoconjugate, said nucleic acid molecule, said vector, said cell and/or said pharmaceutical composition.

In a further aspect, the application also provides the use of said isolated antigen binding protein, said chimeric antigen receptor, said polypeptide molecule, said immunoconjugate, said nucleic acid molecule, said vector, said cell and/or said pharmaceutical composition in the preparation of said reagent or kit for detecting CLDN 18.2.

In the present application, the reagent or kit may be used to detect the presence and/or amount of CLDN18.2 in a sample.

Without intending to be limited by any theory, the following examples are presented merely to illustrate the antigen binding proteins, methods of preparation, uses, and the like of the present application and are not intended to limit the scope of the application.

Examples

EXAMPLE 1 preparation of CLDN18.1 and CLDN18.2 Positive control antibodies

CLDN18.1 positive control antibody a commercially available rabbit monoclonal antibody (abcam, cat#ab 203563) against CLDN18 was selected that recognized both CLDN18.1 and CLDN18.2 and the antigen binding site was located intracellular. In the present application, this antibody was used as a CLDN18.1 positive control antibody and was designated as an Anti-CLDN 18 antibody (Anti-Claudin 18 Anti).

According to 175D10 clone (IMAB 362) antibody heavy and light chain full length amino acid sequence (shown as SEQ ID NO:75 and SEQ ID NO: 76) provided by patent application CN103509114A, the conventional gene synthesis is entrusted to Suzhou Jinweizhi biotechnology limited company and cloned into eukaryotic expression vectors pCMV-k and pCMV-IgG1 (NDL) provided by the company, plasmids comprising pCMV-IMAB362-VL and pCMV-IMAB362-VH are constructed, the two vector plasmids are co-transfected into an Expi293 cell according to the proportion of 3:2 (VL: VH) for transient expression, cell culture supernatant is harvested after 5 days, and the CLDN18.2 positive control antibody is obtained after BCA affinity chromatography purification and BCA quantification.

EXAMPLE 2 construction of highly expressed stable cell lines of CLDN18.1 and CLDN18.2

According to 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 is entrusted to the Suzhou gold intelligence biotechnology limited company and cloned into a lentivirus expression vector pHAGE-full EF1a-IzsGreen provided by the company, the obtained puncture bacteria containing plasmids are activated and then extracted into plasmids, then the lentivirus packaging and infection system of the company are utilized to carry out lentivirus infection on 293T, CHO and SP2/0 cells respectively, after the infection efficiency of the cells obtained after the infection is detected by a FACS, monoclonalization is carried out by a gradient dilution method, and then a monoclonal cell strain which stably and highly expresses the CLDN18.1 and the CLDN18.2 is obtained through FACS screening verification.

Table 1 nucleotide sequences of CLDN18.1 and CLDN18.2

CLDN18 source	Genbank accession number	Nucleotide sequence
Human CLDN18.1	NM_016369.4	SEQ ID NO:56
Human CLDN18.2	NM_001002026.3	SEQ ID NO:57
Mouse CLDN18.1	NM_019815.3	SEQ ID NO:58
Mouse CLDN18.2	NM_001194921.1	SEQ ID NO:59

Cynomolgus monkey CLDN18.1	XM_005545863.2	SEQ ID NO:60
Cynomolgus monkey CLDN18.2	XM_001114708.4	SEQ ID NO:61

For FACS verification of stable high expression CLDN18.2 cell strain, 30000 transfected cells are spread into a V-shaped 96-well plate, then positive antibody zolbetuximab is added and PBS negative control holes are arranged, after incubation for 1 hour at 4 ℃, the 96-well plate is washed 1 time by FACS buffer solution, and then secondary antibody (goat F (ab') 2 anti-human IgG-Fc is added 650 (abcam, cat#ab 98593)); after incubation at 4℃for 30min, the 96-well plates were washed 2 times with FACS buffer, and then cell fluorescence was monitored using an iQue Screen flow cytometer (available from IntelliCyt Co.) and the monoclonal cells with the highest MFI values were selected for expansion and cryopreservation.

FACS validation of stable high expressing CLDN18.1 cell lines cells were divided into two treatments: one was to re-suspend the plates directly with FACS buffer after cell counting, the primary antibody was the CLDN18.2 positive antibody zolbetuximab provided in example 1; the other is to perform fixation and membrane rupture treatment (cypress organism, fixative FXP008 and membrane rupture agent FXP 009) after cell counting so that the antibody can bind to the intracellular antigen site, and the primary antibody is the commercial CLDN18 antibody provided in example 1; and (3) performing on-machine detection, and selecting monoclonal cells which are strongly bound with the commercial CLDN18 antibody and are not bound with zolbetuximab for expansion culture and freezing.

The cell lines which are obtained by construction and are used for stably and highly expressing the CLDN18 are respectively marked as follows: the 293T-human CLDN18.1 (293T-human CLDN 18.1), 293T-human CLDN18.2 (293T-human CLDN 18.2), CHO-human CLDN18.1 (CHO-human CLDN 18.1), CHO-human CLDN18.2 (CHO-human CLDN 18.2), SP2/0-human CLDN18.1 (SP 2/0-human CLDN 18.1), SP2/0-human CLDN18.2 (SP 2/0-human CLDN 18.2), 293T-murine CLDN18.1 (293T-mouseCDN 18.1), 293T-murine CLDN18.2 (293T-mouseCDN 18.2), 293T-cynomolgus CLDN18.1 (293T-macaca CLDN18.1), 293T-cynomolgus CLDN18.2 (293T-macaca DN18.2) are all monoclonal stable cell lines.

EXAMPLE 3 production of CLDN18.2 murine antigen binding protein

1. Immunization of mice

Adopting a first extracellular segment full-length nucleotide sequence of human CLDN18.2, entrusting a Suzhou gold intelligent biotechnology limited company to perform conventional gene synthesis and cloning the gene into a eukaryotic expression vector provided by the company, constructing a plasmid pCMV-CLDN18.2-D1, and extracting the obtained puncture bacterial liquid from the plasmid to obtain DNA immunogen; the cellular immunogen was SP 2/0-human CLDN18.2 cells constructed as described in example 2. In the experiment, 9 Balb/c mice are immunized, 6 mice are in a combined immunization mode of cell antigens and DNA antigens, the other 3 mice are in a whole cell immunization mode, a classical mouse immunization schedule is adopted, serum of the mice is collected after twice immunization, the serum titer is measured by FACS through 293T-human CLDN18.2 cells, the mice meeting the titer requirement are selected for final booster immunization, if the titer is not met, 1 to 2 times of immunization are needed until the serum titer meets the requirement, spleen tissues of the mice are collected under a sterile condition after 3 days of final immunization, the spleen cells are ground into spleen cell suspension, the spleen cells are split red for treatment, and the split mice are frozen in a refrigerator at the temperature of 80 ℃ below zero for later use.

2. Hybridoma cell fusion

Resuscitates mouse myeloma cells SP2/0 in advance, cultures with DMEM containing 10% fetal bovine serum at 37℃on the basis of 5% CO ₂ Culturing and passaging under the condition. On the day of fusion, PEG1450 Solution (Solution) (Sigma Aldrich, cat # 25322-68-3) and high-sugar DMEM medium were pre-warmed in a 37℃water bath. Observing the state of SP2/0 cells by microscopic examination, preferably, the cells are in a logarithmic growth phase, collecting the SP2/0 cells by centrifugation for 5 minutes, re-suspending the cells by a proper amount of preheated DMEM medium, counting trypan blue, requiring the survival rate of the cells to be more than 90%, and then placing the cells into a water bath kettle at 37 ℃ for standby. The spleen cells of the mice were recovered in a 37℃water bath, washed 1 time with 20mL of pre-heated DMEM, 400g for 5 minutes, the supernatant was discarded, 25mL of pre-heated DMEM was added to resuspend the cells, and the cells were counted by a hemocytometer after dilution in a certain proportion. Mix at cell number ratio (splenocytes: SP 2/0=4:1), 400g,5 min, centrifuge, discard supernatant. 1mL of a preheated PEG1450 solution was slowly added to perform cell fusion at 1X 10 per 96-well plate ⁷ The total amount of individual spleen cells was plated in medium containing 10% FBS, 1% diabody, 2% HAT Supplement (Supplement) and 10% Clona cell ^TM High-sugar DMEM Medium of HY Medium C (STEMCELL, CAT#: 03803). The liquid change treatment is carried out the day before the detection, the culture medium after the liquid change is high-sugar DMEM culture medium containing 10% FBS, 1% diabody and 1% HT Supplement (Supplement), and the cell culture supernatant of the 96-well plate is taken after 24 hours for flow detection.

3. Flow-through selection of hybridoma cells

Selection of 293T-human CLDN18.2 and CHO-human CLDN18.2 cells constructed in example 2 the primary clonal hybridoma cells obtained after fusion were subjected to a first alternate primary screening: cells were individually digested and counted, resuspended in flow buffer (PBS containing 0.1% BSA) to a cell density of 1X 10 ⁶ 30 ul/well of the V-bottom 96-well plate was added per ml; adding 50 ul/well hybridoma cell culture supernatant into V-bottom 96-well plate with cell antigen, simultaneously adding positive control (5 ug/ml zolb-ximab antibody, 30 ul/well) and negative control (30 ul/well PBS buffer), incubating at 4deg.C for 1 hr, washing with flow buffer once, adding corresponding secondary antibody, and adding 30ulWell, incubated at 4℃for 30 min, washed twice with streaming buffer, and assayed on-press (iQue Screen flow Meter, intelliCyt Co.) with 25ul of FACS buffer per well after cell lysis. The primary screening shows the positive parent clone of CLDN18.2 to be expanded to a 24-hole cell culture plate, three days later, cell supernatants are taken for FACS rescreening, four cells of CHO-human CLDN18.2, CHO-human CLDN18.1 and 293T-human CLDN18.2 and 293T-human CLDN18.1 are respectively plated, and the positive hybridoma parent clone of CLDN18.2 and the negative hybridoma parent clone of CLDN18.1 are screened. The positive hybridoma maternal clone is subjected to monoclonalization by utilizing a gradient dilution method, and two to three rounds of subcloning and FACS screening are carried out to obtain the hybridoma monoclonal cells secreting the CLDN18.2 specific antibody, which can generate positive combination with 293T-human CLDN18.2 and CHO-human CLDN18.2 cells and negative combination with 293T-human CLDN18.1 and CHO-human CLDN18.1 cells.

The results are shown in table 2, subclones that bind positively to CLDN18.2 from hybridoma cell culture supernatants included: 5C10, 5F3, 1C7 and 14C3 total 4 lines of monoclonal cells.

TABLE 2 flow screening of hybridoma clones

EXAMPLE 4 CLDN18.2 murine antigen binding protein variable region sequencing

The sequences of the heavy chain variable region and the light chain variable region of the CLDN18.2 murine antigen binding protein were obtained by degenerate primer amplification sequencing, and the primers used were referred to PROTOCOL (doi: 10.1038/nprot.2016.102) published by Lotta von Boehmer and were commissioned to be synthesized by Suzhou gold only intelligent company. Specifically, 4 CLDN18.2 positive hybridoma monoclonal cells of example 3 were collected, total cellular RNA was extracted using QIAGEN RNeasy Plus Mini Kit kit, RNA integrity was detected by 1% agarose gel electrophoresis, and RNA concentration was determined by a NanoDrop nucleic acid quantitative analyzer. Using an RNA reverse transcription kit, 1ug of RNA was reverse transcribed into cDNA and stored at-20℃until use. Preparing a PCR reaction system and setting a PCR program according to the operation specification of PCR high-fidelity enzyme (Beijing holohol gold, AP 231) by taking 5ul cDNA as a template, wherein the annealing temperature adopts a gradient cooling method, then directly taking 1ul of the first round PCR product as the template to carry out a second round of PCR, carrying out 1% agarose gel electrophoresis on the whole second round PCR product, cutting off specific target strips with proper size, carrying out glue recovery, connecting a flat end cloning vector (Beijing holohol gold, CB 501), converting Trans1-T1 competent cells, coating a 2YT plate, culturing for 12-16 hours in a 37 ℃ incubator upside down, picking a monoclonal colony in an ultra clean workbench, sequencing by a Suzhou gold intelligent company after colony activation, and obtaining VH and VL gene sequences of 4 hybridoma clones after sequencing, as shown in Table 3.

TABLE 3 VH and VL sequences of 4 hybridoma clones

EXAMPLE 5 preparation of CLDN18.2 antigen binding proteins

Primers were designed using the heavy and light chain variable region sequences obtained by sequencing in example 4 as templates, respectively, and were designated for primer synthesis by Suzhou Jinwei Biotechnology Co., ltd, PCR amplification by high fidelity enzyme, agarose gel electrophoresis and gel recovery, and the obtained plasmid DNA was recovered and subjected to homologous recombination (Vazyme, C112) into the digested eukaryotic expression vectors (pCMV-IgG 1NDL and pCMV-kappa) with human IgG1 constant regions, and designated for sequencing identification of the correct positive recombinant vector by Suzhou Jinwei Biotechnology Co., ltd. And (3) extracting plasmids after the sequencing is correct, co-transfecting the heavy and light chains into an Expi293 cell, centrifuging after 5 days, collecting cell culture supernatant, and purifying by protein A affinity chromatography to obtain the complete human mouse IgG1 and Ig kappa antigen binding protein. These 4 antigen binding proteins were designated C5F3, C1C7, C14C3, C5C10, respectively.

EXAMPLE 6 determination of specific binding Activity of CLDN18.2 antigen binding proteins and CLDN18.2

The specific binding activity of the chimeric antibody to target cells was detected by flow cytometry (FACS) using an iQue screen flow machine (purchased from intelllicyt corporation) using PBS containing 0.1% bsa as a buffer, and three target cells were selected: the binding activity of the stably transformed cell line expressing human CLDN18.2, the stably transformed cell line expressing human CLDN18.1 and the tumor cell line were measured, respectively.

1. Detection of flow-through binding Activity of antigen binding proteins and high expression human CLDN18.2 cells in example 5

The cells were 293T-human CLDN18.2, CHO-human CLDN18.2 and SP 2/0-human CLDN18.2 constructed in example 2, cell digests counted, resuspended in flow buffer and adjusted to 1X 10 ⁶ 30 ul/well of the V-bottom 96-well plate was added per ml; adding primary antibody into 30 ul/hole, starting with 30ug/ml concentration, and diluting with flow buffer solution in three times ratio to form 7 gradients, wherein each antibody is provided with PBS negative control, and the positive control antibody is zolbretuximab obtained by purification in example 1; incubation at 4℃for 1 hour, washing with streaming buffer once, adding secondary antibody (abcam, cat#ab 98593), 30 ul/well, incubation at 4℃for 30 minutes; the flow buffer solution is washed twice, the cells are loose, 25 ul/hole flow buffer solution is added, and the machine is waited for. Raw data were substituted into graphpad8.0 software for mapping and calculation, and the results are shown in fig. 1.

2. Detection of flow-through binding Activity of antigen binding proteins and high expression human CLDN18.1 cells in example 5

The positive control antibody is a commercially available Anti-CLDN 18.2antibody (Anti-claudin 18.2 Anti) (abcam, cat#ab 203563) with an antigen binding site located in the intracellular portion of CLDN18.2 four crotch membrane proteins, and a flow intracellular staining assay was required. Specifically, the cells were 293T-human CLDN18.1, CHO-human CLDN18.1 and SP 2/0-human CLDN18.1 constructed in example 1, and after cell digestion and counting, they were subjected to a fixed membrane disruption treatment, and the treated cells were resuspended to 1X 10 with a streaming buffer ⁶ 30 ul/well of the V-bottom 96-well plate was added per ml; adding primary antibody into 30 ul/hole, starting with 30ug/ml concentration, and diluting with flow buffer solution in three times ratio gradient to form 7 gradients, wherein each antibody is provided with PBS negative control, and the dilution condition of positive control antibody is the same; incubation at 4℃for 1 hour, washing with flow buffer one time, adding secondary antibodies (abcam, cat#ab98593 and Cat#ab 150079), 30 ul/wellIncubation at 4℃for 30 min; the flow buffer solution is washed twice, the cells are loose, 25 ul/hole flow buffer solution is added, and the machine is waited for. Raw data were substituted into graphpad8.0 software for mapping and calculation, and the results are shown in fig. 2.

3. Flow-through binding Activity detection of antigen binding proteins and tumor cell lines in example 5

Tumor cells MC 38-human CLDN18.2 which were stably and highly expressed in human CLDN18.2 and were constructed as described in example 2 were selected, cell digests were counted, resuspended in flow buffer and adjusted to 1X 10 ⁶ 30 ul/well of the V-bottom 96-well plate was added per ml; adding a primary antibody into 30 ul/hole, starting at a concentration of 30ug/ml, and carrying out gradient dilution by using a flow buffer according to a triple ratio to form 7 gradients, wherein each antibody is provided with PBS negative control, and the positive control antibody is zolbetuximab obtained by purification in the example 1, wherein the dilution conditions are the same as above; incubation at 4℃for 1 hour, washing with streaming buffer once, adding secondary antibody (abcam, cat#ab 98593), 30 ul/well, incubation at 4℃for 30 min; the flow buffer solution is washed twice, the cells are loose, 25 ul/hole flow buffer solution is added, and the machine is waited for. Raw data were substituted into graphpad8.0 software for mapping and calculation, and the results are shown in fig. 3.

Through the above operation steps, expression and purification are carried out to obtain 4 human mouse chimeric antigen binding proteins, and the antigen binding activity is verified by flow assay: as shown in fig. 1, all 4 antigen binding proteins exhibited concentration-dependent binding activity to human CLDN18.2 and were mostly stronger than the zolbetuximab positive antibody, with consistent detection results for 3 cells; as shown in fig. 2, 4 antigen binding proteins each bind specifically to human CLDN18.2 but not to human CLDN 18.1; as shown in FIG. 3, the 4 antigen binding proteins are all strongly bound with the mouse colon cancer MC38 cells stably and highly expressing human CLDN18.2, and the binding activity intensity is concentration-dependent and partially stronger than that of the zolbetuximab positive antibody.

EXAMPLE 7 analysis of Cross-species binding Activity of CLDN18.2 antigen binding proteins

The antigen binding proteins were detected by flow cytometry (FACS) using a CytoFLEX flow cytometer (available from BECKMAN COULTER) using PBS containing 0.1% BSA as bufferCross-binding Activity of species of mice (mouseCDN 18.2), cynomolgus monkeys (macaCLDN18.2). Specifically, 293T-mouse CLDN18.2 and 293T-cynomolgus monkey CLDN18.2 cells were stable transgenic cell lines obtained by the construction of example 2, cell digestions were counted, and resuspended to a cell density of 1X 10 with streaming buffer ⁶ 30 ul/well was added to a V-bottom 96-well plate; the primary antibody is diluted in a gradient mode by using a flow buffer solution according to a triple ratio, the initial concentration is 10ug/ml, 6 gradients are formed, PBS negative control is arranged for each antibody, positive control antibody is zolbetuximab, and the dilution conditions are the same; incubation at 4℃for 1 hour, washing with streaming buffer once, adding secondary antibody (abcam, cat#ab 98593), 30 ul/well, incubation at 4℃for 30 min; the flow buffer is washed twice, the cells are loose in vibration, 30 ul/hole flow buffer is added, and the detection is carried out on the machine. Raw data were substituted into graphpad8.0 software for mapping and calculation, and as shown in fig. 4,4 CLDN18.2 chimeric antibodies and positive control antibody zolbretuximab, both bound to mice (mousecldn 18.2), cynomolgus monkey (macaca cldn18.2) and exhibited gradient dependence of binding activity. It was demonstrated that these 4 antigen binding proteins and zolbetuximab not only specifically bind to human CLDN18.2 (human CLDN 18.2), but also have cross-binding activity to human, mouse, cynomolgus monkey species.

Example 8 CDC Activity of CLDN18.2 antigen binding protein

The ability of CLDN18.2 antigen binding proteins to elicit CDC effects against SP 2/0-human CLDN18.2 cells was tested using a cytotoxicity detection kit (Promega, cat#g1780) as follows:

(1) Preparing a culture medium (A: DMEM+2% FBS+1% diabody, B: DMEM+2% FBS+1% diabody+10% rabbit complement);

(2) Target cells SP 2/0-human CLDN18.2 were centrifuged at 400g for 5 min, and the cells were resuspended in medium A above to a target cell density of 4X 10 ⁵ Individual cells/ml, 100 ul/well were added to 96 well cell culture plates;

(3) The antibody is diluted into three different concentrations by the culture medium B, namely 10ug/ml, 2ug/ml and 0.4ug/ml respectively, 100 ul/hole is added into a detection hole, two compound holes are arranged at each concentration point, zolbetuximab is used as a positive control antibody, and human IgG-Fc is used as a negative control antibody;

(4) Complement was Rabbit complement Rabbit compact 3-4 week (Cat # 31061-3) from PelFreez Bio company, and the final concentration of the experiment used for complement was 5%;

(5) Control wells were set as required for the kit, and after incubation of the obtained mixture in an incubator at 37 ℃ for 4 hours, the absorbance of 490nM was recorded by the microplate reader, and the percent of target cell lysis was calculated using the formula given by the kit, graphPad prism 8 analysis data.

As shown in fig. 5a, when SP 2/0-human CLDN18.2 cells were used as killing target cells, all detected antigen binding proteins were able to induce strong CDC effects in a dose dependent manner, i.e. all had significant CDC activity; wherein C5F3, C1C7 and C14C3 all exhibit higher CDC activity than zolbetuximab at any one of the antibody concentrations; C5C10 showed higher CDC activity than zolb et uximab at low antibody concentration (0.4 ug/ml) and slightly weaker CDC activity than zolb et uximab at high antibody concentration (2 ug/ml and 10 ug/ml).

The c5F3 antigen binding protein was further tested for its ability to elicit CDC effects against CHO cells stably overexpressing human at CLDN 18.2. Briefly, CHO-human CLDN18.2 cells were obtained as described in example 1 by construction of the lentiviral transfection system of the company, operating as described above, at 1.2X10 ⁴ The density of each cell was plated at 5% final concentration of rabbit complement, antigen binding protein was diluted in five-fold ratio gradient, i.e., 10ug/ml, 2ug/ml, 0.4ug/ml, 0.08ug/ml, 0.016ug/ml, 0.0032ug/ml, 0.64ng/ml, two wells were set for each concentration, zolbretuximab was used as positive control antibody, human IgG-Fc was used as isotype control antibody, absorbance after co-incubation for 4 hours was measured by a microplate reader, percent target cell lysis was calculated, and GraphPad prism 8 analysis data was processed.

As shown in fig. 5b, the c5F3 antigen binding protein was able to induce strong CDC effects on CHO-human CLDN18.2 cells in a dose-dependent manner and showed higher CDC activity than zolb etuximab.

EXAMPLE 9 in vivo anti-tumor Activity of CLDN18.2 antigen binding protein

The antigen binding protein (C5F 3) of example 5, positive control antibody zolbetuximab and isotype control antibody human IgG-Fc were selected and tested for anti-tumor activity in C57BL/6 mice vaccinated with mouse colon cancer cells. The six antigen binding proteins are purified and obtained by using the transient transfection system of the company Expi293, and the endotoxin of the antigen binding proteins is controlled below 4 EU/mg. The specific embodiment is as follows:

67 female C57BL/6 mice were subcutaneously inoculated with MC 38-human CLDN18.2 cells in the right abdomen at a cell mass of 1.1X10 ⁶ Selecting tumor size of 31.33-116.31mm 7 days after inoculation ³ (average tumor size is 71.59 mm) ³ ) The number of mice in the group was 6, 8 mice per group, and the Day of random grouping of mice was defined as Day0. The mice were given a tail vein dose, once a week, four weeks on a schedule of two groups of 30mg/kg and 7.5mg/kg, with the mice being grouped and given the schedule shown in Table 4. Tumor sizes were weighed and measured three times per week during dosing and were measured by the formula tv= (length x width ² ) Tumor volume was calculated and Tumor Growth Inhibition (TGI) and T/C values were calculated using tumor size. When the tumor volume reaches 2000mm ³ When the experiment was stopped, mice were euthanized.

TABLE 4 grouping and dosing regimen of in vivo efficacy experiments with CLDN18.2 antigen binding proteins

The antitumor effect of tail vein route administration of zolbetuximab and C5F3 was evaluated in female C57BL/6 mice by subcutaneous MC 38-human CLDN18.2 mouse colon cancer model. The results show that in MC 38-human CLDN18.2 tumor-bearing C57BL/6 mice, all the mice given were well tolerated, the body weight remained stable, no adverse reactions were observed, and the changes in body weight and relative changes (%) of the mice are shown in FIGS. 6 and 7, respectively; analysis of tumor volume change curve before Day19 and tumor growth inhibition curve before Day17 showed better tumor inhibition effect compared to phosphate buffer control group, c5F3 and zolbetuximab high dose administration group showed significant difference (< 0.0001), c5F3 tumor inhibition effect (tgi= 41.54%) was slightly weaker than zolbetuximab (tgi= 46.84%) and no significant difference was shown in fig. 8 and 9; analysis of Kaplan-Meier survival curves prior to Day40 showed a higher percentage of mice survival than phosphate buffer control, both the c5F3 high dose and zolbetuximab high dose dosing groups, and exhibited significant differences (×p < 0.05), with the c5F3 high dose group having a slightly weaker percentage of mice survival than the zolbetuximab high dose dosing group, and no significant differences, as shown in fig. 10.

Example 10 cldn18.2 stable expression of specific CARs in T cells

In this example, a CLDN18.2 specific CAR structure was constructed, as shown in FIG. 11A, consisting of a human CD8 signal peptide (SEQ ID NO: 82), an anti-human CLDN18.2 single chain antibody (5F 3-scFv, SEQ ID NO:85; standard-scFv, SEQ ID NO:86, human CD8 hinge region (SEQ ID NO: 77), human CD8 transmembrane region (SEQ ID NO: 78), human 4-1BB intracellular co-stimulatory domain (SEQ ID NO: 79), human CD3 zeta intracellular activating domain (SEQ ID NO: 80) and an added Ori novel element (SEQ ID NO: 84). First, this example explores the expression of CLDN18.2 specific CAR in human T cells and CART cell expansion fold under in vitro conventional culture conditions:

1) Human PBMC cells frozen in liquid nitrogen were resuscitated in a 37 ℃ water bath and rinsed three times (500 g,5min;400g,5min;300g,5 min), the washed human PBMC cells were then subjected to CD3 cationic selection by mixing with CD3 microblades, human (Meinani, 130-050-101), using a magnetic rack, i.e.separating and retaining CD3 ⁺ T cells; CD3 was resuspended in medium containing (4% FBS+X-VIVO (Lonza Co.) +20ng/ml factor 1+10ng/ml factor 2) ⁺ T cell to cell density of 1X 10 ⁶ Adding CD3/CD28 magnetic beads (Sieimer-Fei 40203D) into cells/ml according to the ratio of 1:3, washing the magnetic beads twice with a culture medium, sucking the magnetic beads with a magnetic rack, and standing for 1min to activate T cells; t cell additionMixing with magnetic beads, adding culture medium to 700ul per well of 12-well plate, and counting cells with number of 7X10 ⁵ Holes, density 1X 10 ⁶ cells/ml, sorting when the diary was Day0.

2) Activated T cells were allowed to stand at 37℃CO ₂ After 20 hours of culture in an incubator, adding the corresponding virus supernatant according to the ratio of the complex of virus infection (MOI) of 4, adding polybrene to a final concentration of 10ug/ml, blowing and mixing uniformly, centrifuging for 1 hour at 1200rpm with a horizontal centrifuge, and putting the pore plate back to CO at 37 DEG C ₂ The cells were incubated in the incubator for 24 hours, and the infection was designated Day1.

3) After 24h, cells from each well of the 12-well plate were repeatedly blown up uniformly, transferred to a 1.5ml EP tube, centrifuged at 400g for 5 min, the supernatant removed, and resuspended to a cell density of 7X 10 using 1ml of fresh X-VIVO complete medium ⁵ cells/ml, placed at 37℃in CO ₂ Culturing in an incubator, wherein the culture medium needs to be supplemented to turn yellow; cells were counted every 2 days and cell density was adjusted back to 7X 10 by supplementing fresh X-VIVO complete medium ⁵ cells/ml; statistical counts were recorded, plotted using graphpad8.0 software and CART cell expansion fold under normal culture was calculated.

4) Cell positive rate of CAR-T cells cultured for 8-12 days: myc-Tag is carried in virus used for infecting cells, so that after the virus infects cells, the Myc positive rate is detected by a flow cytometry to obtain the CAR expression positive rate, and the direct standard detection antibody is Myc-Tag (9B 11) Mouse mAb (Alexa)488 Conjugate)(Cell Signaling,2279S)。

Human CLDN18.2 sequence viruses, including 5F3 sequence and Standard sequence viruses, were infected at moi=4, respectively. As a result, FIG. 11B shows that the CAR positive rate was 71.9% and the Standard-CART positive rate was 62.8% in the case of in vitro CAR cell culture on day 8. The CAR positive rate remained essentially unchanged for all 8 to 12 days of in vitro culture, fig. 12C. FIG. 12D shows that 5F3-CART cells were routinely cultured in vitro for 9 days at a cumulative expansion factor of about 69-fold, standard-CART cells were at a cumulative expansion factor of about 58-fold, mock T cells were at a cumulative expansion factor of about 73-fold, and there was no significant difference between the expansion factors.

Example 11 cldn18.2 specific CARs can specifically kill target cells in vitro

In the embodiment, the CHO-hCDNN18.2 cells stably and highly expressing the human CLDN18.2 and the CHO-hCDNN18.1 cells stably and highly expressing the human CLDN18.1 are obtained through slow virus infection and parallel flow type sorting. This example further uses a cytotoxicity detection kit (Promega, cat#g1780) to evaluate the specific killing ability of CAR-T cells in vitro using LDH method, as follows:

CAR-T and Mock T cells routinely cultured for 9 days in example 10 above were centrifuged separately and resuspended in blank X-VIVO medium to a cell density of 1X 105/ml; the target cells are CHO, CHO-hCDN18.2 and CHO-hCDN18.1, the three target cells are respectively digested and counted, and then the three target cells are resuspended in a blank X-VIVO medium to make the cell density 5X 105/ml; then mixed into a sterile v-bottom 96-well plate according to a volumetric system of 100ul target cell suspension +100ul CAR-T/Mock T cell suspension per well. Control wells were set as required for the kit, and after incubation of the obtained mixture in an incubator at 37 ℃ for 24 hours, the absorbance of 490nM was recorded by the microplate reader, and the percent of target cell lysis was calculated using the formula given by the kit, and data was analyzed and processed. The results are shown in FIG. 12,5F3-CART, which specifically induces the lysis of CLDN18.2 positive target cells in vitro, has no lysis effect on negative and CLDN18.1 positive cells, and shows that 5F3-CART cells have the high specific target cell killing activity of human CLDN18.2 in vitro.

EXAMPLE 12 factor secretion by CLDN18.2-specific CAR

The present example used the Human IFN-. Gamma.ELISA kit (R&D, DY 285B) and Human IL-2 ELISA kit (R) &D, DY 202) analysis of the secretion of IFN-gamma and IL-2 by CAR-T in killing target cells. Specifically, according to 1×10 ⁴ Cell mass per well of each cell will be the target cell (CHO-hCDNN 18.2) highly expressing CLDN18.2, the control cell (CHO-hCDNN 1) highly expressing CLDN18.18.1 CHO) and negative cells were inoculated into sterile 96-well plates, respectively, and target cells were targeted according to Effector (effect): CAR-T cells (5F 3-CART and Standard-CART) and non-modified T cells (Mock T cells) were added at a Target) =1:1 ratio equivalent to cells. After 24h incubation, the supernatants were assayed for IL-2 and IFN-gamma content by enzyme-linked immunosorbent assay (ELISA) following the kit instructions. The results are shown in figures 13A and 13B, where 5F3-CART secreted higher levels of factors when incubated with CLDN18.2 positive cells; no significant factor was secreted under co-incubation with negative and CLDN18.1 positive cells, with Standard-CART, and the 5F3-CART factor secretion level was slightly weaker than Standard-CART factor secretion level. Namely, the 5F3-CART cells have the effect of specifically inducing cytokine secretion on tumor cells which highly express CLDN 18.2.

EXAMPLE 13 tumor-inhibiting effect of CLDN18.2-specific CAR-T cells in a mouse model of human gastric cancer

In the embodiment, the human CLDN18.2 gene is firstly introduced into human gastric cancer NCI-N87 cells in a slow virus mode, and then the human gastric cancer cells which highly express the human CLDN18.2, namely N87-hCDN 18.2 cells, are separated by a flow sorter. And constructing a human gastric cancer tumor model by utilizing female B-NDG heavy immunized mice through a subcutaneous injection way, and verifying the tumor inhibition effect of CLDN18.2 specific CAR-T cells in the mice. According to 3X 10 ⁶ N87-hCDN 18.2 cells were inoculated at a dose/dose, and the average tumor size was selected to be 247mm 23 days after tumor inoculation ³ The mice of (a) were randomly divided into 4 groups of 6 mice each, and CAR-T (or Mock T) cells were cultured for 10 days by tail vein feedback, and the mice were grouped and the CART cells were feedback as shown in table 6:

TABLE 6 grouping and CART feedback protocol for in vivo efficacy experiments in human gastric cancer mouse model

Grouping mice	CART cell types	CART cell reinfusion dose/200 ul	Number of mice
G1	Mock T cell	1×10 ⁷	6
G2	Standard-CART	1×10 ⁶	6
G3	5F3-CART	3×10 ⁶	6
G4	5F3-CART	1×10 ⁷	6

As shown in fig. 14A, the body weight and tumor size of the mice were measured 3 times per week, and a total of 37 days was observed after CART cell feedback. As shown in fig. 14B, G4:5F3-CART high dose group (1X 10) ⁷ cells/mice) on day 14 after the return CART, all mice in the group showed a significant decrease in body weight, with a body weight decrease of greater than 20%; and G2: standard-CART group (1×10) ⁶ cells/only) 4 mice began to drop significantly in weight 26 days after back-transfusion CART and died successively; and G3:5F3-CART Low dose group (3X 10) ⁶ cell/only) and G1: mock T cell group (1×10) ⁷ cell/mouse) tumor volume was measured continuously for 37 days after CART feedback, i.e., PG-D37, without a decrease in mouse body weight, and with good mice tolerance. As shown in fig. 14C, compared to G1: mock T cell group, G3:5F3-CART Low dose group (3X 10) ⁶ cells/alone) showed a certain tumor inhibiting effect with very significant statistical differences (p < 0.0001). In conclusion, 5F3-CART was shown to be effective at low doses (3X 10 ⁶ cells/mice) showed tolerance, no abnormal phenomenon such as weight loss, and very remarkable tumor-inhibiting activity.

Claims

An isolated antigen binding protein comprising HCDR3, said HCDR3 comprising the amino acid sequence of SEQ ID No. 64.
The isolated antigen binding protein of claim 1, wherein said HCDR3 comprises an amino acid sequence of any one of SEQ ID No. 5, SEQ ID No. 14 and SEQ ID No. 22.
The isolated antigen binding protein of any one of claims 1-2, comprising HCDR2, said HCDR2 comprising the amino acid sequence of SEQ ID No. 63.
The isolated antigen binding protein of claim 3, wherein said HCDR2 comprises an amino acid sequence of any one of SEQ ID No. 4, SEQ ID No. 13 and SEQ ID No. 21.
The isolated antigen binding protein of any one of claims 1-4, comprising HCDR1, said HCDR1 comprising the amino acid sequence of SEQ ID No. 62.
The isolated antigen binding protein of claim 5, wherein said HCDR1 comprises the amino acid sequence of SEQ ID No. 3 or SEQ ID No. 20.
The isolated antigen binding protein of any one of claims 5-6, comprising H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence of SEQ ID No. 68.
The isolated antigen binding protein of claim 7, wherein the H-FR1 comprises the amino acid sequence set forth in SEQ ID No. 37 or SEQ ID No. 51.
The isolated antigen binding protein of any one of claims 5-8, comprising H-FR2, the H-FR2 being located between the HCDR1 and the HCDR2, and the H-FR2 comprising the amino acid sequence of SEQ ID No. 38.
The isolated antigen binding protein of any one of claims 3-9, comprising H-FR3, the H-FR3 being located between the HCDR2 and the HCDR3, and the H-FR3 comprising the amino acid sequence of SEQ ID No. 69.
The isolated antigen binding protein of claim 10, wherein the H-FR3 comprises an amino acid sequence set forth in any one of SEQ ID No. 39, SEQ ID No. 45, and SEQ ID No. 48.
The isolated antigen binding protein of any one of claims 1-11, comprising H-FR4, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence of SEQ ID No. 40.
The isolated antigen binding protein of any one of claims 1-12, comprising a VH comprising the amino acid sequence shown in SEQ ID No. 73.
The isolated antigen binding protein of claim 13, wherein the VH comprises an amino acid sequence set forth in any one of SEQ ID No. 2, SEQ ID No. 12, SEQ ID No. 19 and SEQ ID No. 27.
The isolated antigen binding protein of any one of claims 1-14, comprising LCDR3, and the LCDR3 comprises the amino acid sequence of SEQ ID No. 67.
The isolated antigen binding protein of claim 15, wherein said LCDR3 comprises an amino acid sequence of any one of SEQ ID No. 10, SEQ ID No. 17, SEQ ID No. 25 and SEQ ID No. 32.
The isolated antigen binding protein of any one of claims 1-16, comprising LCDR2, and the LCDR2 comprises the amino acid sequence of SEQ ID No. 66.
The isolated antigen binding protein of claim 17, wherein said LCDR2 comprises the amino acid sequence of SEQ ID No. 9 or SEQ ID No. 31.
The isolated antigen binding protein of any one of claims 1-18, comprising LCDR1, and wherein the LCDR1 comprises the amino acid sequence of SEQ ID No. 65.
The isolated antigen binding protein of claim 19, wherein said LCDR1 comprises the amino acid sequence of SEQ ID No. 8 or SEQ ID No. 30.
The isolated antigen binding protein of any one of claims 19-20, comprising L-FR1, the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence of SEQ ID No. 70.
The isolated antigen binding protein of claim 21, wherein the L-FR1 comprises an amino acid sequence set forth in any one of SEQ ID No. 41, SEQ ID No. 46, and SEQ ID No. 52.
The isolated antigen binding protein of any one of claims 19-22, comprising L-FR2, the L-FR2 being located between the LCDR1 and the LCDR2, and the L-FR2 comprising the amino acid sequence of SEQ ID No. 42.
The isolated antigen binding protein of any one of claims 17-23, comprising L-FR3, the L-FR3 being located between the LCDR2 and the LCDR3, and the L-FR3 comprising the amino acid sequence of SEQ ID NO: 71.
The isolated antigen binding protein of claim 24, wherein the L-FR3 comprises an amino acid sequence set forth in any one of SEQ ID No. 43, SEQ ID No. 49, and SEQ ID No. 53.
The isolated antigen binding protein of any one of claims 15-25, comprising L-FR4, the N-terminus of L-FR4 is linked to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence of SEQ ID No. 72.
The isolated antigen binding protein of claim 26, wherein the L-FR4 comprises an amino acid sequence set forth in any one of SEQ ID No. 44, SEQ ID No. 47, and SEQ ID No. 50.
The isolated antigen binding protein of any one of claims 1-27, comprising a VL, and said VL comprises the amino acid sequence shown in SEQ ID No. 74.
The isolated antigen binding protein of claim 28, wherein the VL comprises an amino acid sequence set forth in any one of SEQ ID No. 7, SEQ ID No. 16, SEQ ID No. 24, and SEQ ID No. 29.
The isolated antigen binding protein of any one of claims 1-29, comprising any one of the group VH and VL selected from:

1) The VH comprises an amino acid sequence shown in SEQ ID NO. 2, and the VL comprises an amino acid sequence shown in SEQ ID NO. 7;

2) The VH comprises an amino acid sequence shown in SEQ ID NO. 12, and the VL comprises an amino acid sequence shown in SEQ ID NO. 16;

3) The VH comprises an amino acid sequence shown in SEQ ID NO. 19, and the VL comprises an amino acid sequence shown in SEQ ID NO. 24; and

4) The VH comprises the amino acid sequence shown in SEQ ID NO. 27, and the VL comprises the amino acid sequence shown in SEQ ID NO. 29.
The isolated antigen binding protein of any one of claims 1-30, comprising an antibody heavy chain constant region.
The isolated antigen binding protein of claim 31, wherein the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.
The isolated antigen binding protein of any one of claims 31-32, wherein the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.
The isolated antigen binding protein of any one of claims 1-33, comprising an antibody light chain constant region.
The isolated antigen binding protein of claim 34, wherein the antibody light chain constant region is derived from a human igκ constant region.
The isolated antigen binding protein of any one of claims 1-35, comprising an antibody or antigen binding fragment thereof.
The isolated antigen binding protein of claim 36, wherein said antigen binding fragment comprises Fab, fab ', fv fragment, F (ab') ₂ ，F(ab) ₂ scFv, di-scFv and/or dAb.
The isolated antigen binding protein of any one of claims 36-37, wherein the antibody is selected from one or more of the group consisting of: monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
The isolated antigen binding protein of any one of claims 1-38, wherein it is verified in FACS that it does not substantially compete with a reference antibody for binding to CLDN18.2, wherein the reference antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID No. 54 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID No. 55.
The isolated antigen binding protein of any one of claims 1-39, which is capable of specifically binding to CLDN18.2 and does not substantially bind to CLDN18.1.
The isolated antigen binding protein of any one of claims 39-40, wherein the CLDN18.2 comprises mouse CLDN18.2, cynomolgus CLDN18.2, and/or human CLDN18.2.
The isolated antigen binding protein of any one of claims 1-41, which is capable of CDC activity.
The isolated antigen binding protein of any one of claims 1-42, which is capable of inhibiting tumor growth and/or proliferation of tumor cells.
A chimeric antigen receptor comprising a targeting moiety comprising the antigen binding protein of any one of claims 1-43.
The chimeric antigen receptor of claim 44, comprising a co-stimulatory domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B-H3, 2B4, fepsilon RI gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligand, CD40 and MyD88.
The chimeric antigen receptor of any one of claims 44-45, wherein the co-stimulatory domain is an intracellular co-stimulatory signaling region derived from 4-1 BB.
The chimeric antigen receptor according to any one of claims 45-46, wherein the co-stimulatory domain comprises the amino acid sequence set forth in SEQ ID No. 79.
The chimeric antigen receptor of any one of claims 44-47, comprising an intracellular signaling domain derived from one or more proteins selected from the group consisting of: CD3 ζ, CD3 δ, CD3 γ, CD3 ε, CD79a, CD79b, fcεRIγ, fcεRIβ, fcγRIIa, bovine leukemia virus gp30, epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and domains comprising at least one ITAM.
The chimeric antigen receptor of claim 48, wherein the intracellular signaling domain is a signaling domain derived from cd3ζ.
The chimeric antigen receptor according to any one of claims 48-49, wherein the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID No. 80.
The chimeric antigen receptor of any one of claims 44-50, comprising a transmembrane region comprising 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, CD ε, CD3 ζ, 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 of claim 51, wherein the transmembrane region is a transmembrane region derived from CD 8.
The chimeric antigen receptor according to any one of claims 51-52, wherein the transmembrane region comprises the amino acid sequence shown in SEQ ID No. 78.
The chimeric antigen receptor of any one of claims 44-53, comprising a hinge region between the targeting moiety and the transmembrane region, the hinge region comprising 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, fc εRIgamma, BTLA, GITR, DAP, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
The chimeric antigen receptor of claim 54, wherein the hinge region is a CD 8-derived hinge region.
The chimeric antigen receptor according to any one of claims 54-55, wherein the hinge region comprises the amino acid sequence shown in SEQ ID No. 77.
The chimeric antigen receptor of any one of claims 44-56, further comprising a signal peptide.
The chimeric antigen receptor of claim 57, wherein the signal peptide is derived from a signal peptide of a CD8 protein.
The chimeric antigen receptor of any one of claims 57-58, wherein the signal peptide comprises the amino acid sequence set forth in SEQ ID No. 82.
The chimeric antigen receptor of any one of claims 44-59, further comprising a low density lipoprotein receptor-related protein or fragment thereof.
The chimeric antigen receptor of claim 60, 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 1-12 and functional fragments thereof.
The chimeric antigen receptor according to any one of claims 60-61, wherein the low density lipoprotein receptor-related protein or fragment thereof is low density lipoprotein receptor-related protein 5 and/or 6 or fragment thereof.
The chimeric antigen receptor according to any one of claims 60-62, wherein the low density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence set forth in SEQ ID No. 84.
A polypeptide molecule comprising the isolated antigen binding protein of any one of claims 1-43 or the chimeric antigen receptor of any one of claims 44-63.
The polypeptide molecule of claim 64, comprising a fusion protein.
An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-43.
An isolated nucleic acid molecule or molecules encoding the isolated antigen binding protein of any one of claims 1-43, the chimeric antigen receptor of any one of claims 44-63, or the polypeptide molecule of any one of claims 64-65.
The nucleic acid molecule of claim 67 comprising a nucleotide sequence set forth in any one of SEQ ID NO. 1, SEQ ID NO. 6, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 18, SEQ ID NO. 23, SEQ ID NO. 26 and SEQ ID NO. 28.
The nucleic acid molecule of any one of claims 67-68, comprising any one of the group of nucleotide sequences selected from:

1) A nucleotide sequence shown in SEQ ID NO. 1 and a nucleotide sequence shown in SEQ ID NO. 6;

2) A nucleotide sequence shown as SEQ ID NO. 11 and a nucleotide sequence shown as SEQ ID NO. 15;

3) The nucleotide sequence shown as SEQ ID NO. 18 and the nucleotide sequence shown as SEQ ID NO. 23; and

4) The nucleotide sequence shown as SEQ ID NO. 26 and the nucleotide sequence shown as SEQ ID NO. 28.
A vector comprising the nucleic acid molecule of any one of claims 67-69.
A cell comprising the isolated antigen binding protein of any one of claims 1-43, the chimeric antigen receptor of any one of claims 44-63, the polypeptide molecule of any one of claims 64-65, the nucleic acid molecule of any one of claims 67-69, or the vector of claim 50.
The cell of claim 71 which is an immune effector cell.
The cell of any one of claims 71-72, comprising a T cell, B cell, natural killer cell (NK cell), macrophage, NKT cell, monocyte, dendritic cell, granulocyte, lymphocyte, leukocyte, peripheral blood mononuclear cell, embryonic stem cell, lymphoid progenitor cell, and/or pluripotent stem cell.
The cell of any one of claims 71-73, which is a T cell.
The cell of any one of claims 71-74, further comprising and/or expressing a low density lipoprotein receptor-related protein or fragment thereof.
The cell of claim 75, wherein the low density lipoprotein receptor-related protein or fragment thereof comprises one or more of the following selected from the group consisting of: low density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.
The cell of any one of claims 75-76, wherein the low density lipoprotein receptor-related protein or fragment thereof is low density lipoprotein receptor-related protein 5 and/or 6 or fragment thereof.
The cell of any one of claims 75-77, wherein the low density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence set forth in SEQ ID No. 84.
A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-43, the chimeric antigen receptor of any one of claims 44-63, the polypeptide molecule of any one of claims 64-65, the immunoconjugate of claim 66, the nucleic acid molecule of any one of claims 67-69, the vector of claim 70 and/or the cell of any one of claims 71-78, and optionally a pharmaceutically acceptable carrier.
A method of making the isolated antigen binding protein of any one of claims 1-43, comprising culturing the cell of claim 71 under conditions such that the antigen binding protein is expressed.
Use of an isolated antigen binding protein of any one of claims 1-43, a chimeric antigen receptor of any one of claims 44-63, a polypeptide molecule of any one of claims 64-65, an immunoconjugate of claim 66, a nucleic acid molecule of any one of claims 67-69, a vector of claim 70, a cell of any one of claims 71-78, and/or a pharmaceutical composition of claim 79 in the manufacture of a medicament for the prevention, alleviation and/or treatment of a disease and/or disorder.
The use of claim 81, wherein the disease and/or condition comprises cancer.
The use of any one of claims 81-82, wherein the cancer comprises a solid tumor and/or a hematological tumor.
The use of any one of claims 82-83, wherein the cancer comprises gastric and/or colon cancer.
A method of detecting CLDN18.2 in a sample comprising administering the isolated antigen binding protein of any one of claims 44-63, the chimeric antigen receptor of any one of claims 64-65, the immunoconjugate of claim 66, the nucleic acid molecule of any one of claims 67-69, the vector of claim 70, the cell of any one of claims 71-78 and/or the pharmaceutical composition of claim 79.
A reagent or kit for detecting CLDN18.2 in a sample comprising the isolated antigen binding protein of any one of claims 1-43, the chimeric antigen receptor of any one of claims 44-63, the polypeptide molecule of any one of claims 64-65, the immunoconjugate of claim 66, the nucleic acid molecule of any one of claims 67-69, the vector of claim 70, the cell of any one of claims 71-78 and/or the pharmaceutical composition of claim 79.
Use of an isolated antigen binding protein of any one of claims 1-43, a chimeric antigen receptor of any one of claims 44-63, a polypeptide molecule of any one of claims 64-65, an immunoconjugate of claim 66, a nucleic acid molecule of any one of claims 67-69, a vector of claim 70, a cell of any one of claims 71-78, and/or a pharmaceutical composition of claim 79 in the preparation of a kit for detecting the presence and/or amount of CLDN18.2 in a sample.