CN117777290A - Monoclonal antibody specifically recognizing CLDN18.2 and related products, methods and uses thereof - Google Patents

Abstract

The invention discloses a monoclonal antibody specifically recognizing CLDN18.2, and related products, methods and uses thereof, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of a heavy chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the amino acid sequences of CDR1, CDR2 and CDR3 of a light chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11, and experiments prove that CAR-T cells prepared based on the monoclonal antibody can kill cells expressing CLDN18.2 efficiently and specifically, and have good clinical application prospects.

Description

A monoclonal antibody that specifically recognizes CLDN18.2 and related products and methods and use

Technical field

The present invention belongs to the field of biomedicine technology. Specifically, the present invention relates to a monoclonal antibody that specifically recognizes CLDN18.2 and related products, methods and uses thereof.

Background technique

CLDN18.2 (Claudin 18.2) is a protein encoded by the CLDN18 gene, which belongs to the cellular tight junction protein family and can control the flow of molecules between layer cells. The CLDN18.2 protein was initially found to be continuously and stably highly expressed in a variety of gastric cancer tissues, but subsequent studies have shown that it can also be expressed in breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer. Abnormal activation and overexpression in primary malignant tumors are particularly common in digestive system malignancies, including gastric cancer (70%), pancreatic cancer (50%), esophageal cancer (30%), etc. The expression of CLDN18.2 protein is not limited to primary lesions, but is also highly expressed in metastases, and may be involved in the process of malignant tumor cell proliferation and chemotactic metastasis. The abnormal expression of CLDN18.2 is closely related to the occurrence and development of various cancers. The CLDN18.2 protein frequently undergoes abnormal changes during the occurrence and development of various malignant tumors. The CLDN18.2 gene also undergoes abnormal activation, which is highly selective and stable. It is expressed in specific tumor tissues and participates in the proliferation, differentiation and migration of tumor cells, which makes CLDN18.2 a potential target for the development of therapeutic monoclonal antibodies.

CLDN18 (Claudin 18) is a type of integrin membrane protein that exists in epithelial and endothelial tight junctions with a molecular weight of approximately 27.9KD. It forms intercellular tight junctions with other tight junction proteins and regulates the permeability of tissue molecules and ions in the intercellular space. , to maintain the stability of the environment within the organization. There are two isoforms of CLDN18, splice variant 1 (CLDN18.1) and splice variant 2 (CLDN18.2). In normal cells, CLDN18.1 is selectively expressed in lung epithelium, while CLDN18.2 is specifically expressed in normal gastric epithelial differentiated cells and is not expressed in gastric epithelial stem cells with cell division activity. However, in tumor cells, CLDN18.2 is overexpressed in various cancer types. For example, most gastric cancer patients, pancreatic cancer patients, and esophageal cancer patients have high expression of CLDN18.2, and it is also highly expressed in lung cancer and other cancer types. Express. Therefore, finding monoclonal antibodies that specifically bind to CLDN18.2 but not CLDN18.1 is of great significance for the treatment and detection of cancer.

Summary of the invention

In view of this, the purpose of the present invention is to provide a monoclonal antibody that specifically recognizes CLDN18.2 in the field and related products, methods and uses thereof. The monoclonal antibody can specifically bind to CLDN18.2 without binding to CLDN18. .1. The CAR-T cells prepared based on the monoclonal antibody have significant killing effect on cells expressing CLDN18.2.

The above objects of the present invention are achieved through the following technical solutions:

A first aspect of the invention provides a monoclonal antibody that specifically recognizes CLDN18.2.

Further, the monoclonal antibody includes a heavy chain variable region and a light chain variable region;

The amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively;

The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 respectively;

Preferably, the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region are as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 respectively;

Preferably, the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the light chain variable region are shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively;

More preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8;

More preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.

Furthermore, the amino acid sequence of the heavy chain variable region also includes an amino acid sequence having at least 90% homology with SEQ ID NO:8, and the amino acid sequence of the light chain variable region also includes an amino acid sequence having at least 90% homology with SEQ ID NO:16. Amino acid sequences with at least 90% homology.

In some embodiments, the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the heavy chain variable region and light chain variable region of the monoclonal antibody of the invention can be based on the above heavy chain variable region and light chain variable region. The full-length sequence of the chain variable region is defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, and the CDR sequence defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system is also within the protection scope of the present invention.

In some embodiments, the monoclonal antibodies of the invention also encompass amino acid sequence variants of the monoclonal antibodies as described above. For example, it may be desired to improve the binding affinity and/or other biological properties of a monoclonal antibody by introducing appropriate modifications into the nucleotide sequence encoding the monoclonal antibody, or by peptide synthesis to prepare amino acid sequence variants of the monoclonal antibody. body. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the monoclonal antibody, and any combination of deletions, insertions, and substitutions can also be performed to obtain the final monoclonal antibody variant. The resulting monoclonal antibody variants are also included within the scope of the present invention.

In the present invention, the term "monoclonal antibody" refers to an immunoglobulin obtained from a pure line of cells, having the same structure and chemical properties, and being specific for a single antigenic determinant. Monoclonal antibodies differ from conventional polyclonal antibody preparations (usually having different antibodies directed against different determinants) in that each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefit of monoclonal antibodies is that they are derived from hybridoma or recombinantly engineered cell cultures and are not confounded by other immunoglobulins. The modifier "monoclonal" indicates the nature of the antibody as having been obtained from a homogeneous population of antibodies, but this should not be construed as requiring any special method to produce the antibody.

As understood in the art, an antibody is a glycoprotein, or an antigen-binding portion thereof, containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The heavy chain includes the heavy chain variable region (VH) and the heavy chain constant regions (CH1, CH2 and CH3). The light chain includes a light chain variable region (VL) and a light chain constant region (CL). The variable regions of the heavy and light chains contain framework regions (FR) and complementarity determining regions (CDR). The four FRs are relatively conserved, while the CDR regions (CDR1, CDR2, and CDR3) contain hypervariable regions. Antibody structural units are known to comprise tetramers. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having one light chain (approximately 25 kDa) and one heavy chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines the constant region primarily responsible for effector function. FR and CDR are arranged as follows from NH ₂ end to COOH end: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

A second aspect of the invention provides a chimeric antigen receptor.

Further, the chimeric antigen receptor comprises the monoclonal antibody described in the first aspect of the present invention;

Preferably, the chimeric antigen receptor further comprises a hinge region;

Preferably, the chimeric antigen receptor further comprises a transmembrane domain;

Preferably, the chimeric antigen receptor further comprises a costimulatory signaling domain;

Preferably, the chimeric antigen receptor further comprises an intracellular signaling domain;

More preferably, the hinge region includes the hinge region of the following molecules: CD8, CD28, CD34, OX40, CD3ε, IgG1, IgG4, 4-1BB, PD-1, IL-2 receptor, IL-7 receptor, IL -11 receptor;

More preferably, the transmembrane domain includes the transmembrane domain of the following molecules: CD8, CD28, CD34, OX40, CD3ε, IgG1, IgG4, 4-1BB, PD-1, IL-2 receptor, IL-7 Receptor, IL-11 receptor;

More preferably, the costimulatory signal domain includes the costimulatory signal domain of the following molecules: 4-1BB, CD27, CD19, CD4, CD28, CD278, CD8α, CD8β, BAFFR, HVEM, LIGHT, NKp30, CD40, CDS , B7-H3, CD30, TIM1, CD2, CD7, CD226;

More preferably, the intracellular signaling domain includes the intracellular signaling domain of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b , CD278, FcεRI, DAP10, DAP12, CD66d;

Most preferably, the hinge region is a CD8 hinge region;

Most preferably, the transmembrane domain is a CD8 transmembrane domain;

Most preferably, the costimulatory signal domain is a 4-1BB costimulatory signal domain;

Most preferably, the intracellular signaling domain is a CD3ζ intracellular signaling domain;

Most preferably, the chimeric antigen receptor also includes CD8α signal peptide, T2A, GMCSF signal peptide, and EGFR DIII~DIV;

Most preferably, the chimeric antigen receptor is CD8α signal peptide, the monoclonal antibody described in the first aspect of the present invention, CD8 hinge region, CD8 transmembrane domain, 4-1BB costimulatory signal domain, CD3ζ intracellular The signaling domain, T2A, GMCSF signal peptide, EGFR DⅢ~DⅣ are sequentially connected in series;

Most preferably, the amino acid sequence of the CD8α signal peptide is as shown in SEQ ID NO:24; the amino acid sequence of the CD8 hinge region is as shown in SEQ ID NO:25; the amino acid sequence of the CD8 transmembrane domain is as shown in SEQ ID NO:26; the amino acid sequence of the 4-1BB co-stimulatory signal domain is as shown in SEQ ID NO:27; the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO:28; the amino acid sequence of T2A is as shown in SEQ ID NO:29; the amino acid sequence of the GMCSF signal peptide is as shown in SEQ ID NO:30; and the amino acid sequence of EGFR DⅢ～DⅣ is as shown in SEQ ID NO:31.

In the present invention, the term "chimeric antigen receptor (CAR)" generally refers to a fusion protein comprising an extracellular domain capable of binding antigen and at least one intracellular domain. CAR is the core component of chimeric antigen receptor T cells (CAR-T), which may include an antigen (e.g., tumor-specific antigen and/or tumor-associated antigen) binding domain, a transmembrane domain, a costimulatory domain, and Intracellular signaling domain. CAR is an engineered receptor that can implant any specific receptor onto immune effector cells, especially T cells. In CAR, scFv fragments of monoclonal antibodies that specifically recognize tumor antigens can be implanted onto T cells or NK cells. The CAR-encoding nucleic acid can be introduced into T cells, NK cells, or NK T cells using, for example, retroviral vectors. In this manner, large numbers of cancer-specific T cells, NK cells, or NK T cells can be generated for adoptive cell transfer.

A third aspect of the invention provides a nucleic acid molecule.

Further, the nucleic acid molecule includes a nucleotide sequence encoding the monoclonal antibody described in the first aspect of the present invention or the chimeric antigen receptor described in the second aspect of the present invention.

Preferably, the nucleotide sequence encoding the CD8α signal peptide in the chimeric antigen receptor according to the second aspect of the present invention is shown in SEQ ID NO: 32; encoding the chimeric antigen receptor according to the second aspect of the present invention. The nucleotide sequence of the CD8 hinge region in is shown in SEQ ID NO: 33; the nucleotide sequence encoding the CD8 transmembrane domain in the chimeric antigen receptor according to the second aspect of the present invention is shown in SEQ ID NO: 34 is shown; the nucleotide sequence encoding the 4-1BB costimulatory signal domain in the chimeric antigen receptor according to the second aspect of the present invention is shown in SEQ ID NO: 35; encoding the second aspect of the present invention The nucleotide sequence of the CD3ζ intracellular signaling domain in the chimeric antigen receptor is shown in SEQ ID NO: 36; the nucleotide encoding T2A in the chimeric antigen receptor according to the second aspect of the present invention The sequence is shown in SEQ ID NO: 37; the nucleotide sequence encoding the GMCSF signal peptide in the chimeric antigen receptor according to the second aspect of the present invention is shown in SEQ ID NO: 38; the nucleotide sequence encoding the GMCSF signal peptide in the second aspect of the present invention is shown in The nucleotide sequences of EGFR DIII to DIV in the chimeric antigen receptor are shown in SEQ ID NO: 39.

In the present invention, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

Standard molecular biology techniques can be used to obtain the nucleic acid molecules of the invention. Once the DNA fragments encoding the heavy and light chain variable regions are obtained, these DNA fragments are further manipulated by standard recombinant DNA techniques to, for example, convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. . In these operations, a DNA segment encoding a heavy chain variable region and a light chain variable region is operably linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" when used herein is intended to mean connecting two DNA fragments so that the amino acid sequences encoded by the two DNA fragments remain in the same reading frame (in-frame).

A fourth aspect of the invention provides an expression vector.

Further, the expression vector comprises the nucleic acid molecule described in the third aspect of the present invention;

Preferably, the vector includes a DNA vector, an RNA vector, a plasmid, and a virus-derived vector;

More preferably, the virus-derived vectors include lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated virus vectors, poxvirus vectors, and herpes virus vectors.

In some embodiments, the vector may be an expression vector, a cloning vector, or an integration vector. Expression of the nucleic acid molecule sequence according to the third aspect of the present invention is usually achieved by operably linking the nucleic acid molecule sequence according to the third aspect of the present invention to an expression vector. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence. Integration vectors contain components that integrate target sequences into the cellular genome. These vectors can be used to transform appropriate host cells to enable expression of the protein. Vectors typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. The sequence typically includes one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intronic sequence containing the donor and acceptor splice sites, coding Sequences for a leader sequence for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, a multilinker region for insertion of nucleic acid encoding the monoclonal antibody to be expressed, and a selectable labeling element.

In some embodiments, the type of vector is not limited, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids, and may vary depending on the host cell to be introduced. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals, and introduction can be accomplished by standard Techniques such as infection, transfection, transduction or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO precipitation, DEAE _polydextrose , electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In addition, in order to evaluate the expression of the protein of interest (e.g., monoclonal antibody), the vector introduced into the cells may also contain either or both of a selectable marker gene or a reporter gene to facilitate transfection by viral vectors. or infected cell populations to identify and select expressing cells.

A fifth aspect of the invention provides an engineered host cell.

Further, the engineered host cell expresses the monoclonal antibody described in the first aspect of the present invention or the chimeric antigen receptor described in the second aspect of the present invention;

Preferably, the engineered host cell comprises the expression vector according to the fourth aspect of the present invention;

More preferably, the host cells include eukaryotic cells and prokaryotic cells;

Most preferably, the host cells are immune cells;

Most preferably, the engineered host cells comprise engineered immune cells;

Most preferably, the immune cells include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof.

A sixth aspect of the invention provides an immunoconjugate.

Further, the immunoconjugate includes the monoclonal antibody described in the first aspect of the present invention and a conjugate coupled thereto;

Preferably, the conjugate comprises a cytokine, a therapeutic agent, a cytotoxin, an enzyme, a gold nanoparticle/nanorod, a nanomagnetic particle, a viral coat protein or VLP, a radionuclide, or a combination thereof;

More preferably, the cytokines include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN -γ, TNF-β, TNF-α, G-CSF, M-CSF;

More preferably, the therapeutic agent includes an alkylating agent, an antimetabolite, an antitumor antibiotic, a mitotic inhibitor, a chromatin function inhibitor, an antiangiogenic agent, an antiestrogens, an antiandrogens, and an immunomodulator; most preferably, the alkylating agent includes chlorambucil, chlorambucil, melphalan, propranolol, chlorambucil, cyclophosphamide, hexamethylmelamine, carmustine, cisplatin, oxaliplatin, and carboplatin; most preferably, the antimetabolite includes methotrexate, 5-fluorouracil, fluoxetine, capecitabine, cytarabine, fludarabine, 6-mercaptopurine, 2-chlorodeoxyadenosine, 5-azacytidine, and pentostatin; most preferably, the antitumor antibiotic includes daunorubicin, doxorubicin, methoxy-daunomycin, mithramycin, mitomycin C, valrubicin, mitoxantrone hydrochloride, bleomycin, and dactinomycin; most preferably, the mitotic inhibitor includes docetaxel, vinblastine, paclitaxel, and vincristine. , vinblastine, vinorelbine; most preferably, the chromatin function inhibitor includes irinotecan, etoposide, topotecan, etoposide phosphate, teniposide; most preferably, the anti-angiogenic agent includes prinomastat, tanomastat, ilomastat, aprotinin, marimastat, batimastat, CGS-27023A, bromochloropiperaquinone, neovastat, thalidomide; most preferably, the antiestrogens include toremifene, raloxifene, Tamoxifen, anastrozole, letrozole, droloxifene, odoxifene, exemestane; most preferably, the anti-androgen includes nilutamide, bicalutamide, spironolactone, flutamide, finasteride, cyproterone acetate, cimetidine; most preferably, the immunomodulator includes interleukin, tumor necrosis factor, interferon, mushroom polysaccharide, cizosin, roquimek, pidomod, methoxypolyethylene glycol succinamide adenosine deaminase, thymosin preparation;

More preferably, the cytotoxins include MMAE, DM1, MMAF, PBD, DM2, DM4, PNU-159682, IR700, PE-38, PE24, PE-T20, PE-T20-KDEL, PE4E, PE40;

More preferably, the radionuclides include ¹³¹ I, ³² P, ⁸⁹ Sr, ⁹⁰ Y, ²²³ Ra, ¹²⁵ I, ¹⁰³ Pd.

A seventh aspect of the present invention provides a reagent for detecting CLDN18.2 protein.

Further, the reagent includes the monoclonal antibody described in the first aspect of the present invention and a diagnostic agent coupled thereto;

Preferably, the diagnostic agent includes radionuclides, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, and photosensitive diagnostic agents; more preferably, the radioactive nuclide includes 18F, 52Fe, 62Cu, 64Cu, 67Cu, 86Y, 90Y, 89Zr, 120I, 123I, 124I, 125I, 131I, 13N, 15O, 186Re, 188Re, 51Mn, 72As; more preferably, the chemiluminescent agent includes luminol, isoluminol, aromatic acridine imidazole, acridinium salt, oxalate; more preferably, the bioluminescent agent includes luciferin, luciferase, aequorin; more preferably, the paramagnetic ions include chromium (III), manganese (II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium (II), terbium (III), dysprosium (III), holmium (III); more preferably, the enzyme includes horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactase Glycosidase, urease, catalase, or glucoamylase; more preferably, the photosensitive diagnostic agent includes dihydroxysilyl phthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, and photoporphyrin.

The eighth aspect of the present invention provides a pharmaceutical composition or kit.

Further, the pharmaceutical composition includes the monoclonal antibody described in the first aspect of the present invention, the engineered host cell described in the fifth aspect of the present invention, and/or the immunoconjugate described in the sixth aspect of the present invention;

Preferably, the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers and/or excipients;

Further, the pharmaceutically or physiologically acceptable carrier and/or auxiliary material may include a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.

Preferably, the kit includes the monoclonal antibody described in the first aspect of the present invention and/or the reagent described in the seventh aspect of the present invention.

In some embodiments, the kit may also include a sample diluent, a washing solution, a color developing solution, a stop solution, etc. The kit can be used to detect or assist in detecting the presence or absence of CLDN18.2 in a sample, and the sample includes but is not limited to: a serum sample, a blood sample or a tissue sample.

The ninth aspect of the present invention provides any of the following methods:

(1) A method for producing the monoclonal antibody according to the first aspect of the present invention, the method comprising the following steps: cultivating the engineered host cell according to the fifth aspect of the present invention, and obtaining the monoclonal antibody containing the The culture of the culture is separated and purified to obtain the monoclonal antibody described in the first aspect of the present invention;

(2) A method for preparing the engineered host cell according to the fifth aspect of the present invention, the method comprising the following steps: converting the nucleic acid molecule according to the third aspect of the present invention or the fourth aspect of the present invention into The expression vector is introduced into the host cell to obtain the engineered host cell described in the fifth aspect of the present invention;

Preferably, the introduction method includes lipofection, microinjection, electroporation, DNA vector, RNA vector, retroviral vector, lentiviral vector, poxvirus vector, herpes simplex virus vector, adenovirus vector, adenovirus vector, Related viral vectors;

Preferably, the host cell includes eukaryotic cells and prokaryotic cells;

More preferably, the host cells are immune cells;

Most preferably, the immune cells include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof;

Most preferably, the immune cells are T cells;

(3) A method for detecting CLDN18.2 protein for non-diagnostic purposes, the method comprising the following steps: combining the test sample with the monoclonal antibody described in the first aspect of the present invention and/or the seventh aspect of the present invention Contact with reagents detects the presence of antibody-antigen complexes.

The present invention also provides a method for diagnosing whether a subject suffers from a CLDN18.2-related disease, the method comprising the following steps: contacting a test sample derived from the subject with the monoclonal antibody described in the first aspect of the present invention, and detecting the formation of a complex between the monoclonal antibody and the CLDN18.2 protein, wherein the formation of the complex indicates that the subject suffers from a CLDN18.2-related disease.

In some embodiments, the presence and/or expression levels of CLDN18.2 can be determined by, for example, quantitative fluorescence cytometry, immunohistochemistry (IHC), or nucleic acid-based methods. For example, a test sample from a subject can be exposed to the monoclonal antibody described in the first aspect of the present invention, and the expressed CLDN18.2 protein can be detected. Alternatively, the presence and/or expression levels of CLDN18.2 can also be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase PCR, microarrays, SAGE, FISH, etc.

The present invention also provides a method for treating CLDN18.2 related diseases. The method includes the following steps: administering an effective amount of the monoclonal antibody described in the first aspect of the present invention, the fifth aspect of the present invention to a subject in need. The engineered host cell described in the aspect, the immunoconjugate described in the sixth aspect of the present invention, and/or the pharmaceutical composition described in the eighth aspect of the present invention.

In some embodiments, the subject is a subject suffering from a CLDN18.2-related disease, and the means of administration include parenteral, intrapulmonary, intranasal, and, if local treatment is desired, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any appropriate route known to those skilled in the art, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are contemplated herein, including but not limited to: single or multiple administrations at multiple time points, bolus administration, and pulse infusion.

The tenth aspect of the present invention provides any of the following applications:

(1) The monoclonal antibody according to the first aspect of the invention, the chimeric antigen receptor according to the second aspect of the invention, the nucleic acid molecule according to the third aspect of the invention, and the expression vector according to the fourth aspect of the invention , the application of the engineered host cells described in the fifth aspect of the present invention and the immunoconjugate described in the sixth aspect of the present invention in the preparation of anti-tumor drugs;

(2) Application of the monoclonal antibody described in the first aspect of the present invention and the reagent described in the seventh aspect of the present invention in the preparation of detection reagents or kits for detecting CLDN18.2 protein;

(3) The monoclonal antibody according to the first aspect of the invention, the chimeric antigen receptor according to the second aspect of the invention, the nucleic acid molecule according to the third aspect of the invention, and the expression vector according to the fourth aspect of the invention Application in producing anti-tumor engineered host cells;

(4) Application of the monoclonal antibody described in the first aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, and the expression vector described in the fourth aspect of the present invention in the preparation of anti-tumor immunoconjugates;

(5) The monoclonal antibody according to the first aspect of the present invention, the chimeric antigen receptor according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, and the expression vector according to the fourth aspect of the present invention , the application of the engineered host cells described in the fifth aspect of the present invention and the immunoconjugate described in the sixth aspect of the present invention in the preparation of anti-tumor biological preparations;

Preferably, the anti-tumor drugs include anti-tumor antibody drugs and anti-tumor immune cell drugs;

Preferably, the tumor is a tumor expressing CLDN18.2;

More preferably, the tumors include gastric cancer, pancreatic cancer, esophageal cancer, bile duct cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, lung cancer, bone cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, Brain cancer, cervical cancer, uterine cancer, endometrial cancer, colorectal cancer, anal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, glioblastoma, stellate cancer Cytoma, melanoma.

In some embodiments, the CLDN18.2-expressing tumor refers to any cancer or tumor involving CLDN18.2-expressing cancer cells, examples of which include, but are not limited to: non-small cell lung cancer (squamous/ non-squamous), small cell lung cancer, renal cell carcinoma, colorectal cancer, colon cancer, ovarian cancer, breast cancer (including basal breast cancer, ductal cancer, and lobular breast cancer), pancreatic cancer, gastric cancer, bladder cancer, esophagus Carcinoma, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, myeloma, mycosis, Merkel cell carcinoma, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovialoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoid malignancies, basal cell carcinoma , adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary laryngeal carcinoma, bronchial carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, threatening Erm's tumor, cervical cancer, testicular cancer, seminoma, classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, Acute lymphocytic leukemia, acute myelocytic leukemia (acute myelocytic leukemia), chronic myelocytic leukemia (granulocytic leukemia), chronic lymphocytic leukemia, polycythemia vera, mast cell-derived tumors, EBV- Positive and -negative PTLD and diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma, HHV8-associated primary lymphoma, non-Hodgkin lymphoma , multiple myeloma, Waldenstrom macroglobulinemia, heavy chain diseases, myelodysplastic syndromes, hairy cell leukemia and myelodysplasia, primary central nervous system lymphoma, spinal cord axis tumors, brain Stem glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, nerve blastoma and retinoblastoma.

Compared with the existing technology, the present invention has the following advantages and beneficial effects:

The present invention provides the field with a new type of monoclonal antibody that specifically recognizes CLDN18.2. The monoclonal antibody can specifically bind to cells expressing CLDN18.2. The present invention also provides a novel monoclonal antibody that specifically recognizes CLDN18.2. Chimeric antigen receptors, CAR-T cells, pharmaceutical compositions, immunoconjugates, detection reagents, etc. of cloned antibodies have been experimentally verified and found to be highly efficient and specific in CAR-T cells prepared based on the monoclonal antibodies. It specifically kills cells expressing CLDN18.2 and has good clinical application prospects.

Description of the drawings

Figure 1 shows the results of flow cytometry to detect the binding of immune serum to CHO and CHO-CLDN18.2 cells respectively;

Figure 2 shows the results of rechecking the binding of immune serum to CHO, CHO-CLDN18.1 and CHO-CLDN18.2 cells by flow cytometry;

Figure 3 is a schematic structural diagram of a CAR plasmid constructed based on mouse antibody F2H or AB011;

Figure 4 shows the results of specific killing of CLDN18.2 overexpressed CHO cells by CAR-T cells constructed based on mouse antibody F2H or AB011.

Detailed ways

The present invention will be further described below with reference to specific examples, which are only used to explain the present invention and cannot be understood as limiting the present invention. Those of ordinary skill in the art can understand that various changes, modifications, substitutions and modifications can be made to these embodiments without departing from the principles and purposes of the invention. The scope of the invention is defined by the claims and their equivalents. . The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Example 1 Screening of monoclonal antibodies targeting CLDN18.2

1. Experimental methods

(1) Take 5 Balb/c mice and use purified recombinant adenovirus at a dosage of 1×10 ⁷ pfu/mouse to immunize the mice through intramuscular injection. After three immunizations, blood was collected to detect the immune titer (the time interval between each immunization was 21 days).

(2) Dilute the immune serum at 1:1000 and incubate CHO and CHO-CLDN18.2 cells respectively. Confirm that the mice are successfully immunized based on the test results, and perform fusion after shock immunization.

(3) Use the hybridoma fusion supernatant to incubate CHO-CLDN18.2 cells, incubate PE-anti mouse IgG with the secondary antibody, read the fluorescence intensity with a multifunctional microplate reader, and detect the titer of the fusion clone. Select clones with strong binding ability and use CHO-CLDN18.2 and CHO-CLDN18.1 (control) cells for re-examination, and select clones that only bind CHO-CLDN18.2 for multiple subcloning. Select clones with stronger binding and use CHO-CLDN18.2 and CHO cells for flow cytometry re-examination, and select clones that only bind CHO-CLDN18.2 for amplification and sequencing. Among them, the primary antibody: immune serum diluted at 1:1000, 100 μL/tube, and the secondary antibody: PE-anti-mouse IgG, 1:1000.

(4) Obtain hybridoma sequencing results F2H clone

The mouse-derived antibody F2H obtained in this example was sequenced.

2. Experimental results

The results of detecting the binding of immune serum to CHO and CHO-CLDN18.2 cells by flow cytometry are shown in Figure 1, and the results of re-examining the binding of immune serum to CHO, CHO-CLDN18.1 and CHO-CLDN18.2 cells by flow cytometry are shown in Figure 2. The results show that the mouse antibody F2H (F2H-5-E6-B5-B7) obtained in this example can specifically bind to CHO-CLDN18.2 cells. The results of sequencing the mouse antibody F2H obtained in this example are shown in Table 1 below.

Table 1 Sequence of mouse antibody F2H

Example 2 Experimental verification of mouse-derived antibody F2H

1. Experimental methods

(1) Construction of F2H clone nucleic acid sequence

F2H-5-E6-B5-B7 VH: GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTAGTGAAGC CTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGGTATGCCATGTCTTGGGTTCGCCAGTCCAGAGAAGAGGCTGGAGTGGGTCGCAGAAATTAGTAGTGGTGGTAGTAACACCTACTATGTAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACA ACGCCAAGAGCACCCTGTACCTGGAAATGATCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAAGGCCTACTATGGTAACGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 17)

F2H-5-E6-B5-B7 VL: GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGAC AGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACGGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAG ATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTACTGTCAGAATAATTATTATTATCCGCTCATGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:18)

(2) Design of public sequence comparison

The control group used in this example is the currently disclosed monoclonal antibody AB011 against CLDN18.2. This positive control group proves the effectiveness of the mouse-derived antibody F2H screened in the present invention.

The protein sequence of AB011 is as follows:

AB011-VH：QVQLQESGPGLVKPSETLSLTCTVSGFSLTRYGVSWIRQPPGKGLEWIG VIWGEGNTNYNPSLKSRVTISKDSSKSQVSLKLSSVTAADTAVYYCARVNFGNALDYWG QGTLVTVSS(SEQ ID NO:19)

AB011-VL：DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQP PKLLIYWASTRDSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDFIFPLTFGGGTKV EIK(SEQ ID NO:20)

The nucleic acid sequence of AB011 is as follows:

AB011-VH (DNA): CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGC CCAGCGAGACCCTGAGCCTGACCTGCACCGTGTCCGGCTTCAGCCTGACCAGATACGGCGTGAGCTGGATCAGACAGCCTCCCGGCAAGGGCCTGGAGTGGATCGGCGTGATCTGGGGCGAGGGCAACACCAACTACAACCCCAGCCTGAAGAGCAGAGTGACCATCAGCAAGGACAGCAGCAAGAGCCAGGTGTCC CTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAGTGAACTTCGGCAACGCCCTGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC(SEQ IDNO:21)

AB011-VL (DNA): GACATCGTGATGACCCAGTCCCCCGACAGCCTGGCCGTGAG CCTGGGCGAGAGCCACAATCAACTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGACCTGGTATCAGCAGAAGCCCGGCCAGCCCCCTAAGCTGCTGATCTACTGGGCCTCCACCAGAGACTCCGGCGTGCCCGACAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCAT CAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGAATGACTTTATCTTCCCCCTGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAG(SEQ ID NO:22)

(3) Constructing a CAR plasmid. The schematic diagram of the structure of the CAR plasmid constructed based on the mouse antibodies F2H and AB011 is shown in Figure 3, wherein EF1α promoter is the promoter of elongation factor 1α, and its nucleotide sequence is shown in SEQ ID NO:23 in the sequence table, CD8αsp is the CD8α signal peptide, VL is the light chain variable region of the monoclonal antibody, (G4S)3Linker is a linker connecting the light chain and heavy chain variable regions of the monoclonal antibody, VH is the heavy chain variable region of the monoclonal antibody, CD8 Hinge is the CD8 hinge region, CD8 TM is the CD8 transmembrane domain, 4-1BB intracellular is the costimulatory signal domain, CD3ζ is the intracellular signal transduction domain, T2A is the connecting peptide, and GMCSF Signal peptide is the GMCSF signal peptide.

Among them, the CAR constructed based on mouse antibodies F2H and AB011 includes CD8α signal peptide, mouse antibody F2H (light chain variable region-Linker-heavy chain variable region), CD8 hinge region, CD8 transmembrane domain, 4- The 1BB costimulatory signaling domain, CD3ζ intracellular signaling domain, T2A, GMCSF signal peptide, and EGFR DⅢ~DⅣ are sequentially connected in series.

Among them, the CAR constructed based on AB011 includes CD8α signal peptide, positive control antibody AB011 (light chain variable region-Linker-heavy chain variable region), CD8 hinge region, CD8 transmembrane domain, and 4-1BB costimulatory signal structure domain, CD3ζ intracellular signaling domain, T2A, GMCSF signal peptide, and EGFR DⅢ~DⅣ in series.

The specific amino acid sequences and nucleotide sequences corresponding to the CD8α signal peptide, CD8 hinge region, CD8 transmembrane domain, 4-1BB co-stimulatory signal domain, CD3ζ intracellular signal transduction domain, T2A, GMCSF signal peptide, and EGFR DⅢ~DⅣ in the above-mentioned CAR are shown in Tables 2 and 3 below, respectively.

Table 2 Amino acid sequences corresponding to each part in CAR

Table 3 Nucleotide sequences corresponding to each part in CAR

Based on the above-mentioned CAR containing the mouse antibody F2H and the CAR containing AB011, the corresponding CAR-T cells were further constructed, and the healthy human T cells were transduced with 293T packaging lentivirus to construct CAR-T cells, which were cultured and amplified using the X-vivo system containing IL-7 and IL-15, and the killing verification was started on the 8th day. The killing was performed using three effector-target ratios of 1:1, 1:2, and 1:5, and CHO cells overexpressing claudin18.1 and claudin18.2 were used.

Among them, the specific construction method of CAR-T cells is as follows: transfect the lentiviral system plasmid into adherent 293T cells in the logarithmic growth phase, harvest the cell culture supernatant 48-72 hours after transfection, and obtain the CAR after concentration and filtration. Lentivirus is stored at -80°C for later use. Peripheral blood mononuclear cells (PBMC) were isolated from human peripheral blood, T cells were isolated using human CD3/28 magnetic beads, and viral transduction was performed within 72 hours of activation. After 24 hours of transduction, medium was changed and cultured until day 8. Cells were collected by centrifugation and resuspended in physiological saline. Use flow cytometry to identify CAR molecules on the surface of T cells and ensure that the positive rate is greater than 30%. If it is lower than 30%, use EGFR-PE magnetic beads for enrichment and sorting. According to the positive ratio of CAR-T cell harvest, they are mixed with target cells for subsequent killing experiments.

2. Experimental results

The results are shown in Figure 4. The results show that F2H antibody (mouse source) CAR-T has good specific killing of CLDN18.2 and can significantly and specifically kill cells overexpressing CLDN18.2. Compared with the positive control group AB011 CAR-T cells, The killing effects are similar, and both have high killing efficiency.

The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be noted that those of ordinary skill in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims (10)

1. A monoclonal antibody that specifically recognizes CLDN18.2, characterized in that the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 respectively; Preferably, the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region are as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 respectively. ; Preferably, the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the light chain variable region are as shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 respectively. ; More preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8; More preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16. 2. A chimeric antigen receptor, characterized in that, the chimeric antigen receptor comprises the monoclonal antibody of claim 1; Preferably, the chimeric antigen receptor further comprises a hinge region; Preferably, the chimeric antigen receptor further comprises a transmembrane domain; Preferably, the chimeric antigen receptor further comprises a co-stimulatory signaling domain; Preferably, the chimeric antigen receptor further comprises an intracellular signaling domain; More preferably, the hinge region includes the hinge region of the following molecules: CD8, CD28, CD34, OX40, CD3ε, IgG1, IgG4, 4-1BB, PD-1, IL-2 receptor, IL-7 receptor, IL -11 receptor; More preferably, the transmembrane domain includes the transmembrane domain of the following molecules: CD8, CD28, CD34, OX40, CD3ε, IgG1, IgG4, 4-1BB, PD-1, IL-2 receptor, IL-7 Receptor, IL-11 receptor; More preferably, the costimulatory signaling domain includes the costimulatory signaling domains of the following molecules: 4-1BB, CD27, CD19, CD4, CD28, CD278, CD8α, CD8β, BAFFR, HVEM, LIGHT, NKp30, CD40, CDS, B7-H3, CD30, TIM1, CD2, CD7, CD226; More preferably, the intracellular signaling domain includes the intracellular signaling domain of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b , CD278, FcεRI, DAP10, DAP12, CD66d; Most preferably, the hinge region is a CD8 hinge region; Most preferably, the transmembrane domain is a CD8 transmembrane domain; Most preferably, the costimulatory signal domain is a 4-1BB costimulatory signal domain; Most preferably, the intracellular signaling domain is a CD3ζ intracellular signaling domain; Most preferably, the chimeric antigen receptor also includes CD8α signal peptide, T2A, GMCSF signal peptide, and EGFR DIII~DIV; Most preferably, the chimeric antigen receptor is a CD8α signal peptide, the monoclonal antibody of claim 1, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory signal domain, and CD3ζ intracellular signaling. The structural domain, T2A, GMCSF signal peptide, and EGFR DIII~DIV are sequentially connected in series; Most preferably, the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 24; Most preferably, the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO: 25; Most preferably, the amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID NO: 26; Most preferably, the amino acid sequence of the 4-1BB costimulatory signal domain is shown in SEQ ID NO: 27; Most preferably, the amino acid sequence of the CD3ζ intracellular signaling domain is shown in SEQ ID NO: 28; Most preferably, the amino acid sequence of T2A is shown in SEQ ID NO: 29; Most preferably, the amino acid sequence of the GMCSF signal peptide is shown in SEQ ID NO: 30; Most preferably, the amino acid sequence of EGFR DIII to DIV is shown in SEQ ID NO: 31. 3. A nucleic acid molecule, characterized in that the nucleic acid molecule comprises a nucleotide sequence encoding the monoclonal antibody according to claim 1 or the chimeric antigen receptor according to claim 2; Preferably, the nucleotide sequence encoding the CD8α signal peptide in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO: 32; Preferably, the nucleotide sequence encoding the CD8 hinge region in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO: 33; Preferably, the nucleotide sequence encoding the CD8 transmembrane domain in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO: 34; Preferably, the nucleotide sequence encoding the 4-1BB costimulatory signal domain in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO: 35; Preferably, the nucleotide sequence encoding the CD3ζ intracellular signaling domain in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO: 36; Preferably, the nucleotide sequence encoding T2A in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO: 37; Preferably, the nucleotide sequence encoding the GMCSF signal peptide in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO: 38; Preferably, the nucleotide sequence encoding EGFR DIII to DIV in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO: 39. 4. An expression vector, characterized in that the expression vector comprises the nucleic acid molecule according to claim 3; Preferably, the vector includes a DNA vector, an RNA vector, a plasmid, and a virus-derived vector; More preferably, the virus-derived vector includes a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, and a herpesvirus vector. 5. An engineered host cell, characterized in that the engineered host cell expresses the monoclonal antibody of claim 1 or the chimeric antigen receptor of claim 2; Preferably, the engineered host cell comprises the expression vector of claim 4; More preferably, the host cells include eukaryotic cells and prokaryotic cells; Most preferably, the host cells are immune cells; Most preferably, the engineered host cells comprise engineered immune cells; Most preferably, the immune cells include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, or any combination thereof. 6. An immunoconjugate, characterized in that the immunoconjugate comprises the monoclonal antibody according to claim 1 and a conjugate conjugated thereto; Preferably, the conjugate includes cytokines, therapeutic agents, cytotoxins, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, radionuclides, or combinations thereof; More preferably, the cytokines include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-γ, TNF-β, TNF-α, G-CSF, M-CSF; More preferably, the therapeutic agents include alkylating agents, antimetabolites, anti-tumor antibiotics, mitotic inhibitors, chromatin function inhibitors, anti-angiogenic agents, anti-estrogens, anti-androgens, and immunomodulators; Most preferably, the alkylating agent includes bischloroethylmethylamine, chlorambucil, phenylalanine, bromopropazine, methacrylate, cyclophosphamide, hexamethonium melamine, carbazole mustard, cisplatin, oxaliplatin, carboplatin; Most preferably, the antimetabolites include methotrexate, 5-fluorouracil, fluoroside, capecitabine, cytarabine, fludarabine, 6-mercaptopurine, 2-chlorodeoxyadenosine , 5-azacytidine, pentostatin; Most preferably, the anti-tumor antibiotics include daunorubicin, doxorubicin, doxorubicin, mithramycin, mitomycin C, valrubicin, mitoxantrone hydrochloride, and belet Mycin, dactinomycin; Most preferably, the mitotic inhibitor includes docetaxel, vinblastine, paclitaxel, vincristine, vindesamide, and vinorelbine; Most preferably, the chromatin function inhibitor includes irinotecan, etopoxa, topotecan, etopoxa phosphate, and thiopodophyllin; Most preferably, the anti-angiogenic agent includes prolinostat, tanonostat, ilomastat, proximide, mamastat, batimastat, CGS-27023A, bromoclopiquantel , neovalastat, thalidomide; Most preferably, the anti-estrogens include toremifene, raloxifene, tamoxifen, anatozole, letrozole, droxifene, oxydoxifene, and exemestane; Most preferably, the anti-androgens include nilutamide, bicalutamide, spironolactone, flutamide, finasteride, cyproterone acetate, and cimetidine; Most preferably, the immunomodulatory agent includes interleukin, tumor necrosis factor, interferon, mushroom polysaccharide, cizorose, roquinamac, pidotimod, methoxypolyethylene glycol succinamide adenosine deaminase, Thymosin preparations; More preferably, the cytotoxins include MMAE, DM1, MMAF, PBD, DM2, DM4, PNU-159682, IR700, PE-38, PE24, PE-T20, PE-T20-KDEL, PE4E, PE40; More preferably, the radionuclides include ¹³¹ I, ³² P, ⁸⁹ Sr, ⁹⁰ Y, ²²³ Ra, ¹²⁵ I, ¹⁰³ Pd. 7. A reagent for detecting CLDN18.2 protein, characterized in that the reagent comprises the monoclonal antibody of claim 1 and a diagnostic agent coupled thereto; Preferably, the diagnostic agent includes radionuclides, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, and photosensitive diagnostic agents; More preferably, the radionuclides include 18F, 52Fe, 62Cu, 64Cu, 67Cu, 86Y, 90Y, 89Zr, 120I, 123I, 124I, 125I, 131I, 13N, 15O, 186Re, 188Re, 51Mn, and 72As; More preferably, the chemiluminescent agent includes luminol, isoluminol, aromatic acridinium esters, imidazole, acridinium salts, and oxalate esters; More preferably, the bioluminescent agent includes luciferin, luciferase, and aequorin; More preferably, the paramagnetic ions include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), Samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III); More preferably, the enzyme comprises horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase; More preferably, the photosensitive diagnostic agent includes dihydroxysilyl phthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, and photoporphyrin. 8. A pharmaceutical composition or kit, characterized in that the pharmaceutical composition comprises the monoclonal antibody according to claim 1, the engineered host cell according to claim 5 and/or the engineered host cell according to claim 6. The above-mentioned immunoconjugate; Preferably, the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers and/or excipients; Preferably, the kit comprises the monoclonal antibody according to claim 1 and/or the reagent according to claim 7. 9. Any one of the following methods, characterized in that the method includes: (1) A method for producing the monoclonal antibody of claim 1, characterized in that the method includes the following steps: culturing the engineered host cell of claim 5 to obtain the monoclonal antibody containing the The culture, isolate and purify the monoclonal antibody according to claim 1 from the culture; (2) A method for preparing the engineered host cell according to claim 5, characterized in that the method includes the following steps: converting the nucleic acid molecule according to claim 3 or the expression vector according to claim 4 into Introduced into a host cell to obtain the engineered host cell described in claim 5; Preferably, the introduction method includes lipofection, microinjection, electroporation, DNA vector, RNA vector, retroviral vector, lentiviral vector, poxvirus vector, herpes simplex virus vector, adenovirus vector, adenovirus vector, Related viral vectors; Preferably, the host cells include eukaryotic cells and prokaryotic cells; More preferably, the host cells are immune cells; Most preferably, the immune cells include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof; Most preferably, the immune cells are T cells; (3) A method for detecting CLDN18.2 protein for non-diagnostic purposes, characterized in that the method includes the following steps: combining the sample to be tested with the monoclonal antibody of claim 1 and/or the monoclonal antibody of claim 7 Contact with reagents detects the presence of antibody-antigen complexes. 10. The application of any of the following aspects, characterized in that the application includes: (1) Use of the monoclonal antibody of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the expression vector of claim 4, the engineered host cell of claim 5, or the immunoconjugate of claim 6 in the preparation of anti-tumor drugs; (2) Application of the monoclonal antibody of claim 1 and the reagent of claim 7 in the preparation of detection reagents or kits for detecting CLDN18.2 protein; (3) The monoclonal antibody of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, and the expression vector of claim 4 are used in the preparation of engineered anti-tumor products. Application in host cells; (4) Use of the monoclonal antibody according to claim 1, the nucleic acid molecule according to claim 3, and the expression vector according to claim 4 in the preparation of an anti-tumor immunoconjugate; (5) Use of the monoclonal antibody of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the expression vector of claim 4, the engineered host cell of claim 5, or the immunoconjugate of claim 6 in the preparation of anti-tumor biological agents; Preferably, the anti-tumor drugs include anti-tumor antibody drugs and anti-tumor immune cell drugs; Preferably, the tumor is a tumor expressing CLDN18.2; More preferably, the tumors include gastric cancer, pancreatic cancer, esophageal cancer, bile duct cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, lung cancer, bone cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, Brain cancer, cervical cancer, uterine cancer, endometrial cancer, colorectal cancer, anal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, glioblastoma, stellate cancer Cytoma, melanoma.