A monoclonal antibody that specifically recognizes CLDN18.2 and related products, methods and uses thereof

Abstract

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

Description

Technical Field

[0001] 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 Art

[0002] CLDN18.2 (Claudin 18.2) is a protein encoded by the CLDN18 gene. It belongs to the family of tight junction proteins 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 abnormally activated and overexpressed in a variety of primary malignant tumors such as breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer. It is particularly prone to 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 metastatic lesions, and may be involved in the process of malignant tumor cell proliferation and chemotaxis and 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 becomes abnormally activated and is highly selectively and stably expressed in specific tumor tissues, participating in the proliferation, differentiation and migration of tumor cells. This makes CLDN18.2 a potential target for the development of therapeutic monoclonal antibodies.

[0003] CLDN18 (Claudin 18) is a type of integrin membrane protein present in the tight junctions of epithelial and endothelial cells, with a molecular weight of about 27.9KD. Together with other tight junction proteins, it forms tight junctions between cells, regulates the permeability of tissue molecules and ions in the intercellular space, and maintains the stability of the tissue environment. There are two subtypes of CLDN18, splice variant 1 (CLDN18.1) and splice variant 2 (CLDN18.2). In normal cells, CLDN18.1 is selectively expressed in the epithelium of the lung, while CLDN18.2 is specifically expressed in differentiated cells of normal gastric epithelium and is not expressed in gastric epithelial stem cells with cell division activity. However, in tumor cells, CLDN18.2 is overexpressed in many types of cancer, such as most patients with gastric cancer, pancreatic cancer, and esophageal cancer have high expression of CLDN18.2, and it is also highly expressed in cancer types such as lung cancer. Therefore, finding a monoclonal antibody that specifically binds to CLDN18.2 but not CLDN18.1 is of great significance for the treatment and detection of cancer. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a monoclonal antibody that specifically recognizes CLDN18.2 and related products, methods and uses thereof in the art. The monoclonal antibody can specifically bind to CLDN18.2 but not to CLDN18.1, and the CAR-T cells prepared based on the monoclonal antibody have a significant killing effect on cells expressing CLDN18.2.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] The first aspect of the present invention provides a monoclonal antibody that specifically recognizes CLDN18.2.

[0007] Further, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0008] 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;

[0009] 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;

[0010] Preferably, the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively;

[0011] 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;

[0012] More preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8;

[0013] More preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:16.

[0014] Furthermore, the amino acid sequence of the heavy chain variable region also includes an amino acid sequence that has 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 that has at least 90% homology with SEQ ID NO:16.

[0015] 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 described in the present invention can be obtained based on the full-length sequences of the above-mentioned heavy chain variable region and light chain variable region according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. The CDR sequences defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system are also within the scope of protection of the present invention.

[0016] In some embodiments, the monoclonal antibodies of the present invention also encompass amino acid sequence variants of the monoclonal antibodies as described above. For example, it may be desirable to improve the binding affinity and / or other biological properties of the monoclonal antibodies, and amino acid sequence variants of the monoclonal antibodies may be prepared by introducing suitable modifications into the nucleotide sequence encoding the monoclonal antibodies, or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues within the amino acid sequence of the monoclonal antibodies, and any combination of deletion, insertion and substitution may also be performed to obtain final monoclonal antibody variants, and the monoclonal antibody variants obtained based on this are also included in the protection scope of the present invention.

[0017] 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 specific for a single antigenic determinant. Monoclonal antibodies are different from conventional polyclonal antibody preparations (usually with different antibodies against different determinants), and each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained through hybridoma or recombinant engineered cell culture and are not mixed with other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a homogeneous antibody population, but this should not be interpreted as requiring any special method to produce the antibody.

[0018] As understood in the art, an antibody is a glycoprotein or antigen binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, and CH3). The light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The variable regions of the heavy and light chains comprise a framework region (FR) and a complementarity determining region (CDR). The four FRs are relatively conservative, while the CDR regions (CDR1, CDR2, and CDR3) comprise hypervariable regions. Antibody structural units are known to comprise tetramers. Each tetramer is composed of two identical polypeptide chain pairs, each pair having a light chain (about 25 kDa) and a heavy chain (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that are primarily responsible for antigen recognition. The carboxyl terminal portion of each chain defines a constant region that is primarily responsible for effector function. The FRs and CDRs are arranged from the NH2 terminus to the COOH terminus as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0019] A second aspect of the present invention provides a chimeric antigen receptor.

[0020] Further, the chimeric antigen receptor comprises the monoclonal antibody described in the first aspect of the present invention;

[0021] Preferably, the chimeric antigen receptor further comprises a hinge region;

[0022] Preferably, the chimeric antigen receptor further comprises a transmembrane domain;

[0023] Preferably, the chimeric antigen receptor further comprises a co-stimulatory signaling domain;

[0024] Preferably, the chimeric antigen receptor further comprises an intracellular signaling domain;

[0025] 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;

[0026] More preferably, the transmembrane domain includes the transmembrane domains of the following molecules: CD8, CD28, CD34, OX40, CD3ε, IgG1, IgG4, 4-1BB, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor;

[0027] 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;

[0028] 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;

[0029] Most preferably, the hinge region is the CD8 hinge region;

[0030] Most preferably, the transmembrane domain is a CD8 transmembrane domain;

[0031] Most preferably, the costimulatory signaling domain is a 4-1BB costimulatory signaling domain;

[0032] Most preferably, the intracellular signaling domain is a CD3ζ intracellular signaling domain;

[0033] Most preferably, the chimeric antigen receptor further comprises CD8α signal peptide, T2A, GMCSF signal peptide, EGFR DⅢ~DⅣ;

[0034] Most preferably, the chimeric antigen receptor is a CD8α signal peptide, the monoclonal antibody described in the first aspect of the present invention, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory signal domain, a CD3ζ intracellular signaling domain, T2A, a GMCSF signal peptide, and EGFR DⅢ to DⅣ connected in series in sequence;

[0035] 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.

[0036] In the present invention, the term "chimeric antigen receptor (CAR)" generally refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. CAR is a core component of a chimeric antigen receptor T cell (CAR-T), which may include an antigen (e.g., a tumor-specific antigen and / or a tumor-associated antigen) binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. CAR is an engineered receptor that can implant any specific receptor into immune effector cells, especially T cells. In CAR, the scFv fragment of a monoclonal antibody that specifically recognizes a tumor antigen can be implanted into a T cell or a NK cell. A nucleic acid encoding CAR can be introduced into a T cell, a NK cell, or a NK T cell using, for example, a retroviral vector. In this way, a large number of cancer-specific T cells, NK cells, or NK T cells can be generated for adoptive cell transfer.

[0037] The third aspect of the present invention provides a nucleic acid molecule.

[0038] Furthermore, the nucleic acid molecule comprises 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.

[0039] Preferably, the nucleotide sequence encoding the CD8α signal peptide in the chimeric antigen receptor described in the second aspect of the present invention is as shown in SEQ ID NO:32; the nucleotide sequence encoding the CD8 hinge region in the chimeric antigen receptor described in the second aspect of the present invention is as shown in SEQ ID NO:33; the nucleotide sequence encoding the CD8 transmembrane domain in the chimeric antigen receptor described in the second aspect of the present invention is as shown in SEQ ID NO:34; the nucleotide sequence encoding the 4-1BB co-stimulatory signal domain in the chimeric antigen receptor described in the second aspect of the present invention is as shown in SEQ ID NO:35; the nucleotide sequence encoding the CD3ζ intracellular signaling domain in the chimeric antigen receptor described in the second aspect of the present invention is as shown in SEQ ID NO:36; the nucleotide sequence encoding T2A in the chimeric antigen receptor described in the second aspect of the present invention is as shown in SEQ ID NO:37; the nucleotide sequence encoding the GMCSF signal peptide in the chimeric antigen receptor described in the second aspect of the present invention is as shown in SEQ ID NO:38; the nucleotide sequence encoding EGFR DⅢ~DⅣ in the chimeric antigen receptor described in the second aspect of the present invention is as shown in SEQ ID NO:39.

[0040] In the present invention, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.

[0041] Standard molecular biology techniques can be used to obtain nucleic acid molecules of the present invention. Once the DNA fragments encoding the variable region of heavy chain and the variable region of light chain are obtained, these DNA fragments are further operated by standard recombinant DNA techniques, such as to convert the variable region gene into a full-length antibody chain gene, a Fab fragment gene or a scFv gene. In these operations, the DNA fragments encoding the variable region of heavy chain and the variable region of light chain are operably connected to another DNA fragment encoding another protein, such as an antibody constant region or a flexible joint. Wherein, the term "operable connection" is intended to represent the connection of two DNA fragments when used herein so that the amino acid sequence encoded by the two DNA fragments remains in the same reading frame (in-frame).

[0042] The fourth aspect of the present invention provides an expression vector.

[0043] Further, the expression vector comprises the nucleic acid molecule described in the third aspect of the present invention;

[0044] Preferably, the vector includes a DNA vector, an RNA vector, a plasmid, or a vector of viral origin;

[0045] 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.

[0046] In some embodiments, the vector can be an expression vector, a cloning vector or an integration vector. Usually, the expression of the nucleic acid molecule sequence described in the third aspect of the present invention is achieved by operably connecting the nucleic acid molecule sequence described in the third aspect of the present invention to the expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Integration vectors contain components that integrate the target sequence into the cell genome. These vectors can be used to transform appropriate host cells to enable them to express proteins. The vector usually contains sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequence usually includes one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, a replication origin, a transcription termination sequence, a complete intron sequence containing donor and acceptor splicing sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multi-linker region and a selectable marker element for inserting a nucleic acid encoding a monoclonal antibody to be expressed.

[0047] In some embodiments, the type of the carrier is not limited, for example, plasmid, phagemid, phage derivative, animal virus and clay, can be changed according to the host cell to be introduced. Viral vector technology is well known in the art and described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals, and can be introduced by standard techniques, for example, infection, transfection, transduction or conversion. Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE polydextrose, electroporation, protoplast fusion, liposome transfection, cell microinjection and viral vectors. Viruses that can be used as carriers include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. In addition, in order to evaluate the expression of target protein (for example, monoclonal antibody), the carrier introduced into the cell may also include any one or two of selectable marker genes or reporter genes, so as to identify and select expression cells from the cell group seeking to be transfected or infected by viral vectors.

[0048] A fifth aspect of the present invention provides an engineered host cell.

[0049] 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;

[0050] Preferably, the engineered host cell comprises the expression vector described in the fourth aspect of the present invention;

[0051] More preferably, the host cell includes eukaryotic cells and prokaryotic cells;

[0052] Most preferably, the host cell is an immune cell;

[0053] Most preferably, the engineered host cell comprises an engineered immune cell;

[0054] 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.

[0055] The sixth aspect of the present invention provides an immunoconjugate.

[0056] Furthermore, the immunoconjugate comprises the monoclonal antibody described in the first aspect of the present invention and a conjugate conjugated thereto;

[0057] 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;

[0058] 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;

[0059] 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, an immunomodulator; most preferably, the alkylating agent includes chlorambucil, chlorambucil, melphalan, propranolol, phenylbutyric acid, cyclophosphamide, hexamethylmetabolite, carmustine, cisplatin, oxaliplatin, carboplatin; most preferably, the antimetabolite includes methotrexate. Preferably, the anti-tumor antibiotics include daunorubicin, doxorubicin, idarubicin, mithramycin, valrubicin, mitoxantrone hydrochloride, bleomycin, and dactinomycin; Most preferably, the mitotic inhibitors include 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, roquine, pidomod, methoxypolyethylene glycol succinamide adenosine deaminase, thymosin preparation;

[0060] More preferably, the cytotoxins include MMAE, DM1, MMAF, PBD, DM2, DM4, PNU-159682, IR700, PE-38, PE24, PE-T20, PE-T20-KDEL, PE4E, PE40;

[0061] More preferably, the radionuclide comprises 131 I. 32 P. 89 Sr. 90 Y. 223 Ra, 125 I. 103 Pd.

[0062] The seventh aspect of the present invention provides a reagent for detecting CLDN18.2 protein.

[0063] Further, the reagent comprises the monoclonal antibody described in the first aspect of the present invention and a diagnostic agent coupled thereto;

[0064] Preferably, the diagnostic agent comprises a radionuclide, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, or a photosensitive diagnostic agent; more preferably, the radionuclide comprises 18F, 52Fe, 62Cu, 64Cu, 67Cu, 86Y, 90Y, 89Zr, 120I, 123I, 124I, 125I, 131I, 13N, 15O, 186Re, 188Re, 51Mn, 72As; more preferably, the chemiluminescent agent comprises luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt, oxalate; more preferably, the bioluminescent agent comprises luciferin, luciferase, jellyfish Photoprotein; 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-galactosidase, urease, catalase, or glucoamylase; more preferably, the photosensitive diagnostic agent includes dihydroxysilicon phthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, photoporphyrin.

[0065] The eighth aspect of the present invention provides a pharmaceutical composition or kit.

[0066] Further, the pharmaceutical composition comprises 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;

[0067] Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers and / or excipients;

[0068] Further, the pharmaceutically or physiologically acceptable carrier and / or excipient may comprise a sterile injectable liquid (e.g., 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), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., a phosphate buffer solution), Ringer's solution, and any combination thereof.

[0069] Preferably, the kit comprises 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.

[0070] 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.

[0071] A ninth aspect of the present invention provides any of the following methods:

[0072] (1) A method for producing the monoclonal antibody of the first aspect of the present invention, the method comprising the following steps: culturing the engineered host cell of the fifth aspect of the present invention to obtain a culture containing the monoclonal antibody, and isolating and purifying the monoclonal antibody of the first aspect of the present invention from the culture;

[0073] (2) A method for preparing the engineered host cell according to the fifth aspect of the present invention, the method comprising the following steps: introducing the nucleic acid molecule according to the third aspect of the present invention or the expression vector according to the fourth aspect of the present invention into a host cell to obtain the engineered host cell according to the fifth aspect of the present invention;

[0074] Preferably, the introduction method comprises lipofection, microinjection, electroporation, DNA vector, RNA vector, retroviral vector, lentiviral vector, poxvirus vector, herpes simplex virus vector, adenovirus vector, adeno-associated virus vector;

[0075] Preferably, the host cell includes eukaryotic cells and prokaryotic cells;

[0076] More preferably, the host cell is an immune cell;

[0077] 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;

[0078] Most preferably, the immune cells are T cells;

[0079] (3) A method for detecting CLDN18.2 protein for non-diagnostic purposes, the method comprising the following steps: contacting a sample to be tested with 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, and detecting the presence of an antibody-antigen complex.

[0080] 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.

[0081] In some embodiments, the presence and / or expression level of CLDN18.2 can be determined by, for example, quantitative fluorescent cytometry, immunohistochemistry (IHC), or nucleic acid-based methods. For example, the test sample from the subject can be exposed to the monoclonal antibody described in the first aspect of the present invention to detect the expressed CLDN18.2 protein. Alternatively, the presence and / or expression level of CLDN18.2 can also be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, etc.

[0082] The present invention also provides a method for treating CLDN18.2-related diseases, the method comprising the following steps: administering an effective amount of 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, 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 to a subject in need.

[0083] In some embodiments, the subject is a subject with a CLDN18.2-related disease, and the administration method includes parenteral, intrapulmonary and intranasal, and if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any appropriate route known to those skilled in the art, for example, by injection, such as intravenous or subcutaneous injection, which depends in part on whether the administration is short-term or long-term. A variety of dosing schedules are contemplated herein, including but not limited to: single or multiple administrations at multiple time points, push administration and pulse infusion.

[0084] The tenth aspect of the present invention provides any of the following applications:

[0085] (1) Use of the monoclonal antibody of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the expression vector of the fourth aspect of the present invention, the engineered host cell of the fifth aspect of the present invention, or the immunoconjugate of the sixth aspect of the present invention in the preparation of anti-tumor drugs;

[0086] (2) Use of the monoclonal antibody described in the first aspect of the present invention or the reagent described in the seventh aspect of the present invention in the preparation of a detection reagent or kit for detecting CLDN18.2 protein;

[0087] (3) Use of the monoclonal antibody of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, or the expression vector of the fourth aspect of the present invention in the preparation of an anti-tumor engineered host cell;

[0088] (4) Use 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, or the expression vector described in the fourth aspect of the present invention in the preparation of an anti-tumor immunoconjugate;

[0089] (5) Use of the monoclonal antibody of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the expression vector of the fourth aspect of the present invention, the engineered host cell of the fifth aspect of the present invention, and the immunoconjugate of the sixth aspect of the present invention in the preparation of anti-tumor biological agents;

[0090] Preferably, the anti-tumor drugs include anti-tumor antibody drugs and anti-tumor immune cell drugs;

[0091] Preferably, the tumor is a tumor expressing CLDN18.2;

[0092] More preferably, the tumor includes 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, astrocytoma, melanoma.

[0093] In some embodiments, the tumor expressing CLDN18.2 refers to any cancer or tumor involving cancer cells expressing CLDN18.2, 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 carcinoma, and lobular breast cancer), pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, myeloma, mycotic fungus, Merkel cell carcinoma, hepatocellular carcinoma (HCC), fibrosarcoma , myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovial tumors, 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 cancer, bronchial cancer, hepatocarcinoma, bile duct cancer, choriocarcinoma, Wilm'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 myeloid 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-related primary lymphoma, non-Hodgkin lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia, primary central nervous system lymphoma, spinal cord axial tumors, brain stem glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, neuroblastoma and retinoblastoma.

[0094] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0095] The present invention provides a novel monoclonal antibody that specifically recognizes CLDN18.2 in the art. The monoclonal antibody can specifically bind to cells expressing CLDN18.2. The present invention also provides a chimeric antigen receptor, CAR-T cell, pharmaceutical composition, immunoconjugate, detection reagent, etc. comprising the monoclonal antibody. Experimental verification shows that the CAR-T cell prepared based on the monoclonal antibody can efficiently and specifically kill cells expressing CLDN18.2, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 The results of flow cytometry detection of the binding of immune serum to CHO and CHO-CLDN18.2 cells;

[0097] Figure 2 The results of retesting the binding of immune serum to CHO, CHO-CLDN18.1 and CHO-CLDN18.2 cells by flow cytometry;

[0098] Figure 3 Schematic diagram of the structure of the CAR plasmid constructed based on the mouse antibody F2H or AB011;

[0099] Figure 4 The results of the specific killing of CHO cells overexpressing CLDN18.2 by CAR-T cells constructed based on mouse antibodies F2H or AB011. DETAILED DESCRIPTION

[0100] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods used in the following embodiments are conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0101] Example 1 Screening of monoclonal antibodies targeting CLDN18.2

[0102] 1. Experimental methods

[0103] (1) Five Balb / c mice were fed with purified recombinant adenovirus at a rate of 1 × 10 7 pfu / mouse, and mice were immunized by intramuscular injection. Blood was collected after three immunizations to detect the immune titer (each immunization was separated by 21 days).

[0104] (2) The immune serum was diluted at 1:1000 and incubated in CHO and CHO-CLDN18.2 cells, respectively. The successful immunization of mice was confirmed based on the test results, and fusion was performed after shock immunization.

[0105] (3) CHO-CLDN18.2 cells were incubated with hybridoma fusion supernatant, PE-anti mouse IgG was incubated with secondary antibody, and the fluorescence intensity was read by a multifunctional microplate reader to detect the titer of the fusion clone. The clones with stronger binding were selected for re-examination using CHO-CLDN18.2 and CHO-CLDN18.1 (control) cells, and clones that only bind to CHO-CLDN18.2 were selected for multiple subcloning. The clones with stronger binding were selected for flow cytometry re-examination using CHO-CLDN18.2 and CHO cells, and clones that only bind to CHO-CLDN18.2 were selected for amplification and sequencing. Among them, primary antibody: 1:1000 diluted immune serum, 100μL / tube, secondary antibody: PE-anti-mouse IgG, 1:1000.

[0106] (4) Obtaining hybridoma sequencing results F2H clone

[0107] The mouse antibody F2H obtained in this example was sequenced.

[0108] 2. Experimental results

[0109] The results of flow cytometry detection of immune serum binding to CHO and CHO-CLDN18.2 cells are shown in the figure. Figure 1 The results of flow cytometry re-examination of the binding of immune serum to CHO, CHO-CLDN18.1 and CHO-CLDN18.2 cells are shown in the figure. 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.

[0110] Table 1 Sequence of mouse antibody F2H

[0111]

[0112] Example 2 Experimental verification of mouse antibody F2H

[0113] 1. Experimental methods

[0114] (1) Construction of F2H clone nucleic acid sequence

[0115] F2H-5-E6-B5-B7 VH:GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTAGTGAAGC CTGGAGGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGGTATGCCATGTCTTGGGTTCGCCAGTCTCCAGAGAAGAGGCTGGAGTGGGTCGCAGAAATTAGTAGTGGTGGTAGTAACACCTACTATGTAGACACTGTGACGGGCC GATTCACCATCTCCAGAGACAACGCCAAGAGCACCCTGTACCTGGAAATGATCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAAGGCCTACTATGGTAACGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO:17)

[0116] F2H-5-E6-B5-B7 VL:GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGAC AGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACGGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCCTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTC CCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATAATTATTATCCGCTCATGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:18)

[0117] (2) Design of public sequence comparison

[0118] The control group used in this example is the currently disclosed monoclonal antibody AB011 against CLDN18.2. The positive control group is used to demonstrate the effectiveness of the mouse antibody F2H screened by the present invention.

[0119] The protein sequence of AB011 is as follows:

[0120] AB011-VH: QVQLQESGPGLVKPSETLSLTCTVSGFSLTRYGVSWIRQPPGKGLEWIGVIWGEGNTNYNPSLKSRVTISKDSSKSQVSLKLSSVTAADTAVYYCARVNFGNALDYWGQGTLVTVSS (SEQ ID NO:19)

[0121] AB011-VL: DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRDSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDFIFPLTFGGGTKVEIK (SEQ ID NO:20)

[0122] The nucleic acid sequence of AB011 is as follows:

[0123] AB011-VH (DNA): CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGTCCGGCTTCAGCCTGACCAGATACGGCGTGAGCTGGATCAGACAGCCTCCCGGCAAGGGCCTGGAGTGGATCGGCGTGATCTGGGGCGAGGGCAACACCAACTACAACCCCAGCCTGAAGAGCAGAGTGACCATCAGCAAGGACAGCAGCAAGAGCCAGGTGTCCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAGTGAACTTCGGCAACGCCCTGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC (SEQ IDNO:21)

[0124] AB011-VL(DNA):GACATCGTGATGACCCAGTCCCCCGACAGCCTGGCCGTGAG CCTGGGCGAGAGAGCCACAATCAACTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGACCTGGTATCAGCAGAAGCCCGGCCAGCCCCCTAAGCTGCTGATCTACTGGGCCTCCACCAGAGACTCCGGCGTG CCCGACAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGAATGACTTTATCTTCCCCTGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAG(SEQ ID NO:22)

[0125] (3) Construction of CAR plasmid. The schematic diagram of the structure of the CAR plasmid constructed based on mouse antibodies F2H and AB011 is shown in the figure. Figure 3 As shown, 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 CD8α signal peptide, VL is the light chain variable region of the monoclonal antibody, (G4S)3Linker is a linker (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.

[0126] Among them, the CAR constructed based on mouse antibodies F2H and AB011 is a series of CD8α signal peptide, mouse antibody F2H (light chain variable region-Linker-heavy chain variable region), 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.

[0127] Among them, the CAR constructed based on AB011 is a series of CD8α signal peptide, positive control antibody AB011 (light chain variable region-Linker-heavy chain variable region), 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.

[0128] 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.

[0129] Table 2 Amino acid sequences corresponding to each part of CAR

[0130]

[0131] Table 3 Nucleotide sequences corresponding to each part of CAR

[0132]

[0133]

[0134] 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.

[0135] Among them, the specific construction method of CAR-T cells is as follows: the lentiviral system plasmid is transfected into adherent 293T cells in the logarithmic growth phase, and the cell culture supernatant after transfection for 48-72 hours is harvested. After concentration and filtration, the CAR lentivirus is obtained and stored at -80°C for standby use. Peripheral blood mononuclear cells (PBMC) are separated from human peripheral blood, and T cells are isolated using human CD3 / 28 magnetic beads. Virus transduction is performed within 72 hours of activation, and the medium is changed and cultured for 24 hours after transduction until the 8th day. The cells are collected by centrifugation and resuspended in normal saline. Flow cytometry is used to identify CAR molecules on the surface of T cells to ensure that the positive rate is greater than 30%. If it is lower, EGFR-PE magnetic beads are used for enrichment and sorting. According to the positive ratio of CAR-T cells harvested, they are mixed with target cells for subsequent killing experiments.

[0136] 2. Experimental results

[0137] Results Figure 4 The results showed that F2H antibody (mouse source) CAR-T has good specific killing effect on CLDN18.2, and can significantly and specifically kill cells overexpressing CLDN18.2. The killing effect is comparable to that of the positive control group AB011 CAR-T cells, and both have high killing efficiency.

[0138] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

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.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region are shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively.

3. The monoclonal antibody according to claim 1, characterized in that 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.

4. The monoclonal antibody according to claim 2, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

8.

5. The monoclonal antibody according to claim 3, characterized in that The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

6. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the monoclonal antibody of claim 1.

7. The chimeric antigen receptor according to claim 6, characterized in that The chimeric antigen receptor also comprises a hinge region.

8. The chimeric antigen receptor according to claim 7, characterized in that The chimeric antigen receptor also comprises a transmembrane domain.

9. The chimeric antigen receptor according to claim 8, characterized in that The chimeric antigen receptor also comprises a co-stimulatory signaling domain.

10. The chimeric antigen receptor according to claim 9, characterized in that The chimeric antigen receptor also comprises an intracellular signaling domain.

11. The chimeric antigen receptor according to claim 7, characterized in that 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 or IL-11 receptor.

12. The chimeric antigen receptor according to claim 8, characterized in that 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 or IL-11 receptor.

13. The chimeric antigen receptor according to claim 9, characterized in that The co-stimulatory signaling domain includes the co-stimulatory signaling 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 or CD226.

14. The chimeric antigen receptor according to claim 10, characterized in that 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 or CD66d.

15. The chimeric antigen receptor according to claim 11, characterized in that The hinge region is the CD8 hinge region.

16. The chimeric antigen receptor according to claim 12, characterized in that The transmembrane domain is the CD8 transmembrane domain.

17. The chimeric antigen receptor according to claim 13, characterized in that The co-stimulatory signaling domain is a 4-1BB co-stimulatory signaling domain.

18. The chimeric antigen receptor according to claim 14, characterized in that The intracellular signaling domain is a CD3ζ intracellular signaling domain.

19. The chimeric antigen receptor according to claim 14, characterized in that The chimeric antigen receptor also comprises CD8α signal peptide, T2A, GMCSF signal peptide, and EGFR D Ⅲ~D Ⅳ.

20. The chimeric antigen receptor according to claim 19, characterized in that The chimeric antigen receptor is obtained by sequentially connecting CD8α signal peptide, the monoclonal antibody, CD8 hinge region, CD8 transmembrane domain, 4-1BB costimulatory signal domain, CD3ζ intracellular signal transduction domain, T2A, GMCSF signal peptide, and EGFR D Ⅲ~D Ⅳ.

21. The chimeric antigen receptor according to claim 20, characterized in that The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO:

24.

22. The chimeric antigen receptor according to claim 20, characterized in that The amino acid sequence of the CD8 hinge region is shown in SEQ ID NO:

25.

23. The chimeric antigen receptor according to claim 20, characterized in that The amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID NO:

26.

24. The chimeric antigen receptor according to claim 20, characterized in that The amino acid sequence of the 4-1BB co-stimulatory signal domain is shown in SEQ ID NO:

27.

25. The chimeric antigen receptor according to claim 20, characterized in that The amino acid sequence of the CD3ζ intracellular signaling domain is shown in SEQ ID NO:

28.

26. The chimeric antigen receptor according to claim 20, characterized in that The amino acid sequence of T2A is shown in SEQ ID NO:

29.

27. The chimeric antigen receptor according to claim 20, characterized in that The amino acid sequence of the GMCSF signal peptide is shown in SEQ ID NO:

30.

28. The chimeric antigen receptor according to claim 20, characterized in that The amino acid sequence of the EGFR D III~D IV is shown in SEQ ID NO:

31.

29. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding the monoclonal antibody of any one of claims 1 to 5 or the chimeric antigen receptor of any one of claims 6 to 28.

30. The nucleic acid molecule according to claim 29, characterized in that The nucleotide sequence encoding the CD8α signal peptide in the chimeric antigen receptor is shown in SEQ ID NO:

32.

31. The nucleic acid molecule according to claim 29, characterized in that The nucleotide sequence encoding the CD8 hinge region in the chimeric antigen receptor is shown in SEQ ID NO:

33.

32. The nucleic acid molecule according to claim 29, characterized in that The nucleotide sequence encoding the CD8 transmembrane domain in the chimeric antigen receptor is shown in SEQ ID NO:

34.

33. The nucleic acid molecule according to claim 29, characterized in that The nucleotide sequence encoding the 4-1BB co-stimulatory signaling domain in the chimeric antigen receptor is shown in SEQ ID NO:

35.

34. The nucleic acid molecule according to claim 29, characterized in that The nucleotide sequence encoding the CD3ζ intracellular signaling domain in the chimeric antigen receptor is shown in SEQ ID NO:

36.

35. The nucleic acid molecule according to claim 29, characterized in that The nucleotide sequence encoding T2A in the chimeric antigen receptor is shown in SEQ ID NO:

37.

36. The nucleic acid molecule according to claim 29, characterized in that The nucleotide sequence encoding the GMCSF signal peptide in the chimeric antigen receptor is shown in SEQ ID NO:

38.

37. The nucleic acid molecule according to claim 29, characterized in that The nucleotide sequence encoding EGFR DⅢ~DⅣ in the chimeric antigen receptor is shown in SEQ ID NO:

39.

38. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule of any one of claims 29-37.

39. The expression vector according to claim 38, characterized in that The vector includes a DNA vector, an RNA vector or a virus-derived vector.

40. The expression vector according to claim 39, characterized in that The DNA vector is a plasmid.

41. The expression vector according to claim 39, characterized in that The virus-derived vector includes a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector or a herpesvirus vector.

42. An engineered host cell, characterized in that The engineered host cell expresses the monoclonal antibody of any one of claims 1-5 or the chimeric antigen receptor of any one of claims 6-28.

43. The engineered host cell of claim 42, wherein The engineered host cell comprises the expression vector of any one of claims 38-41.

44. The engineered host cell of claim 43, wherein The host cell includes a eukaryotic cell or a prokaryotic cell.

45. The engineered host cell of claim 44, wherein The host cell is an immune cell.

46. ​​The engineered host cell of claim 45, wherein The engineered host cells include engineered immune cells.

47. The engineered host cell of claim 46, wherein The immune cells include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof.

48. An immunoconjugate, characterized in that The immunoconjugate comprises the monoclonal antibody according to any one of claims 1 to 5 and a conjugate conjugated thereto.

49. The immunoconjugate according to claim 48, characterized in that The conjugates include cytokines, therapeutic agents, cytotoxins, enzymes, gold nanoparticles / nanorods, nanomagnetic particles, viral coat proteins or VLPs, radionuclides or combinations thereof.

50. The immunoconjugate according to claim 49, characterized in that 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 or M-CSF.

51. The immunoconjugate according to claim 49, characterized in that 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 or an immunomodulator.

52. The immunoconjugate according to claim 51, characterized in that The alkylating agents include chlorambucil, chlorambucil, melphalan, propidium bromide, phenylbutyric acid mustard, cyclophosphamide, hexamethylmetazobactam, carmustine, cisplatin, oxaliplatin or carboplatin.

53. The immunoconjugate according to claim 51, characterized in that The antimetabolites include methotrexate, 5-fluorouracil, fluticasone, capecitabine, cytarabine, fludarabine, 6-mercaptopurine, 2-chlorodeoxyadenosine, 5-azacytidine or pentostatin.

54. The immunoconjugate according to claim 51, characterized in that The antitumor antibiotics include daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin C, valrubicin, mitoxantrone hydrochloride, bleomycin or dactinomycin.

55. The immunoconjugate according to claim 51, characterized in that The mitotic inhibitors include docetaxel, vinblastine, paclitaxel, vincristine, vinblastine or vinorelbine.

56. The immunoconjugate according to claim 51, characterized in that The chromatin function inhibitor includes irinotecan, etoposide, topotecan, etoposide phosphate or teniposide.

57. The immunoconjugate according to claim 51, characterized in that The anti-angiogenic agents include prinomastat, tanomastat, ilomastat, aprotinoxate, marimastat, batimastat, CGS-27023A, bromochloropiperaquinone, neivastat or thalidomide.

58. The immunoconjugate according to claim 51, characterized in that The antiestrogens include toremifene, raloxifene, tamoxifen, anastrozole, letrozole, droloxifene, odoxifene or exemestane.

59. The immunoconjugate according to claim 51, characterized in that The antiandrogen includes nilutamide, bicalutamide, spironolactone, flutamide, finasteride, cyproterone acetate or cimetidine.

60. The immunoconjugate according to claim 51, characterized in that The immunomodulators include interleukins, tumor necrosis factors, interferons, mushroom polysaccharides, cisozin, roquine, pidomod, methoxypolyethylene glycol succinamide adenosine deaminase or thymosin preparations.

61. The immunoconjugate according to claim 49, characterized in that The cytotoxins include MMAE, DM1, MMAF, PBD, DM2, DM4, PNU-159682, IR700, PE-38, PE24, PE-T20, PE-T20-KDEL, PE4E or PE40.

62. The immunoconjugate according to claim 49, characterized in that The radionuclides include 131 I. 32 P. 89 Sr. 90 Y. 223 Ra, 125 I or 103 Pd.

63. A reagent for detecting CLDN18.2 protein, characterized in that The reagent comprises the monoclonal antibody according to any one of claims 1 to 5 and a diagnostic agent coupled thereto.

64. The reagent according to claim 63, characterized in that The diagnostic agent includes a radionuclide, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme or a photosensitive diagnostic agent.

65. The reagent according to claim 64, characterized in that The radionuclides include 18F, 52Fe, 62Cu, 64Cu, 67Cu, 86Y, 90Y, 89Zr, 120I, 123I, 124I, 125I, 131I, 13N, 15O, 186Re, 188Re, 51Mn or 72As.

66. The reagent according to claim 64, characterized in that The chemiluminescent agent includes luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt or oxalate ester.

67. The reagent according to claim 64, characterized in that The bioluminescent agent includes luciferin, luciferase or aequorin.

68. The reagent according to claim 64, characterized in that 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) or holmium (III).

69. The reagent according to claim 64, characterized in that The enzymes include horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase or glucoamylase.

70. The reagent according to claim 64, characterized in that The photosensitive diagnostic agent includes dihydroxysilicon phthalocyanine, methylene blue, protoporphyrin, hematoporphyrin or photoporphyrin.

71. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the monoclonal antibody of any one of claims 1-5, the engineered host cell of any one of claims 42-47 and / or the immunoconjugate of any one of claims 48-62.

72. The pharmaceutical composition according to claim 71, characterized in that The pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers and / or excipients.

73. A kit, characterized in that The kit comprises the monoclonal antibody according to any one of claims 1 to 5 and / or the reagents according to claims 63 to 70.

74. A method for producing the monoclonal antibody of any one of claims 1-5, characterized in that The method comprises the following steps: culturing the engineered host cell according to any one of claims 42 to 47 to obtain a culture containing the monoclonal antibody, and isolating and purifying the monoclonal antibody according to any one of claims 1 to 5 from the culture.

75. A method for preparing the engineered host cell of any one of claims 42-47, characterized in that The method comprises the following steps: introducing the nucleic acid molecule of any one of claims 29-37 or the expression vector of any one of claims 38-41 into a host cell to obtain the engineered host cell of any one of claims 42-47.

76. The method according to claim 75, characterized in that The introduction method includes lipofection, microinjection, electroporation, DNA vector, RNA vector, retroviral vector, lentiviral vector, poxvirus vector, herpes simplex virus vector, adenovirus vector or adeno-associated virus vector.

77. The method according to claim 75, characterized in that The host cell includes a eukaryotic cell or a prokaryotic cell.

78. The method according to claim 77, characterized in that The host cell is an immune cell.

79. The method according to claim 78, characterized in that The immune cells include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof.

80. The method according to claim 79, characterized in that The immune cells are T cells.

81. A method for detecting CLDN18.2 protein for a non-diagnostic purpose, characterized in that: The method comprises the following steps: contacting the sample to be tested with the monoclonal antibody described in any one of claims 1 to 5 and / or the reagent described in any one of claims 63 to 70, and detecting the presence of the antibody-antigen complex.

82. Use of the monoclonal antibody of any one of claims 1-5, the chimeric antigen receptor of any one of claims 6-28, the nucleic acid molecule of any one of claims 29-37, the expression vector of any one of claims 38-41, the engineered host cell of any one of claims 42-47 and / or the immunoconjugate of any one of claims 48-62 in the preparation of an anti-tumor drug; The tumor is a tumor expressing CLDN18.2; The tumor includes gastric cancer, pancreatic cancer, esophageal cancer, bile duct cancer, breast cancer, liver cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, colorectal cancer or melanoma.

83. Use of the monoclonal antibody according to any one of claims 1 to 5 or the reagent according to any one of claims 63 to 70 in preparing a detection reagent for detecting CLDN18.2 protein.

84. Use of the monoclonal antibody according to any one of claims 1 to 5 or the reagent according to any one of claims 63 to 70 in preparing a kit for detecting CLDN18.2 protein.

85. Use of the monoclonal antibody of any one of claims 1-5, the chimeric antigen receptor of any one of claims 6-28, the nucleic acid molecule of any one of claims 29-37, or the expression vector of any one of claims 38-41 in preparing an anti-tumor engineered host cell; The tumor is a tumor expressing CLDN18.2; The tumor includes gastric cancer, pancreatic cancer, esophageal cancer, bile duct cancer, breast cancer, liver cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, colorectal cancer or melanoma.

86. Use of the monoclonal antibody according to any one of claims 1 to 5, the nucleic acid molecule according to any one of claims 29 to 37, or the expression vector according to any one of claims 38 to 41 in the preparation of an anti-tumor immunoconjugate; The tumor is a tumor expressing CLDN18.2; The tumor includes gastric cancer, pancreatic cancer, esophageal cancer, bile duct cancer, breast cancer, liver cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, colorectal cancer or melanoma.

87. Use of the monoclonal antibody of any one of claims 1-5, the chimeric antigen receptor of any one of claims 6-28, the nucleic acid molecule of any one of claims 29-37, the expression vector of any one of claims 38-41, the engineered host cell of any one of claims 42-47, or the immunoconjugate of any one of claims 48-62 in the preparation of an anti-tumor biological agent; The tumor is a tumor expressing CLDN18.2; The tumor includes gastric cancer, pancreatic cancer, esophageal cancer, bile duct cancer, breast cancer, liver cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, colorectal cancer or melanoma.

Images