CN117777289A - Humanized antibodies targeting CLDN18.2, derived products and their use in tumor therapy - Google Patents
Humanized antibodies targeting CLDN18.2, derived products and their use in tumor therapy Download PDFInfo
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Abstract
The invention discloses a humanized antibody targeting CLDN18.2, a derivative product and application thereof in tumor treatment, wherein the derivative product comprises a chimeric antigen receptor, a nucleic acid molecule, a vector, a genetically engineered host cell, a biological agent, a pharmaceutical composition, a detection reagent and a kit, and the invention lays a foundation for development of medicaments for treating CLDN18.2 positive related diseases, treatment of CLDN18.2 positive related diseases, mechanism research of CLDN18.2 positive related diseases and the like.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and in particular relates to a humanized antibody targeting CLDN18.2, a derivative product and application thereof in tumor treatment.
Background
Global cancer statistics report data from the world health organization international cancer research center (International Agency for Research on Cancer, IARC) and global cancer epidemiological database (Global Cancer Statistics, GLOBOCAN) show that the overall incidence of global malignancies presents a continuously increasing trend, severely threatening the health of humans. At present, malignant tumors are mainly treated by surgery, chemotherapy and radiotherapy in clinic, but satisfactory curative effects are difficult to achieve. In recent years, antitumor targeted drugs have made breakthrough progress in the treatment of various malignant tumors, making them a great emerging treatment method after surgery, chemotherapy and radiotherapy. However, the currently developed targeted drugs still have a plurality of defects, and trastuzumab approved by the U.S. FDA in 2010 has been successful in Her2 positive breast cancer, but has not ideal therapeutic effects on gastric and esophageal tumors. Similarly, inhibitors of the epidermal growth factor receptor (Epidermal growth factor receptor, EGFR) and mammalian target of rapamycin (Mammalian target of rapamycin, mTOR) have also proven ineffective in phase III clinical trials of multiple gastric and esophageal tumors. The CLDN18.2 protein is a target protein which is highly specifically and stably expressed on the cell surface of various malignant tumors (such as gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, breast cancer and the like), and is expected to become an ideal molecular target for tumor targeted therapy for expressing the CLDN 18.2.
The CLDN18.2 (Claudin 18.2) protein is a transmembrane protein, belongs to one of Claudins (CLDNs) family members, has both an N-terminal end and a C-terminal end located in cells, and is expressed on cell membranes as an important structural component for cell tight junctions. The CLDN18 gene is located in 3q22.3 of human chromosome 3, and the first exon of the gene has two choices, so that two different shear mutants are formed, and two protein subtypes of 69 different amino acid sequences at the N-terminal can be expressed, namely CLDN18.1 protein and CLDN18.2 protein. The difference of the amino acids can prevent the epitope of the CLDN18.2 protein from generating immune cross reaction with the CLDN18.1 protein. The CLDN18.1 protein is a specific antigen selectively expressed by alveolar epithelial cells, is only highly expressed in normal alveolar tissues, but is not found in other normal tissues including tissues such as pancreatic ducts; the CLDN18.2 protein is also a highly selective marker protein, but its distribution is totally different from that of CLDN18.1 protein. CLDN18.2 protein is highly restricted in expression in normal healthy tissues, is not expressed in undifferentiated gastric stem cells, is expressed only in differentiated gastric mucosal epithelial cells, and is expressed to a very limited extent. The two variants CLDN18.1 and CLDN18.2 differ only in the N-terminal 69 amino acid sequence, being present in the first extracellular loop, and the remaining sequences being identical. Whereas CLDN18.1 is expressed in normal lung tissue, it is of great importance to screen antibodies that specifically bind to CLDN18.2 while not binding to CLDN 18.1.
Disclosure of Invention
The invention provides a humanized antibody targeting CLDN18.2, a derivative product and application thereof in tumor treatment, wherein the humanized antibody targeting CLDN18.2 can specifically bind to CLDN18.2 and does not bind to CLDN18.1, has good affinity and specificity to CLDN18.2, and a CAR-T cell prepared based on the humanized antibody has obvious killing effect on cells expressing CLDN18.2 and can be used for preventing and/or treating diseases and/or symptoms related to the expression of CLDN 18.2.
The above object of the present invention is achieved by the following technical solutions:
in a first aspect the invention provides a humanized antibody targeting CLDN 18.2.
Further, the humanized antibody comprises a heavy chain variable region VH and a light chain variable region VL;
the VH comprises three complementarity determining regions HCDR1, HCDR2, HCDR3; the VL comprises three complementarity determining regions LCDR1, LCDR2, LCDR3;
the amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; the amino acid sequences of the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;
preferably, the humanized antibody comprises VHVL; more preferably, the VHVL comprises VH1VL1, VH1VL2, VH1VL3, VH1VL4, VH3VL1, VH3VL2, VH3VL3, VH3VL4, VH4VL1, VH4VL2, VH4VL3, VH4VL4;
Most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of the VH1 are respectively shown as SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19; most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of the VH3 are respectively shown as SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22 and SEQ ID NO. 23; most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of the VH4 are respectively shown as SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26 and SEQ ID NO. 27;
most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of VL1 are shown as SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30 and SEQ ID NO. 31 respectively; most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of VL2 are shown as SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34 and SEQ ID NO. 35 respectively; most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of VL3 are shown as SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38 and SEQ ID NO. 39 respectively; most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of VL4 are shown as SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42 and SEQ ID NO. 43 respectively;
Most preferably, the amino acid sequence of VH1 is as shown in SEQ ID NO. 8 or has at least 90% homology with SEQ ID NO. 8; most preferably, the amino acid sequence of VH3 is as shown in SEQ ID NO. 9 or has at least 90% homology with SEQ ID NO. 9; most preferably, the amino acid sequence of VH4 is as shown in SEQ ID NO. 10 or has at least 90% homology with SEQ ID NO. 10;
most preferably, the amino acid sequence of VL1 is as shown in SEQ ID NO. 12 or an amino acid sequence having at least 90% homology with SEQ ID NO. 12; most preferably, the amino acid sequence of VL2 is as shown in SEQ ID NO. 13 or an amino acid sequence having at least 90% homology with SEQ ID NO. 13; most preferably, the amino acid sequence of VL3 is as shown in SEQ ID NO. 14 or an amino acid sequence having at least 90% homology with SEQ ID NO. 14; most preferably, the amino acid sequence of VL4 is as shown in SEQ ID NO. 15 or an amino acid sequence having at least 90% homology with SEQ ID NO. 15.
In certain embodiments, the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the heavy and light chain variable regions of a humanized antibody of the invention can be obtained based on the full length sequences of the heavy and light chain variable regions described above according to the Kabat, IMGT, chothia, abM or Contact numbering system definition, and CDR sequences defined according to the Kabat, IMGT, chothia, abM or Contact numbering system are also within the scope of the invention.
In certain embodiments, humanized antibodies of the invention generally refer to humanized non-human antibodies to reduce immunogenicity to humans, while preserving the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody sequence and the FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
In a second aspect, the invention provides a chimeric antigen receptor that targets CLDN 18.2.
Further, the chimeric antigen receptor comprises the humanized antibody of the first aspect of the present invention;
preferably, the chimeric antigen receptor further comprises a hinge region; more preferably, the hinge region comprises a hinge region of: CD8, 4-1BB, igG1, igG4, PD-1, CD28, CD34, OX40, CD3 ε; most preferably, the hinge region is a CD8 hinge region;
Preferably, the chimeric antigen receptor further comprises a transmembrane domain; more preferably, the transmembrane domain comprises the transmembrane domain of: CD8, 4-1BB, igG1, igG4, PD-1, CD28, CD34, OX40, CD3 ε; most preferably, the transmembrane domain is a CD8 transmembrane domain;
preferably, the chimeric antigen receptor further comprises a costimulatory signaling domain; more preferably, the costimulatory signaling domain comprises the costimulatory signaling domain of: 4-1BB, HVEM, CD, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278; most preferably, the costimulatory signaling domain is a 4-1BB costimulatory signaling domain;
preferably, the chimeric antigen receptor further comprises an intracellular signaling domain; more preferably, the intracellular signaling domain comprises an intracellular signaling domain of: cd3ζ, cd3γ, cd3δ, cd3ε, CD278, CD21, CD22, fcεri, fcrγ, fcrβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d; most preferably, the intracellular signaling domain is a cd3ζ intracellular signaling domain;
Most preferably, the chimeric antigen receptor further comprises a CD8 a signal peptide, a T2A, GMCSF signal peptide, EGFR diniii-diniv; most preferably, the chimeric antigen receptor is selected from any one of the following groups:
(1) The chimeric antigen receptor is obtained by sequentially and serially connecting the CD8 alpha signal peptide, the humanized antibody of the first aspect of the invention, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory signal domain, a CD3 zeta intracellular signaling domain, a T2A, GMCSF signal peptide and EGFR DIII-DIV;
(2) A derivative chimeric antigen receptor formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence of the chimeric antigen receptor described in (1);
most preferably, the amino acid sequence of the CD8 alpha signal peptide is shown as SEQ ID NO. 45; the amino acid sequence of the CD8 hinge region is shown as SEQ ID NO. 46; the amino acid sequence of the CD8 transmembrane domain is shown as SEQ ID NO. 47; the amino acid sequence of the 4-1BB co-stimulatory signaling domain is shown as SEQ ID NO. 48; the amino acid sequence of the CD3 zeta intracellular signal transduction domain is shown as SEQ ID NO. 49; the amino acid sequence of the T2A is shown as SEQ ID NO. 50; the amino acid sequence of the GMCSF signal peptide is shown as SEQ ID NO. 51; the amino acid sequences of EGFR DIII-DIV are shown as SEQ ID NO. 52.
In a third aspect the invention provides a nucleic acid molecule encoding a humanized antibody according to the first aspect of the invention or a chimeric antigen receptor according to the second aspect of the invention;
preferably, the nucleotide sequence encoding the CD8 alpha signal peptide in the chimeric antigen receptor according to the second aspect of the invention is shown in SEQ ID NO. 53; the nucleotide sequence of the CD8 hinge region of the chimeric antigen receptor of the second aspect of the invention is shown as SEQ ID NO. 54; the nucleotide sequence of the CD8 transmembrane domain in the chimeric antigen receptor of the second aspect of the invention is shown as SEQ ID NO. 55; the nucleotide sequence of the 4-1BB costimulatory signal domain in the chimeric antigen receptor of the second aspect of the invention is shown as SEQ ID NO. 56; the nucleotide sequence of the CD3 zeta intracellular signaling domain in the chimeric antigen receptor according to the second aspect of the invention is shown as SEQ ID NO. 57; the nucleotide sequence of the T2A in the chimeric antigen receptor of the second aspect of the invention is shown as SEQ ID NO. 58; the nucleotide sequence of the GMCSF signal peptide in the chimeric antigen receptor of the second aspect of the invention is shown in SEQ ID NO. 59; the nucleotide sequence of EGFR DIII-DIV in the chimeric antigen receptor of the second aspect of the invention is shown as SEQ ID NO. 60.
In certain embodiments, the person skilled in the art may vary the class and sequence of combinations of signal peptides, hinge and transmembrane regions, co-stimulatory signal domains and intracellular signaling domains, as the case may be or as desired, as long as the chimeric antigen receptor has the CDR sequences or heavy chain variable region sequences of the heavy chain variable region of the humanized antibody described above, and/or the CDR sequences or light chain variable region sequences of the light chain variable region of the humanized antibody described above, depending on the form of the modification, which are within the scope of the invention.
In a fourth aspect the invention provides a vector comprising a nucleic acid molecule according to the third aspect of the invention;
preferably, the vector comprises a DNA vector, an RNA vector, a plasmid, a viral-derived vector, a transposon vector, a CRISPR/Cas9 vector; more preferably, the viral-derived vector comprises a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector.
In certain embodiments, the nucleic acid molecules provided herein can be cloned into a variety of types of vectors. For example, the nucleic acid molecule may be cloned into vectors including, but not limited to: plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.
In a fifth aspect, the invention provides a genetically engineered host cell.
Further, the genetically engineered host cell comprises a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention;
preferably, the host cell comprises a prokaryotic cell, a eukaryotic cell; more preferably, the prokaryotic cell comprises bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, rickettsia; more preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, yeast cells; most preferably, the host cell is an immune cell; most preferably, the immune cells comprise T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof; most preferably, the immune cells are T cells.
In certain embodiments, the immune cells include, but are not limited to: such as leukocytes, lymphocytes (T cells, B cells, natural Killer (NK) cells) derived from Hematopoietic Stem Cells (HSCs) produced in the bone marrow, and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). In a specific embodiment of the invention, the genetically engineered host cell is a T cell expressing the chimeric antigen receptor of the second aspect of the invention.
In a sixth aspect, the invention provides a population of host cells.
Further, the population of host cells comprises genetically engineered host cells of the fifth aspect of the invention;
preferably, the population of host cells further comprises host cells which do not comprise a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention; more preferably, the host cell comprises a prokaryotic cell, a eukaryotic cell; most preferably, the prokaryotic cell comprises bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, rickettsia; most preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, yeast cells; most preferably, the host cell is an immune cell; most preferably, the immune cells comprise T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof; most preferably, the immune cells are T cells.
In a seventh aspect, the invention provides a biological or pharmaceutical composition for the treatment and/or prevention of CLDN 18.2-positive related diseases.
Further, the biological agent comprises the humanized antibody of the first aspect of the invention, the genetically engineered host cell of the fifth aspect of the invention, and/or the population of host cells of the sixth aspect of the invention;
Preferably, the biological agent further comprises a pharmaceutically acceptable carrier, diluent and/or excipient; more preferably, the biological agent comprises a monospecific antibody medicament comprising a humanized antibody according to the first aspect of the invention, a bispecific antibody medicament comprising a humanized antibody according to the first aspect of the invention, an antibody-conjugated medicament comprising a humanized antibody according to the first aspect of the invention, a CAR-T cell medicament comprising a genetically engineered host cell according to the fifth aspect of the invention and/or a population of host cells according to the sixth aspect of the invention;
preferably, the pharmaceutical composition comprises the humanized antibody of the first aspect of the invention, the genetically engineered host cell of the fifth aspect of the invention, the population of host cells of the sixth aspect of the invention and/or the biological agent; more preferably, the pharmaceutical composition further comprises a therapeutic agent for the adjuvant treatment and/or prevention of CLDN 18.2-positive related diseases; most preferably, the therapeutic agent comprises an alkylating agent, an antimetabolite, an antitumor antibiotic, a mitotic inhibitor, a chromatin function inhibitor, an anti-angiogenic agent, an antiestrogen, an antiandrogen, an immunomodulator, a cytotoxin; most preferably, the alkylating agent comprises mechlorethamine, chlorambucil, melphalan, bromopropylamine, melphalan, fosfestivaline, cyclophosphamide, hexamethylmelamine, ifosfamide, fosetyl-amine, triamthos, carmustine, streptavidin, infliximab, azomethine, cisplatin, oxaliplatin, carboplatin; most preferably, the antimetabolite comprises methotrexate, 5-fluorouracil, fluoroglycoside, 5-fluorodeoxyuracil, capecitabine, cytarabine, fludarabine, 6-mercaptopurine, 2-chlorodeoxyadenosine, 5-azacytidine, 2-difluorodeoxycytidine, cladribine, deoxy Ke Fumei, pennistin; most preferably, the antitumor antibiotic comprises daunorubicin, doxorubicin, norubicin, mithramycin, mitomycin C, valubicin, mitoxantrone hydrochloride, bleomycin, dactinomycin, mithramycin, and procarbazine; most preferably, the mitotic inhibitor comprises taxane, vinblastine, paclitaxel, vincristine, vinamidone, vinorelbine; most preferably, the chromatin function inhibitor comprises irinotecan, etoposide, topotecan, etoposide phosphate, etoposide; most preferably, the anti-angiogenic agent comprises praline stat, tamnustat, ilomastat, propylimine, pamphlet, CGS-27023A, bromclopidoquine, novalastat, BMS-275291, thalidomide; most preferably, the antiestrogens include toremifene, raloxifene, tamoxifen, anastrozole, letrozole, droloxifene, oxybenzofene, exemestane; most preferably, the anti-androgens comprise nilutamide, bicalutamide, busulfan, flutamide, finasteride, cyproterone acetate, cimetidine; most preferably, the immunomodulator comprises interleukin, tumor necrosis factor, interferon, mushroom polysaccharide, sirolimus, luo Kuimei g, pidotimod, methoxypolyethylene glycol succinamide adenosine deaminase, thymus peptide preparation; most preferably, the cytotoxin comprises DM4, MMAE, DM1, ozogamicin, dxd, SN-38, MMAF, PBD, DM2, amanitin, PNU-159582, IR700, PE-38, PE4E, PE40, PE25, PE35;
Most preferably, the CLDN 18.2-positive related disease comprises a solid tumor, a hematological tumor, or a combination thereof; most preferably, the CLDN 18.2-positive-related disease comprises gastric cancer, pancreatic cancer, esophageal cancer, cholangiocarcinoma, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchogenic 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.
In certain embodiments, the biological agents of the present invention may comprise buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; an adjuvant; and a preservative. The biological agents of the invention are preferably formulated for intravenous administration.
In certain embodiments, the pharmaceutical compositions of the present invention further comprise one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipient combinations. Such compositions may comprise: buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids, such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and (3) a preservative. The compositions of the present invention may be formulated for oral, intravenous, topical, enteral and/or parenteral administration. In certain embodiments, the compositions of the present invention are formulated for intravenous administration.
In an eighth aspect, the invention provides a detection reagent or kit for detecting CLDN18.2 protein.
Further, the detection reagent comprises the humanized antibody of the first aspect of the present invention;
preferably, the detection reagent further comprises a diagnostic reagent coupled to the humanized antibody; more preferably, the diagnostic reagent comprises a radionuclide, chemiluminescent, bioluminescent, paramagnetic ion, enzyme, photosensitive diagnostic agent; most preferably, the radionuclides include 18F, 52Fe, 62Cu, 64Cu, 67Cu, 86Y, 90Y, 89Zr, 120I, 123I, 124I, 125I, 131I, 13N, 15O, 55Co, 72As, 51Mn; most preferably, the chemiluminescent agent comprises luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, oxalic esters; most preferably, the bioluminescent agent comprises fluorescein, luciferase, aequorin; most preferably, the paramagnetic ion comprises 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); most preferably, the enzyme comprises horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-D-galactosidase, urease, catalase, or glucoamylase; most preferably, the photosensitive diagnostic agent comprises dihydroxysilicon phthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, photoporphyrin;
Preferably, the kit comprises a monoclonal antibody according to the first aspect of the invention and/or the detection reagent; more preferably, the kit further comprises a lysis medium for dissolving the sample to be tested, universal reagents required for detection and a buffer; more preferably, the kit further comprises instructions for use.
A ninth aspect of the invention provides a method of any one of:
(1) A method for non-diagnostic and non-therapeutic destination detection of CLDN18.2 in a sample to be tested, said method comprising the steps of: contacting a sample to be tested with the humanized antibody according to the first aspect of the invention, and detecting the formation of a complex of the humanized antibody and CLDN 18.2;
preferably, the humanized antibody is a humanized antibody labeled with a label available for detection; more preferably, the labels useful for detection include fluorescent pigments, avidin, paramagnetic atoms, radioisotopes; most preferably, the fluorescent pigment is fluorescein, rhodamine, texas red, phycoerythrin, phycocyanin, allophycocyanin, polymannuin-chlorophyll protein; most preferably, the avidin is biotin, ovalbumin, streptavidin, vitellin, avidin-like; most preferably, the radioisotope is radioiodine, radiocesium, radioiridium, radiocobalt;
(2) A method of producing a humanized antibody according to the first aspect of the invention, the method comprising the steps of: culturing the genetically engineered host cell of the fifth aspect of the invention and/or the population of host cells of the sixth aspect of the invention, and isolating the humanized antibody of the first aspect of the invention from the culture;
(3) A method of preparing a genetically engineered host cell according to the fifth aspect of the invention and/or a population of host cells according to the sixth aspect of the invention, the method comprising the steps of: providing a host cell to be engineered, introducing a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention into said host cell, obtaining said genetically engineered host cell or population of host cells;
preferably, the method of introducing comprises calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, electroporation, TALEN method, ZFN method, liposome-mediated transfection, lentiviral infection, retroviral infection, adenovirus infection, transposon technology, CRISPR-Cas9 technology;
preferably, the host cell comprises an immune cell; more preferably, the immune cells comprise T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof; most preferably, the immune cells are T cells;
(4) A method of stimulating an immune response in a mammalian target cell population or tissue, the method comprising the steps of: administering to the mammal an effective amount of a genetically engineered host cell according to the fifth aspect of the invention and/or a population of host cells according to the sixth aspect of the invention;
(5) A method of specifically inhibiting CLDN18.2 activity in vitro comprising the steps of: introducing a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention into a cell of an organism, and inhibiting the activity of CLDN18.2 by expressing a humanized antibody according to the first aspect of the invention.
The present invention also provides a method of treating or delaying progression of a CLDN 18.2-positive related disease in a subject in need thereof, the method comprising the steps of: administering to a subject in need thereof an effective amount of a humanized antibody according to the first aspect of the invention, a genetically engineered host cell according to the fifth aspect of the invention, a population of host cells according to the sixth aspect of the invention, a biological agent according to the seventh aspect of the invention, or a pharmaceutical composition, in some embodiments the subject comprises a mammal, in particular embodiments of the invention the subject is preferably a human.
The invention also provides a method of diagnosing whether a subject has a CLDN 18.2-positive related disease, the method comprising the steps of: contacting a test sample from a subject with a humanized antibody according to the first aspect of the invention, and detecting whether a complex is formed between the antibody and CLDN18.2 in the test sample. In some embodiments, the test sample is a test biological sample derived from a subject, including but not limited to: a blood sample, a tissue sample, etc., the subject comprising a mammal selected from the group consisting of: human, animal, non-human primate, dog, cat, sheep, mouse, horse or cow, in some embodiments, the subject is preferably a human.
A tenth aspect of the invention provides any one of the following applications:
(1) Use of a humanized antibody according to the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, a genetically engineered host cell according to the fifth aspect of the invention, a population of host cells according to the sixth aspect of the invention, a biological agent or a pharmaceutical composition according to the seventh aspect of the invention for the preparation of a medicament for the treatment and/or prevention of a CLDN 18.2-positive related disease;
(2) Use of a humanized antibody according to the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, a genetically engineered host cell according to the fifth aspect of the invention, a population of host cells according to the sixth aspect of the invention for the preparation of a biological agent for the treatment and/or prevention of a CLDN 18.2-positive related disease;
(3) Use of a humanized antibody according to the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention for the preparation of a genetically engineered host cell for the treatment and/or prevention of a CLDN 18.2-positive related disease;
(4) Use of a humanized antibody according to the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention for the preparation of a population of host cells for the treatment and/or prevention of a CLDN 18.2-positive related disease;
(5) The humanized antibody of the first aspect of the invention and the nucleic acid molecule of the third aspect of the invention are used for preparing a detection reagent for detecting the CLDN18.2 protein or the antigen fragment thereof;
(6) Use of a humanized antibody according to the first aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a detection reagent or kit according to the eighth aspect of the invention, in the preparation of a product for detecting CLDN18.2 protein or an antigenic fragment thereof;
(7) Use of a humanized antibody according to the first aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a detection reagent or kit according to the eighth aspect of the invention, for the preparation of a product for diagnosis and/or co-diagnosis of a CLDN18.2 positive-related disease;
preferably, the CLDN 18.2-positive related disease comprises a solid tumor, a hematological tumor, or a combination thereof; more preferably, the CLDN 18.2-positive-related disease comprises gastric cancer, pancreatic cancer, esophageal cancer, cholangiocarcinoma, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchogenic 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. In certain embodiments, the CLDN 18.2-positive related diseases described herein are not limited to the above-described diseases, and any diseases and/or conditions associated with expression of CLDN18.2 are within the scope of the present invention.
Drawings
FIG. 1 is a graph showing the results of detecting binding of humanized antibodies to CHO-CLDN18.2 cells by flow cytometry;
FIG. 2 is a graph showing the results of detecting binding of humanized antibodies to CHO-CLDN18.1 cells by flow cytometry;
FIG. 3 is a graph showing the results of detecting the binding of humanized antibodies to CHO cells by flow cytometry;
FIG. 4 is a graph of humanized antibody affinity validation results;
FIG. 5 is a schematic structural diagram of a CAR lentiviral expression vector plasmid;
FIG. 6 is a graph showing the results of specific killing of CAR-T cells based on humanized antibody construction against CHO cells overexpressing CLDN 18.2.
Detailed Description
The invention is further illustrated below in conjunction with specific examples, which are intended to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents. The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biological materials, etc. used in the examples described below are commercially available unless otherwise specified.
Example 1 preparation of humanized antibodies targeting CLDN18.2
Humanization of antibody sequences: in order to further reduce the murine nature of the antibody, humanized antibodies, i.e., humanized versions of heavy and light chains of murine antibody F2H sequences specifically recognizing CLDN18.2, were prepared by grafting the CDRs (CDR 1, CDR2 and CDR 3) of a murine antibody to the framework regions of a human antibody, the sequences of murine antibody F2H being shown in Table 1 below, and the information on the degree of humanization T20 score (https:// dm.lakepharma.com/bioinformation) being shown in Table 2 below, wherein the sequences of the framework regions (FR 1, FR2, FR3 and FR 4) of humanized VH1, VH3, VH4, VL1, VL2, VL3, VL4 are shown in Table 3 below.
TABLE 1 sequence of murine antibody F2H
TABLE 2 humanized altered sequences of antibody heavy and light chain variable regions
TABLE 3 sequences of framework regions in humanized altered antibody heavy and light chains
Example 2 affinity validation of humanized antibodies targeting CLDN18.2
1. Experimental method
(1) And (3) carrying out gene synthesis on the heavy chain and the light chain of the humanized antibody designed above after codon optimization, respectively carrying out gene synthesis, subcloning the heavy chain into a pcDNA3.4-IgG1 expression vector, and subcloning the light chain into a pcDNA3.4-IgKc expression vector. After the vector is verified by sequencing, endotoxin-removing plasmids are prepared. Humanized VH and VL antibody expression vectors (VH 1VL1, VH1VL2, VH1VL3, VH1VL4, VH3VL1, VH3VL2, VH3VL3, VH3VL4, VH4VL1, VH4VL2, VH4VL3, VH4VL 4) were transiently transfected into 293 cells to express antibodies, and the humanized antibodies prepared in example 1 were examined for binding to target proteins (examined cells: CHO-CLDN18.2, CHO-CLDN18.1, CHO) using flow cytometry fluorescence sorting (FACS). Wherein Chimeric represents the human murine Chimeric antibody constructed from the original murine antibodies VH and VL, as a control.
(2) According to the detection result of the humanized antibody, the purified antibody expressed by the VH4VL1, the VH1VL1, the VH4VL4 and the VH3VL1 is selected for detecting the affinity. The results from the flow assays were statistically averaged for MFI values, with higher MFI values representing higher affinity of the corresponding humanized antibody for antigen CLDN 18.2.
2. Experimental results
The results of detecting the binding of the humanized antibodies to CHO-CLDN18.2, CHO-CLDN18.1 and CHO cells by flow cytometry are shown in fig. 1-3, and the results show that the humanized antibodies (VH 1VL1, VH1VL2, VH1VL3, VH1VL4, VH3VL1, VH3VL2, VH3VL3, VH3VL4, VH4VL1, VH4VL2, VH4VL3 and VH 4) prepared by the present invention can specifically bind to CHO-CLDN18.2 cells but not to CHO-CLDN18.1 cells or CHO cells. The results of the affinity validation are shown in fig. 4, which shows that humanized antibodies (VH 4VL1, VH1VL1, VH4VL4, VH3VL 1) and chimeric antibodies (F2H) have identical affinities and higher affinity for both antigen CLDN 18.2.
Example 3 validation of killing effect of humanized antibodies targeting CLDN18.2
1. Experimental method
(1) Construction of CAR lentiviral expression vector plasmids: the CAR virus expression vector plasmid is constructed based on the humanized antibodies (VH 4VL1, VH1VL1, VH4VL4, VH3VL 1) and murine antibody F2H, and the specific construction method of the CAR lentiviral expression vector plasmid is as follows: the humanized antibody chimeric sequence is obtained by sequentially connecting a CD8 alpha signal peptide, a humanized antibody (light chain variable region-Linker-heavy chain variable region), a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory signal domain, a CD3 zeta intracellular signal transduction domain, a T2A, GMCSF signal peptide and EGFR DIII-DIV in series. The synthetic sequence is inoculated into a pCDH-EF1 alpha lentiviral expression plasmid by homologous recombination mode by using two enzyme cutting sites BstBI and XbaI, the schematic diagram of the plasmid is shown in figure 5, and the nucleotide sequence of the EF1 alpha promoter is shown as SEQ ID NO. 44 in a sequence table.
Similarly, the CAR constructed based on the murine antibody F2H is obtained by sequentially connecting a CD8 alpha signal peptide, a murine antibody F2H (light chain variable region-Linker-heavy chain variable region), a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory signal domain, a CD3 zeta intracellular signaling domain, a T2A, GMCSF signal peptide and EGFR D III-D IV in series.
Specific amino acid sequences and nucleotide sequences corresponding to the CD8 alpha signal peptide, the CD8 hinge region, the CD8 transmembrane domain, the 4-1BB costimulatory signal domain, the CD3 zeta intracellular signaling domain, the T2A, GMCSF signal peptide, and EGFR DIII through DIV in the CAR are shown in Table 4 and Table 5 below, respectively.
Table 4 amino acid sequences corresponding to portions of CARs
Table 5 nucleotide sequences corresponding to portions of CARs
(2) Corresponding CAR-T cells are further constructed based on the CAR containing the humanized antibody and the CAR containing the murine antibody F2H, the CAR-T cells are constructed by transducing healthy human T cells with 293T packaging lentivirus, and the CAR-T cells are cultured and amplified by an X-vivo system containing IL-7 and IL-15 until the 8 th day to start killing verification. Killing CHO cells overexpressed with claudin18.1 and claudin18.2 using two effect target ratios of 1:1, 1:2.
The specific construction method of the CAR-T cell comprises the following steps: the lentivirus system plasmid is transfected to adherent 293T cells in logarithmic phase, cell culture supernatant transfected for 48-72 hours is harvested, and CAR lentivirus is obtained after concentration and filtration and is stored at-80 ℃ for standby. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from human peripheral blood, T cells were isolated using human CD3/28 magnetic beads and virus transduction was performed within 72 hours of activation, and after 24 hours of transduction, the culture was changed to day 8, and cells were collected by centrifugation and resuspended in physiological saline. And (3) identifying the CAR molecules on the surface of the T cells by using flow cells, wherein the positive rate is ensured to be more than 30%, and enrichment and separation are performed by using EGFR-PE magnetic beads if the positive rate is lower than the positive rate. Subsequent killing experiments were performed with the target cells mixed according to the positive proportion of CAR-T cell harvest.
2. Experimental results
The result is shown in fig. 6, and the result shows that the F2H antibody (murine) CAR-T has good specific killing to CLDN18.2, and can still keep the target recognition and killing specificity after the F2H sequence is humanized, namely, the humanized antibody CAR-T prepared by the invention can remarkably and specifically kill cells over-expressing CLDN18.2, and has higher killing efficiency.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.
Claims (10)
1. A humanized antibody targeting CLDN18.2, comprising a heavy chain variable region VH and a light chain variable region VL;
the VH comprises three complementarity determining regions HCDR1, HCDR2, HCDR3;
the VL comprises three complementarity determining regions LCDR1, LCDR2, LCDR3;
the amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3;
the amino acid sequences of the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;
Preferably, the humanized antibody comprises VHVL;
more preferably, the VHVL comprises VH1VL1, VH1VL2, VH1VL3, VH1VL4, VH3VL1, VH3VL2, VH3VL3, VH3VL4, VH4VL1, VH4VL2, VH4VL3, VH4VL4;
most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of the VH1 are respectively shown as SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19;
most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of the VH3 are respectively shown as SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22 and SEQ ID NO. 23;
most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of the VH4 are respectively shown as SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26 and SEQ ID NO. 27;
most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of VL1 are shown as SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30 and SEQ ID NO. 31 respectively;
most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of VL2 are shown as SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34 and SEQ ID NO. 35 respectively;
most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of VL3 are shown as SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38 and SEQ ID NO. 39 respectively;
Most preferably, the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of VL4 are shown as SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42 and SEQ ID NO. 43 respectively;
most preferably, the amino acid sequence of VH1 is as shown in SEQ ID NO. 8 or has at least 90% homology with SEQ ID NO. 8;
most preferably, the amino acid sequence of VH3 is as shown in SEQ ID NO. 9 or has at least 90% homology with SEQ ID NO. 9;
most preferably, the amino acid sequence of VH4 is as shown in SEQ ID NO. 10 or has at least 90% homology with SEQ ID NO. 10;
most preferably, the amino acid sequence of VL1 is as shown in SEQ ID NO. 12 or an amino acid sequence having at least 90% homology with SEQ ID NO. 12;
most preferably, the amino acid sequence of VL2 is as shown in SEQ ID NO. 13 or an amino acid sequence having at least 90% homology with SEQ ID NO. 13;
most preferably, the amino acid sequence of VL3 is as shown in SEQ ID NO. 14 or an amino acid sequence having at least 90% homology with SEQ ID NO. 14;
most preferably, the amino acid sequence of VL4 is as shown in SEQ ID NO. 15 or an amino acid sequence having at least 90% homology with SEQ ID NO. 15.
2. A chimeric antigen receptor that targets CLDN18.2, comprising the humanized antibody of claim 1;
preferably, the chimeric antigen receptor further comprises a hinge region;
more preferably, the hinge region comprises a hinge region of: CD8, 4-1BB, igG1, igG4, PD-1, CD28, CD34, OX40, CD3 ε;
most preferably, the hinge region is a CD8 hinge region;
preferably, the chimeric antigen receptor further comprises a transmembrane domain;
more preferably, the transmembrane domain comprises the transmembrane domain of: CD8, 4-1BB, igG1, igG4, PD-1, CD28, CD34, OX40, CD3 ε;
most preferably, the transmembrane domain is a CD8 transmembrane domain;
preferably, the chimeric antigen receptor further comprises a costimulatory signaling domain;
more preferably, the costimulatory signaling domain comprises the costimulatory signaling domain of: 4-1BB, HVEM, CD, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278;
most preferably, the costimulatory signaling domain is a 4-1BB costimulatory signaling domain;
preferably, the chimeric antigen receptor further comprises an intracellular signaling domain;
More preferably, the intracellular signaling domain comprises an intracellular signaling domain of: cd3ζ, cd3γ, cd3δ, cd3ε, CD278, CD21, CD22, fcεri, fcrγ, fcrβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d;
most preferably, the intracellular signaling domain is a cd3ζ intracellular signaling domain;
most preferably, the chimeric antigen receptor further comprises a CD8 a signal peptide, a T2A, GMCSF signal peptide, EGFR diniii-diniv;
most preferably, the chimeric antigen receptor is selected from any one of the following groups:
(1) A chimeric antigen receptor obtained by sequentially concatenating the CD8 a signal peptide, the humanized antibody of claim 1, the CD8 hinge region, the CD8 transmembrane domain, the 4-1BB costimulatory signal domain, the CD3 zeta intracellular signaling domain, the T2A, GMCSF signal peptide, and EGFR dinil-diniv;
(2) A derivative chimeric antigen receptor formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence of the chimeric antigen receptor described in (1);
most preferably, the amino acid sequence of the CD8 alpha signal peptide is shown as SEQ ID NO. 45;
most preferably, the amino acid sequence of the CD8 hinge region is shown as SEQ ID NO. 46;
Most preferably, the amino acid sequence of the CD8 transmembrane domain is shown as SEQ ID NO. 47;
most preferably, the amino acid sequence of the 4-1BB costimulatory signal domain is shown as SEQ ID NO. 48;
most preferably, the amino acid sequence of the CD3ζ intracellular signaling domain is depicted as SEQ ID NO. 49;
most preferably, the amino acid sequence of T2A is shown as SEQ ID NO. 50;
most preferably, the amino acid sequence of the GMCSF signal peptide is shown in SEQ ID NO. 51;
most preferably, the amino acid sequences of EGFR DIII through DIV are shown in SEQ ID NO: 52.
3. A nucleic acid molecule encoding the humanized antibody of claim 1 or the chimeric antigen receptor of claim 2;
preferably, the nucleotide sequence encoding the CD8 alpha signal peptide in the chimeric antigen receptor of claim 2 is shown as SEQ ID NO. 53;
preferably, the nucleotide sequence encoding the CD8 hinge region in the chimeric antigen receptor of claim 2 is set forth in SEQ ID NO. 54;
preferably, the nucleotide sequence encoding the CD8 transmembrane domain of the chimeric antigen receptor of claim 2 is shown in SEQ ID NO. 55;
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. 56;
Preferably, the nucleotide sequence encoding the CD3 zeta intracellular signaling domain in the chimeric antigen receptor according to claim 2 is shown in SEQ ID No. 57;
preferably, the nucleotide sequence encoding T2A in the chimeric antigen receptor of claim 2 is set forth in SEQ ID NO. 58;
preferably, the nucleotide sequence encoding the GMCSF signal peptide in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO 59;
preferably, the nucleotide sequence encoding EGFR DIII-DIV in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO 60.
4. A vector comprising the nucleic acid molecule of claim 3;
preferably, the vector comprises a DNA vector, an RNA vector, a plasmid, a viral-derived vector, a transposon vector, a CRISPR/Cas9 vector;
more preferably, the viral-derived vector comprises a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector.
5. A genetically engineered host cell, comprising the nucleic acid molecule of claim 3 or the vector of claim 4;
preferably, the host cell comprises a prokaryotic cell, a eukaryotic cell;
More preferably, the prokaryotic cell comprises bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, rickettsia;
more preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, yeast cells;
most preferably, the host cell is an immune cell;
most preferably, the immune cells comprise T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof;
most preferably, the immune cells are T cells.
6. A population of host cells, wherein the population of host cells comprises the genetically engineered host cell of claim 5;
preferably, the population of host cells further comprises host cells that do not comprise the nucleic acid molecule of claim 3 or the vector of claim 4;
more preferably, the host cell comprises a prokaryotic cell, a eukaryotic cell;
most preferably, the prokaryotic cell comprises bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, rickettsia;
most preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, yeast cells;
Most preferably, the host cell is an immune cell;
most preferably, the immune cells comprise T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof;
most preferably, the immune cells are T cells.
7. A biological agent or pharmaceutical composition for the treatment and/or prevention of a CLDN 18.2-positive related disease, characterized in that the biological agent comprises the humanized antibody of claim 1, the genetically engineered host cell of claim 5 and/or the host cell population of claim 6;
preferably, the biological agent further comprises a pharmaceutically acceptable carrier, diluent and/or excipient;
more preferably, the biological agent comprises a monospecific antibody medicament comprising the humanized antibody of claim 1, a bispecific antibody medicament comprising the humanized antibody of claim 1, an antibody-conjugated medicament comprising the humanized antibody of claim 1, a CAR-T cell medicament comprising the genetically engineered host cell of claim 5 and/or the host cell population of claim 6;
Preferably, the pharmaceutical composition comprises the humanized antibody of claim 1, the genetically engineered host cell of claim 5, the host cell population of claim 6 and/or the biological agent;
more preferably, the pharmaceutical composition further comprises a therapeutic agent for the adjuvant treatment and/or prevention of CLDN 18.2-positive related diseases;
most preferably, the therapeutic agent comprises an alkylating agent, an antimetabolite, an antitumor antibiotic, a mitotic inhibitor, a chromatin function inhibitor, an anti-angiogenic agent, an antiestrogen, an antiandrogen, an immunomodulator, a cytotoxin;
most preferably, the alkylating agent comprises mechlorethamine, chlorambucil, melphalan, bromopropylamine, melphalan, fosfestivaline, cyclophosphamide, hexamethylmelamine, ifosfamide, fosetyl-amine, triamthos, carmustine, streptavidin, infliximab, azomethine, cisplatin, oxaliplatin, carboplatin;
most preferably, the antimetabolite comprises methotrexate, 5-fluorouracil, fluoroglycoside, 5-fluorodeoxyuracil, capecitabine, cytarabine, fludarabine, 6-mercaptopurine, 2-chlorodeoxyadenosine, 5-azacytidine, 2-difluorodeoxycytidine, cladribine, deoxy Ke Fumei, pennistin;
Most preferably, the antitumor antibiotic comprises daunorubicin, doxorubicin, norubicin, mithramycin, mitomycin C, valubicin, mitoxantrone hydrochloride, bleomycin, dactinomycin, mithramycin, and procarbazine;
most preferably, the mitotic inhibitor comprises taxane, vinblastine, paclitaxel, vincristine, vinamidone, vinorelbine;
most preferably, the chromatin function inhibitor comprises irinotecan, etoposide, topotecan, etoposide phosphate, etoposide;
most preferably, the anti-angiogenic agent comprises praline stat, tamnustat, ilomastat, propylimine, pamphlet, CGS-27023A, bromclopidoquine, novalastat, BMS-275291, thalidomide;
most preferably, the antiestrogens include toremifene, raloxifene, tamoxifen, anastrozole, letrozole, droloxifene, oxybenzofene, exemestane;
most preferably, the anti-androgens comprise nilutamide, bicalutamide, busulfan, flutamide, finasteride, cyproterone acetate, cimetidine;
most preferably, the immunomodulator comprises interleukin, tumor necrosis factor, interferon, mushroom polysaccharide, sirolimus, luo Kuimei g, pidotimod, methoxypolyethylene glycol succinamide adenosine deaminase, thymus peptide preparation;
Most preferably, the cytotoxin comprises DM4, MMAE, DM1, ozogamicin, dxd, SN-38, MMAF, PBD, DM2, amanitin, PNU-159582, IR700, PE-38, PE4E, PE40, PE25, PE35;
most preferably, the CLDN 18.2-positive related disease comprises a solid tumor, a hematological tumor, or a combination thereof;
most preferably, the CLDN 18.2-positive-related disease comprises gastric cancer, pancreatic cancer, esophageal cancer, cholangiocarcinoma, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchogenic 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.
8. A detection reagent or kit for detecting CLDN18.2 protein, comprising the humanized antibody of claim 1;
preferably, the detection reagent further comprises a diagnostic reagent coupled to the humanized antibody;
more preferably, the diagnostic reagent comprises a radionuclide, chemiluminescent, bioluminescent, paramagnetic ion, enzyme, photosensitive diagnostic agent;
Most preferably, the radionuclides include 18F, 52Fe, 62Cu, 64Cu, 67Cu, 86Y, 90Y, 89Zr, 120I, 123I, 124I, 125I, 131I, 13N, 15O, 55Co, 72As, 51Mn;
most preferably, the chemiluminescent agent comprises luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, oxalic esters;
most preferably, the bioluminescent agent comprises fluorescein, luciferase, aequorin;
most preferably, the paramagnetic ion comprises 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);
most preferably, the enzyme comprises horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-D-galactosidase, urease, catalase, or glucoamylase;
most preferably, the photosensitive diagnostic agent comprises dihydroxysilicon phthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, photoporphyrin;
preferably, the kit comprises the monoclonal antibody of claim 1 and/or the detection reagent;
more preferably, the kit further comprises a lysis medium for dissolving the sample to be tested, universal reagents required for detection and a buffer;
More preferably, the kit further comprises instructions for use.
9. A method of any one of:
(1) A method for non-diagnostic and non-therapeutic destination detection of CLDN18.2 in a sample to be tested, said method comprising the steps of: contacting a sample to be tested with the humanized antibody of claim 1, detecting the formation of a complex of the humanized antibody with CLDN 18.2;
preferably, the humanized antibody is a humanized antibody labeled with a label available for detection;
more preferably, the labels useful for detection include fluorescent pigments, avidin, paramagnetic atoms, radioisotopes;
most preferably, the fluorescent pigment is fluorescein, rhodamine, texas red, phycoerythrin, phycocyanin, allophycocyanin, polymannuin-chlorophyll protein;
most preferably, the avidin is biotin, ovalbumin, streptavidin, vitellin, avidin-like;
most preferably, the radioisotope is radioiodine, radiocesium, radioiridium, radiocobalt;
(2) A method of producing the humanized antibody of claim 1, comprising the steps of: culturing the genetically engineered host cell of claim 5 and/or the population of host cells of claim 6, and isolating the humanized antibody of claim 1 from the culture;
(3) A method of making the genetically engineered host cell of claim 5 and/or the population of host cells of claim 6, comprising the steps of: providing a host cell to be engineered, introducing the nucleic acid molecule of claim 3 or the vector of claim 4 into said host cell, obtaining said genetically engineered host cell or host cell population;
preferably, the method of introducing comprises calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, electroporation, TALEN method, ZFN method, liposome-mediated transfection, lentiviral infection, retroviral infection, adenovirus infection, transposon technology, CRISPR-Cas9 technology;
preferably, the host cell comprises an immune cell;
more preferably, the immune cells comprise T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof;
most preferably, the immune cells are T cells;
(4) A method of stimulating an immune response in a target cell population or tissue of a mammal, the method comprising the steps of: administering to a mammal an effective amount of the genetically engineered host cell of claim 5 and/or the host cell population of claim 6;
(5) A method of specifically inhibiting CLDN18.2 activity in vitro comprising the steps of: introducing the nucleic acid molecule of claim 3 or the vector of claim 4 into a cell of an organism to inhibit the activity of CLDN18.2 by expressing the humanized antibody of claim 1.
10. The following are applied in any aspect:
(1) Use of a humanized antibody according to claim 1, a chimeric antigen receptor according to claim 2, a nucleic acid molecule according to claim 3, a vector according to claim 4, a genetically engineered host cell according to claim 5, a host cell population according to claim 6, a biological agent or a pharmaceutical composition according to claim 7 for the preparation of a medicament for the treatment and/or prevention of CLDN 18.2-positive related diseases;
(2) Use of the humanized antibody of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the genetically engineered host cell of claim 5, the host cell population of claim 6 for the preparation of a biological agent for the treatment and/or prevention of a CLDN 18.2-positive-related disease;
(3) Use of the humanized antibody of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4 for the preparation of a genetically engineered host cell for the treatment and/or prevention of CLDN 18.2-positive related diseases;
(4) Use of the humanized antibody of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4 for the preparation of a population of host cells for the treatment and/or prevention of CLDN 18.2-positive related diseases;
(5) Use of the humanized antibody of claim 1, the nucleic acid molecule of claim 3, for the preparation of a detection reagent for detecting CLDN18.2 protein or an antigenic fragment thereof;
(6) Use of the humanized antibody of claim 1, the nucleic acid molecule of claim 3, the detection reagent or kit of claim 8 in the preparation of a product for detecting CLDN18.2 protein or an antigenic fragment thereof;
(7) Use of the humanized antibody of claim 1, the nucleic acid molecule of claim 3, the detection reagent or kit of claim 8 for the preparation of a product for the diagnosis and/or co-diagnosis of CLDN 18.2-positive related diseases;
Preferably, the CLDN 18.2-positive related disease comprises a solid tumor, a hematological tumor, or a combination thereof;
more preferably, the CLDN 18.2-positive-related disease comprises gastric cancer, pancreatic cancer, esophageal cancer, cholangiocarcinoma, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchogenic 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.
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