Fully human B7H3 antibody, chimeric antigen receptor and application of fully human B7H3 antibody and chimeric antigen receptor in tumor treatment

Abstract

The invention discloses a fully human B7H3 antibody, a chimeric antigen receptor and application of the fully human B7H3 antibody and the chimeric antigen receptor in tumor treatment, the antibody has high specificity and affinity to B7H3 and can be specifically combined with a B7H3 positive cell line, and a CAR-T cell constructed based on the antibody has high specific killing efficiency to the B7H3 positive cell line and can be used for treating tumor cells. The invention provides a research and development basis for the development of immunotherapy drugs based on B7H3 targets.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a fully human B7H3 antibody and a chimeric antigen receptor and their application in treating tumors. Background Art

[0002] B7H3 (CD276) is a type I transmembrane protein that belongs to the B7 immune co-stimulatory and co-inhibitory family. It has an immunosuppressive function and can reduce type I interferon (IFN) released by T cells and reduce the cytotoxicity of NK cells. B7H3 protein is limitedly expressed in normal tissues (such as prostate, breast, placenta, liver, colon and lymphoid organs), but is abnormally highly expressed in most malignant tumors. The expression of B7H3 can be detected in non-small cell lung cancer cell lines and tumor tissues. In tumor tissues expressing B7H3, the number of infiltrating lymphoid cells is significantly reduced, which is positively correlated with lymph node metastasis. The high expression of B7H3 in tumor cells is usually closely related to the decrease of tumor infiltrating lymphocytes, accelerated cancer progression and clinical outcomes of malignant tumors (neurological tumors, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, prostate cancer, ovarian cancer, lung cancer and clear cell renal carcinoma, etc.). Due to its widespread expression in a variety of tumors, B7H3 has become a potential target for cancer immunotherapy. However, there is currently a lack of immunotherapy products with related specificity, such as antibodies and chimeric antigen receptor-immune cells, that can be used to effectively treat diseases expressing B7H3. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide a fully human B7H3 antibody AHF23046 and a chimeric antigen receptor and their use in treating tumors in the art. The antibody AHF23046 can specifically bind to B7H3, and the CAR-T cells prepared based on the antibody AHF23046 have a significant killing effect on cells expressing B7H3, and have broad application prospects. On this basis, the following invention has been completed.

[0004] Antibodies that specifically recognize B7H3

[0005] In one aspect, the present invention provides an antibody that specifically recognizes B7H3, the antibody comprising a heavy chain variable region and a light chain variable region;

[0006] 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;

[0007] The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively;

[0008] Optionally, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4 or has at least 85% homology with SEQ ID NO:4; optionally, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9 or has at least 85% homology with SEQ ID NO:9.

[0009] The antibody corresponding to the amino acid sequence having at least 85% homology with the above-mentioned amino acid sequence, that is, the amino acid sequence obtained by replacing the amino acid at any one or more positions with any other amino acid on the basis of the corresponding amino acid sequence, is within the protection scope of the present invention as long as the amino acid sequence has at least 85% homology with the amino acid sequence of the above-mentioned antibody of the present invention.

[0010] In certain embodiments, the antibody also includes functional variants of the above antibodies, specifically, the functional variants include but are not limited to: derivatives that are substantially similar in primary structural sequence but include those not present in the parent antibody of the present invention, such as chemical and / or biochemical modifications in vitro or in vivo. These modifications include, for example, acetylation, acylation, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, cross-linking, formation of disulfide bonds, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolytic treatment, phosphorylation, and such functional variants are also included in the protection scope of the present invention.

[0011] Alternatively, the functional variant may be an antibody that includes an amino acid sequence containing one or more amino acid substitutions, insertions, deletions, or a combination thereof, compared to the amino acid sequence of the parent antibody. Further, the functional variant may include a truncation of the amino acid sequence at one or both of the amino terminus or carboxyl terminus. Compared to the parent antibody, the functional variant of the present invention may have the same or different, higher or lower binding affinity, but still be able to specifically bind to B7H3. For example, compared to the parent antibody, the functional variant of the present invention may have an increased or decreased binding affinity to B7H3.

[0012] Chimeric Antigen Receptor (CAR)

[0013] In another aspect, the present invention provides a chimeric antigen receptor, which comprises the antibody as described above;

[0014] Optionally, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, and a co-stimulatory signaling domain; optionally, the chimeric antigen receptor further comprises any one or more of a signal peptide, a self-cleaving peptide, a safety switch, an immunomodulatory molecule, or a cytokine; optionally, the transmembrane domain is selected from the transmembrane domain of any one of the following molecules: CD8, CD28, CD34, CD3ε, PD-1, IgG1, 4-1BB, IgG4, OX40, IL-2 receptor, IL-7 receptor; optionally, the intracellular signaling domain is selected from the intracellular signaling domain of any one of the following molecules : CD3ζ, CD3γ, CD3δ, FcRγ, FcRβ, TCRζ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD66d, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12; Optionally, the hinge region is selected from the hinge region of any one of the following molecules: CD8, 4-1BB, OX40, CD3ε, IgG1, CD28, CD34, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the costimulatory signaling domain is selected from the costimulatory signaling domain of any one of the following molecules: Stimulation signal domain: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8α, CD8β, BAFFR, HVEM, CD40, OX40, CD30, TIM1, CD2, CD7, CD226; Optionally, the signal peptide is selected from the signal peptide of any one of the following molecules: α chain and β chain of T cell receptor, CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD16, CD22, CD33, CD134, CD137, CD15 4. GITR, GM-CSF; Optionally, the self-cleaving peptide is selected from any one of the following self-cleaving peptides: T2A, P2A, E2A, F2A; Optionally, the safety switch is selected from any one of the following safety switches: tEGFR, iCaspase-9, RQR8; Optionally, the immunomodulatory molecule or cytokine is selected from any one of the following immunomodulatory molecules or cytokines: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, IL-15, IL-18, IL-21, LEC, OX40L;

[0015] Optionally, the chimeric antigen receptor is a signal peptide, a heavy chain variable region of the antibody as described above, a linker, a light chain variable region of the antibody as described above, a CD8 hinge region and a transmembrane domain, a CD28 costimulatory signal domain, a CD3ζ intracellular signaling domain, a T2A self-cleaving peptide, and a tEGFR safety switch connected in series in sequence; optionally, the linker is (G4S)3; optionally, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:11; optionally, the amino acid sequence of the linker is as shown in SEQ ID NO:15; optionally, the amino acid sequence of the CD8 hinge region and the transmembrane domain is as shown in SEQ ID NO:19; optionally, the amino acid sequence of the CD28 costimulatory signal domain is as shown in SEQ ID NO:21; optionally, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO:23; optionally, the amino acid sequence of the T2A self-cleaving peptide is as shown in SEQ ID NO:25; optionally, the amino acid sequence of the tEGFR safety switch is as shown in SEQ ID NO:27; optionally, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:29.

[0016] In certain embodiments, the signal peptide, linker, CD8 hinge region and transmembrane domain, CD28 co-stimulatory signal domain, CD3ζ intracellular signaling domain, T2A self-cleaving peptide, and tEGFR safety switch described in the present invention are not limited to the above sequences. As long as the amino acid sequence has at least 85% homology with the above amino acid sequence, it is within the protection scope of the present invention.

[0017] In certain embodiments, the linker is a linker conventionally used in the art, and the linker is not limited to (G4S)3, and can be (GGGGS) n 、(GGGS) n 、(SSSSG) n 、(GSGSA) n 、(GGSGG) n Or any one of the other linkers, wherein n can be any integer between 1-10.

[0018] Nucleic acid molecules

[0019] In another aspect, the present invention provides a nucleic acid molecule, the sequence of which is the coding sequence of the antibody as described above or the chimeric antigen receptor as described above;

[0020] Optionally, in the antibody as described above, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 14; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO: 18; optionally, in the chimeric antigen receptor as described above, the nucleotide sequence encoding the signal peptide is shown in SEQ ID NO: 12; the nucleotide sequence encoding the linker is shown in SEQ ID NO: 16; the nucleotide sequence encoding the CD8 hinge region and transmembrane domain is shown in SEQ ID NO: 20; the nucleotide sequence encoding the CD28 co-stimulatory signal domain is shown in SEQ ID NO: 22; the nucleotide sequence encoding the CD3ζ intracellular signaling domain is shown in SEQ ID NO: 24; the nucleotide sequence encoding the T2A self-cleaving peptide is shown in SEQ ID NO: 26; the nucleotide sequence encoding the tEGFR safety switch is shown in SEQ ID NO: 28;

[0021] Optionally, the nucleotide sequence encoding the chimeric antigen receptor as described above is shown in SEQ ID NO:30.

[0022] A vector comprising the nucleic acid molecule

[0023] In another aspect, the present invention provides a vector comprising the nucleic acid molecule as described above, wherein the vector is a cloning vector or an expression vector;

[0024] Optionally, the vector is a DNA vector, an RNA vector, a plasmid or a vector of viral origin; optionally, the vector of viral origin is a retroviral vector, an adenoviral vector, an adeno-associated viral vector or a lentiviral vector.

[0025] In certain embodiments, the present invention has no particular limitation on the vector, and its selection depends on the desired function. Non-limiting examples of vectors include plasmid vectors, vectors of viral origin, phage vectors and other conventional vectors used in, for example, genetic engineering. Various plasmids and vectors can be constructed based on methods well known to those skilled in the art.

[0026] In certain embodiments, the expression vector according to the present invention is capable of directing the replication and expression of the nucleic acid molecule of the present invention in a host, and thus ensuring the expression of the antibody or chimeric antigen receptor targeting B7H3 of the present invention encoded thereby in a selected host. The expression vector may be, for example, a cloning vector, a binary vector or an integrative vector. Expression includes transcription of the nucleic acid molecule, for example, transcription into a translatable mRNA.

[0027] Engineered host cells

[0028] In another aspect, the present invention provides an engineered host cell, the engineered host cell comprising the vector as described above;

[0029] Optionally, the host cell is Escherichia coli, yeast cell, insect cell or mammalian cell; optionally, the host cell is an immune cell; optionally, the immune cell is a T cell, B cell, NK cell, iNKT cell, CTL cell, dendritic cell, myeloid cell, monocyte, macrophage or any combination thereof.

[0030] In the present invention, the host cell refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells, etc. In a specific embodiment of the present invention, the host cell is preferably an immune cell, and the immune cell includes but is not limited to T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof, preferably T cells. In addition, the host cell of the present invention may include a single cell or a cell group, that is, the engineered host cell of the present invention includes a single engineered host cell and an engineered host cell group.

[0031] Immunoconjugates

[0032] In another aspect, the present invention provides an immunoconjugate, wherein the immunoconjugate comprises the antibody as described above and a conjugate conjugated thereto;

[0033] Optionally, the conjugate is 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 any combination thereof. Optionally, the cytokine includes 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; Optionally, the therapeutic agent includes an alkylating agent, an antimetabolite, an antitumor antibiotic, a mitotic inhibitor, a chromatin function inhibitor, an antiangiogenic agent, an anti-estrogen, an anti-androgen, an immunomodulator;

[0034] Optionally, the alkylating agent includes bischloroethyl methylamine, chlorambucil, phenylalanine mustard, propidium bromide, phenyl mustard, cyclophosphamide, hexamethylmelamine, carmustine, cisplatin, oxaliplatin, and carboplatin; Optionally, the antimetabolite includes methotrexate, 5-fluorouracil, fluorouracil, capecitabine, cytarabine, fludarabine, 6-mercaptopurine, 2-chlorodeoxyadenosine, 5-azacytidine, and pentostatin; Optionally, the antitumor antibiotic includes daunorubicin, doxorubicin, Deoxydinocycline, mithramycin, mitomycin C, valrubicin, mitoxantrone hydrochloride, bleomycin, dactinomycin; optionally, the mitotic inhibitor includes docetaxel, vinblastine, paclitaxel, vincristine, vinblastine, vinorelbine; optionally, the chromatin function inhibitor includes irinotecan, etoposide, topotecan, etoposide phosphate, teniposide; optionally, the anti-angiogenic agent includes prinomastat, tanomastat, ilomastat, propylamine Amine, Marimastat, Batimastat, CGS-27023A, Bromochloropiperaquinone, Neovastat, Thalidomide; Optionally, the anti-estrogen includes toremifene, raloxifene, tamoxifen, anastrozole, letrozole, droloxifene, odoxifene, exemestane; Optionally, the anti-androgen includes nilutamide, bicalutamide, spironolactone, flutamide, finasteride, cyproterone acetate, cimetidine; Optionally, the immunomodulator includes interleukin, tumor Tumor necrosis factor, interferon, mushroom polysaccharide, sizofosan, roquine, pidomod, methoxy polyethylene glycol succinamide adenosine deaminase, thymosin preparation; Optionally, the cytotoxin includes MMAE, DM1, MMAF, PBD, DM2, DM4, PNU-159682, IR700, PE-38, PE24, PE-T20, PE-T20-KDEL, PE4E, PE40; Optionally, the radionuclide includes 131 I. 32 P. 89 Sr. 90 Y. 223 Ra, 125 I. 103 Pd.

[0035] Detection Reagents

[0036] In another aspect, the present invention provides a detection reagent, which comprises the antibody as described above and a diagnostic agent coupled thereto;

[0037] Optionally, the diagnostic agent is a radionuclide, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme or a photosensitive diagnostic agent. Optionally, the radionuclide includes 18 F. 52 Fe, 62 Cu, 64 Cu,67 Cu, 86 Y. 90 Y. 89 Zr, 120 I. 123 I. 124 I. 125 I. 131 I. 13 N. 15 O. 186 Re, 188 Re, 51 Mn, 72 As; optionally, the chemiluminescent agent includes luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, oxalates; optionally, the bioluminescent agent includes luciferin, luciferase, aequorin; optionally, 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); optionally, the enzyme includes horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase; optionally, the photosensitive diagnostic agent includes dihydroxysilicon phthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, photoporphyrin.

[0038] Pharmaceutical composition, biological preparation or detection kit

[0039] In another aspect, the present invention provides a pharmaceutical composition, a biological preparation or a detection kit, wherein the pharmaceutical composition comprises the antibody as described above, the engineered host cell as described above and / or the immunoconjugate as described above;

[0040] The biological preparation comprises an antibody as described above, an engineered host cell as described above, and / or an immunoconjugate as described above;

[0041] The detection kit comprises the above-mentioned antibody and / or the above-mentioned detection reagent.

[0042] Furthermore, the pharmaceutical composition or biological preparation also includes one or more pharmaceutically or physiologically acceptable carriers and / or excipients.

[0043] In some embodiments, the detection kit further comprises a container, instructions for use, a buffer, etc. In other embodiments, the detection kit further comprises a lysis medium for dissolving the sample to be tested, universal reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0044] In certain embodiments, the pharmaceutical composition or pharmaceutical preparation described in the present invention may also contain other therapeutic agents that can be used to treat and / or prevent (or assist in the treatment and / or prevention) of diseases expressing B7H3. The active ingredients in the pharmaceutical composition or pharmaceutical preparation (such as the aforementioned antibodies, the aforementioned engineered host cells, the aforementioned immunoconjugates) and / or other therapeutic agents may be administered simultaneously, separately or sequentially.

[0045] In certain embodiments, 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 liquids are 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., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0046] Application in the preparation of drugs or testing products

[0047] In another aspect, the present invention provides any of the following applications:

[0048] (1) Use of the above-described antibody, the above-described chimeric antigen receptor, the above-described nucleic acid molecule, the above-described vector, the above-described engineered host cell and / or the above-described immunoconjugate in the preparation of a drug for treating and / or preventing a disease expressing B7H3;

[0049] (2) Use of the above-mentioned antibody and / or the above-mentioned detection reagent in the preparation of a detection product for detecting B7H3 protein;

[0050] (3) Use of the above-described antibody, the above-described chimeric antigen receptor, the above-described nucleic acid molecule and / or the above-described vector in the preparation of an engineered host cell for treating and / or preventing a disease expressing B7H3;

[0051] (4) Use of the above-described antibody, the above-described nucleic acid molecule and / or the above-described vector in the preparation of an immunoconjugate for treating and / or preventing a disease expressing B7H3;

[0052] (5) Use of the above-described antibody, the above-described chimeric antigen receptor, the above-described nucleic acid molecule, the above-described vector, the above-described engineered host cell and / or the above-described immunoconjugate in the preparation of a biological agent for treating and / or preventing a disease expressing B7H3;

[0053] Optionally, the drug for treating and / or preventing a disease expressing B7H3 is an antibody drug or an immune cell drug;

[0054] Optionally, the disease expressing B7H3 is ovarian cancer, prostate cancer, liver cancer, lung cancer, head and neck cancer, kidney cancer, cervical cancer, breast cancer, colorectal cancer, pituitary tumor, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, skin cancer, myeloma, B cell lymphoma, T cell lymphoma, bladder cancer, osteosarcoma, glioma or myeloid leukemia.

[0055] Production method, preparation method and detection method

[0056] In another aspect, the present invention provides any of the following methods:

[0057] (1) A method for producing the antibody as described above, the method comprising: culturing the engineered host cell as described above to obtain a culture containing the antibody, and isolating and purifying the antibody as described above from the culture;

[0058] (2) A method for preparing the engineered host cell as described above, the method comprising: introducing the nucleic acid molecule as described above or the vector as described above into a host cell to obtain the engineered host cell as described above;

[0059] (3) A method for detecting B7H3 protein for non-diagnostic purposes, the method comprising: contacting a sample to be tested with the antibody and / or the detection reagent as described above, and detecting the presence of an antibody-antigen complex.

[0060] The present invention also provides a method for diagnosing whether a subject suffers from a disease expressing B7H3, the method comprising: contacting a test sample derived from the subject with the antibody, the detection reagent and / or the detection kit as described above of the present invention, detecting the formation of a complex between the antibody or the detection reagent and B7H3 or detecting the amount of the complex, wherein the formation of the complex indicates the presence of B7H3 or cells expressing B7H3.

[0061] In certain embodiments, the sample to be tested refers to a collection (such as fluid, cell or tissue) separated from a subject, as well as a fluid, cell or tissue present in a subject. Exemplary samples to be tested are biological fluids, such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues or secretory organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage fluid, synovial fluid, liquid solutions in contact with a subject or biological source, such as culture medium (including conditioned culture medium), lavage fluid, etc., tissue biopsy specimens, fine needle aspiration, surgically removed tissues, organ cultures or cell cultures.

[0062] In certain embodiments, the subject includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. In certain embodiments, the subject is a human.

[0063] The present invention also provides a method for treating and / or preventing a disease expressing B7H3, the method comprising: administering to a subject in need thereof a therapeutically and / or preventively effective amount of the antibody, engineered host cell, immunoconjugate, pharmaceutical composition and / or pharmaceutical preparation of the present invention as described above.

[0064] In certain embodiments, the mode of administration 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, 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 administration time schemes are contemplated herein, including but not limited to: single or multiple administrations at multiple time points, push administration and pulse infusion.

[0065] In the present invention, the disease expressing B7H3 refers to any disease or condition related to the expression of B7H3, including but not limited to: ovarian cancer, prostate cancer, liver cancer, lung cancer, head and neck cancer, kidney cancer, cervical cancer, breast cancer, colorectal cancer, pituitary tumor, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, skin cancer, myeloma, B cell lymphoma, T cell lymphoma, bladder cancer, osteosarcoma, glioma or myeloid leukemia. As long as the disease or condition is related to the expression of B7H3, it is within the protection scope of the present invention.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] (1) The present invention provides an antibody AHF23046 targeting B7H3, wherein the amino acid sequences of HCDR1-3 corresponding to the antibody AHF23046 are shown in SEQ ID NOs: 1-3, respectively, and the amino acid sequences of LCDR1-3 are shown in SEQ ID NOs: 6-8, respectively. The antibody AHF23046 has strong specificity and high affinity for B7H3 and can specifically bind to B7H3-positive cell lines, thereby providing a new idea for the development of effective immunotherapy drugs targeting B7H3.

[0068] (2) The present invention provides a chimeric antigen receptor (CAR) comprising the antibody AHF23046, and also provides a CAR-T cell targeting B7H3 prepared based on the CAR. The CAR-T cell can specifically recognize B7H3-positive tumor cell lines and has a high specific killing efficiency against B7H3-positive cell lines, providing a potential treatment option for diseases expressing B7H3 in the art, which has important application prospects and clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 :Flow cytometry detection of B7H3 expression in prostate cancer cells, where A: prostate cancer cell DU145, B: prostate cancer cell DU145 B7H3 KO ;

[0070] Figure 2 : Flow cytometry technology to detect antibody specificity, where AB figure: prostate cancer cell DU145, CD figure: prostate cancer cell DU145 B7H3 KO ;

[0071] Figure 3 : Antibody-mediated NK cell ADCC killing effect;

[0072] Figure 4 : Preparation and detection of fully human B7H3 CAR-T cells, including: Figure A: CAR-T positive rate flow cytometry, Figure B: CAR-T positive rate statistical chart;

[0073] Figure 5 : Tumor sphere experiment detects the killing effect of CAR-T cells prepared based on the fully human B7H3 antibody described in the present application on tumor cells, wherein Figure A: ovarian cancer cell SKOV3, Figure B: prostate cancer cell DU145, Figure C: liver cancer cell A549;

[0074] Figure 6 : RTCA detection of B7H3 by CAR-T prepared based on the fully human B7H3 antibody described in this application +The killing effect of tumor cells, including: Figure A: ovarian cancer cell SKOV3, Figure B: prostate cancer cell DU145, Figure C: liver cancer cell A549, Figure D: lung cancer cell H1299. DETAILED DESCRIPTION

[0075] The present invention is further described below in conjunction with specific embodiments. The specific embodiments are only used to explain the present invention and cannot be construed as limiting the present invention. It will be appreciated 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 purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer. The reagents, biomaterials, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0076] Example 1 Monoclonal antibody AHF23046 targeting B7H3

[0077] The present invention constructs a fully human monoclonal antibody AHF23046 that specifically targets B7H3. The sequence information of the AHF23046 antibody is shown in Table 1 below. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 9. In addition, this example demonstrates through experiments that the AHF23046 antibody has a good affinity for B7H3.

[0078] Table 1 Sequence of AHF23046 antibody

[0079]

[0080] Example 2 Flow cytometry to detect the specificity of antibody AHF23046

[0081] 1. Detection of B7H3 expression in prostate cancer cells by flow cytometry

[0082] In this example, flow cytometry was used to detect the expression of B7H3 in prostate cancer cells. B7H3 KO After co-incubation of cells, flow cytometry was performed. The specific experimental steps are as follows:

[0083] (1) Collect prostate cancer DU145 and DU145 B7H3 KO Cells were incubated with antibody AHF23046 (1:200) at 4°C for 30 min;

[0084] (2) Wash with PBS, centrifuge and discard the supernatant, resuspend the cell pellet in 300 μL PBS, and filter into a flow tube;

[0085] (3) Streaming on-machine testing.

[0086] 2. Detection of antibody specificity by flow cytometry

[0087] In order to determine whether the B7H3 antibody AHF23046 used can specifically bind to the B7H3 antigen expressed by cells, in this example, ① antibody AHF23046; ② commercial B7H3 antibody (Enoblituzumab MGA271) were diluted in a concentration gradient and then mixed with DU145 and DU145 B7H3 KO After co-incubation of cells, flow cytometry was performed. The specific experimental steps are as follows:

[0088] (1) Select cells DU145 and DU145 B7H3 KO , 2-5×10 5 / Sample;

[0089] (2) Antibody selection: ① Antibody AHF23046; ② Commercialized B7H3 antibody (Enoblituzumab MGA271);

[0090] (3) Antibody gradient: 0, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL (antibody diluent: PBS containing 2% FBS), incubate at 37°C for 30 min, add 1 mL PBS for washing, centrifuge, and discard the supernatant as much as possible;

[0091] (4) Secondary antibody: AF647-IgG (1:500), incubate at 4°C for 30 min, add 1 mL PBS to wash, centrifuge, discard the supernatant, add 300 μL PBS containing 0.5% BSA to resuspend, and detect on flow cytometer (collect 10,000 target cell populations).

[0092] 3. Experimental results

[0093] Flow cytometry analysis of prostate cancer cells DU145 and DU145 B7H3 KO The corresponding results of B7H3 expression are as follows Figure 1 The results showed that B7H3 was highly expressed in DU145 cells. B7H3 KO Not expressed in.

[0094] The results of antibody specificity detection by flow cytometry are as follows Figure 2As shown, the results show that, compared with DU145 B7H3 KO In comparison, antibody AHF23046 showed strong specific binding ability to DU145 expressing B7H3, and was superior to commercial antibodies.

[0095] Example 3: The fully human B7H3 antibody AHF23046 can kill DU145 cells through ADCC effect

[0096] 1. Experimental methods

[0097] This example uses RTCA experiment to detect the killing effect of DU145 cells after being treated with antibody AHF23046 and added with NK cells. The specific experimental steps are as follows:

[0098] (1) Prostate cancer DU145 cells were collected and seeded into RTCA plates at 20,000 cells per well;

[0099] (2) When the cell index of tumor cells grows to about 1, add 50 μL of antibodies (final concentration 5 μg / mL), including ① antibody AHF23046; ② commercial B7H3 antibody (Enoblituzumab MGA271); ③ IgG1 isotype control (InVivoMAb MOPC-21); add 50 μL of culture medium to the remaining wells and incubate with antibodies at 37°C for 1 h;

[0100] (3) Add 50 μL of NK cell suspension at a 1:1 effector-target ratio and continue culturing and observation for 2-3 days;

[0101] (4) The RTCA instrument automatically records the cell growth index. After 2-3 days, RTCA is stopped and a curve reflecting the cell growth index is automatically generated.

[0102] 2. Experimental results

[0103] The results are as follows Figure 3 As shown, the results showed that when the effector-target ratio was 1:1, in DU145 cells, compared with the NK cell alone group, the NK cell combined with the antibody significantly enhanced the NK cell's tumor-killing effect. And the effect was basically equivalent to that of commercial antibodies. The above results show that the fully human B7H3 antibody AHF23046 can kill DU145 cells through the ADCC effect.

[0104] Example 4 Preparation and detection of fully human B7H3 CAR-T cells

[0105] 1. Experimental methods

[0106] The CAR expression plasmid was constructed using the sequence of the above-mentioned antibody AHF23046, and the virus was packaged. The collected retroviral fluid was used to infect T cells to prepare CAR-T, named AHF23046.CAR-T. After 48 hours of infection, the cells were taken for flow staining to detect the positive rate. Among them, the coding sequence of the fully human chimeric antigen receptor (CAR) targeting B7H3 is shown in Table 2 below. The specific experimental steps are as follows:

[0107] (1) The constructed plasmid was packaged for retrovirus, and the viral supernatant was harvested at 48 h and 72 h, respectively. After centrifugation and filtration, it was divided into 1.5 mL EP tubes and stored at -80 °C;

[0108] (2) After T cell activation, 0.5×10 6 / mL was divided into 1.5mL EP tubes, 1mL virus solution (AHF23046.CAR-T) was added to resuspend the cells, and then slowly added to the 24-well plate coated with Retronectin in advance, and centrifuged at 30℃, 1500g for 2h. After centrifugation, the virus solution was discarded, complete culture medium was added, and the plate was placed in an incubator for culture;

[0109] (3) After 48 hours, take 0.2×10 6 / mL cells were used for CAR positive rate detection, PE-protein L and FITC-CD3 flow cytometry antibodies were diluted to a final concentration of 1μg / μL, added to the cells at 50μL / sample, incubated at 4℃ in the dark for 30min, washed with 1mL PBS, centrifuged and discarded the supernatant;

[0110] (4) Resuspend the cell pellet in 300 μL PBS, filter it into a flow cytometer, and detect it on a flow cytometer.

[0111] 2. Experimental results

[0112] The results are as follows Figure 4 As shown, the results showed that compared with uninfected T cells, the CAR-T positive rate was stable between 70% and 90%. The above results indicate that this example successfully prepared CAR-T cells targeting B7H3.

[0113] Table 2 Coding sequences of fully human chimeric antigen receptors (CARs) targeting B7H3

[0114]

[0115]

[0116] Example 5 Tumor sphere experiment to detect the killing effect of CAR-T cells prepared based on fully human B7H3 antibody (AHF23046) on tumor cells

[0117] 1. Experimental methods

[0118] In order to verify the killing ability of the CAR-T cells on B7H3-positive tumors, this example used a 3D tumor experiment to observe the killing effect of AHF23046.CAR-T cells on ovarian cancer SKOV3, prostate cancer DU145, and lung cancer A549 cells that highly expressed B7H3 and had GFP fluorescence. The specific experimental steps are as follows:

[0119] (1) Add 0.15 g agarose to 10 mL DMEM and autoclave at 121°C for 20 min.

[0120] (2) Add 50 μL of hot agarose solution to each well of a 96-well plate and cool to room temperature;

[0121] (3) 200 μL of GFP-fluorescent tumor cell suspension was inoculated into a 96-well plate covered with agarose at a cell density of 1.2×10 4 / mL, and further cultured for 3-4 days;

[0122] (4) Observe the size of the tumor sphere under a microscope. When the diameter is about 300 μm, take a photo and record it;

[0123] (5) Collect effector cells (AHF23046.CAR-T), stain the CAR-T effector cells with DIL cell membrane dye (1:500), stain for 20 min at room temperature in the dark, add 10 mL of PBS to wash twice, and discard the supernatant;

[0124] (6) Resuspend the pellet using the culture medium of the effector cells, count the pellet, and add 50 μL of the effector cell suspension according to the initial cell:target ratio of 1:1;

[0125] (7) Take photos and record observations under a fluorescence microscope.

[0126] 2. Experimental results

[0127] The results are as follows Figure 5 As shown, the results showed that compared with T cells, the presence of CAR significantly improved the killing ability of T cells against solid tumors (ovarian cancer, prostate cancer, liver cancer). The above results show that CAR-T cells prepared based on the fully human B7H3 antibody (AHF23046) have a good killing effect on tumor cells.

[0128] Example 6 RTCA detection of CAR-T cells against B7H3 + Tumor cell killing effect

[0129] 1. Experimental methods

[0130] In order to verify the killing ability of CAR-T cells against B7H3-positive tumors, RTCA was used to detect the killing effect of AHF23046.CAR-T cells on tumor cells with high expression of B7H3 (ovarian cancer SKOV3, prostate cancer DU145, lung cancer A549 and lung cancer H1299 cells with high expression of B7H3) at an effector-target ratio of 2:1. The specific experimental steps are as follows:

[0131] (1) Collect tumor cells and inoculate 20,000 cells per well in RTCA plates;

[0132] (2) When the cell index of tumor cell growth reaches about 1, add the corresponding AHF23046.CAR-T cells;

[0133] (3) The RTCA instrument automatically records the cell growth index;

[0134] (4) After 2-3 days, stop RTCA and automatically generate a curve reflecting the cell growth index.

[0135] 2. Experimental results

[0136] The results are as follows Figure 6 As shown, the results showed that compared with T cells, the presence of CAR significantly improved the ability of T cells to kill solid tumors. The above results once again proved that CAR-T cells prepared based on the fully human B7H3 antibody (AHF23046) have a good killing effect on tumor cells.

Claims

1. An antibody that specifically recognizes B7H3, characterized in that: The 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:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; Optionally, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4 or has at least 85% homology to SEQ ID NO:4; Optionally, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9 or has at least 85% homology to SEQ ID NO:

9.

2. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the antibody of claim 1; Optionally, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, and a co-stimulatory signaling domain; Optionally, the chimeric antigen receptor further comprises any one or more of a signal peptide, a self-cleaving peptide, a safety switch, an immunomodulatory molecule or a cytokine; Optionally, the transmembrane domain is selected from the transmembrane domain of any one of the following molecules: CD8, CD28, CD34, CD3ε, PD-1, IgG1, 4-1BB, IgG4, OX40, IL-2 receptor, IL-7 receptor; Optionally, the intracellular signaling domain is selected from the intracellular signaling domain of any one of the following molecules: CD3ζ, CD3γ, CD3δ, FcRγ, FcRβ, TCRζ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD66d, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12; Optionally, the hinge region is selected from the hinge region of any one of the following molecules: CD8, 4-1BB, OX40, CD3ε, IgG1, CD28, CD34, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the costimulatory signaling domain is selected from the costimulatory signaling domains of any one of the following molecules: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8α, CD8β, BAFFR, HVEM, CD40, OX40, CD30, TIM1, CD2, CD7, CD226; Optionally, the signal peptide is selected from the signal peptides of any one of the following molecules: α chain and β chain of T cell receptor, CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD16, CD22, CD33, CD134, CD137, CD154, GITR, GM-CSF; Optionally, the self-cleaving peptide is selected from any one of the following self-cleaving peptides: T2A, P2A, E2A, F2A; Optionally, the safety switch is selected from any one of the following safety switches: tEGFR, iCaspase-9, RQR8; Optionally, the immunomodulatory molecule or cytokine is selected from any one of the following immunomodulatory molecules or cytokines: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, IL-15, IL-18, IL-21, LEC, OX40L; Optionally, the chimeric antigen receptor is obtained by sequentially connecting a signal peptide, a heavy chain variable region of the antibody according to claim 1, a linker, a light chain variable region of the antibody according to claim 1, a CD8 hinge region and a transmembrane domain, a CD28 co-stimulatory signal domain, a CD3ζ intracellular signaling domain, a T2A self-cleaving peptide, and a tEGFR safety switch in series; Optionally, the Linker is (G4S)3; Optionally, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 11; Optionally, the amino acid sequence of the Linker is shown in SEQ ID NO: 15; Optionally, the amino acid sequence of the CD8 hinge region and transmembrane domain is as shown in SEQ ID NO: 19; Optionally, the amino acid sequence of the CD28 costimulatory signal domain is as shown in SEQ ID NO: 21; Optionally, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO: 23; Optionally, the amino acid sequence of the T2A self-cleaving peptide is as shown in SEQ ID NO: 25; Optionally, the amino acid sequence of the tEGFR safety switch is as shown in SEQ ID NO: 27; Optionally, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:

29.

3. A nucleic acid molecule, characterized in that The sequence of the nucleic acid molecule is the coding sequence of the antibody according to claim 1 or the chimeric antigen receptor according to claim 2; Optionally, in the antibody of claim 1, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 14; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO: 18; Optionally, in the chimeric antigen receptor of claim 2, the nucleotide sequence encoding the signal peptide is shown in SEQ ID NO: 12; the nucleotide sequence encoding the linker is shown in SEQ ID NO: 16; the nucleotide sequence encoding the CD8 hinge region and transmembrane domain is shown in SEQ ID NO: 20; the nucleotide sequence encoding the CD28 co-stimulatory signal domain is shown in SEQ ID NO: 22; the nucleotide sequence encoding the CD3ζ intracellular signaling domain is shown in SEQ ID NO: 24; the nucleotide sequence encoding the T2A self-cleaving peptide is shown in SEQ ID NO: 26; the nucleotide sequence encoding the tEGFR safety switch is shown in SEQ ID NO: 28; Optionally, the nucleotide sequence encoding the chimeric antigen receptor of claim 2 is shown as SEQ ID NO:

30.

4. A vector comprising the nucleic acid molecule according to claim 3, characterized in that: The vector is a cloning vector or an expression vector; Optionally, the vector is a DNA vector, an RNA vector, a plasmid or a virus-derived vector; Optionally, the virus-derived vector is a retroviral vector, an adenoviral vector, an adeno-associated viral vector or a lentiviral vector.

5. An engineered host cell, characterized in that The engineered host cell comprises the vector of claim 4; Optionally, the host cell is Escherichia coli, yeast cell, insect cell or mammalian cell; Optionally, the host cell is an immune cell; Optionally, the immune cells are T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof.

6. An immunoconjugate, characterized in that: The immunoconjugate comprises the antibody of claim 1 and a conjugate conjugated thereto; Optionally, the conjugate is 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 any combination thereof.

7. A detection reagent, characterized in that: The detection reagent comprises the antibody of claim 1 and a diagnostic agent coupled thereto; Optionally, the diagnostic agent is a radionuclide, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme or a photosensitive diagnostic agent.

8. A pharmaceutical composition, biological preparation or detection kit, characterized in that: The pharmaceutical composition comprises the antibody of claim 1, the engineered host cell of claim 5 and / or the immunoconjugate of claim 6; The biological agent comprises the antibody of claim 1, the engineered host cell of claim 5 and / or the immunoconjugate of claim 6; The detection kit comprises the antibody according to claim 1 and / or the detection reagent according to claim 7.

9. Any of the following applications: (1) Use of the antibody of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the engineered host cell of claim 5 and / or the immunoconjugate of claim 6 in the preparation of a medicament for treating and / or preventing a disease expressing B7H3; (2) Use of the antibody according to claim 1 and / or the detection reagent according to claim 7 in the preparation of a detection product for detecting B7H3 protein; (3) Use of the antibody of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3 and / or the vector of claim 4 in the preparation of an engineered host cell for treating and / or preventing a disease expressing B7H3; (4) Use of the antibody according to claim 1, the nucleic acid molecule according to claim 3 and / or the vector according to claim 4 in the preparation of an immunoconjugate for treating and / or preventing a disease expressing B7H3; (5) Use of the antibody of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the engineered host cell of claim 5 and / or the immunoconjugate of claim 6 in the preparation of a biological agent for treating and / or preventing a disease expressing B7H3; Optionally, the drug for treating and / or preventing a disease expressing B7H3 is an antibody drug or an immune cell drug; Optionally, the disease expressing B7H3 is ovarian cancer, prostate cancer, liver cancer, lung cancer, head and neck cancer, kidney cancer, cervical cancer, breast cancer, colorectal cancer, pituitary tumor, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, skin cancer, myeloma, B cell lymphoma, T cell lymphoma, bladder cancer, osteosarcoma, glioma or myeloid leukemia.

10. Any of the following methods: (1) A method for producing the antibody according to claim 1, comprising: Cultivating the engineered host cell of claim 5 to obtain a culture containing the antibody, and isolating and purifying the antibody of claim 1 from the culture; (2) A method for preparing the engineered host cell according to claim 5, the method comprising: introducing the nucleic acid molecule according to claim 3 or the vector according to claim 4 into a host cell to obtain the engineered host cell according to claim 5; (3) A method for detecting B7H3 protein for non-diagnostic purposes, the method comprising: contacting a sample to be tested with the antibody of claim 1 and / or the detection reagent of claim 7, and detecting the presence of an antibody-antigen complex.

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