B7H3-targeting CAR-gamma delta T as well as related products and application of B7H3-targeting CAR-gamma delta T

Abstract

The invention discloses CAR-gamma delta T targeting B7H3 and related products and application of the CAR-gamma delta T. CAR in the CAR-gamma delta T targeting B7H3 comprises a humanized monoclonal antibody AHF23050 specifically targeting B7H3. Experiments prove that the CAR-gamma delta T targeting B7H3 has a remarkable killing effect on tumor cells with high expression of B7H3, and the CAR-gamma delta T targeting B7H3 has the advantages that the CAR-gamma delta T targeting B7H3 can be used for killing tumor cells with high expression of B7H3; the compound can be used for effective treatment of related diseases with high expression of B7H3.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a CAR-γδT targeting B7H3 and related products and applications thereof. Background Art

[0002] γδT cells are a special type of immune cell that has both acquired and innate immune response characteristics. γδT cells co-express TCR-type γ and δ chains and NKG 2D (one of the main functional receptors expressed on NK cells). Therefore, γδT cells can simulate the functions of both T cells and NK cells. Unlike traditional αβT cells whose TCRs are composed of α and β chains, αβT cells recognize antigen-derived peptides presented by MHC molecules (called human leukocyte antigen HLA in humans), while γδT cells are able to recognize and kill pathogens independently of MHC. At the same time, γδT cells can release various cytokines to activate other types of immune cells, such as NK cells, macrophages, and CD8 + Cytotoxic lymphocytes. Although the research on CAR-γδT cells is still in its early stages, it has been considered a potential tumor immunotherapy strategy that can provide better treatment options for cancer patients.

[0003] B7H3 (also known as CD276) is a human cell surface molecule and one of the new members of the B7 family. It plays an important role in immunoregulation and anti-tumor immunity. It is a member of the B7 family, including B7-1 (CD80), B7-2 (CD86), PD-L1 (CD274), PD-L2 (CD273), etc. The role of B7H3 is similar to that of other immune checkpoint molecules, and it can play a key role in T cell activation and immune response. B7H3 is usually highly expressed in tumors, inflammation, and autoimmune diseases. It can regulate the activity and proliferation of T cells by binding to T cell ligands (such as CD28 or CTLA-4). At the same time, B7H3 can also directly participate in regulating the growth and metastasis of tumor cells, affecting the immune response and escape mechanism of the tumor microenvironment. Therefore, B7H3 has become a hot target in the field of tumor treatment. At present, a variety of B7H3 antibodies, CAR-T and other biological agents are under development to enhance the effect of tumor immunotherapy.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a CAR-γδT targeting B7H3 and related products and applications in the art. The CAR in the CAR-γδT targeting B7H3 comprises a humanized monoclonal antibody AHF23050 that specifically targets B7H3. The present invention proves through experiments that the CAR-γδT targeting B7H3 has a significant killing effect on tumor cells that highly express B7H3.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] CAR-γδ T cells targeting B7H3

[0008] In one aspect, the present invention provides a CAR-γδT cell targeting B7H3, wherein the CAR-γδT cell is a γδT cell expressing a chimeric antigen receptor targeting B7H3;

[0009] The chimeric antigen receptor targeting B7H3 comprises a monoclonal antibody targeting B7H3; the monoclonal antibody targeting B7H3 comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1-3, and the light chain variable region comprises LCDR1-3; the amino acid sequences of HCDR1-3 are respectively shown in SEQ ID NO:1-3; the amino acid sequences of LCDR1-3 are respectively shown in SEQ ID NO:6-8.

[0010] Further, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4 or has at least 80% 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 80% homology with SEQ ID NO:9.

[0011] Further, the chimeric antigen receptor targeting B7H3 also comprises a transmembrane domain, an intracellular signaling domain, a hinge region, and a co-stimulatory signaling domain; optionally, the chimeric antigen receptor targeting B7H3 also comprises any one or more of a signal peptide, a self-cleaving peptide, a safety switch, an immunomodulatory molecule, or a cytokine. Further, the transmembrane domain is selected from the transmembrane domain of any one of the following molecules: CD8, CD28, 4-1BB, CD34, CD3ε, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-7 receptor, IL-11 receptor; optionally, the intracellular signaling domain is selected from the intracellular signaling domain of any one of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, Fcε RI, DAP10, DAP12, CD66d; Optionally, the hinge region is selected from the hinge region of any one of the following molecules: CD8, CD28, CD34, 4-1BB, OX40, CD3ε, IgG1, 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: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, 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, CD16, CD22, CD33, CD4, CD5, CD8, CD9, CD28, CD37, CD45, CD64, CD80, CD86, 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.

[0012] Furthermore, the chimeric antigen receptor targeting B7H3 is obtained by sequentially connecting a signal peptide, a heavy chain variable region of the monoclonal antibody targeting B7H3, a linker, a light chain variable region of the monoclonal antibody targeting B7H3, 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;

[0013] 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 transmembrane domain is as shown in SEQ ID NO:19; optionally, the amino acid sequence of the CD28 co-stimulatory 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 targeting B7H3 is as shown in SEQ ID NO:29.

[0014] In some embodiments, the sequences of HCDR1-3 and LCDR1-3 of the heavy chain variable region and light chain variable region of the monoclonal antibody targeting B7H3 described in the present invention can be obtained based on the above heavy chain variable region and light chain variable region sequences according to the Kabat, IMGT, Chothia, AbM or Contact numbering system definitions, and the CDR sequences defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems are also within the scope of protection of the present invention.

[0015] In the present invention, the chimeric antigen receptor and CAR refer to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. CAR is a core component of a chimeric antigen receptor immune cell (CAR-immune cell), which may include an antigen (e.g., a tumor-specific antigen and / or a tumor-associated antigen) binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. CAR is an engineered receptor that can implant any specific receptor into an immune effector cell. In CAR, a scFv fragment of a monoclonal antibody that specifically recognizes a tumor antigen can be implanted into an immune cell. For example, a retroviral vector can be used to introduce a nucleic acid encoding CAR into an immune cell. In this way, a large number of cancer-specific immune cells can be generated for adoptive cell transfer.

[0016] CAR-γδT cell population targeting B7H3

[0017] On the other hand, the present invention provides a CAR-γδT cell population targeting B7H3, wherein the CAR-γδT cell population comprises the CAR-γδT cells of the present invention as described above; optionally, the CAR-γδT cell population further comprises γδT cells that do not express the chimeric antigen receptor targeting B7H3.

[0018] Pharmaceutical composition

[0019] In another aspect, the present invention provides a pharmaceutical composition comprising the CAR-γδT cell of the present invention as described above or the CAR-γδT cell population of the present invention as described above.

[0020] In some embodiments, the pharmaceutical composition described in the present invention may also contain other drugs that can be used to treat and / or prevent (adjuvant treatment and / or prevention) diseases related to high expression of B7H3. Exemplarily, the other drugs include but are not limited to: alkylating agents, anti-metabolites, anti-tumor antibiotics, mitotic inhibitors, chromatin function inhibitors, anti-angiogenic agents, antiestrogens, antiandrogens, and immunomodulators.

[0021] In some embodiments, the alkylating agent includes chlorambucil, chlorambucil, melphalan, propioperazine, chlorambucil, cyclophosphamide, hexamethylmelamine, carmustine, cisplatin, oxaliplatin, carboplatin; the antimetabolite includes methotrexate, 5-fluorouracil, fluoxetine, capecitabine, cytarabine, fludarabine, 6-mercaptopurine, 2-chlorodeoxyadenosine, 5-azacytidine, pentostatin; the anti-tumor antibiotic includes daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin C, valrubicin, mitoxantrone hydrochloride, bleomycin, dactinomycin; the mitotic inhibitor includes docetaxel, vinblastine, paclitaxel, vincristine, vinblastine, vinorelbine; the chromatin function inhibitor includes irinotecan , etoposad, topotecan, etoposad phosphate, teniposide; the anti-angiogenic agents include prinomastat, tanomastat, ilomastat, aprotinin, marimastat, batimastat, CGS-27023A, bromochloropiperaquinone, neivastat, thalidomide; the antiestrogens include toremifene, raloxifene, tamoxifen, anastrozole, letrozole, droloxifene, odoxifene, exemestane; the anti-androgens include nilutamide, bicalutamide, spironolactone, flutamide, finasteride, cyproterone acetate, cimetidine; the immunomodulators include interleukins, tumor necrosis factor, interferon, mushroom polysaccharides, sizose, roquimek, pidomod, methoxypolyethylene glycol succinamide adenosine deaminase, and thymosin preparations.

[0022] In some embodiments, the pharmaceutical composition of the present invention further comprises one or more pharmaceutically or physiologically acceptable carriers, diluents or excipient combinations. Such combinations may include: buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives.

[0023] In some embodiments, suitable pharmaceutically acceptable carriers, diluents or excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995), and these substances are used to help the stability of the formulation or to help improve the activity or its biological effectiveness or to produce an acceptable taste or smell in the case of oral administration as needed. The preparations that can be used in such pharmaceutical compositions can be in the form of the original compound itself, or optionally in the form of a pharmaceutically acceptable salt thereof. The pharmaceutical composition thus prepared can be administered in any appropriate manner known to those skilled in the art as needed. When using the pharmaceutical composition, a safe and effective amount of the drug of the present application is administered to a person.

[0024] Biologics

[0025] In another aspect, the present invention provides a biological preparation, which comprises the pharmaceutical composition of the present invention as described above; optionally, the dosage form of the biological preparation is an injection, a lyophilized agent, an oral agent, a cream, a gel, drops or a patch.

[0026] In some embodiments, the present invention has no particular restrictions on the specific dosage form of the biological agent, and any biological agent (not limited to dosage form) containing the CAR-γδT cells of the present invention as described above or the CAR-γδT cell population of the present invention as described above as an active ingredient is within the scope of protection of the present invention.

[0027] In some embodiments, the pharmaceutical compositions or biological preparations provided by the present invention can be formulated into pharmaceutical compositions or biological preparations for topical, intravenous, oral, enteral and / or parenteral administration according to actual needs.

[0028] In some embodiments, the suitable dosage of the pharmaceutical composition or biological preparation provided by the present invention can be prescribed in a variety of ways according to factors such as the formulation method, administration method, patient's age, weight, gender, morbidity, diet, administration time, administration route, excretion rate and reaction sensitivity. A skilled physician can usually easily determine the prescription and the dosage that is effective for the desired treatment or prevention.

[0029] Method for preparing the CAR-γδT cell

[0030] On the other hand, the present invention provides a method for preparing a CAR-γδT cell as described above, the method comprising introducing a nucleic acid molecule encoding a chimeric antigen receptor targeting B7H3 as described above of the present invention or an expression vector comprising a nucleic acid molecule encoding a chimeric antigen receptor targeting B7H3 as described above of the present invention into a γδT cell.

[0031] In some embodiments, the specific type of the expression vector is not limited, for example, plasmid, phagemid, phage derivative, animal virus and clay, can be changed according to the host cell to be introduced. Viral vector technology is well known in the art and described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals, and can be introduced by standard techniques, for example, infection, transfection, transduction or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE polydextrose, electroporation, protoplast fusion, liposome transfection, cell microinjection and viral vectors. Viruses that can be used as expression vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. In addition, in order to evaluate the expression of target protein (for example, chimeric antigen receptor), the vector introduced into the cell may also include any one or two of selectable marker genes or reporter genes, so as to identify and select expression cells from the cell group seeking to be transfected or infected by viral vectors.

[0032] In some embodiments, the nucleic acid molecule or expression vector can be introduced into γδT cells by various suitable methods, not limited to the methods listed in this application, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, non-viral vector-mediated transfection (such as liposomes) or viral vector-mediated transfection (such as lentiviral infection, retroviral infection, adenoviral infection), and other physical, chemical or biological means for transfer into cells, such as transposon technology, CRISPR-Cas9 and other technologies.

[0033] Methods for treating and / or preventing diseases related to high expression of B7H3

[0034] On the other hand, the present invention also provides a method for treating and / or preventing diseases related to high expression of B7H3, the method comprising the following steps: administering a therapeutically and / or preventively effective amount of the CAR-γδT cells, CAR-γδT cell populations, pharmaceutical compositions or biological preparations of the present invention as described above to a subject in need thereof.

[0035] In some embodiments, the subject is a subject suffering from a disease related to high expression of B7H3 or a subject at risk of a disease related to high expression of B7H3, and the administration method includes parenteral, intrapulmonary and intranasal, and if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any appropriate route known to those skilled in the art, for example, by injection, such as intravenous or subcutaneous injection, which depends in part on whether the administration is short-term or long-term. A variety of dosing schedules are contemplated herein, including but not limited to: single or multiple administrations at multiple time points, push administration and pulse infusion.

[0036] In some embodiments, the subject includes, but is not limited to, humans, non-human animals, wherein non-human animals include rabbits, rats, mice, monkeys or other lower primates. In a preferred embodiment, the subject is a human.

[0037] Application of the CAR-γδT cell or CAR-γδT cell population

[0038] In another aspect, the present invention provides the following application:

[0039] (1) Use of the CAR-γδT cells as described above in the present invention or the CAR-γδT cell population as described above in the present invention in the preparation of a drug for treating and / or preventing diseases related to high expression of B7H3;

[0040] (2) Use of the CAR-γδT cells as described above in the present invention or the CAR-γδT cell population as described above in the present invention in the preparation of a pharmaceutical composition for treating and / or preventing diseases related to high expression of B7H3;

[0041] (3) Use of the CAR-γδT cells, the CAR-γδT cell populations or the pharmaceutical compositions described above in the preparation of biological agents for treating and / or preventing diseases related to high expression of B7H3;

[0042] Optionally, the disease related to high expression of B7H3 is prostate cancer, breast cancer, ovarian cancer, liver cancer, pancreatic cancer, lymphoma, lung cancer, kidney cancer, breast cancer, colorectal cancer, esophageal cancer, oral cancer, gastric cancer, endometrial cancer, bladder cancer, osteosarcoma, myeloma, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, glioma or myeloid leukemia.

[0043] In some embodiments, the disease associated with high expression of B7H3 refers to any disease or condition involving high expression of B7H3. Examples of the disease associated with high expression of B7H3 include, but are not limited to, prostate cancer, breast cancer, ovarian cancer, liver cancer, pancreatic cancer, lymphoma, lung cancer, kidney cancer, breast cancer, colorectal cancer, esophageal cancer, oral cancer, gastric cancer, endometrial cancer, bladder cancer, osteosarcoma, myeloma, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, neurosis, Glioma, myeloid leukemia (acute myeloid leukemia, chronic myeloid leukemia), non-Hodgkin's lymphoma, Hodgkin's lymphoma, testicular cancer, pituitary cancer, skin cancer, bone cancer, B-cell lymphoma, T-cell lymphoma, thymoma, anal cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, angiosarcoma, hemangioendothelioma, thyroid cancer, soft tissue sarcoma, gastrointestinal cancer, intrahepatic bile duct cancer, joint cancer, nasal cancer, and any other diseases related to high B7H3 expression that are now known or discovered later. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : The results of γδT cell purity induced by two schemes on different days, among which, Figure A: flow cytometry representative figure, Figure B: statistical figure;

[0045] Figure 2 :Difference in cell proliferation fold between the two schemes;

[0046] Figure 3 : Result diagram of γδT cell memory phenotype induced by two schemes at different days, among which, Figure A: representative flow cytometry diagram, Figure B: CD62L + γδT cell proportion statistics;

[0047] Figure 4 : Preparation of B7H3.CAR-γδT cell CAR positive rate detection result diagram, wherein, Figure A: flow cytometry representative diagram, Figure B: statistical diagram;

[0048] Figure 5 : The killing effect of B7H3.CAR-γδT cells on prostate cancer cells, wherein, Figure A: The killing effect of B7H3.CAR-γδT cells on prostate cancer cells PC-3 and DU-145 was detected by RTCA technology, and Figure B: The killing rate statistics;

[0049] Figure 6 : Results of cytokine release ability of B7H3.CAR-γδT cells when co-cultured with prostate cancer cells PC-3 and DU-145;

[0050] Figure 7 :The killing effect of B7H3.CAR-γδT cells on breast cancer cells;

[0051] Figure 8 :The killing effect of B7H3.CAR-γδT cells on ovarian cancer cells. DETAILED DESCRIPTION

[0052] 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.

[0053] Example 1 Monoclonal antibody AHF23050 targeting B7H3

[0054] The present invention constructs a fully human monoclonal antibody AHF23050 that specifically targets B7H3. The sequence information of the AHF23050 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 AHF23050 antibody has a good affinity for B7H3.

[0055] Table 1 Sequence of AHF23050 antibody

[0056]

[0057] Example 2 Preparation of fully human B7H3.CAR-γδT targeting B7H3

[0058] 1. Experimental methods

[0059] (1) Preparation of γδT

[0060] 1) Isolation of PBMCs: Collect peripheral blood from donors, dilute whole blood with an equal amount of saline, add lymphocyte separation solution and diluted blood into a centrifuge tube at a ratio of 1:2, centrifuge at 2000 rpm for 20 minutes, collect buffy coat cells, wash twice with saline, and centrifuge at 1500 rpm / 8 minutes to obtain peripheral blood mononuclear cells (PBMCs);

[0061] 2) Induction of γδT cells: Resuspend PBMCs in lymphocyte culture medium and adjust the concentration to 1×10 6 / mL, added with 100 μM zoledronic acid (ZOL), stimulated at 37°C for 4 h, then washed, resuspended for culture and supplemented with cytokine 200 U / mL IL-2 or IL-2 combined with 5 ng / mL IL-21, seeded the cells in 24-well plates, placed in a 37°C, 5% CO2 incubator, observed the cell status daily, changed half the medium every other day, and expanded and cultured for 14-21 days.

[0062] 3) For the preparation of B7H3.CAR-γδT cells: the cells were collected and resuspended in lymphocyte culture medium containing IL-7 and IL-15, inoculated on CD3Ab and CD28Ab pre-coated plates, and infected with viruses after activation for 48 hours.

[0063] (2) Construction of CAR expression vector

[0064] 1) synthesizing a scFv coding sequence targeting human B7H3, wherein the scFv comprises a heavy chain VH and a light chain VL (as described in Example 1), which are connected by a G4S short peptide;

[0065] 2) The retroviral vector MSCV and the synthesized scFv targeting human B7H3 were double-digested with Nco I and Mlu I to recover the fragments, and the recovered target fragments were ligated with T4 ligase and then transformed into Stbl3 competent cells;

[0066] 3) Pick a single clone for plasmid extraction, identify it by enzyme digestion and send it for sequencing to confirm that the correct plasmid is B7H3.CAR;

[0067] In the above construction method, the coding sequence of the fully human chimeric antigen receptor targeting B7H3 is shown in Table 2 below.

[0068] Table 2 Coding sequences of fully human chimeric antigen receptors (CARs) targeting B7H3

[0069]

[0070]

[0071]

[0072] (3) Retroviral packaging

[0073] Mix 6 μg of the shuttle plasmid MSCV-B7H3.CAR containing the CAR structure and 4 μg of the auxiliary plasmid pCL-Ampho in 300 μL of opti-MEM medium, add 30 μL of Genejuice transfection reagent dropwise to another 300 μL of opti-MEM medium, flick to mix, let stand at room temperature for 5 minutes, add the mixture containing the transfection reagent dropwise to the plasmid mixture, shake to mix, let stand at room temperature for 15 minutes, then add PEI and the plasmid mixture dropwise to the pre-laid 293T cell culture dish, shake to mix, collect the supernatant after 48-72 hours, filter through a 0.45 μm filter, and store in an ultra-low temperature refrigerator for later use.

[0074] (4) Virus infection of γδT cells

[0075] The B7H3.CAR virus solution was added with 10μM HEPES and 6-8μg / mL polybrene, mixed, and the activated γδT cells were resuspended with the virus solution, and then added to a 24-well plate pre-coated with RetroNectin. After centrifugation at 1500g and 30°C for 2 hours, the supernatant was removed and X-Vivo medium containing 5% fetal bovine serum, 200U / mL IL-2, 10ng / mL IL-7 and 5ng / mL IL-15 was added. The culture was continued for expansion to obtain fully human B7H3.CAR-γδT cells targeting B7H3.

[0076] (5) Detection of CAR expression efficiency

[0077] 48-72 hours after virus infection, take 2×10 5 For cell staining, first add 1 μg / mL B7H3-Fc protein and incubate at 4°C for 30 minutes. Wash the cells and then add AF647-anti-human IgG antibody and incubate at 4°C in the dark for 30 minutes. Wash the cells and finally add PerCP-CD3 and PE-γδTCR antibodies and incubate at 4°C in the dark for 30 minutes. After washing, detect on the machine.

[0078] 2. Experimental results

[0079] The γδT cells obtained by the scheme provided by the present invention were detected by flow cytometry. Figure 1 AB results showed that both induction schemes could successfully induce high-purity γδT cells. Figure 2 The results showed that compared with the ZOL+IL-2 induction regimen, the cell proliferation rate in the IL-21 group was higher. Figure 3AB results showed that compared with the ZOL+IL-2 induction regimen, the IL-21 group had higher proportions of γδT cell TCM and TEM on days 7, 10, and 14 of induction. Figure 4 The results showed that B-H3.CAR-γδT cells were successfully prepared, and the CAR transfection efficiency was about 80%.

[0080] Example 3B7H3. Verification of the ability of CAR-γδT cells to kill tumor cells in vitro

[0081] 1. Experimental methods

[0082] First, add 50 μL of DMEM complete medium to the E-Plate detection plate of the xCELLigence cell function analyzer to measure the background impedance value; collect prostate cancer cells PC-3 and DU-145 in the logarithmic phase and adjust the cell suspension concentration to 1×10 5 / mL, add 100μL of cell suspension to the E-Plate detection plate, let it stand at room temperature for 30 minutes and then place it on the detection table; when the target cell proliferation is in the plateau phase, 50μL of effector cells without transfection of γδT and B7H3.CAR-γδT are added to the experimental wells according to the effector-target ratio of 2 / 1, 1 / 1, and 1 / 2. At the same time, tumor cells alone are set as the control group, and the cell-mediated cell killing effect curve is observed in real time.

[0083] 2. Experimental results

[0084] The experimental results are shown in Figure 5 AB, the results show that B7H3.CAR-γδT cells can effectively and specifically kill B7H3-positive prostate cancer cells. The same method confirmed that B7H3.CAR-γδT cells can effectively and specifically kill breast cancer and ovarian cancer cells. The corresponding experimental results are shown in Figure 7 , Figure 8 .

[0085] Example 4 Detection of the ability of fully human B7H3.CAR-γδT cells to release cytokines targeting B7H3

[0086] 1. Experimental methods

[0087] ELISA kit was used to detect cytokine release ability: 2×10 5 B7H3.CAR-γδT cells and 2×10 5 Prostate cells PC-3 and DU-145 were mixed at an effector-target ratio of 1:1 and added to a 24-well plate for co-incubation. The wells were set up in duplicate and the culture supernatant was collected after 24 hours. The contents of IL-2, Granzyme B, IFN-γ, and TNF-α were detected using ELISA kits.

[0088] 2. Experimental results

[0089] The experimental results are shown in Figure 6 The results showed that compared with untransfected γδT, the B7H3.CAR-γδT cells prepared by the present invention can secrete higher levels of cytokines IL-2, Granzyme B, IFN-γ, and TNF-α. The above results once again prove that the B7H3.CAR-γδT cells can efficiently and specifically kill B7H3-positive tumor cell lines.

Claims

1. A CAR-γδT cell targeting B7H3, characterized in that: The CAR-γδT cells are γδT cells expressing a chimeric antigen receptor targeting B7H3; The chimeric antigen receptor targeting B7H3 comprises a monoclonal antibody targeting B7H3; The monoclonal antibody targeting B7H3 comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises HCDR1-3, and the light chain variable region comprises LCDR1-3; The amino acid sequences of HCDR1-3 are shown in SEQ ID NOs: 1-3 respectively; The amino acid sequences of LCDR1-3 are shown in SEQ ID NOs: 6-8, respectively.

2. The CAR-γδT cell according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4 or has at least 80% 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 80% homology to SEQ ID NO:

9.

3. The CAR-γδT cell according to claim 1, characterized in that The chimeric antigen receptor targeting B7H3 further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, and a co-stimulatory signaling domain; Optionally, the chimeric antigen receptor targeting B7H3 further comprises any one or more of a signal peptide, a self-cleaving peptide, a safety switch, an immunomodulatory molecule or a cytokine.

4. The CAR-γδT cell according to claim 3, characterized in that The transmembrane domain is selected from any one of the following transmembrane domains: CD8, CD28, 4-1BB, CD34, CD3ε, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the intracellular signaling domain is selected from the intracellular signaling domain of any one of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d; Optionally, the hinge region is selected from the hinge region of any one of the following molecules: CD8, CD28, CD34, 4-1BB, OX40, CD3ε, IgG1, 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, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, 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, CD16, CD22, CD33, CD4, CD5, CD8, CD9, CD28, CD37, CD45, CD64, CD80, CD86, 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.

5. The CAR-γδT cell according to claim 1, characterized in that The chimeric antigen receptor targeting B7H3 is obtained by sequentially connecting a signal peptide, a heavy chain variable region of the monoclonal antibody targeting B7H3, a linker, a light chain variable region of the monoclonal antibody targeting B7H3, a CD8 hinge region and a transmembrane domain, a CD28 costimulatory signal domain, a CD3ζ intracellular signal transduction domain, a T2A self-cleaving peptide, and a tEGFR safety switch; 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 targeting B7H3 is as shown in SEQ ID NO:

29.

6. A CAR-γδT cell population targeting B7H3, characterized in that: The CAR-γδT cell population comprises the CAR-γδT cells of any one of claims 1 to 5; Optionally, the CAR-γδT cell population further comprises γδT cells that do not express the chimeric antigen receptor targeting B7H3.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the CAR-γδT cell of any one of claims 1 to 5 or the CAR-γδT cell population of claim 6.

8. A biological agent, characterized in that: The biological preparation comprises the pharmaceutical composition of claim 7; Optionally, the biological preparation is in the form of an injection, a lyophilized preparation, an oral preparation, a cream, a gel, drops or a patch.

9. A method for preparing the CAR-γδT cell according to any one of claims 1 to 5, characterized in that: The method comprises introducing into γδT cells a nucleic acid molecule encoding a chimeric antigen receptor targeting B7H3 as claimed in any one of claims 1 to 5 or an expression vector comprising a nucleic acid molecule encoding a chimeric antigen receptor targeting B7H3 as claimed in any one of claims 1 to 5.

10. Any of the following applications: (1) Use of the CAR-γδT cell according to any one of claims 1 to 5 or the CAR-γδT cell population according to claim 6 in the preparation of a drug for treating and / or preventing a disease related to high expression of B7H3; (2) Use of the CAR-γδT cell according to any one of claims 1 to 5 or the CAR-γδT cell population according to claim 6 in the preparation of a pharmaceutical composition for treating and / or preventing a disease related to high expression of B7H3; (3) Use of the CAR-γδT cell according to any one of claims 1 to 5, the CAR-γδT cell population according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a biological agent for treating and / or preventing a disease related to high expression of B7H3; Optionally, the disease associated with high B7H3 expression is prostate cancer, breast cancer, ovarian cancer, liver cancer, pancreatic cancer, lymphoma, lung cancer, kidney cancer, colorectal cancer, esophageal cancer, oral cancer, gastric cancer, endometrial cancer, bladder cancer, osteosarcoma, myeloma, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, glioma or myeloid leukemia.

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