Anti-B7H3 fully human monoclonal antibody and derivative product and application thereof

Abstract

The invention discloses an anti-B7H3 fully human monoclonal antibody and a derivative product and application thereof, the derivative product comprises a B7H3-targeted chimeric antigen receptor, the chimeric antigen receptor comprises an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory signal domain and an intracellular signal transduction domain, the antigen binding structural domain is an anti-B7H3 full human monoclonal antibody, the antibody has an ADCC effect, the chimeric antigen receptor provided by the invention has a specific targeting effect on B7H3 positive tumor cells, the killing effect of T cells expressing the chimeric antigen receptor is remarkable, and the chimeric antigen receptor has important significance in the field of tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically, relates to a fully human monoclonal antibody AHF23037 against B7H3, and more specifically, relates to a fully human monoclonal antibody AHF23037 against B7H3 and its derivative products and applications. Background Art

[0002] B7H3 (B7 homolog 3protein, also known as CD276) belongs to the B7 / CD28 immunoglobulin superfamily. It is a protein with a unidirectional transmembrane structure, composed of 316 amino acids, with a signal peptide at the amino terminus, including the extracellular immunoglobulin-like variable region (IgV), constant region (IgC), transmembrane region and 45 amino acids of cytoplasmic region. It is expressed in monocytes, dendritic cells and activated T cells. B7H3 is expressed at a low level in normal tissues, but is overexpressed in many cancers, especially non-small cell lung cancer, renal cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer and pancreatic cancer. B7H3 plays an important role in tumor immune escape by inhibiting T cells, and has been shown to promote tumor progression and metastasis of cancer cells. In addition, B7H3 is expressed not only in cancer cells, but also in tumors or surrounding blood vessels. B7H3 is also associated with autoimmune diseases. In rheumatism and other autoimmune diseases, B7H3 plays an important role in the interaction between fibroblast-like synoviocytes and activated T cells, and B7H3 acts as a co-stimulatory factor when macrophages release cytokines, so it is related to the occurrence of sepsis. In summary, due to its widespread expression in a variety of tumors, B7H3 has become a potential target for cancer immunotherapy, but there are currently few reports on immunotherapy targeting B7H3. Summary of the invention

[0003] In view of the above-mentioned technical problems existing in the prior art, the present invention provides a fully human anti-B7H3 monoclonal antibody AHF23037 and its derivative products and applications. The derivative products include a chimeric antigen receptor targeting B7H3. The chimeric antigen receptor adopts the anti-B7H3 monoclonal antibody AHF23037 with specific binding ability to human B7H3 as the antigen binding domain, which has important application prospects in the field of tumor treatment.

[0004] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0005] In a first aspect, the present invention provides a fully human antibody targeting B7H3, the antibody comprising a heavy chain variable region VH and a light chain variable region VL;

[0006] The VH comprises three complementarity determining regions HCDR1, HCDR2, and HCDR3;

[0007] The VL comprises three complementary determining regions LCDR1, LCDR2, and LCDR3;

[0008] The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively;

[0009] The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 respectively.

[0010] Optionally, the amino acid sequence of the VH is shown in SEQ ID NO: 4;

[0011] Optionally, the amino acid sequence of the VL is as shown in SEQ ID NO:9.

[0012] In some embodiments, the amino acid sequence of the VH is as shown in SEQ ID NO: 4 or an amino acid sequence having at least 90% homology to SEQ ID NO: 4, and the amino acid sequence of the VL is as shown in SEQ ID NO: 9 or an amino acid sequence having at least 90% homology to SEQ ID NO: 9. In some embodiments, the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the VH and VL of the antibody described in the present invention can be obtained based on the above VH and VL 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 system definitions are also included in the protection scope of the present invention.

[0013] In some embodiments, the fully human antibody described in the present invention is an antibody produced by the human immune system, with high specificity and affinity, and the amino acid sequences that make up the antibody are all from humans. This antibody, which is completely encoded by human antibody genes, has low immunogenicity and good clinical effects. Fully human antibodies are antibodies obtained from the human immune system by biotechnology. Compared with traditional animal-derived antibodies, fully human antibodies have lower immunogenicity and can be better used in human treatment. Fully human antibodies are transformed by genetic engineering technology and can be efficiently expressed in human cells for the prevention and treatment of diseases. Fully human antibodies not only reduce the risk of human immune response during the research and development process, but also show better efficacy and safety in practical applications.

[0014] In a second aspect, the present invention provides a chimeric antigen receptor targeting B7H3, wherein the chimeric antigen receptor comprises the antibody described in the first aspect of the present invention;

[0015] 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, 4-1BB, CD34, CD3ε, PD-1, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor; optionally, the intracellular signaling domain is selected from any one of the following The intracellular signal transduction domain of the following molecules: CD3ζ, CD3δ, CD3ε, FcRγ, CD3γ, FcRβ, CD8, CD21, TCRζ, CD4, CD5, CD22, CD79; optionally, the hinge region is selected from the hinge region of any one of the following molecules: CD8, IgG4, 4-1BB, PD-1, CD34, OX40, CD28, CD3ε, IgG1, IL-2 receptor, IL-7 receptor, IL-11 receptor; optionally, the costimulatory signal domain is selected from the costimulatory signal domain of any one of the following molecules: The signal peptide is selected from the signal peptides of any one of the following molecules: CD3ζ, CD3ε, CD16, CD22, α chain and β chain of T cell receptor, CD33, CD37, CD28, CD45, CD64, CD80; Optionally, the ... The cleavage peptide is selected from any one of the following self-cleavage peptides: T2A, E2A, P2A, 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;

[0016] Optionally, the chimeric antigen receptor is a signal peptide, a heavy chain variable region of the antibody described in the first aspect of the present invention, a linker, a light chain variable region of the antibody described in the first aspect of the present invention, 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.

[0017] In a third aspect, the present invention provides a nucleic acid molecule encoding the antibody of the first aspect of the present invention or the chimeric antigen receptor of the second aspect of the present invention;

[0018] Optionally, the nucleotide sequence of the heavy chain variable region of the antibody according to the first aspect of the present invention is as shown in SEQ ID NO: 14; Optionally, the nucleotide sequence of the light chain variable region of the antibody according to the first aspect of the present invention is as shown in SEQ ID NO: 18; Optionally, the nucleotide sequence of the signal peptide in the chimeric antigen receptor according to the second aspect of the present invention is as shown in SEQ ID NO: 12; Optionally, the nucleotide sequence of Linker in the chimeric antigen receptor according to the second aspect of the present invention is as shown in SEQ ID NO: 16; Optionally, the nucleotide sequence of the CD8 hinge region and transmembrane domain in the chimeric antigen receptor according to the second aspect of the present invention is as shown in SEQ ID NO: 20; Optionally, the nucleotide sequence of the CD28 co-stimulatory signaling domain in the chimeric antigen receptor according to the second aspect of the present invention is as shown in SEQ ID NO: 22; Optionally, the nucleotide sequence of the CD3ζ intracellular signaling domain in the chimeric antigen receptor according to the second aspect of the present invention is as shown in SEQ ID NO: 24; Optionally, the nucleotide sequence of the T2A self-cleaving peptide in the chimeric antigen receptor according to the second aspect of the present invention is as shown in SEQ ID NO:26; optionally, the nucleotide sequence of the tEGFR safety switch in the chimeric antigen receptor described in the second aspect of the present invention is shown in SEQ ID NO:28; optionally, the nucleotide sequence of the chimeric antigen receptor described in the second aspect of the present invention is shown in SEQ ID NO:30.

[0019] In some embodiments, those skilled in the art can change the combination category and sequence of the signal peptide, hinge region and transmembrane region, co-stimulatory signal domain and intracellular signal transduction domain according to actual conditions or needs. Regardless of the form of the change, as long as the chimeric antigen receptor has the CDR sequence or heavy chain variable region sequence of the heavy chain variable region of the above-mentioned fully human antibody of the present invention, and / or the CDR sequence or light chain variable region sequence of the light chain variable region of the above-mentioned fully human antibody, it falls within the protection scope of the present invention.

[0020] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the third aspect of the present invention;

[0021] Optionally, the vector is a DNA vector, an RNA vector, a plasmid, a viral-derived vector, a transposon vector or a CRISPR / Cas9 vector; optionally, the viral-derived vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a retroviral vector.

[0022] In some embodiments, nucleic acid molecules provided by the invention can be cloned into many types of vectors. For example, this nucleic acid molecule can be cloned into such a vector, which includes but is not limited to: plasmid, phagemid, phage derivative, animal virus and cosmid. Specific vector of interest comprises expression vector, replication vector, probe generation vector and sequencing vector.

[0023] In a fifth aspect, the present invention provides a genetically engineered host cell, wherein the genetically engineered host cell comprises the expression vector according to the fourth aspect of the present invention;

[0024] Optionally, the host cell is an immune cell; optionally, the immune cell is a T cell, a NK cell, an iNKT cell, a B cell, a CTL cell, a monocyte, a myeloid cell, a dendritic cell, a macrophage or any combination thereof.

[0025] In some embodiments, the host cell is a eukaryotic cell, and the eukaryotic cell includes a mammalian cell, an insect cell, a plant cell, a yeast cell; alternatively, the host cell is an immune cell; alternatively, the immune cell includes a T cell, a NK cell, an iNKT cell, a B cell, a CTL cell, a monocyte, a myeloid cell, a dendritic cell, a macrophage, or any combination thereof; alternatively, the immune cell is a T cell. In some embodiments, the immune cell includes, but is not limited to, for example, leukocytes, lymphocytes (T cells, B cells, natural killer (NK) cells) derived from hematopoietic stem cells (HSC) produced in the bone marrow, and cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells) derived from bone marrow. In a specific embodiment of the present invention, the genetically engineered host cell is a T cell expressing the chimeric antigen receptor described in the second aspect of the present invention.

[0026] In a sixth aspect, the present invention provides a host cell population, wherein the host cell population comprises the genetically engineered host cell according to the fifth aspect of the present invention;

[0027] Optionally, the host cell population further comprises host cells that do not comprise the expression vector described in the fourth aspect of the present invention.

[0028] In a seventh aspect, the present invention provides a pharmaceutical composition or a biological preparation, which comprises the antibody described in the first aspect of the present invention, the genetically engineered host cell described in the fifth aspect of the present invention, or the host cell population described in the sixth aspect of the present invention.

[0029] In some embodiments, the pharmaceutical composition or biological preparation further comprises a pharmaceutically acceptable carrier, diluent and / or excipient; optionally, the pharmaceutical composition or biological preparation includes a monospecific antibody drug containing the antibody described in the first aspect of the invention, a bispecific antibody drug containing the antibody described in the first aspect of the invention, an antibody-drug conjugate containing the antibody described in the first aspect of the invention, a CAR-T cell drug containing the genetically engineered host cell described in the fifth aspect of the invention and / or the host cell population described in the sixth aspect of the invention.

[0030] In some embodiments, the pharmaceutical composition or biologic described herein may comprise a buffer such as neutral buffered saline, sulfate buffered saline, and the like; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant; and a preservative.

[0031] In some embodiments, the pharmaceutical composition or biological preparation of the present invention further comprises 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, 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. The pharmaceutical composition or biological preparation of the present invention may be formulated for oral, intravenous, topical, enteral and / or parenteral administration.

[0032] In an eighth aspect, the present invention provides a detection reagent or a detection kit, wherein the detection reagent or the detection kit comprises the antibody described in the first aspect of the present invention.

[0033] In some embodiments, the detection reagent further comprises a diagnostic reagent coupled to the antibody; optionally, the diagnostic reagent comprises a radionuclide, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, or a photosensitive diagnostic agent; the present invention has no particular limitation on the specific type of the diagnostic reagent, and any conventional diagnostic reagent known to those skilled in the art is within the scope of protection of the present invention.

[0034] In some embodiments, the detection kit further comprises a lysis medium for dissolving the sample to be tested, universal reagents and buffer required for detection; more preferably, the kit further comprises instructions for use.

[0035] In a ninth aspect, the present invention provides any of the following methods:

[0036] (1) A method for detecting B7H3 in a sample for non-diagnostic and non-therapeutic purposes, the method comprising: contacting the sample with the antibody of the first aspect of the present invention, and detecting the formation of a complex between the antibody and B7H3;

[0037] In some embodiments, the antibody is an antibody labeled with a detectable marker; optionally, the detectable marker includes a fluorescent pigment, avidin, a paramagnetic atom, or a radioisotope; optionally, the fluorescent pigment is fluorescein, rhodamine, Texas red, phycoerythrin, phycocyanin, allophycocyanin, or peridinin-chlorophyll protein; optionally, the avidin is biotin, ovalbumin, streptavidin, yolk avidin, or avidin-like; optionally, the radioisotope is radioactive iodine, radioactive cesium, radioactive iridium, or radioactive cobalt;

[0038] (2) A method for producing the antibody of the first aspect of the present invention, the method comprising: culturing the genetically engineered host cell of the fifth aspect of the present invention or the host cell population of the sixth aspect of the present invention, and isolating the antibody of the first aspect of the present invention from the culture;

[0039] (3) A method for preparing the genetically engineered host cell of the fifth aspect of the present invention or the host cell population of the sixth aspect of the present invention, the method comprising: providing a host cell to be modified, introducing the nucleic acid molecule of the third aspect of the present invention or the expression vector of the fourth aspect of the present invention into the host cell, to obtain the genetically engineered host cell or host cell population;

[0040] In some embodiments, the method of introduction includes calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, liposome-mediated transfection, lentiviral infection, retroviral infection, adenoviral infection, transposon technology, CRISPR-Cas9 technology;

[0041] (4) A method for stimulating a mammalian target cell population or tissue to produce an immune response, the method comprising: administering to the mammal an effective amount of the genetically engineered host cell of the fifth aspect of the present invention or the host cell population of the sixth aspect of the present invention;

[0042] (5) A method for specifically inhibiting B7H3 activity in vitro, the method comprising: introducing the nucleic acid molecule described in the third aspect of the present invention or the expression vector described in the fourth aspect of the present invention into a cell of an organism, and inhibiting the activity of B7H3 by expressing the antibody described in the first aspect of the present invention.

[0043] In addition, the present invention also provides a method for treating a B7H3-positive related disease or delaying its progression in a subject in need thereof, the method comprising: administering to a subject in need thereof an effective amount of the antibody described in the first aspect of the present invention, the genetically engineered host cell described in the fifth aspect of the present invention, the host cell population described in the sixth aspect of the present invention, the pharmaceutical composition or biological preparation described in the seventh aspect of the present invention, in some embodiments, the subject comprises a mammal, and in a specific embodiment of the present invention, the subject is preferably a human.

[0044] In addition, the present invention also provides a method for diagnosing whether a subject suffers from a B7H3-positive related disease, the method comprising: contacting a test sample derived from a subject with the antibody described in the first aspect of the present invention, and detecting whether a complex is formed between the antibody and B7H3 in the test sample. In some embodiments, the test sample is a biological sample derived from a subject, including but not limited to a blood sample, a tissue sample, etc. In some embodiments, the subject includes a mammal, the mammal is selected from: a human, an animal, a non-human primate, a dog, a cat, a sheep, a mouse, a horse or a cow, and in some embodiments, the subject is preferably a human.

[0045] In a tenth aspect, the present invention provides any of the following applications:

[0046] (1) Use of the antibody of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the expression vector of the fourth aspect of the present invention, the genetically engineered host cell of the fifth aspect of the present invention and / or the host cell population of the sixth aspect of the present invention in the preparation of a medicament for treating and / or preventing a B7H3-positive related disease;

[0047] (2) Use of the antibody of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the expression vector of the fourth aspect of the present invention, the genetically engineered host cell of the fifth aspect of the present invention and / or the host cell population of the sixth aspect of the present invention in the preparation of a biological agent for treating and / or preventing a B7H3-positive related disease;

[0048] (3) Use of the antibody of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention and / or the expression vector of the fourth aspect of the present invention in the preparation of a genetically engineered host cell for treating and / or preventing a B7H3-positive related disease;

[0049] (4) Use of the antibody of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention and / or the expression vector of the fourth aspect of the present invention in preparing a host cell population for treating and / or preventing B7H3-positive related diseases;

[0050] (5) Use of the antibody described in the first aspect of the present invention and / or the nucleic acid molecule described in the third aspect of the present invention in the preparation of a detection reagent for detecting B7H3 protein or an antigenic fragment thereof;

[0051] (6) Use of the antibody according to the first aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention and / or the detection reagent or detection kit according to the eighth aspect of the present invention in the preparation of a product for detecting B7H3 protein or an antigenic fragment thereof;

[0052] (7) Use of the antibody according to the first aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention and / or the detection reagent or detection kit according to the eighth aspect of the present invention in the preparation of a product for diagnosis and / or auxiliary diagnosis of B7H3-positive related diseases;

[0053] Optionally, the B7H3-positive related disease is ovarian cancer, prostate cancer, liver cancer, lung cancer, kidney cancer, breast cancer, glioblastoma, colorectal cancer, urothelial carcinoma, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, bladder cancer, autoimmune disease, osteosarcoma or myeloid leukemia.

[0054] In some embodiments, the B7H3-positive related diseases described in the present invention are not limited to the above diseases, and any disease and / or condition related to the expression of B7H3 is within the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] 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 ;

[0056] Figure 2 : Flow cytometry technology to detect antibody specificity, where AB figure: prostate cancer cell DU145, CD figure: prostate cancer cell DU145 B7H3 KO ;

[0057] Figure 3 : Antibody-mediated NK cell ADCC killing effect;

[0058] 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;

[0059] 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;

[0060] 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

[0061] 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.

[0062] Example 1 Monoclonal antibody AHF23037 targeting B7H3

[0063] The present invention constructs a fully human monoclonal antibody AHF23037 that specifically targets B7H3. The sequence information of the AHF23037 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 AHF23037 antibody has a good affinity for B7H3.

[0064] Table 1 Sequence of AHF23037 antibody

[0065]

[0066]

[0067] Example 2 Flow cytometry to detect the specificity of antibody AHF23037

[0068] 1. Detection of B7H3 expression in prostate cancer cells by flow cytometry

[0069] 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:

[0070] (1) Collect prostate cancer DU145 and DU145 B7H3 KO Cells were incubated with antibody AHF23037 (1:200) at 4°C for 30 min;

[0071] (2) Wash with PBS, centrifuge and discard the supernatant, resuspend the cell pellet in 300 μL PBS, and filter into a flow tube;

[0072] (3) Streaming on-machine testing.

[0073] 2. Detection of antibody specificity by flow cytometry

[0074] In order to determine whether the B7H3 antibody AHF23037 used can specifically bind to the B7H3 antigen expressed by cells, in this example, ① antibody AHF23037; 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:

[0075] (1) Select cells DU145 and DU145 B7H3 KO , 2-5×10 5 / Sample;

[0076] (2) Antibody selection: ① Antibody AHF23037; ② Commercialized B7H3 antibody (Enoblituzumab MGA271);

[0077] (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;

[0078] (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).

[0079] 3. Experimental results

[0080] 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.

[0081] The results of antibody specificity detection by flow cytometry are as follows Figure 2 As shown, the results show that, compared with DU145 B7H3 KO In comparison, antibody AHF23037 showed strong specific binding ability to DU145 expressing B7H3, and was superior to commercial antibodies.

[0082] Example 3: The fully human B7H3 antibody AHF23037 can kill DU145 cells through ADCC effect

[0083] 1. Experimental methods

[0084] This example uses RTCA experiment to detect the killing effect of DU145 cells after being treated with antibody AHF23037 and added with NK cells. The specific experimental steps are as follows:

[0085] (1) Prostate cancer DU145 cells were collected and seeded into RTCA plates at 20,000 cells per well;

[0086] (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 AHF23037; 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;

[0087] (3) Add 50 μL of NK cell suspension at a 1:1 effector-target ratio and continue culturing and observation for 2-3 days;

[0088] (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.

[0089] 2. Experimental results

[0090] 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 AHF23037 can kill DU145 cells through the ADCC effect.

[0091] Example 4 Preparation and detection of fully human B7H3 CAR-T cells

[0092] 1. Experimental methods

[0093] The CAR expression plasmid was constructed using the sequence of the above-mentioned antibody AHF23037, and viral packaging was performed. The collected retroviral fluid was used to infect T cells to prepare CAR-T, named AHF23037.CAR-T. After 48 hours of infection, the cells were taken for flow cytometry 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:

[0094] (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;

[0095] (2) After T cell activation, 0.5×10 6 / mL was divided into 1.5mL EP tubes, 1mL virus solution (AHF23037.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;

[0096] (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;

[0097] (4) Resuspend the cell pellet in 300 μL PBS, filter it into a flow cytometer, and detect it on a flow cytometer.

[0098] 2. Experimental results

[0099] 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.

[0100] Table 2 Coding sequences of fully human chimeric antigen receptors (CARs) targeting B7H3

[0101]

[0102]

[0103] Example 5 Tumor sphere experiment to detect the killing effect of CAR-T cells prepared based on fully human B7H3 antibody (AHF23037) on tumor cells

[0104] 1. Experimental methods

[0105] 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 AHF23037.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:

[0106] (1) Add 0.15 g agarose to 10 mL DMEM and autoclave at 121°C for 20 min.

[0107] (2) Add 50 μL of hot agarose solution to each well of a 96-well plate and cool to room temperature;

[0108] (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;

[0109] (4) Observe the size of the tumor sphere under a microscope. When the diameter is about 300 μm, take a photo and record it;

[0110] (5) Collect effector cells (AHF23037.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;

[0111] (6) Resuspend the pellet in 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;

[0112] (7) Take photos and record observations under a fluorescence microscope.

[0113] 2. Experimental results

[0114] 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 (AHF23037) have a good killing effect on tumor cells.

[0115] Example 6 RTCA detection of CAR-T cells against B7H3 + Tumor cell killing effect

[0116] 1. Experimental methods

[0117] In order to verify the killing ability of CAR-T cells against B7H3-positive tumors, RTCA was used to detect the killing effect of AHF23037.CAR-T cells on tumor cells that highly express B7H3 (ovarian cancer SKOV3, prostate cancer DU145, lung cancer A549 and lung cancer H1299 cells that highly express B7H3) at an effector-target ratio of 2:1. The specific experimental steps are as follows:

[0118] (1) Collect tumor cells and inoculate 20,000 cells per well in RTCA plates;

[0119] (2) When the cell index of tumor cell growth reaches about 1, add the corresponding AHF23037.CAR-T cells;

[0120] (3) The RTCA instrument automatically records the cell growth index;

[0121] (4) After 2-3 days, stop RTCA and automatically generate a curve reflecting the cell growth index.

[0122] 2. Experimental results

[0123] 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 (AHF23037) have a good killing effect on tumor cells.

Claims

1. A fully human antibody targeting B7H3, characterized in that: The antibody comprises a heavy chain variable region VH and a light chain variable region VL; The VH comprises three complementarity determining regions HCDR1, HCDR2, and HCDR3; The VL comprises three complementary determining regions LCDR1, LCDR2, and LCDR3; The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively; The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 respectively. Optionally, the amino acid sequence of the VH is shown in SEQ ID NO: 4; Optionally, the amino acid sequence of the VL is as shown in SEQ ID NO:

9.

2. A chimeric antigen receptor targeting B7H3, 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 domains of any one of the following molecules: CD8, 4-1BB, CD34, CD3ε, PD-1, CD28, 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, CD3δ, CD3ε, FcRγ, CD3γ, FcRβ, CD8, CD21, TCRζ, CD4, CD5, CD22, CD79; Optionally, the hinge region is selected from the hinge region of any one of the following molecules: CD8, IgG4, 4-1BB, PD-1, CD34, OX40, CD28, CD3ε, IgG1, 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, CD30, CD40, TIM1, SLAMF7, NKp30, NKp46; Optionally, the signal peptide is selected from the signal peptides of any one of the following molecules: CD3ζ, CD3ε, CD16, CD22, α chain and β chain of T cell receptor, CD33, CD37, CD28, CD45, CD64, CD80; Optionally, the self-cleaving peptide is selected from any one of the following self-cleaving peptides: T2A, E2A, P2A, 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 encoding the antibody of claim 1 or the chimeric antigen receptor of claim 2; Optionally, the nucleotide sequence of the heavy chain variable region of the antibody of claim 1 is shown in SEQ ID NO: 14; Optionally, the nucleotide sequence of the light chain variable region of the antibody of claim 1 is shown in SEQ ID NO: 18; Optionally, the nucleotide sequence of the signal peptide in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO: 12; Optionally, the nucleotide sequence of Linker in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO: 16; Optionally, the nucleotide sequence of the CD8 hinge region and transmembrane domain in the chimeric antigen receptor of claim 2 is as shown in SEQ ID NO: 20; Optionally, the nucleotide sequence of the CD28 co-stimulatory signal domain in the chimeric antigen receptor of claim 2 is as shown in SEQ ID NO: 22; Optionally, the nucleotide sequence of the CD3ζ intracellular signaling domain in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO: 24; Optionally, the nucleotide sequence of the T2A self-cleaving peptide in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO: 26; Optionally, the nucleotide sequence of the tEGFR safety switch in the chimeric antigen receptor of claim 2 is shown in SEQ ID NO: 28; Optionally, the nucleotide sequence of the chimeric antigen receptor according to claim 2 is shown in SEQ ID NO:

30.

4. An expression vector comprising the nucleic acid molecule of claim 3; Optionally, the vector is a DNA vector, an RNA vector, a plasmid, a virus-derived vector, a transposon vector or a CRISPR / Cas9 vector; Optionally, the virus-derived vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a retroviral vector.

5. A genetically engineered host cell, characterized in that: The genetically engineered host cell comprises the expression vector of claim 4; Optionally, the host cell is an immune cell; Optionally, the immune cells are T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages or any combination thereof.

6. A host cell population, characterized in that The host cell population comprises the genetically engineered host cell of claim 5; Optionally, the host cell population further comprises host cells that do not comprise the expression vector of claim 4.

7. A pharmaceutical composition or biological preparation, characterized in that: The pharmaceutical composition or biological preparation comprises the antibody of claim 1, the genetically engineered host cell of claim 5 or the host cell population of claim 6.

8. A detection reagent or a detection kit, characterized in that: The detection reagent or detection kit comprises the antibody according to claim 1.

9. Any of the following methods: (1) A method for detecting B7H3 in a sample for non-diagnosis and non-therapeutic purposes, characterized in that: The method comprises: contacting a sample to be tested with the antibody of claim 1, and detecting formation of a complex between the antibody and B7H3; (2) A method for producing the antibody of claim 1, characterized in that the method comprises: culturing the genetically engineered host cell of claim 5 or the host cell population of claim 6, and isolating the antibody of claim 1 from the culture; (3) A method for preparing the genetically engineered host cell of claim 5 or the host cell population of claim 6, characterized in that the method comprises: providing a host cell to be modified, introducing the nucleic acid molecule of claim 3 or the expression vector of claim 4 into the host cell, to obtain the genetically engineered host cell or host cell population; (4) A method for stimulating a target cell population or tissue of a mammal to produce an immune response, characterized in that the method comprises: administering an effective amount of the genetically engineered host cell of claim 5 or the host cell population of claim 6 to the mammal; (5) A method for specifically inhibiting B7H3 activity in vitro, characterized in that the method comprises: introducing the nucleic acid molecule according to claim 3 or the expression vector according to claim 4 into a cell of an organism, and inhibiting the activity of B7H3 by expressing the antibody according to claim 1.

10. 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 expression vector of claim 4, the genetically engineered host cell of claim 5 and / or the host cell population of claim 6 in the preparation of a drug for treating and / or preventing a B7H3-positive related disease; (2) Use of the antibody of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the expression vector of claim 4, the genetically engineered host cell of claim 5 and / or the host cell population of claim 6 in the preparation of a biological agent for treating and / or preventing a B7H3-positive related disease; (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 expression vector of claim 4 in the preparation of a genetically engineered host cell for treating and / or preventing a B7H3-positive related disease; (4) Use of the antibody of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3 and / or the expression vector of claim 4 in preparing a host cell population for treating and / or preventing B7H3-positive related diseases; (5) Use of the antibody according to claim 1 and / or the nucleic acid molecule according to claim 3 in the preparation of a detection reagent for detecting B7H3 protein or an antigenic fragment thereof; (6) Use of the antibody according to claim 1, the nucleic acid molecule according to claim 3 and / or the detection reagent or detection kit according to claim 8 in the preparation of a product for detecting B7H3 protein or an antigenic fragment thereof; (7) Use of the antibody according to claim 1, the nucleic acid molecule according to claim 3 and / or the detection reagent or detection kit according to claim 8 in the preparation of a product for diagnosis and / or auxiliary diagnosis of B7H3-positive related diseases; Optionally, the B7H3-positive related disease is ovarian cancer, prostate cancer, liver cancer, lung cancer, kidney cancer, breast cancer, glioblastoma, colorectal cancer, urothelial carcinoma, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, bladder cancer, autoimmune disease, osteosarcoma or myeloid leukemia.

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