A fully human monoclonal antibody against b7h3 and derivative products and applications thereof

Abstract

The application discloses an anti-B7H3 full human monoclonal antibody and a derivative product and application thereof, wherein the derivative product comprises a chimeric antigen receptor targeting B7H3, and 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, wherein the antigen binding domain is the anti-B7H3 full human monoclonal antibody, and the antibody has an ADCC effect; the chimeric antigen receptor provided by the application has specific targeting effect on B7H3 positive tumor cells, and T cells expressing the chimeric antigen receptor have significant killing effect, and thus have important significance in the tumor treatment field.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and in particular relates to an anti-B7H3 full human monoclonal antibody AHF23037, and more particularly to an anti-B7H3 full human monoclonal antibody AHF23037 and derivative products and applications thereof. BACKGROUND

[0002] B7H3 (B7 homolog 3 protein, also known as CD276) belongs to the B7 / CD28 immunoglobulin superfamily and is a protein with a single transmembrane structure, which is composed of 316 amino acids, has a signal peptide at the amino terminal, includes an extracellular immunoglobulin-like variable region (IgV), a constant region (IgC), a transmembrane region and a cytoplasmic region of 45 amino acids, and is expressed in monocytes, dendritic cells and activated T cells. B7H3 has a low expression level in normal tissues, but is overexpressed in various cancers, especially non-small cell lung cancer, renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer and pancreatic cancer. B7H3 plays an important role in tumor immune escape through its inhibitory effect on T cells, and has been shown to promote tumor progression and cancer cell metastasis. In addition, B7H3 is not only expressed in cancer cells, but also in tumor 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 costimulatory factor when macrophages release cytokines, thus being associated with the occurrence of sepsis. In summary, due to its wide expression in various tumors, B7H3 has become a potential target for cancer immunotherapy, but there are few reports on immunotherapy targeting B7H3. SUMMARY

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

[0004] The present application achieves the above-mentioned application purposes by adopting the following technical solutions:

[0005] In a first aspect, the present application provides a full human antibody targeting B7H3, which comprises a heavy chain variable region VH and a light chain variable region VL.

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

[0007] the VL comprises three complementarity determining regions LCDR1, LCDR2, LCDR3;

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

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

[0010] Optionally, the amino acid sequence of the VH is as 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 with 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 with SEQ ID NO: 9. In some embodiments, the sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the VH and VL of the antibody of the present application can be obtained based on the above-mentioned VH and VL sequences according to the Kabat, IMGT, Chothia, AbM or Contact numbering system definition, and the CDR sequences defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system are also included in the protection scope of the present application.

[0013] In some embodiments, the fully human antibody of the present application is an antibody produced by the human immune system, which has high specificity and affinity, and the amino acid sequences constituting the antibody are all from humans. Such an antibody encoded entirely by human antibody genes has small immunogenicity and good clinical effect. The fully human antibody is an antibody obtained from the human immune system through biotechnology, and compared with traditional animal-derived antibodies, the fully human antibody has lower immunogenicity and can be better applied to human treatment. The fully human antibody is modified by genetic engineering technology and can be efficiently expressed in human cells for disease prevention and treatment. The fully human antibody not only reduces the risk of human immune response in the research and development process, but also shows better efficacy and safety in actual application.

[0014] In a second aspect, the present application provides a chimeric antigen receptor targeting B7H3, wherein the chimeric antigen receptor comprises the antibody of the first aspect of the present application.

[0015] Optionally, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a costimulatory 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 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 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 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 domain of any one of 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 peptide of any one of CD3ζ, CD3ε, CD16, CD22, α and β chains of T cell receptor, CD33, CD37, CD28, CD45, CD64, CD80; optionally, the self-cleaving peptide is selected from the self-cleaving peptide of any one of T2A, E2A, P2A, F2A; optionally, the safety switch is selected from the safety switch of any one of tEGFR, iCaspase-9, RQR8; optionally, the immunomodulatory molecule or cytokine is selected from the immunomodulatory molecule or cytokine of any one of 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 of the first aspect of the application, a Linker, a light chain variable region of the antibody of the first aspect of the application, a CD8 hinge and transmembrane domain, a CD28 costimulatory signaling domain, a CD3 zeta intracellular signaling domain, a T2A self-cleaving peptide, a tEGFR safety switch, in sequence; optionally, the Linker is (G4S)3; optionally, the amino acid sequence of the signal peptide is shown as SEQ ID NO: 11; optionally, the amino acid sequence of the Linker is shown as SEQ ID NO: 15; optionally, the amino acid sequence of the CD8 hinge and transmembrane domain is shown as SEQ ID NO: 19; optionally, the amino acid sequence of the CD28 costimulatory signaling domain is shown as SEQ ID NO: 21; optionally, the amino acid sequence of the CD3 zeta intracellular signaling domain is shown as SEQ ID NO: 23; optionally, the amino acid sequence of the T2A self-cleaving peptide is shown as SEQ ID NO: 25; optionally, the amino acid sequence of the tEGFR safety switch is shown as SEQ ID NO: 27; optionally, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO: 29.

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

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

[0019] In some embodiments, the skilled person in the art can change the combination category and sequence of the signal peptide, hinge and transmembrane region, costimulatory signaling domain and intracellular signaling domain according to the actual situation or needs, regardless of the form of change, as long as the chimeric antigen receptor has the CDR sequence of the heavy chain variable region or the heavy chain variable region sequence of the fully human antibody of the present application, and / or the CDR sequence of the light chain variable region or the light chain variable region sequence of the fully human antibody of the present application, which all fall within the protection scope of the present application.

[0020] In a fourth aspect, the present application provides an expression vector comprising the nucleic acid molecule of the third aspect of the present application.

[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, the nucleic acid molecules provided herein can be cloned into a number of types of vectors. For example, the nucleic acid molecules can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particular vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0023] In a fifth aspect, the present application provides a genetically engineered host cell, comprising the expression vector of the fourth aspect of the present application.

[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, including a mammalian cell, an insect cell, a plant cell, a yeast cell; optionally, the host cell is an immune cell; optionally, 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; optionally, the immune cell is a T cell. In some embodiments, the immune cell includes, but is not limited to, white blood cells, lymphocytes (T cells, B cells, natural killer (NK) cells), and myeloid-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells) derived, for example, from hematopoietic stem cells (HSCs) produced in the bone marrow. In particular embodiments of the present application, the genetically engineered host cell is a T cell expressing the chimeric antigen receptor of the second aspect of the present application.

[0026] In a sixth aspect, the present application provides a population of host cells, comprising the genetically engineered host cell of the fifth aspect of the present application.

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

[0028] In a seventh aspect, the present application provides a pharmaceutical composition or biological agent, comprising the antibody of the first aspect of the present application, the genetically engineered host cell of the fifth aspect of the present application, or the population of host cells of the sixth aspect of the present application.

[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 comprises a mono-specific antibody drug comprising the antibody of the first aspect of the application, a bi-specific antibody drug comprising the antibody of the first aspect of the application, an antibody conjugate drug comprising the antibody of the first aspect of the application, a CAR-T cell drug comprising the genetically engineered host cell of the fifth aspect of the application and / or the population of host cells of the sixth aspect of the application.

[0030] In some embodiments, the pharmaceutical composition or biological preparation of the application can comprise a buffer such as neutral buffered saline, sulfate buffered saline, and the like; a carbohydrate such as dextrose, mannose, sucrose or dextrans, mannitol; a protein; a polypeptide or 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 application further comprises one or more pharmaceutically or physiologically acceptable carriers, diluents or excipient combinations. Such compositions can comprise: a buffer, e.g., neutral buffered saline, phosphate buffered saline and the like; a carbohydrate, e.g., dextrose, mannose, sucrose or dextrans, mannitol; a protein; a polypeptide or amino acid, e.g., glycine; an antioxidant; a chelating agent, e.g., EDTA or glutathione; an adjuvant, e.g., aluminum hydroxide; and a preservative. The pharmaceutical composition or biological preparation of the application can be formulated for oral, intravenous, topical, enteral, and / or parenteral administration.

[0032] In a eighth aspect, the present application provides a detection reagent or a detection kit, which comprises the antibody of the first aspect of the application.

[0033] In some embodiments, the detection reagent further comprises a diagnostic agent coupled to the antibody; optionally, the diagnostic agent comprises a radionuclide, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, a photosensitive diagnostic agent; the specific type of the diagnostic agent is not particularly limited in the present application, and any conventional diagnostic agent known to those skilled in the art is within the scope of the present application.

[0034] In some embodiments, the detection kit further comprises a lysis medium for dissolving the sample to be tested, general reagents and buffers required for detection; more preferably, the kit further comprises an instruction manual.

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

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

[0037] In some embodiments, the antibody is an antibody labeled with a detectable label; optionally, the detectable label comprises a fluorochrome, avidin, paramagnetic atom, radioisotope; optionally, the fluorochrome is fluorescein, rhodamine, Texas red, phycoerythrin, phycocyanin, allophycocyanin, peridinin-chlorophyll protein; optionally, the avidin is biotin, egg white avidin, streptavidin, ovomucoid avidin, avidin; optionally, the radioisotope is radioactive iodine, radioactive cesium, radioactive iridium, radioactive cobalt;

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

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

[0040] In some embodiments, the method of introduction comprises calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, liposome-mediated transfection, lentivirus infection, retrovirus infection, adenovirus infection, transposon technology, CRISPR-Cas9 technology;

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

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

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

[0044] In addition, the present application also provides a method for diagnosing whether a subject has a B7H3-positive related disease, the method comprising: contacting a test sample derived from a subject with the antibody of the first aspect of the present application, and detecting whether a complex is formed between the antibody and B7H3 in the test sample. In some embodiments, the test sample is a test biological sample derived from a subject, including but not limited to: a blood sample, a tissue sample, etc. In some embodiments, the subject comprises a mammal 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 application provides the use of any of the following:

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

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

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

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

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

[0052] (7) use of the antibody of the first aspect of the present application, the nucleic acid molecule of the third aspect of the present application and / or the detection reagent or kit of the eighth aspect of the present application in the preparation of a product for diagnosing and / or aiding diagnosis of a B7H3-positive related disease;

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

[0054] In some embodiments, the B7H3-positive related disease of the present application is not limited to the above-mentioned diseases, and any disease and / or disorder related to the expression of B7H3 is within the protection scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 Figure 1: Detection of B7H3 expression in prostate cancer cells by flow cytometry, wherein A: prostate cancer cell DU145, B: prostate cancer cell DU145 B7H3 KO ;

[0056] Figure 2 Figure 2: Detection of antibody specificity by flow cytometry, wherein A-B: prostate cancer cell DU145, C-D: prostate cancer cell DU145 B7H3 KO ;

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

[0058] Figure 4Preparation and detection of full human B7H3 CAR-T cells, wherein, A: CAR-T positive rate flow chart, B: CAR-T positive rate histogram;

[0059] Figure 5 Tumor sphere experiment for detecting the killing effect of CAR-T cells prepared based on the full human B7H3 antibody on tumor cells, wherein, A: ovarian cancer cell SKOV3, B: prostate cancer cell DU145, C: liver cancer cell A549;

[0060] Figure 6 RTCA detection of the killing effect of CAR-T based on the full human B7H3 antibody on B7H3 + tumor cells, wherein, A: ovarian cancer cell SKOV3, B: prostate cancer cell DU145, C: liver cancer cell A549, D: lung cancer cell H1299. DETAILED DESCRIPTION

[0061] The present application will be further described below in conjunction with specific examples, which are only used to explain the present application and cannot be understood as limiting the present application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these examples without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents. The experimental methods in the following examples are not specified, and are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturer. The reagents, biological materials, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0062] Example 1 Monoclonal antibody AHF23037 targeting B7H3

[0063] The present application constructs a full human monoclonal antibody AHF23037 specifically targeting B7H3, and the sequence information of the AHF23037 antibody is shown in Table 1 below, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 9. In addition, the present example proves that the AHF23037 antibody has good affinity to B7H3 through experiments.

[0064] Table 1 Sequence of AHF23037 antibody

[0065]

[0066]

[0067] Example 2 Flow cytometry technology for detecting the specificity of antibody AHF23037

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

[0069] This example first detects B7H3 expression in prostate cancer cells by flow cytometry, and incubates antibody AHF23037 with DU145 and DU145 B7H3 KO cells, and then detects by flow cytometry. The specific experimental steps are as follows:

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

[0071] (2) Add PBS to wash, centrifuge to discard the supernatant, resuspend the cell pellet in 300 μL PBS, and filter through a filter screen into a flow tube;

[0072] (3) Detect by flow cytometry.

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

[0074] In order to determine that the B7H3 antibody AHF23037 used can specifically bind to the B7H3 antigen expressed by the cells, this example respectively dilutes ① antibody AHF23037; ② commercial B7H3 antibody (Enoblituzumab MGA271) at a concentration gradient, and then incubates with DU145 and DU145 B7H3 KO cells, and then detects by flow cytometry. The specific experimental steps are as follows:

[0075] (1) Select cells DU145 and DU145 B7H3 KO , 2-5 x 10 5 per sample;

[0076] (2) Select antibodies: ① antibody AHF23037; ② commercial 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 dilution: PBS containing 2% FBS), incubate at 37°C for 30 min, add 1 mL PBS to wash, centrifuge, and discard the supernatant as much as possible;

[0078] (4) Secondary antibody: AF647-IgG (1:500), incubated at 4℃ for 30 min, washed with 1 mL PBS, centrifuged, discarded supernatant, resuspended in 300 μL PBS containing 0.5% BSA, and detected by flow cytometry (10,000 target cells were collected).

[0079] 3. Experimental Results

[0080] Flow cytometry analysis of prostate cancer cells DU145 and DU145 B7H3 KO The expression status of B7H3 and the corresponding results are as follows Figure 1 As shown, the results indicate that B7H3 is highly expressed in DU145 cells. B7H3 KO It is not expressed in Chinese.

[0081] Results of flow cytometry detection of antibody specificity, such as Figure 2 As shown, the results indicate that, compared to DU145 B7H3 KO In comparison, antibody AHF23037 exhibits a highly specific binding ability to DU145 expressing B7H3, which is superior to commercially available antibodies.

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

[0083] 1. Experimental Methods

[0084] This embodiment uses an RTCA assay to detect the killing effect of NK cells treated with antibody AHF23037 on DU145 cells. The specific experimental steps are as follows:

[0085] (1) Collect prostate cancer DU145 cells and seed them into RTCA plates at a density of 20,000 cells per well;

[0086] (2) When the cell index of tumor cells reaches approximately 1, add 50 μL of antibody (final concentration 5 μg / mL), 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 the antibody at 37℃ for 1 h;

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

[0088] (4) RTCA instrument automatically records cell growth index, and stops RTCA after 2-3 days to automatically generate a curve of reaction cell growth index.

[0089] 2. Experimental results

[0090] The results are shown in Figure 3 The results show that when the effector-target ratio is 1:1, the killing effect of NK cells on tumors is significantly enhanced after the combination of NK cells and antibodies in DU145 cells compared with the group of NK cells alone. The effect is basically equivalent to that of commercial antibodies. The above results show that the fully human B7H3 antibody AHF23037 can kill DU145 cells through ADCC effect.

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

[0092] 1. Experimental method

[0093] The CAR expression plasmid is constructed using the sequence of the above antibody AHF23037, and the virus is packaged, and the collected retrovirus liquid is used to infect T cells to prepare CAR-T, which is named AHF23037.CAR-T. The positive rate is detected by flow cytometry staining 48h after infection. The coding sequence of the fully human chimeric antigen receptor (CAR) targeting B7H3 is shown in Table 2. The specific experimental steps are as follows:

[0094] (1) The constructed plasmid is subjected to reverse transcription virus packaging, and the virus supernatant is harvested at 48h and 72h, respectively, centrifuged and filtered, then divided into 1.5mL EP tubes and stored at -80℃;

[0095] (2) After T cell activation, 0.5×10 6 / mL is divided into 1.5mL EP tubes, 1mL virus liquid (AHF23037.CAR-T) is added to resuspend the cells, and then slowly added into the 24-well plate coated with Retronectin in advance, and centrifuged at 30℃, 1500g for 2h. After centrifugation, the virus liquid is discarded, complete culture medium is added, and the culture is placed in an incubator for culture;

[0096] (3) 0.2×10 6 / mL cells are taken after 48h for CAR positive rate detection. PE-proteinL and FITC-CD3 flow cytometry antibodies are diluted to a final concentration of 1μg / μL, and 50μL / sample is added to the cells, 4℃, avoiding light for 30min, 1mL PBS is added for washing, and centrifuged to discard the supernatant;

[0097] (4) 300μL PBS is used to resuspend the cell pellet, and filtered into a flow cytometry tube, and then detected by flow cytometry.

[0098] 2. Experimental results

[0099] Results are shown in Figure 4 Figure 2, which show that the positive rate of CAR-T is stable at 70-90% compared with uninfected T cells, which indicates that the CAR-T cells targeting B7H3 are successfully prepared in this embodiment.

[0100] Table 2 Coding sequence of full human chimeric antigen receptor (CAR) targeting B7H3

[0101]

[0102]

[0103] Example 5 Spheroid experiment for detecting the killing effect of CAR-T cells prepared based on full human B7H3 antibody (AHF23037) on tumor cells

[0104] 1. Experimental method

[0105] In order to verify the killing ability of the CAR-T cells on B7H3 positive tumors, in this embodiment, the killing effect of AHF23037.CAR-T cells on ovarian cancer SKOV3, prostate cancer DU145, lung cancer A549 cells which highly express B7H3 and have GFP fluorescence was observed by 3D tumor experiment. The specific experimental steps are as follows:

[0106] (1) 0.15 g of agarose was added to 10 mL of DMEM, and sterilized at 121°C for 20 minutes;

[0107] (2) 50 μL of hot agarose solution was added to each well of a 96-well plate, and cooled to room temperature;

[0108] (3) 200 μL of tumor cell suspension with GFP fluorescence was inoculated into the 96-well plate covered with agarose, and the cell density was 1.2 x 10 4 / mL, and further cultured for 3-4 days;

[0109] (4) The size of the spheroid was observed under a microscope, and when the diameter was about 300 μm, it was photographed and recorded;

[0110] (5) The effector cells (AHF23037.CAR-T) were collected, and the CAR-T effector cells were stained with DIL cell membrane dye (1:500) at room temperature for 20 min in the dark, washed twice with 10 mL of PBS, and the supernatant was discarded;

[0111] (6) The effector cell culture medium was used to resuspend the precipitate, counted, and 50 μL of effector cell suspension was added according to the initial cell 1:1 effector target ratio;

[0112] (7) Take photos under fluorescence microscope to record observation.

[0113] 2. Experimental results

[0114] The results are shown in Table 1, and the results show that the presence of CAR significantly improves the killing ability of T cells to solid tumors (ovarian cancer, prostate cancer, liver cancer) compared with T cells. The above results show that the CAR-T cells prepared based on the full human B7H3 antibody (AHF23037) have good killing effect on tumor cells. Figure 5 Example 6 RTCA detection of CAR-T killing effect on B7H3 + tumor cells

[0115] 1. Experimental method

[0116] In order to verify the killing ability of CAR-T cells to B7H3 positive tumors, the killing effect of AHF23037.CAR-T cells on tumor cells (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 to target ratio of 2:1 was detected by RTCA. The specific experimental steps are as follows:

[0117] (1) Collect tumor cells, and inoculate 20,000 cells per well in an RTCA plate;

[0118] (2) When the cell index of the tumor cells grows to about 1, add the corresponding AHF23037.CAR-T cells;

[0119] (3) The RTCA instrument automatically records the cell growth index;

[0120] (4) After 2-3 days, stop RTCA, and automatically generate a curve of the cell growth index of the reaction.

[0121] 2. Experimental results

[0122] The results are shown in Table 1, and the results show that the presence of CAR significantly improves the killing ability of T cells to solid tumors (ovarian cancer, prostate cancer, liver cancer) compared with T cells. The above results show that the CAR-T cells prepared based on the full human B7H3 antibody (AHF23037) have good killing effect on tumor cells.

[0123] Figure 6 The results are shown in Table 1, and the results show that the presence of CAR significantly improves the killing ability of T cells to solid tumors (ovarian cancer, prostate cancer, liver cancer) compared with T cells. The above results show that the CAR-T cells prepared based on the full human B7H3 antibody (AHF23037) have 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, HCDR3; The VL comprises three complementarity determining regions LCDR1, LCDR2, LCDR3; The amino acid sequences of the HCDR1, HCDR2, HCDR3 are respectively as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3; The amino acid sequences of the LCDR1, LCDR2, LCDR3 are respectively as shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:

8.

2. The antibody of claim 1, wherein The amino acid sequence of the VH is as shown in SEQ ID NO:

4.

3. The antibody of claim 1, wherein The amino acid sequence of the VL is as shown in SEQ ID NO:

9.

4. A chimeric antigen receptor targeting B7H3, characterized in that, The chimeric antigen receptor comprises the antibody of any one of claims 1-3. The antibody is obtained in series from the heavy chain variable region of the antibody of claim 1, a Linker, the light chain variable region of the antibody of claim 1. The chimeric antigen receptor further comprises a hinge region and a transmembrane domain, an intracellular signaling domain, a costimulatory signal domain. The chimeric antigen receptor further comprises a signal peptide, a self-cleavage peptide, a safety switch. The hinge region and the transmembrane domain are CD8 hinge region and transmembrane domain. The intracellular signaling domain is CD3ζ intracellular signaling domain. The costimulatory signal domain is CD28 costimulatory signal domain. The self-cleavage peptide is T2A self-cleavage peptide. The safety switch is tEGFR safety switch.

5. The chimeric antigen receptor of claim 4, wherein, The chimeric antigen receptor is obtained in series from a signal peptide, the heavy chain variable region of the antibody of claim 1, a Linker, the light chain variable region of the antibody of claim 1, CD8 hinge region and transmembrane domain, CD28 costimulatory signal domain, CD3ζ intracellular signaling domain, T2A self-cleavage peptide, tEGFR safety switch.

6. The chimeric antigen receptor of claim 5, wherein, The Linker is (G4S)3. The amino acid sequence of the signal peptide is as shown in SEQ ID NO:

11. The amino acid sequence of the Linker is as shown in SEQ ID NO:

15. The amino acid sequence of the CD8 hinge region and transmembrane domain is as shown in SEQ ID NO:

19. The amino acid sequence of the CD28 costimulatory signal domain is as shown in SEQ ID NO:

21. The amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO:

23. The amino acid sequence of the T2A self-cleavage peptide is as shown in SEQ ID NO:

25. The amino acid sequence of the tEGFR safety switch is as shown in SEQ ID NO:

27.

7. A nucleic acid molecule encoding the antibody of any one of claims 1-3 or the chimeric antigen receptor of any one of claims 4-6.

8. The nucleic acid molecule of claim 7, wherein, The nucleotide sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:

14.

9. The nucleic acid molecule of claim 7, wherein, The nucleotide sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:

18.

10. The nucleic acid molecule of claim 7, wherein The nucleotide sequence of the signal peptide in the chimeric antigen receptor is shown as SEQ ID NO: 12; The nucleotide sequence of the Linker in the chimeric antigen receptor is shown as SEQ ID NO: 16; The nucleotide sequence of the CD8 hinge and transmembrane domain in the chimeric antigen receptor is shown as SEQ ID NO: 20; The nucleotide sequence of the CD28 co-stimulatory signaling domain in the chimeric antigen receptor is shown as SEQ ID NO: 22; The nucleotide sequence of the CD3 zeta intracellular signaling domain in the chimeric antigen receptor is shown as SEQ ID NO: 24; The nucleotide sequence of the T2A self-cleaving peptide in the chimeric antigen receptor is shown as SEQ ID NO: 26; The nucleotide sequence of the tEGFR safety switch in the chimeric antigen receptor is shown as SEQ ID NO:

28.

11. An expression vector comprising the nucleic acid molecule of any one of claims 7-10.

12. The expression vector of claim 11, wherein, The vector is a DNA vector, an RNA vector, or a vector of viral origin.

13. The expression vector of claim 12, wherein, The vector is a plasmid.

14. The expression vector of claim 12, wherein, The vector is a transposon vector or a CRISPR / Cas9 vector.

15. The expression vector of claim 12, wherein, The vector of viral origin is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.

16. A genetically engineered host cell, characterized in that, The genetically engineered host cell comprises the expression vector of any one of claims 11-15.

17. The genetically engineered host cell of claim 16, wherein, The host cell is an immune cell; The immune cell is a T cell, an NK cell, or an iNKT cell; The expression vector comprised in the genetically engineered host cell encodes the chimeric antigen receptor of any one of claims 4-6.

18. A population of host cells, wherein, The population of host cells comprises the genetically engineered host cell of claim 16 or 17.

19. The population of host cells of claim 18, wherein, The population of host cells further comprises host cells that do not comprise the expression vector of any one of claims 11-15.

20. A pharmaceutical composition comprising, The pharmaceutical composition comprises the antibody of any one of claims 1-3, the genetically engineered host cell of claim 16 or 17, or the population of host cells of claim 18 or 19.

21. A biological agent, characterized in that, The biological agent comprises the antibody of any one of claims 1-3, the genetically engineered host cell of claim 16 or 17, or the population of host cells of claim 18 or 19.

22. A detection reagent, characterized by comprising: The detection reagent comprises the antibody of any one of claims 1-3.

23. A test kit, characterized in that, The detection kit comprises the antibody of any one of claims 1-3.

24. A method of detecting B7H3 in a test sample for non-diagnostic and non- therapeutic purposes, comprising: contacting the test sample with an antibody of claim 1 under conditions that allow the antibody to bind to B7H3; and detecting the presence of the antibody bound to B7H3. The method comprises contacting a sample to be tested with the antibody of any one of claims 1-3, and detecting the formation of a complex of the antibody and B7H3.

25. A method of producing the antibody of any one of claims 1-3, wherein, The method comprises culturing the genetically engineered host cell of claim 16, and isolating the antibody of any one of claims 1-3 from the culture.

26. A method of making the genetically engineered host cell of claim 16 or 17 or the population of host cells of claim 18 or 19, wherein, The method comprises providing a host cell to be engineered, introducing the nucleic acid molecule of any one of claims 7-10 or the expression vector of any one of claims 11-15 into the host cell, and obtaining the genetically engineered host cell or population of host cells.

27. Use of the antibody of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4-6, the nucleic acid molecule of any one of claims 7-10, the expression vector of any one of claims 11-15, the genetically engineered host cell of claim 16 or 17, and / or the population of host cells of claim 18 or 19 in the manufacture of a medicament for treating and / or preventing a B7H3-positive associated disease. The B7H3-positive associated disease is prostate cancer, ovarian cancer, liver cancer, or lung cancer.

28. Use of the antibody of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4-6, the nucleic acid molecule of any one of claims 7-10, the expression vector of any one of claims 11-15, the genetically engineered host cell of claim 16 or 17, and / or the population of host cells of claim 18 or 19 in the manufacture of a biological preparation for treating and / or preventing a B7H3-positive associated disease. The B7H3-positive associated disease is prostate cancer, ovarian cancer, liver cancer, or lung cancer.

29. Use of the antibody of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4-6, the nucleic acid molecule of any one of claims 7-10, and / or the expression vector of any one of claims 11-15 in the manufacture of a genetically engineered host cell for treating and / or preventing a B7H3-positive associated disease. The host cell is an immune cell; The immune cell is a T cell, a NK cell, or an iNKT cell; The B7H3-positive associated disease is prostate cancer, ovarian cancer, liver cancer, or lung cancer.

30. Use of the antibody of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4-6, the nucleic acid molecule of any one of claims 7-10, and / or the expression vector of any one of claims 11-15 in the manufacture of a population of host cells for treating and / or preventing a B7H3-positive associated disease. The host cell is an immune cell; The immune cell is a T cell, a NK cell, or an iNKT cell; The B7H3-positive associated disease is prostate cancer, ovarian cancer, liver cancer, or lung cancer.

31. Use of the antibody of any one of claims 1-3 and / or the nucleic acid molecule of any one of claims 7-10 in the manufacture of a detection reagent for detecting a B7H3 protein or an antigenic fragment thereof.

32. Use of the antibody of any one of claims 1-3, the nucleic acid molecule of any one of claims 7-10, and / or the detection reagent of claim 22 in the manufacture of a detection kit for detecting a B7H3 protein or an antigenic fragment thereof.

33. Use of the antibody of any one of claims 1-3, the nucleic acid molecule of any one of claims 7-10, and / or the detection reagent of claim 22 in the manufacture of a product for diagnosing and / or aiding in the diagnosis of a B7H3-positive associated disease. The B7H3 positive related disease is prostate cancer, ovarian cancer, liver cancer or lung cancer.

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