B7H3-targeting fully humanized antibody, chimeric antigen receptor, and related products and medical application of B7H3-targeting fully humanized antibody and chimeric antigen receptor

Abstract

The invention discloses a B7H3-targeted fully-humanized antibody, a chimeric antigen receptor as well as related products and medical application thereof, and provides a brand-new B7H3-targeted fully-humanized monoclonal antibody AHF23050, and the antibody AHF23050 can be specifically combined with cells expressing B7H3, so that the B7H3-targeted fully-humanized monoclonal antibody AHF23050 can be used for detecting B7H3. The invention further provides a chimeric antigen receptor containing the antibody AHF23050, a CAR-T cell, a pharmaceutical composition, a pharmaceutical preparation, an immunoconjugate, a detection reagent and the like, and experimental verification shows that the CAR-T cell can efficiently and specifically kill tumor cells expressing B7H3 and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically, relates to a fully human antibody targeting B7H3, a chimeric antigen receptor and related products and medical uses thereof. Background Art

[0002] B7H3 (CD276) belongs to the B7 superfamily and is a transmembrane glycoprotein. Its extracellular domain structure is divided into two types, one is a monovalent 2Ig-B7-H3, and the other is a bivalent 4Ig-B7-H3 composed of two repeating units. Related studies have shown that B7H3 can inhibit T cell proliferation and cytokine release by interacting with an unknown receptor. Although the receptor of B7H3 is unknown, there have been more and more reports on the negative regulation of B7H3 and receptors in tumor immunity in recent years. Tumor cells express B7H3 to avoid CD8 + T cell immune surveillance. Related studies have shown that B7H3 knockout mice or anti-B7H3 antibodies can significantly inhibit tumor growth, and this inhibitory effect depends on CD8 + The functions of T and NK cells, the above studies all indicate that B7H3 can be used as an effective therapeutic target for tumor immunotherapy.

[0003] In addition, the non-immunological effects of B7H3 have also attracted the attention of more and more researchers. Not only does B7H3 play a non-immune role in various diseases and functions such as bone differentiation, inflammatory response, and metabolism, but in tumor cells, B7H3 can also function independently by regulating tumor cell biological signals without relying on immune effects. B7H3 can directly participate in tumor progression, chemotherapeutic drug sensitivity, and glycolysis of various tumor cells through various signaling pathways, including PI3K / Akt, Jak / STAT, NF-κB-p65 / MAPK-p38, and Ras / Raf / MEK signals. B7H3 can be detected and abnormally expressed in tumor tissues of patients with various cancer types, and predict poor prognosis. Therefore, B7H3 can be used as a candidate cancer treatment target. It can be seen that further research and development of anti-B7H3 monoclonal antibodies that can be used to treat B7H3-positive related diseases has important scientific significance and clinical value. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a fully human antibody targeting B7H3, a chimeric antigen receptor and related products and medical uses thereof in the art.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a monoclonal antibody targeting B7H3.

[0007] Further, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1-3, and the light chain variable region comprises LCDR1-3; the HCDR1-3 are HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO:4; and the LCDR1-3 are LCDR1-3 in the light chain variable region as shown in SEQ ID NO:9.

[0008] Further, the amino acid sequences of the HCDR1-3 are respectively as shown in SEQ ID NO:1-3 or have at least 70% homology with SEQ ID NO:1-3, respectively; optionally, the amino acid sequences of the LCDR1-3 are respectively as shown in SEQ ID NO:6-8 or have at least 70% homology with SEQ ID NO:6-8, respectively; optionally, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4 or has at least 70% homology with SEQ ID NO:4; optionally, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9 or has at least 70% homology with SEQ ID NO:9.

[0009] In the present invention, antibody sequences corresponding to the amino acid sequence corresponding to the heavy chain variable region, the amino acid sequence corresponding to the light chain variable region, the amino acid sequence corresponding to HCDR1-3 and / or the amino acid sequence corresponding to LCDR1-3 of the monoclonal antibody provided by the present invention that have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology are all within the protection scope of the present invention.

[0010] The amino acid sequences of HCDR1-3 and LCDR1-3 corresponding to the monoclonal antibody described in the present invention are not limited to the amino acid sequences shown above. The amino acid sequences or nucleotide sequences corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 obtained by defining CDR1, CDR2, and CDR3 in the heavy chain variable region and light chain variable region described above of the present invention using any CDR numbering scheme are all within the protection scope of the present invention.

[0011] In some embodiments, the CDR numbering scheme includes, but is not limited to, any one or any combination of two or more (two or more) of the IMGT numbering scheme, the Chothia numbering scheme, the Kabat numbering scheme, the Contact numbering scheme, the Martin (enhanced Chothia) numbering scheme, the AbM numbering scheme, and the Aho numbering scheme.

[0012] A second aspect of the present invention provides a bispecific antibody.

[0013] Furthermore, the bispecific antibody comprises the monoclonal antibody described in the first aspect of the present invention; optionally, the bispecific antibody further comprises a second antibody that specifically binds to other antigens.

[0014] In the present invention, the bispecific antibody refers to an antibody having two different antigen binding regions defined by different antibody sequences. It can be understood as binding to different targets, but also includes binding to different epitopes of a target. It includes full-length bispecific antibodies and antigen-binding fragments thereof. Bispecific antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Bispecific antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, as long as these antibodies exhibit the desired biological activity, they all belong to the scope of the bispecific antibody.

[0015] The third aspect of the present invention provides a chimeric antigen receptor targeting B7H3.

[0016] Further, the chimeric antigen receptor comprises the monoclonal antibody described in the first aspect of the present invention; 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;

[0017] Optionally, the transmembrane domain is selected from the transmembrane domain of any one of the following molecules: CD8, CD28, 4-1BB, CD34, CD3ε, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the intracellular signaling domain is selected from the intracellular signaling domain of any one of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, Fcε RI, DAP10, DAP12, CD66d; Optionally, the hinge region is selected from the hinge region of any one of the following molecules: CD8, CD28, CD34, 4-1BB, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the costimulatory signaling domain is selected from the costimulatory signaling domain of any one of the following molecules: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, CD226; Optionally, the signal peptide is selected from the signal peptide of any one of the following molecules: α chain and β chain of T cell receptor, CD3ζ, CD3ε, CD16, CD22, CD33, CD4, CD5, CD8, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, GM-CSF; optionally, the self-cleaving peptide is selected from any one of the following self-cleaving peptides: T2A, P2A, E2A, F2A; optionally, the safety switch is selected from any one of the following safety switches: tEGFR, iCaspase-9, RQR8; optionally, the immunomodulatory molecule or cytokine is selected from any one of the following immunomodulatory molecules or cytokines: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, IL-15, IL-18, IL-21, LEC, OX40L;

[0018] Optionally, the chimeric antigen receptor is obtained by sequentially connecting a signal peptide, a heavy chain variable region of the monoclonal antibody described in the first aspect of the present invention, a linker, a light chain variable region of the monoclonal antibody described in the first aspect of the present invention, 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;

[0019] Optionally, the Linker is (G4S)3; optionally, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:11; optionally, the amino acid sequence of the Linker is as shown in SEQ ID NO:15; optionally, the amino acid sequence of the CD8 hinge region and transmembrane domain is as shown in SEQ ID NO:19; optionally, the amino acid sequence of the CD28 co-stimulatory signal domain is as shown in SEQ ID NO:21; optionally, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQID 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.

[0020] The fourth aspect of the present invention provides a nucleic acid molecule.

[0021] Further, the nucleic acid molecule comprises a nucleotide sequence encoding the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, or the chimeric antigen receptor of the third aspect of the present invention; optionally, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody of 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 monoclonal antibody of 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 of the third aspect of the present invention is as shown in SEQ ID NO: 12; optionally, the nucleotide sequence of Linker in the chimeric antigen receptor of the third 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 of the third 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 of the third 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 of the third aspect of the present invention is as shown in SEQ ID NO: NO:24; optionally, the nucleotide sequence of the T2A self-cleaving peptide in the chimeric antigen receptor described in the third aspect of the present invention is shown in SEQ ID NO:26; optionally, the nucleotide sequence of the tEGFR safety switch in the chimeric antigen receptor described in the third aspect of the present invention is shown in SEQ ID NO:28; optionally, the nucleotide sequence of the chimeric antigen receptor described in the third aspect of the present invention is shown in SEQ ID NO:30.

[0022] The fifth aspect of the present invention provides an expression vector.

[0023] Further, the expression vector comprises the nucleic acid molecule described in the fourth aspect of the present invention; optionally, the vector is a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector or a virus-derived vector; optionally, the virus-derived vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a poxvirus vector or a herpesvirus vector.

[0024] In some embodiments, the expression vectors of the present invention refer to nucleic acid molecules capable of transporting another nucleic acid to which it is attached. One type of vector is a plasmid, which is a circular double-stranded DNA loop into which additional DNA segments can be attached. Another type of vector is a viral vector, in which additional DNA segments can be attached to the viral genome.

[0025] The sixth aspect of the present invention provides a recombinant host cell.

[0026] Further, the recombinant host cell comprises the expression vector described in the fifth aspect of the present invention; optionally, the host cell is a eukaryotic cell and / or a prokaryotic cell; optionally, the host cell is an immune cell; optionally, the immune cell is a T cell, a B cell, a NK cell, an iNKT cell, a CTL cell, a dendritic cell, a myeloid cell, a monocyte, a macrophage or any combination thereof.

[0027] In some embodiments, the present invention has no particular limitation on the specific type of the host cell. Any host cell that can carry the expression vector described in the fifth aspect of the present invention is within the protection scope of the present invention. Exemplarily, the host cell includes but is not limited to: T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof, CHO-K1, DUkXB11, Pro-5, CHO-S, NSO, SP2 / 0-Ag14, YB2 / 0 or CHO-3E7, etc.

[0028] A seventh aspect of the present invention provides any of the following products:

[0029] (1) A diagnostic conjugate, comprising the monoclonal antibody according to the first aspect of the present invention and / or the bispecific antibody according to the second aspect of the present invention;

[0030] (2) A detection reagent, comprising the monoclonal antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention and / or the diagnostic conjugate;

[0031] (3) A detection product, comprising the monoclonal antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the diagnostic conjugate and / or the detection reagent;

[0032] (4) A therapeutic conjugate, comprising the monoclonal antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, and / or the recombinant host cell according to the sixth aspect of the present invention;

[0033] (5) A pharmaceutical composition comprising the monoclonal antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the recombinant host cell according to the sixth aspect of the present invention and / or the therapeutic conjugate;

[0034] (6) A pharmaceutical preparation comprising the monoclonal antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the recombinant host cell according to the sixth aspect of the present invention, the therapeutic conjugate and / or the pharmaceutical composition;

[0035] (7) A drug kit comprising a therapeutically effective amount of the monoclonal antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the recombinant host cell according to the sixth aspect of the present invention, the therapeutic conjugate, the pharmaceutical composition and / or the pharmaceutical preparation;

[0036] Optionally, the diagnostic conjugate is a complex formed by directly or indirectly binding the monoclonal antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention to a diagnostic agent; optionally, the diagnostic agent is a radionuclide, a contrast agent and / or a photosensitive diagnostic agent; optionally, the radionuclide includes a γ-emitting isotope, a β-emitting isotope and / or a positron-emitting isotope; optionally, the contrast agent includes an iodine compound, a barium compound, a gallium compound and / or a thallium compound; optionally, the photosensitive diagnostic agent includes a chemiluminescent compound and / or a bioluminescent compound; in some embodiments, specific examples of the radionuclide include, but are not limited to: 110 In, 111 In, 177 Lu, 18 F. 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y. 90 Y. 89 Zr, 94 mTc, 94 Tc,99 mTc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52 mMn, 55 Co, 72 As, 75 Br, 76 Br, 82 mRb, 83 Sr, 51 Cr, 57 Co, 58 Co, 59 Fe, 75 Se, 97 Ru, 114 mIn, 169 Yb, 197 Hg and / or 201Tl; In some embodiments, the contrast agent is a radioactive contrast agent, which may be a paramagnetic ion, including but not limited to: chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), erbium (III), lanthanum (III), gold (III), lead (II) and / or bismuth (III); In some embodiments, the contrast agent is an ultrasound enhancement agent (e.g., liposomes); In some embodiments, the contrast agent is a radiopaque substance of an iodine compound, a barium compound, a gallium compound and / or a thallium compound. The radiopaque substances include, but are not limited to, barium, diatrizoate, ethiodized oil, gallium citrate, iocaic acid, iodamide, choline, iodoxic acid, iogulamide, iohexol, iopamidol, iopanoic acid, iopsidic acid, iodoxic acid, iodose acid, iodine acid, iosulfamide meglumine, iothalamic acid, iotetic acid, iotalamic acid, iodixic acid, hydroxydiatrizoate, ipodate, meglumine, meglumine, metrizoate, iodone and / or thallium chloride; in some embodiments, the photosensitive diagnostic agent includes a chemiluminescent compound and / or a bioluminescent compound, and the bioluminescent compound includes, but is not limited to, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, luciferin, luciferase and / or aequorin. The chemiluminescent compound: luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt and / or oxalate;

[0037] Optionally, the detection product is a detection kit, a detection test strip or a detection chip;

[0038] Optionally, the therapeutic conjugate is a complex formed by directly or indirectly binding the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, or the recombinant host cell described in the sixth aspect of the present invention to a therapeutic agent; optionally, the therapeutic agent is a radionuclide, an immunomodulator, a hormone, a hormone antagonist, an oligonucleotide, an enzyme, an enzyme inhibitor, a photosensitizer, a cytotoxic agent and / or an angiogenesis inhibitor;

[0039] Optionally, the medicine kit is packaged in a container and also includes a label affixed to or packaged in the container, which label describes the contents of the container and provides instructions and / or instructions for using the contents of the container to treat and / or prevent B7H3-positive related diseases; optionally, the medicine kit may also include a second therapeutic agent, which is a drug that can be used to treat and / or prevent (or assist in the treatment and / or prevention of) B7H3-positive related diseases (the present invention has no particular limitation on the specific type of the second therapeutic agent).

[0040] An eighth aspect of the present invention provides any of the following methods:

[0041] (1) A method for producing the monoclonal antibody of the first aspect of the present invention or the bispecific antibody of the second aspect of the present invention, the method comprising the following steps: culturing the recombinant host cell of the sixth aspect of the present invention, and isolating the monoclonal antibody of the first aspect of the present invention or the bispecific antibody of the second aspect of the present invention from the culture product of the recombinant host cell;

[0042] (2) A method for preparing the recombinant host cell according to the sixth aspect of the present invention, the method comprising the following steps: introducing the expression vector according to the fifth aspect of the present invention into a host cell to obtain the recombinant host cell according to the sixth aspect of the present invention;

[0043] (3) A method for detecting B7H3 protein in a sample for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: contacting the sample with the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the diagnostic conjugate, the detection reagent and / or the detection product of the seventh aspect of the present invention, and detecting the formation of an immune complex between the B7H3 protein and the antibody;

[0044] (4) A method for inhibiting B7H3 protein activity in vitro, the method comprising the following steps: contacting a sample to be tested with the monoclonal antibody described in the first aspect of the present invention and / or the bispecific antibody described in the second aspect of the present invention.

[0045] In addition, the present invention also provides a method for diagnosing whether a subject suffers from a B7H3-positive related disease, the method comprising the following steps: contacting a test sample derived from the subject with the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the diagnostic conjugate, detection reagent and / or detection product described in the seventh aspect of the present invention, detecting the formation of antibody immune complexes, and judging whether the subject suffers from a B7H3-positive related disease or the risk of the subject suffering from a B7H3-positive related disease based on the result.

[0046] In addition, the present invention also provides a method for treating and / or preventing a B7H3-positive related disease in a subject in need thereof, the method comprising the following steps: administering to a subject in need thereof an effective amount of the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the recombinant host cell described in the sixth aspect of the present invention, the therapeutic conjugate described in the seventh aspect of the present invention, the pharmaceutical composition described in the seventh aspect of the present invention and / or the pharmaceutical preparation described in the seventh aspect of the present invention.

[0047] In the present invention, the B7H3-positive related diseases include any diseases related to B7H3 expression, including but not limited to: ovarian cancer, prostate cancer, liver cancer, lung cancer, kidney cancer, breast cancer, colorectal cancer, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, bladder cancer, osteosarcoma, glioma or myeloid leukemia.

[0048] In the present invention, the subject refers to any animal, preferably a mammal, including but not limited to humans, higher primates, domestic and farm animals, and zoo animals, competitive animals or pets, such as horses, pigs, cows, dogs, cats and ferrets, etc. In a specific embodiment of the present invention, the subject is preferably a human.

[0049] In the present invention, the administration refers to a method of administering a certain dose of a compound (e.g., the monoclonal antibody, bispecific antibody, recombinant host cell described in the present invention) or a composition (e.g., the therapeutic conjugate, pharmaceutical composition or pharmaceutical preparation described in the present invention) to a subject in need.

[0050] In some embodiments, the present invention is not particularly limited to the mode of administration, and the mode of administration includes, but is not limited to, intralesional, intramuscular, intravenous, intradermal, transdermal, intraarterial, intraperitoneal, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intrarectal, surface, subcutaneous, subconjunctival, intravesical, transmucosal, pericardial, umbilical, intraocular, oral, topical, by inhalation, by injection, by infusion, by continuous infusion, by catheter, by irrigation, with cream, or with lipid composition. The selection of the specific method of administration and the selection of the dosage form of the pharmaceutical preparation can vary according to a variety of factors (e.g., the compound or composition administered and the severity of the illness, disease, or condition treated).

[0051] A ninth aspect of the present invention provides any of the following applications:

[0052] (1) Use of the monoclonal antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention and / or the nucleic acid molecule according to the fourth aspect of the present invention in the preparation of a diagnostic conjugate for detecting B7H3 protein;

[0053] (2) Use of the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the nucleic acid molecule of the fourth aspect of the present invention and / or the diagnostic conjugate of the seventh aspect of the present invention in the preparation of a detection reagent for detecting B7H3 protein;

[0054] (3) Use of the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, the diagnostic conjugate described in the seventh aspect of the present invention, and / or the detection reagent described in the seventh aspect of the present invention in the preparation of a detection product for detecting B7H3 protein;

[0055] (4) Use of the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the nucleic acid molecule of the fourth aspect of the present invention, the diagnostic conjugate of the seventh aspect of the present invention, the detection reagent of the seventh aspect of the present invention and / or the detection product of the seventh aspect of the present invention in detecting B7H3 protein for non-diagnostic and non-therapeutic purposes;

[0056] (5) Use of the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the nucleic acid molecule of the fourth aspect of the present invention, the diagnostic conjugate of the seventh aspect of the present invention, the detection reagent of the seventh aspect of the present invention and / or the detection product of the seventh aspect of the present invention in the preparation of a diagnostic product for diagnosing or assisting in the diagnosis of B7H3-positive related diseases;

[0057] (6) Use of the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the chimeric antigen receptor of the third aspect of the present invention, the nucleic acid molecule of the fourth aspect of the present invention, the expression vector of the fifth aspect of the present invention, and the recombinant host cell of the sixth aspect of the present invention in the preparation of a therapeutic conjugate for treating and / or preventing B7H3-positive related diseases;

[0058] (7) Use of the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the chimeric antigen receptor of the third aspect of the present invention, the nucleic acid molecule of the fourth aspect of the present invention, the expression vector of the fifth aspect of the present invention, the recombinant host cell of the sixth aspect of the present invention and / or the therapeutic conjugate of the seventh aspect of the present invention in the preparation of a pharmaceutical composition for treating and / or preventing B7H3-positive related diseases;

[0059] (8) Use of the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the chimeric antigen receptor of the third aspect of the present invention, the nucleic acid molecule of the fourth aspect of the present invention, the expression vector of the fifth aspect of the present invention, the recombinant host cell of the sixth aspect of the present invention, the therapeutic conjugate of the seventh aspect of the present invention and / or the pharmaceutical composition of the seventh aspect of the present invention in the preparation of a pharmaceutical preparation for treating and / or preventing a B7H3-positive related disease;

[0060] Optionally, the B7H3-positive related disease is ovarian cancer, prostate cancer, liver cancer, lung cancer, kidney cancer, breast cancer, colorectal cancer, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, bladder cancer, osteosarcoma, glioma or myeloid leukemia.

[0061] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0062] The present invention provides a novel fully human monoclonal antibody AHF23050 targeting B7H3, wherein the antibody AHF23050 can specifically bind to cells expressing B7H3. The present invention also provides a chimeric antigen receptor, a CAR-T cell, a pharmaceutical composition, a pharmaceutical preparation, an immunoconjugate, a detection reagent, etc. comprising the antibody AHF23050. The present invention has been experimentally verified to have found that the CAR-T cell can efficiently and specifically kill tumor cells expressing B7H3, and has broad prospects for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] 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 ;

[0064] Figure 2 : Flow cytometry technology to detect antibody specificity, where AB figure: prostate cancer cell DU145, CD figure: prostate cancer cell DU145 B7H3 KO ;

[0065] Figure 3 : Antibody-mediated NK cell ADCC killing effect;

[0066] 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;

[0067] 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: lung cancer cell A549;

[0068] 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: lung cancer cell A549, Figure D: lung cancer cell H1299. DETAILED DESCRIPTION

[0069] 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.

[0070] Example 1 Monoclonal antibody AHF23050 targeting B7H3

[0071] The present invention constructs a fully human monoclonal antibody AHF23050 that specifically targets B7H3. The sequence information of the AHF23050 antibody is shown in Table 1 below. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 9. In addition, this example demonstrates through experiments that the AHF23050 antibody has a good affinity for B7H3.

[0072] Table 1 Sequence of AHF23050 antibody

[0073]

[0074]

[0075] Example 2 Flow cytometry to detect the specificity of antibody AHF23050

[0076] 1. Detection of B7H3 expression in prostate cancer cells by flow cytometry

[0077] 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:

[0078] (1) Collect prostate cancer DU145 and DU145 B7H3 KO Cells were incubated with antibody AHF23050 (1:200) at 4°C for 30 min;

[0079] (2) Wash with PBS, centrifuge and discard the supernatant, resuspend the cell pellet in 300 μL PBS, and filter into a flow tube;

[0080] (3) Streaming on-machine testing.

[0081] 2. Detection of antibody specificity by flow cytometry

[0082] In order to determine whether the B7H3 antibody AHF23050 used can specifically bind to the B7H3 antigen expressed by cells, in this example, ① antibody AHF23050; 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:

[0083] (1) Select cells DU145 and DU145 B7H3 KO , 2-5×10 5 / Sample;

[0084] (2) Antibody selection: ① Antibody AHF23050; ② Commercialized B7H3 antibody (Enoblituzumab MGA271);

[0085] (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;

[0086] (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).

[0087] 3. Experimental results

[0088] 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.

[0089] 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 AHF23050 showed strong specific binding ability to DU145 expressing B7H3, and was superior to commercial antibodies.

[0090] Example 3: The fully human B7H3 antibody AHF23050 can kill DU145 cells through ADCC effect

[0091] 1. Experimental methods

[0092] This example uses RTCA experiment to detect the killing effect of DU145 cells after being treated with antibody AHF23050 and added with NK cells. The specific experimental steps are as follows:

[0093] (1) Prostate cancer DU145 cells were collected and seeded into RTCA plates at 20,000 cells per well;

[0094] (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 AHF23050; 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;

[0095] (3) Add 50 μL of NK cell suspension at a 1:1 effector-target ratio and continue culturing and observation for 2-3 days;

[0096] (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.

[0097] 2. Experimental results

[0098] 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 AHF23050 can kill DU145 cells through the ADCC effect.

[0099] Example 4 Preparation and detection of fully human B7H3 CAR-T cells

[0100] 1. Experimental methods

[0101] The CAR expression plasmid was constructed using the sequence of the above-mentioned antibody AHF23050, and viral packaging was performed. The collected retroviral fluid was used to infect T cells to prepare CAR-T, named AHF23050.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:

[0102] (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;

[0103] (2) After T cell activation, 0.5×10 6 / mL was divided into 1.5mL EP tubes, 1mL virus solution (AHF23050.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;

[0104] (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;

[0105] (4) Resuspend the cell pellet in 300 μL PBS, filter it into a flow cytometer, and detect it on a flow cytometer.

[0106] 2. Experimental results

[0107] 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.

[0108] Table 2 Coding sequences of fully human chimeric antigen receptors (CARs) targeting B7H3

[0109]

[0110]

[0111]

[0112] Example 5 Tumor sphere experiment to detect the killing effect of CAR-T cells prepared based on fully human B7H3 antibody (AHF23050) on tumor cells

[0113] 1. Experimental methods

[0114] 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 AHF23050.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:

[0115] (1) Add 0.15 g agarose to 10 mL DMEM and autoclave at 121°C for 20 min.

[0116] (2) Add 50 μL of hot agarose solution to each well of a 96-well plate and cool to room temperature;

[0117] (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;

[0118] (4) Observe the size of the tumor sphere under a microscope. When the diameter is about 300 μm, take a photo and record it;

[0119] (5) Collect effector cells (AHF23050.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;

[0120] (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;

[0121] (7) Take photos and record observations under a fluorescence microscope.

[0122] 2. Experimental results

[0123] 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 (AHF23050) have a good killing effect on tumor cells.

[0124] Example 6 RTCA detection of CAR-T cells against B7H3 + Tumor cell killing effect

[0125] 1. Experimental methods

[0126] In order to verify the killing ability of CAR-T cells against B7H3-positive tumors, RTCA was used to detect the killing effect of AHF23050.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:

[0127] (1) Collect tumor cells and inoculate 20,000 cells per well in RTCA plates;

[0128] (2) When the cell index of tumor cell growth reaches about 1, add the corresponding AHF23050.CAR-T cells;

[0129] (3) The RTCA instrument automatically records the cell growth index;

[0130] (4) After 2-3 days, stop RTCA and automatically generate a curve reflecting the cell growth index.

[0131] 2. Experimental results

[0132] 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 (AHF23050) have a good killing effect on tumor cells.

Claims

1. A monoclonal antibody targeting B7H3, characterized in that: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-3, and the light chain variable region comprises LCDR1-3; The HCDR1-3 are the HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO:4; The LCDR1-3 is LCDR1-3 in the light chain variable region as shown in SEQ ID NO:

9.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequences of HCDR1-3 are shown in SEQ ID NO:1-3, respectively, or have at least 70% homology with SEQ ID NO:1-3, respectively; Optionally, the amino acid sequences of LCDR1-3 are as shown in SEQ ID NO: 6-8, respectively, or have at least 70% homology with SEQ ID NO: 6-8, respectively; Optionally, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4 or has at least 70% homology to SEQ ID NO:4; Optionally, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9 or has at least 70% homology to SEQ ID NO:

9.

3. A bispecific antibody, characterized in that: The bispecific antibody comprises the monoclonal antibody according to claim 1 or 2.

4. A chimeric antigen receptor targeting B7H3, characterized in that: The chimeric antigen receptor comprises the monoclonal antibody of claim 1 or 2; Optionally, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, and a co-stimulatory signaling domain; Optionally, the chimeric antigen receptor further comprises any one or more of a signal peptide, a self-cleaving peptide, a safety switch, an immunomodulatory molecule or a cytokine; Optionally, the transmembrane domain is selected from the transmembrane domain of any one of the following molecules: CD8, CD28, 4-1BB, CD34, CD3ε, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the intracellular signaling domain is selected from the intracellular signaling domain of any one of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d; Optionally, the hinge region is selected from the hinge region of any one of the following molecules: CD8, CD28, CD34, 4-1BB, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the costimulatory signaling domain is selected from the costimulatory signaling domains of any one of the following molecules: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, CD226; Optionally, the signal peptide is selected from the signal peptides of any one of the following molecules: α chain and β chain of T cell receptor, CD3ζ, CD3ε, CD16, CD22, CD33, CD4, CD5, CD8, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, GM-CSF; Optionally, the self-cleaving peptide is selected from any one of the following self-cleaving peptides: T2A, P2A, E2A, F2A; Optionally, the safety switch is selected from any one of the following safety switches: tEGFR, iCaspase-9, RQR8; Optionally, the immunomodulatory molecule or cytokine is selected from any one of the following immunomodulatory molecules or cytokines: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, IL-15, IL-18, IL-21, LEC, OX40L; Optionally, the chimeric antigen receptor is obtained by sequentially connecting a signal peptide, a heavy chain variable region of the monoclonal antibody according to claim 1 or 2, a linker, a light chain variable region of the monoclonal antibody according to claim 1 or 2, 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; 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.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding the monoclonal antibody of claim 1 or 2, the bispecific antibody of claim 3, or the chimeric antigen receptor of claim 4; Optionally, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody of claim 1 or 2 is shown in SEQ ID NO: 14; Optionally, the nucleotide sequence of the light chain variable region of the monoclonal antibody of claim 1 or 2 is shown in SEQ ID NO: 18; Optionally, the nucleotide sequence of the signal peptide in the chimeric antigen receptor of claim 4 is shown in SEQ ID NO: 12; Optionally, the nucleotide sequence of Linker in the chimeric antigen receptor of claim 4 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 4 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 4 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 4 is shown in SEQ ID NO: 24; Optionally, the nucleotide sequence of the T2A self-cleaving peptide in the chimeric antigen receptor of claim 4 is shown in SEQ ID NO: 26; Optionally, the nucleotide sequence of the tEGFR safety switch in the chimeric antigen receptor of claim 4 is shown in SEQ ID NO: 28; Optionally, the nucleotide sequence of the chimeric antigen receptor according to claim 4 is shown in SEQ ID NO:

30.

6. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 5; Optionally, the vector is a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector or a vector of viral origin; Optionally, the virus-derived vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a poxvirus vector or a herpesvirus vector.

7. A recombinant host cell, characterized in that The recombinant host cell comprises the expression vector of claim 6; Optionally, the host cell is a eukaryotic cell and / or a prokaryotic cell; Optionally, the host cell is an immune cell; Optionally, the immune cells are T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof.

8. Any of the following products, characterized in that: The products include: (1) A diagnostic conjugate comprising the monoclonal antibody according to claim 1 or 2 and / or the bispecific antibody according to claim 3; (2) A detection reagent comprising the monoclonal antibody according to claim 1 or 2, the bispecific antibody according to claim 3 and / or the diagnostic conjugate; (3) A detection product, comprising the monoclonal antibody according to claim 1 or 2, the bispecific antibody according to claim 3, the diagnostic conjugate and / or the detection reagent; (4) A therapeutic conjugate, comprising the monoclonal antibody of claim 1 or 2, the bispecific antibody of claim 3, and / or the recombinant host cell of claim 7; (5) A pharmaceutical composition comprising the monoclonal antibody according to claim 1 or 2, the bispecific antibody according to claim 3, the recombinant host cell according to claim 7 and / or the therapeutic conjugate; (6) A pharmaceutical preparation comprising the monoclonal antibody according to claim 1 or 2, the bispecific antibody according to claim 3, the recombinant host cell according to claim 7, the therapeutic conjugate and / or the pharmaceutical composition; (7) A drug kit comprising a therapeutically effective amount of the monoclonal antibody according to claim 1 or 2, the bispecific antibody according to claim 3, the recombinant host cell according to claim 7, the therapeutic conjugate, the pharmaceutical composition and / or the pharmaceutical preparation; Optionally, the diagnostic conjugate is a complex formed by the monoclonal antibody of claim 1 or 2 or the bispecific antibody of claim 3 directly or indirectly bound to a diagnostic agent; Optionally, the diagnostic agent is a radionuclide, a contrast agent and / or a photosensitive diagnostic agent; Optionally, the detection product is a detection kit, a detection test strip or a detection chip; Optionally, the therapeutic conjugate is a complex formed by the monoclonal antibody of claim 1 or 2, the bispecific antibody of claim 3, or the recombinant host cell of claim 7 directly or indirectly binding to a therapeutic agent; Optionally, the kit is packaged in a container and further comprises a label affixed to or packaged in the container, the label describing the contents of the container and providing instructions and / or instructions for using the contents of the container to treat and / or prevent B7H3-positive related diseases.

9. Any of the following methods, characterized in that: The method comprises: (1) A method for producing the monoclonal antibody of claim 1 or 2 or the bispecific antibody of claim 3, the method comprising the following steps: culturing the recombinant host cell of claim 7, and isolating the monoclonal antibody of claim 1 or 2 or the bispecific antibody of claim 3 from the culture product of the recombinant host cell; (2) A method for preparing the recombinant host cell according to claim 7, the method comprising the following steps: introducing the expression vector according to claim 6 into a host cell to obtain the recombinant host cell according to claim 7; (3) A method for detecting B7H3 protein in a sample for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: contacting the sample with the monoclonal antibody of claim 1 or 2, the bispecific antibody of claim 3, the diagnostic conjugate, the detection reagent and / or the detection product of claim 8, and detecting the formation of an immune complex between the B7H3 protein and the antibody; (4) A method for inhibiting B7H3 protein activity in vitro, the method comprising the following steps: contacting a sample to be tested with the monoclonal antibody according to claim 1 or 2 and / or the bispecific antibody according to claim 3.

10. Any of the following applications, characterized in that: The applications include: (1) Use of the monoclonal antibody according to claim 1 or 2, the bispecific antibody according to claim 3 and / or the nucleic acid molecule according to claim 5 in the preparation of a diagnostic conjugate for detecting B7H3 protein; (2) Use of the monoclonal antibody according to claim 1 or 2, the bispecific antibody according to claim 3, the nucleic acid molecule according to claim 5 and / or the diagnostic conjugate according to claim 8 in the preparation of a detection reagent for detecting B7H3 protein; (3) Use of the monoclonal antibody of claim 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 5, the diagnostic conjugate of claim 8 and / or the detection reagent of claim 8 in the preparation of a detection product for detecting B7H3 protein; (4) Use of the monoclonal antibody of claim 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 5, the diagnostic conjugate of claim 8, the detection reagent of claim 8 and / or the detection product of claim 8 in detecting B7H3 protein for non-diagnostic and non-therapeutic purposes; (5) Use of the monoclonal antibody of claim 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 5, the diagnostic conjugate of claim 8, the detection reagent of claim 8 and / or the detection product of claim 8 in the preparation of a diagnostic product for diagnosing or assisting in the diagnosis of B7H3-positive related diseases; (6) Use of the monoclonal antibody according to claim 1 or 2, the bispecific antibody according to claim 3, the chimeric antigen receptor according to claim 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6 and / or the recombinant host cell according to claim 7 in the preparation of a therapeutic conjugate for treating and / or preventing B7H3-positive related diseases; (7) Use of the monoclonal antibody of claim 1 or 2, the bispecific antibody of claim 3, the chimeric antigen receptor of claim 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the recombinant host cell of claim 7 and / or the therapeutic conjugate of claim 8 in the preparation of a pharmaceutical composition for treating and / or preventing B7H3-positive related diseases; (8) Use of the monoclonal antibody of claim 1 or 2, the bispecific antibody of claim 3, the chimeric antigen receptor of claim 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the recombinant host cell of claim 7, the therapeutic conjugate of claim 8 and / or the pharmaceutical composition of claim 8 in the preparation of a pharmaceutical preparation for treating and / or preventing B7H3-positive related diseases; Optionally, the B7H3-positive related disease is ovarian cancer, prostate cancer, liver cancer, lung cancer, kidney cancer, breast cancer, colorectal cancer, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, bladder cancer, osteosarcoma, glioma or myeloid leukemia.

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