An anti-B7H3 nanobody and its application

Abstract

This invention belongs to the field of biomedical technology and discloses an anti-B7H3 nanobody and its applications. Specifically, it discloses an anti-B7H3 nanobody with a defined amino acid sequence. This invention provides an anti-B7H3 nanobody obtained by immunizing alpacas. It exhibits high affinity and specificity and can be used for immunoblotting, enzyme-linked immunosorbent assay (ELISA) kits, flow cytometry detection and counting, as well as for the treatment of tumors, autoimmune diseases, and other conditions.

Description

[0001] This application is a divisional application of Chinese application No. 2024119349338, filed on December 26, 2024, entitled "An anti-B7H3 nanobody and its application". Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to an anti-B7H3 nanobody and its application. Background Technology

[0003] Immune checkpoint blockade therapy, represented by PD-1 / PD-L1 and CTLA-4, has achieved remarkable efficacy in the treatment of solid tumors. However, the fact that less than 30% of patients benefit from it indicates the existence of other immune checkpoint molecules-mediated immunosuppression. B7H3 (also known as CD276) is a transmembrane glycoprotein, a member of the B7 family of immunomodulators, expressed on APC antigen-presenting cells. Unlike CTLA-4 / PD-1, which is mainly expressed on T cells and mediates their immunosuppression or exhaustion, B7H3 protein is not only inducibly expressed on immune cells but also constitutively highly expressed on various tumor cells and tumor-associated vascular systems, such as glioblastoma (GBM), neuroblastoma (NB), non-small cell lung cancer, cervical cancer, liver cancer, bladder cancer, ovarian cancer, osteosarcoma, hematologic malignancies, and renal cell carcinoma. B7H3 not only activates multiple signaling pathways to directly promote the malignant phenotype of tumor cells, but also plays an important role in the biological processes of tumorigenesis, proliferation, and migration. Some studies have shown that B7H3 plays an important role in various autoimmune diseases, such as lupus erythematosus, sepsis, arthritis, pancreatitis, and type 1 diabetes.

[0004] Traditional monoclonal antibodies are more susceptible to epitope loss due to chemical processing, while alpaca antibodies can withstand denaturation temperatures of 60-80°C. Furthermore, to ensure correct structural folding and post-translational modifications such as glycosylation during production, traditional monoclonal antibodies often utilize mammalian cell lines, resulting in high production costs and long production times. Non-fully human monoclonal antibodies also exhibit greater immunogenicity.

[0005] The high expression of B7H3 protein in tumor tissues and tumor-associated vascular tissues, and its low expression or absence in normal tissues, makes it an ideal tumor antigen target for CAR-T cell design. Current clinical trials of B7H3-targeted CAR-T cells have shown that B7H3-targeted CAR-T cells exhibit strong anti-tumor activity in various solid tumors and hematologic malignancies, with no significant toxicity detected. One type of B7H3-CAR-T cell (TAA-06) has already received orphan drug designation from the FDA for the treatment of neuroblastoma.

[0006] Studies have shown that combined blocking of B7H3 and PD-L1 can significantly increase tumor antigen-specific CD8. + T-cell anti-tumor effects. Phase I clinical trial results showed that enobritumab combined with CTLA-4 antibody or PD-1 antibody was more effective than either antibody alone. The combination therapy significantly enhanced intratumoral recruitment and anti-tumor activity of TILs, with no dose-dependent toxicity or serious adverse reactions observed. The overall response rate of enobritumab combined with pembrolizumab reached 36% and 33% in patients with newly diagnosed / metastatic non-small cell lung cancer and recurrent / metastatic head and neck squamous cell carcinoma, respectively.

[0007] Nanobodies (VHHs) share the same structural domains as conventional VH antibodies, namely four conserved framework regions (FR1 / 2 / 3 / 4) and three complement-determining regions (CDR1 / 2 / 3). Nanobodies have a molecular weight only 10% that of traditional antibodies, retaining the complete antigen-binding capacity of HCAbs, and exhibiting high specificity, good affinity, and high stability. Because nanobodies lack an Fc fragment, they cannot produce ADCC / CDC cytotoxic effects like conventional antibodies. Therefore, VHH antibodies are often fused with an Fc fragment to construct Fc-VHH fusion antibodies to enhance ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) activities. Compared to conventional antibodies, these forms of nanobodies can be widely used in the treatment of various diseases. Summary of the Invention

[0008] The first objective of this invention is to provide an anti-B7H3 nanobody.

[0009] A second aspect of the present invention is to provide a fusion antibody.

[0010] A third aspect of the present invention aims to provide biomaterials related to the anti-B7H3 nanobody of the first aspect of the present invention or the fusion antibody of the second aspect of the present invention.

[0011] A fourth aspect of the present invention is to provide an immunoconjugate.

[0012] The fifth aspect of this invention is to provide a medicine.

[0013] The sixth aspect of this invention aims to provide the application of the anti-B7H3 nanobody of the first aspect of this invention, the fusion antibody of the second aspect of this invention, the biomaterial of the third aspect of this invention, the immunoconjugate of the fourth aspect of this invention, or the drug of the fifth aspect of this invention.

[0014] The seventh aspect of this invention is to provide a reagent kit or medicine box.

[0015] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an anti-B7H3 nanobody, the anti-B7H3 nanobody comprising a complementarity-determining region (CDR), the CDR comprising complementarity-determining region CDR1, complementarity-determining region CDR2 and complementarity-determining region CDR3, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 of the anti-B7H3 nanobody are as shown in SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, respectively.

[0016] In some embodiments of the present invention, CDR1, CDR2 and CDR3 are separated by the backbone regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0017] In some embodiments of the present invention, the amino acid sequence of the anti-B7H3 nanobody is as shown in SEQ ID NO:46 or is an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:46 after substitution, deletion or addition of one or more amino acids.

[0018] A second aspect of the present invention provides a fusion antibody comprising a first domain and a second domain; The first structural domain is the anti-B7H3 nanobody of the first aspect of the present invention; The second domain has the effect of prolonging the in vivo half-life and / or binding to effector cells.

[0019] In some embodiments of the present invention, the second structural domain includes (but is not limited to): Immunoglobulin Fc region (such as the human immunoglobulin Fc region); and / or Serum albumin (such as human HSA) or fragments thereof, serum albumin-binding domains (such as anti-serum albumin antibodies, including nanobodies), polyethylene glycol, polyethylene glycol-liposome complexes, or combinations thereof; and / or Molecules that have an affinity for T cell surface molecules and / or are able to bind to surface molecules (such as CD3) present on T cells.

[0020] In some embodiments of the present invention, the human immunoglobulin Fc region includes mutations for altering Fc-mediated effector functions, the effector functions including one or more combinations of CDC activity, ADCC activity, and ADCP activity.

[0021] In some embodiments of the present invention, the immunoglobulin is one or more selected from IgG, IgA1, IgA2, IgD, IgE, and IgM.

[0022] In some embodiments of the present invention, the IgG is selected from one or more combinations of IgG1, IgG2, IgG3 or IgG4 subtypes.

[0023] In some embodiments of the present invention, the amino acid sequence of the Fc region of the immunoglobulin is as shown in SEQ ID NO:56; or an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:56 after substitution, deletion or addition of one or more amino acids.

[0024] In some embodiments of the present invention, the first structural domain is connected to the N-terminus or C-terminus of the second structural domain.

[0025] In some embodiments of the present invention, the first and second domains are hinged together (e.g., the anti-B7H3 nanobody is directly fused to the hinge region of Fc) or connected via a linker peptide.

[0026] In some embodiments of the present invention, the linker peptide is a flexible linker.

[0027] In some embodiments of the present invention, the flexible linker amino acid sequence includes, but is not limited to, GSAS and (GGCAGCGCCAGC). n (GGCGGCGGCAGC) n (GGCGGCGGCGGCAGC) n YAPVDV, (GGGS) n (GGSG) n (GGGGS) n (G) n , where 1≤n≤5, and n is an integer.

[0028] In some embodiments of the present invention, the amino acid sequence of the fusion antibody is as shown in SEQ ID NO:1-8.

[0029] A third aspect of the present invention provides biomaterials related to the anti-B7H3 nanobody of the first aspect of the present invention or the fusion antibody of the second aspect of the present invention; said biomaterials are any one of a1) to a12): a1) A nucleic acid molecule encoding the anti-B7H3 nanobody of the first aspect of the present invention or the fusion antibody of the second aspect of the present invention; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecule described in a1); a4) A recombinant vector containing the expression cassette described in a2); a5) Recombinant microorganisms containing the nucleic acid molecules described in a1); a6) Recombinant microorganisms containing the expression cassette described in a2); a7) Recombinant microorganisms containing the recombinant vector described in a3); a8) Recombinant microorganisms containing the recombinant vector described in a4); a9) Transgenic cell lines containing the nucleic acid molecules described in a1); a10) Transgenic cell lines containing the expression cassette described in a2); a11) Transgenic cell lines containing the recombinant vector described in a3); a12) Transgenic cell lines containing the recombinant vector described in a4).

[0030] In some embodiments of the present invention, the nucleic acid molecule encoding the anti-B7H3 nanobody of the first aspect of the present invention is shown in SEQ ID NO:47.

[0031] In some embodiments of the present invention, the nucleic acid molecule encoding the fusion antibody of the second aspect of the present invention is shown in SEQ ID NO:15.

[0032] In some embodiments of the present invention, the transgenic animal cell line does not contain reproductive material.

[0033] In some embodiments of the present invention, the expression cassette includes a 5' transcription control region, an open reading frame encoding the fusion antibody of the first aspect of the present invention, a translation control signal, a 3' untranslated region (3'UTR), and a transcription termination signal.

[0034] In some embodiments of the present invention, the 5' transcriptional control region includes a promoter (which may be a universal promoter, such as a viral promoter (SV40 promoter) or a mammalian "housekeeper" promoter), a transcription start site, an enhancer, and / or a silencing element.

[0035] In some embodiments of the invention, the 3'UTR may encode AU-rich elements, which are common regulators of mRNA stability via the 3'-5' exogenous pathway and are typically located in the 3'UTR. AU-rich elements may comprise one or more repeats of the sequence AUUUA. It may also comprise one or more so-called US2B elements with the sequence AUAUAU.

[0036] In some embodiments of the present invention, the vector includes a promoter that is operatively linked to the nucleic acid molecule.

[0037] In some embodiments of the present invention, the vector is independently selected from non-pathogenic viral vectors and viral vectors.

[0038] In some embodiments of the present invention, the viral vector includes at least one of lentiviral vector, adenovirus vector, baculovirus vector, retrovirus vector, poxvirus vector, Sendai virus vector, and herpes simplex virus vector.

[0039] In some embodiments of the present invention, the non-viral vector includes at least one of plasmid vectors, cationic polymer vectors, chitosan, polyethyleneimine, nanoparticle vectors, and liposomes.

[0040] In some embodiments of the present invention, the vector is a plasmid vector, a phage particle, a viral vector, a cell vector, a bacteriophage, a phage, an F phage, or an artificial chromosome.

[0041] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0042] In some embodiments of the present invention, the recombinant expression vector uses pET-28a(+) as the original expression vector.

[0043] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new plant or animal varieties.

[0044] In some embodiments of the present invention, the prokaryotic cells include bacteria well known in the art, such as Escherichia coli, Streptomyces, and Bacillus subtilis, which are capable of expressing the target protein.

[0045] In some embodiments of the present invention, the eukaryotic cells include at least one of yeast cells, mammalian cells, plant cells, and insect cells.

[0046] A fourth aspect of the present invention provides an immunoconjugate comprising: (A) The anti-PD-L1 nanobody of the first aspect of the present invention or the fusion antibody of the second aspect of the present invention; and (B) Functional molecules that are connected to (A) (including but not limited to covalent linkage, coupling, attachment, and adsorption).

[0047] In some embodiments of the present invention, (B) includes cytotoxins, radioisotopes, bioactive proteins, molecules targeting tumor surface markers, molecules that inhibit tumors, molecules or detectable markers targeting immune cell surface markers, extracellular hinge regions based on chimeric antigen receptor technology, transmembrane regions (such as the transmembrane regions of CD8 or CD28), and intracellular signaling regions (such as the CD3ζ chain, FcεRIγ tyrosine activation motif, intracellular signaling regions of co-stimulatory signaling molecules CD27, CD28, CD137, CD134, MyD88, CD40, etc.), or combinations thereof.

[0048] In some embodiments of the present invention, the molecules that target tumor surface markers are antibodies or ligands that bind to tumor surface markers.

[0049] In some embodiments of the present invention, the tumor-inhibiting molecules are anti-tumor cytokines (such as IL-12, IL-15, IFN-beta, TNFalpha) or anti-tumor toxins.

[0050] In some embodiments of the present invention, the detectable marker is selected from radioactive isotopes, fluorescent substances, chemiluminescent substances, colored substances, or any combination thereof.

[0051] In some embodiments of the present invention, (B) is selected from: fluorescent substances, chemiluminescent markers, colored substances, radioactive isotopes, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes, chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0052] In a fifth aspect, the present invention provides a medicament comprising an anti-B7H3 nanobody of the first aspect of the present invention, a fusion antibody of the second aspect of the present invention, a biomaterial of the third aspect of the present invention, or an immunoconjugate of the fourth aspect of the present invention.

[0053] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0054] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one selected from fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, humectants, flavoring agents, solubilizers, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, fragrances, and solvents.

[0055] In some embodiments of the present invention, the drug further includes combination drugs, including but not limited to immune effector molecules, cells, cytotoxic substances, and multi-kinase inhibitors.

[0056] A sixth aspect of the present invention provides the use of the anti-B7H3 nanobody of the first aspect of the present invention, the fusion antibody of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, the immunoconjugate of the fourth aspect of the present invention, or the drug of the fifth aspect of the present invention in any one of (1) to (4): (1) To prepare preparations, kits or boxes for the diagnosis, treatment or prevention of cancer; (2) To prepare preparations or kits for the treatment or prevention of autoimmune diseases; (3) Quantitative / qualitative detection of B7H3 expression level; (4) Prepare reagents / kits for quantitative / qualitative detection of B7H3 expression.

[0057] It can also be used for targeted delivery of traditional chemotherapy drugs (Chinese herbal monomers, microRNA, etc.).

[0058] In some embodiments of the present invention, the cancer described in (1) includes at least one of glioblastoma, neuroblastoma, non-small cell lung cancer, cervical cancer, liver cancer, bladder cancer, prostate cancer, colon cancer, melanoma, ovarian cancer, osteosarcoma, neuroblastoma, hematologic malignancy, and kidney cancer.

[0059] In some embodiments of the present invention, the autoimmune disease described in (2) includes at least one of lupus erythematosus, sepsis, arthritis, pancreatitis and type I diabetes.

[0060] In some embodiments of the present invention, the kit / kit described in (4) includes reagents for immunoblotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry for counting.

[0061] A seventh aspect of the present invention provides a kit or pharmaceutical kit comprising an anti-B7H3 nanobody of the first aspect of the present invention, a fusion antibody of the second aspect of the present invention, a biomaterial of the third aspect of the present invention, an immunoconjugate of the fourth aspect of the present invention, or a drug of the fifth aspect of the present invention.

[0062] In some embodiments of the present invention, the kit can be used for immunoblotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry for cell counting.

[0063] The beneficial effects of this invention are: The anti-B7H3 nanobody obtained by immunizing alpacas in this invention has high affinity and strong specificity. It can be used for immunoblotting, enzyme-linked immunosorbent assay kits, flow cytometry detection and counting, and can also be used for the treatment of diseases such as tumors, autoimmune diseases and chronic infections. It has the potential to be developed as an immune checkpoint inhibitor. Attached Figure Description

[0064] Figure 1 This is a schematic diagram of the structure of the fusion antibody of the present invention.

[0065] Figure 2 The antigen lentiviral vector atlas (A) and the flow cytometry report of the B7H3 target antigen cell line construction were used for the construction of the B7H3 target antigen cell line (B).

[0066] Figure 3 The results of flow cytometry analysis of the cell affinity of the supernatant of fusion antibodies FP878, FP879, FP880, FP881, FP882 and FP883 for the target antigens are presented.

[0067] Figure 4 The results of flow cytometry analysis of the cell affinity of the supernatant of fusion antibodies FP884 and FP885 for the target antigen are shown.

[0068] Figure 5 The results of flow cytometry analysis of the affinity of each fusion antibody for non-target antigens on cell clearing cells are shown.

[0069] Figure 6 Flow cytometry report for quantitatively detecting the binding ability of fusion antibodies FP878, FP879, FP880 or FP881 to target cells.

[0070] Figure 7 Flow cytometry report for quantitatively detecting the binding ability of fusion antibodies FP882, FP883, FP884 or FP885 to target cells.

[0071] Figure 8 To quantitatively detect the binding ability of each fusion antibody to Anti-B7H3 in target cells—MFI.

[0072] Figure 9 To quantitatively detect the binding ability of each fusion antibody to Anti-B7H3 in target cells—the proportion of positive cells. Detailed Implementation

[0073] The present invention will be further described in detail below through specific embodiments.

[0074] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0075] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0076] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0077] Example 1 Plasmid Construction Multiple candidate nanobody sequences were obtained by immunizing alpacas with the B7H3 target antigen amino acid sequence. Using multi-fragment homologous recombination technology, the VHH region nucleic acid sequence of the nanobody was ligated with a human IgG Fc nucleic acid sequence, with the hinge region as the ligation region. The plasmids after homologous recombination were identified by enzyme digestion and then sequenced to determine the fusion antibody sequence structure. Figure 1 As shown in the figure. Eight fusion antibodies, namely FP878, FP879, FP880, FP881, FP882, FP883, FP884 and FP885, were constructed, and their amino acid sequences are shown in SEQ ID NO:1 to 8, and their nucleotide sequences are shown in SEQ ID NO:9 to 16.

[0078] The amino acid sequence of VHH of the fusion antibody FP878 is shown in SEQ ID NO:17, the nucleotide sequence is shown in SEQ ID NO:18, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO:19 to 21, respectively.

[0079] QVQLVESGGGLVQAGGSLRLSCAASGRTSGNYIMGWFRQAPGKEREFVAAINWTPGTTYYANSVKGRFTISGDNAKNTVYLQMNGLRAEDTAVYSCGARLGWNIQNTNYPYWGQGTQVTVS (SEQ ID NO: 17); ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGGACTTCCGGTAACTATATCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCAGCTATTAA CTGGACTCCTGGTACTACATACTATGCAAACTCCGTGAAGGGCCGATTCACCATCTCCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACGGCCTGAGAGCTGAGGACACGGCCGTTTATTCCTGTGGAGCTCGGTTGGGTTGGAATATTCAAAATACGAATTATCCCTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCC (SEQ ID NO:18); CDR1: GRTSGNYI (SEQ ID NO: 19); CDR2: INWTPGTT (SEQ ID NO: 20); CDR3: GARLGWNIQNTNYPY (SEQ ID NO: 21).

[0080] The amino acid sequence of VHH of the fusion antibody FP879 is shown in SEQ ID NO:22, the nucleotide sequence is shown in SEQ ID NO:23, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO:24 to 26 respectively.

[0081] QVQLVESGGGLVQAGGSLRLSCAASGRTSGNYIQGWFRQAPGKEREFVAAINWSAGTTYYADSVKGRFTISRDNAKNTVYLQMTSLRAEDTAVYSCGARPGWNIQNTNYQHWGQGTQVTVS (SEQ ID NO: 22); ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGGACTTCCGGTAACTATATCCAGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCAGCTATTAA CTGGAGGTGCTGGTACTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGACTAGCCTGAGAGCTGAGGACACGGCCGTTTATTCCTGTGGAGCTCGGCCGGGTTGGAATATTCAAAATACGAATTATCAACACTGGGGCCAGGGGGACCCAGGTCACCGTCTCC (SEQ ID NO:23); CDR1: GRTSGNYI (SEQ ID NO: 19); CDR2: INWSAGTT (SEQ ID NO: 24); CDR3: GARPGWNIQNTNYQH (SEQ ID NO: 25).

[0082] The amino acid sequence of VHH of the fusion antibody FP880 is shown in SEQ ID NO:26, the nucleotide sequence is shown in SEQ ID NO:27, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO:28 to 30 respectively.

[0083] QVQLVESGGGLVQPGGSLRLSCGASGRTFSRDTMAWFRQAPGKEREFVAGISWLGATTYYADSVKGRFTISRDNAQNTVYLQMNAPKPEDTAEYYCAASSRLDPRVRTTDMDYWGKGTLVTVS (SEQ ID NO: 26); ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGGTCTCTGAGACTCTCCTGTGGAGCCTCTGGACGCACCTTCAGTAGGGATACCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGGTATTAGCTG GCTTGGTGCGACCACATACTATGCAGACTCCGTAAAGGGCCGATTCACCATCTCCAGAGACAACGCCCAGAACACGGTGTATCTGCAAATGAACGCACCGAAACCTGAGGACACGGCCGAATATTACTGTGCAGCAAGTTCCCGGCTAGATCCCCGAGTACGTACTACGGACATGGACTACTGGGGCAAAGGGACCCTGGTCACCGTCTCC (SEQ ID NO:27); CDR1: GRTFSRDT (SEQ ID NO:28); CDR2: ISWLGATT (SEQ ID NO:29); CDR3: AASSRLDPRVRTTDMDY (SEQ ID NO:30).

[0084] The amino acid sequence of VHH of the fusion antibody FP881 is shown in SEQ ID NO:31, the nucleotide sequence is shown in SEQ ID NO:32, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO:33 to 35 respectively.

[0085] QVQLVESGGGLVQAGGSLRLSCAASGATFSSLLLGWFRQAPGKEREFVALRSGIGAITYYADSVEDRFTISRDSTKNTVYLQMNDLKPEDTAVFYCAALPLGSFTYSRPRSYDYWGQGTQVTVS (SEQ ID NO:31); ATGGAAACCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGCCACCTTCAGCAGTCTCTCTTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTGGCACTTCGTAGCGGGAT TGGTGCAATCACATACTATGCAGACTCCGTGGAGGACCGATTCACCATCTCCAGAGACAGCACCAAGAACACGGTGTATCTGCAAATGAACGACCTGAAACCTGAGGACACGGCCGTTTTTTACTGTGCAGCGCGACCCCTGGGTGGTAGTTTCACATATAGCCGTCCCAGATCTTATGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCC (SEQ ID NO:32); CDR1: GATFSSLL (SEQ ID NO:33); CDR2: RSGIGAIT (SEQ ID NO:34); CDR3: AARPLGSFTYSRPRSYDY (SEQ ID NO:35).

[0086] The amino acid sequence of VHH of the fusion antibody FP882 is shown in SEQ ID NO:36, the nucleotide sequence is shown in SEQ ID NO:37, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO:38 to 40, respectively.

[0087] QVQLVESGGGLVQAGGSLRLSCAASGRTSGAYVMGWFRQAPGKEREFVAAIQWTAGTTYYADSAEGRFIISRDNAKNTVYLEMNSLRTEDAAVYSCGARPGWNVQSTNYPYWGQGTQVTVS (SEQ ID NO:36); ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGGACTTCCGGTGCCTATGTCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATTCA GTGGACTGCTGGTACTACATACTATGCAGACTCCGCGGAGGGCCGATTCATCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGGAAATGAACAGCCTGAGAACTGAGGACGCGGCCGTTTATTCCTGTGGAGCTCGGCCGGGTTGGAATGTTCAAAGTACAAATTATCCCTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCC (SEQ ID NO:37); CDR1: GRTSGAYV (SEQ ID NO:38); CDR2: IQWTAGTT (SEQ ID NO:39); CDR3: GARPGWNVQSTNYPY (SEQ ID NO:40).

[0088] The amino acid sequence of VHH of the fusion antibody FP883 is shown in SEQ ID NO:41, the nucleotide sequence is shown in SEQ ID NO:42, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO:43 to 45 respectively.

[0089] QLQLVESGGGLVQSGGSLRLSCTASGYTLNYYAIGWFRQAPGKEREGISCITSIGTGGSTNYADSVKGRFTISRDSAKNTVYLQTYSLKPEDTAVYYCAAAPRFGSSCNRSGGMYDYWGQGTQVTVS (SEQ ID NO:41); ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGTCTGGGGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATACACTTTGAATTACTACGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGATCTCATGTATCACTAGTATTGG TACCGGTGGTAGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAACACGGTGTATCTCCAAACGTATAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCGGCCGCCCCTCGCTTCGGTAGTAGCTGTAACCGGTCAGGGGGAATGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCC (SEQ ID NO:42); CDR1: GYTLNYYA (SEQ ID NO:43); CDR2: ITSIGTGGST (SEQ ID NO:44); CDR3: AAAPRFGSSCNRSGGMYDY (SEQ ID NO:45).

[0090] The amino acid sequence of VHH of the fusion antibody FP884 is shown in SEQ ID NO:46, the nucleotide sequence is shown in SEQ ID NO:47, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO:48-50, respectively.

[0091] QLQLVESGGRSVQAGGSLRLSCTASGGTLSSFSNYAMAWFRQAPGKEREFVIAIGRSSGSLSYATTADSVRGRFTMSRDNGRNTAYLHMINLKPEDTAVYYCAARSGADADAPRVDNEYYYWGQGTQVTVS (SEQ ID NO:46); ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGTTGCAGCTCGTGGAGTCTGGGGGACGATCGGTGCAGGCTGGGGGTTCTCTGAGACTCTCCTGTACAGCCTCTGGAGGCACCCTCAGTAGCTTCAGTAACTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAACGTGAGTTTGTTATCGCTATTGGGAGGAGTAGTGGTAGTTTAAGTTATGCAACCACTGCAGACTCCGTGAGGGGCCGATTCACCATGTCCAGAGACAACGGCAGGAACACGGCGTATCTGCACATGATTAACCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCCGCTAGGAGTGGTGCCGACGCCGACGCGCCGCGGGTCGACAATGAATATTACTATTGGGGCCAGGGGACCCAGGTCACCGTCTCC (SEQ ID NO: 47); CDR1: GGTLSSFSNYA (SEQ ID NO: 48); CDR2: IGRSSGSLSYA (SEQ ID NO: 49); CDR3: AARSGADADAPRVDNEYYY (SEQ ID NO: 50).

[0092] The amino acid sequence of the VHH of fusion antibody FP885 is shown as SEQ ID NO: 51, the nucleotide sequence is shown as SEQ ID NO: 52, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown sequentially as SEQ ID NOs: 53 to 55.

[0093] QVQLVESGGGLAQAGGSLRLSCDASGRTVNEYNMGWLRQAPGKGREFVGAISWSGQIGYADPVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCTAARRWMGIVSPTDYEYRGRGTQVTVS (SEQ ID NO: 51); ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGATTGGCGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGACGCCTCTGGACGCACCGTCAATGAGTACAACATGGGCTGGCTCCGCCAGGCTCCAGGGAAGGGGCGCGAGTTTGTTGGAGCTATCAGCTGGAGTGGTCAAATAGGTTATGCAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGATGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGCACAGCCGCGCGTAGATGGATGGGAATTGTCTCACCAACCGACTATGAGTACCGGGGCCGCGGGACCCAGGTCACCGTCTCC (SEQ ID NO: 52); CDR1: GRTVNEYN (SEQ ID NO: 53); CDR2: ISWSGQI (SEQ ID NO: 54); CDR3: TAARRWMGIVSPTDYEY (SEQ ID NO: 55).

[0094] The humanized IgG Fc amino acid sequence is set forth in SEQ ID NO: 56, and the nucleotide sequence is set forth in SEQ ID NO: 57.

[0095] EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 56); (SEQ ID NO:57).

[0096] The specific experimental procedures for constructing plasmids for each antibody are as follows: (1) Preparation and procedure of single-fragment amplification reaction system Prepare the single-fragment amplification reaction system as shown in Table 1, and perform the reaction according to the reaction procedure shown in Table 2. The primers for PCR amplification of the VHH region of the Anti-B7H3 nanobody are shown in Table 3, and the amplified fragment size is 408 bp.

[0097] hIgG1-Fc fragment amplification primers: F-IGg1: CTGAGCTGCTGGGAGGAccg (SEQ ID NO:58) (70% GC Tm=68); R-IGg1:AAAAGGCGCAACCCGCTAGCtcatttacccggagacagggag (SEQ ID NO:59) (54%GC Tm=67); Table 1 Single-fragment amplification reaction system

[0098] Table 2 Reaction Procedure

[0099] Table 3 Primers for PCR amplification of the VHH region of Anti-B7H3 nanobody

[0100] (2) Multi-fragment homologous recombination preparation system and procedure The above two fragments were homologously recombinated with the 8044bp lentiviral vector backbone recovered after double digestion with NotI and NheI. The system preparation is shown in Table 4.

[0101] Prepare the multi-fragment homologous recombination reaction system as shown in Table 4. Perform the multi-fragment recombination reaction at 50°C for 15 min; then cool to 4°C or immediately place on ice to cool.

[0102] Table 4. Multi-fragment homologous recombination reaction system

[0103] (3) Construction of lentiviral vectors for overexpressing target antigen genes 1) Fragment PCR: The amino acid sequence of the B7H3 target antigen (SEQ ID NO:63) was constructed into a lentiviral shuttle plasmid. The PCR amplification primers are as follows: F-EF1-B7H3:catttcaggtgtcgtgaagcggccgcgccaccatgctgcgtcggcgg (SEQ ID NO: 64); P2A-B7H3:gAAgTTaGTAGCTCCggatccggctatttcttgtccatcatc (SEQ ID NO:65) The large fragment 7802bp / 718bp was recovered by NotI-BamHI double digestion; and a lentiviral expression vector for B7H3 target antigen was constructed by combining it with the single fragment GIBSON of B7H3 target antigen. Figure 2 (A)

[0104] 2) Multi-fragment homologous recombination preparation system and procedure Prepare the multi-fragment homologous recombination reaction system as shown in Table 4, and carry out the reaction according to the following reaction procedure: multi-fragment recombination reaction, 50℃, 15min; cool to 4℃ or immediately place on ice to cool.

[0105] Example 2 Plasmid Extraction (1) Transform the vector with the correct sequence alignment into a plasmid at 37°C for 14 h. (2) Pick single colonies, culture a small amount of bacterial solution (4 mL Amp+ LB (100 mg / L Amp+)) at 37℃ and 220 rpm for 10 h. (3) Large-scale culture of bacterial culture: The next day, the seed culture was inoculated into 250 mL of Amp+ LB (100 mg / L Amp+), and cultured at 37 ℃ and 220 rpm for 14 h. (4) Plasmid extraction was performed using the Tiangen plasmid large-scale extraction kit.

[0106] Example 3: Antibody Production (1) Observe the 293T cells used for packaging under a microscope and screen out cells with a cell density of 80% to 95%.

[0107] (2) Prepare 10% packaged culture medium. Open a new bottle of 500 mL DMEM culture medium, add 55 mL FBS, then add 550 μL of 1 mol / L sodium pyruvate solution and 550 μL of 25 mmol / L chloroquine phosphate solution, and mix well.

[0108] (3) Take out the selected cells from the incubator, pour the old culture medium in the culture flask into the waste liquid tank, add 17 mL of 10% packaged culture medium, and then put it back into the carbon dioxide incubator to adapt.

[0109] (4) Calculate the required amount of plasmid (prepared in Example 2, 30 μg plasmid per bottle) and 0.125 mol / L calcium chloride solution to prepare a DNA-CaCl2 mixture, based on the number of culture flasks to be transfected. Then, add an equal volume of 2×HBS solution dropwise to the DNA-CaCl2 mixture while vortexing the mixture. After the addition is complete, allow it to stand at room temperature for 20 min.

[0110] (5) Remove the cells obtained in step (3) from the incubator, add 8 mL of calcium phosphate-DNA precipitation complex to each bottle, put them back into the incubator, and change the medium with 5% packaged culture medium for 4-6 hours.

[0111] For the preparation of 5% packaged culture medium, open a new 500 mL bottle of DMEM medium, add the cell adhesion-promoting factor complex ITX-100× gibco 51500056 and 27.5 mL gibco KnockOut™ serum substitute (catalog number 10828028), then add 550 μL of 1 mol / L sodium pyruvate solution and 550 μL of 25 mmol / L chloroquine phosphate solution, and mix well.

[0112] (6) Take out the 293T cells obtained in step (5) from the incubator, discard the old culture medium, add 25 mL of 5% packaged culture medium to each bottle, and then put them back into the incubator to continue culturing. After culturing for 72 hours, harvest the supernatant to obtain the fusion antibody.

[0113] Example 4: Preparation of Lentiviral Virus Overexpressing Target Antigen (1) Observe the 293T cells used for packaging under a microscope and screen out cells with a cell density of 80% to 95%.

[0114] (2) Prepare 10% packaged culture medium. Open a new 500mL bottle of DMEM culture medium, add 55mL of FBS, then add 550μL of 1mol / L sodium pyruvate solution and 550μL of 25mmol / L chloroquine phosphate solution, and mix well.

[0115] (3) Take out the selected cells from the incubator, pour the old culture medium in the culture flask into the waste liquid tank, add 17 mL of 10% packaged culture medium, and then put it back into the carbon dioxide incubator to adapt.

[0116] (4) Calculate the added expression plasmid, packaging helper plasmid, coating plasmid, and 0.125 mol / L calcium chloride solution, and prepare a DNA-CaCl2 mixture. Then, take an equal volume of 2×HBS solution and add it dropwise to the DNA-CaCl2 mixture while vortexing the mixture. After the addition is complete, let it stand at room temperature for 20 minutes until a white precipitate forms.

[0117] (5) Remove the cells obtained in step (2) from the incubator, add 8 mL of calcium phosphate-DNA precipitation complex to each bottle, and mark the virus name and operation date on the bottle. Put it back into the incubator and change the medium with 5% packaged culture medium for 4-6 hours.

[0118] (6) Prepare 5% packaged culture medium. Take a 500mL bottle of DMEM culture medium, add 27.5mL FBS and 27.5mL serum substitute, add 550μL 1mol / L sodium pyruvate solution and 550μL 25mmol / L chloroquine phosphate solution, and mix well.

[0119] (7) Take out the 293T cells obtained in step (5) from the incubator, discard the old culture medium, add 25mL of 5% packaged culture medium to each bottle, and then put them back into the incubator to continue culturing. Harvest the virus after culturing for 42-48 hours.

[0120] Example 5: Construction of target antigen overexpression cell lines Resuscitating the Jurakat cell line: Remove the Jurakat cells to be resuscitated from the liquid nitrogen tank, thaw them rapidly in a 37°C constant temperature water bath, and then use a 10mL pipette to transfer the cell suspension to RM1640 medium containing 10mL of 10% FBS. Centrifuge at 1000rpm for 5min, discard the supernatant after centrifugation, resuspend the cell pellet in 10mL of fresh RM1640 medium containing 10% FBS, transfer it to T25, and incubate in a 37°C CO2 incubator. Passage the cells every two days at a ratio of 1:3. Cells that had been stably cultured for three generations from the start of resuscitation were seeded into 24-well plates at 0.8 × E5 / well. The harvested lentiviral supernatant (Example 4) was transduced at 10 μL, 20 μL, and 40 μL. EGFP expression was detected by flow cytometry after 72 hours. The positive rate was greater than 85%, and the positive rate of the B7H3 target antigen overexpressing cells involved in this invention was 86.6%. Figure 2 The B7H3 jurkat cell line was successfully constructed and can be used for subsequent antibody supernatant affinity property detection.

[0121] Example 6 Antibody supernatant specificity test (1) Collect 1 mL of the antibody supernatant obtained 72 h after transfection (Example 3) into a 1.5 mL centrifuge tube.

[0122] (2) Centrifuge at 12000 rpm for 5 min to remove cell debris, then clarify using a 0.22 μm filter for later use; (3) Cell staining: Take B7H3 jurkat cells (Example 5) and jurkat cells to be stained by flow cytometry, wash with sodium chloride / PBS, add 50 μL and 200 μL of antibody supernatant from step (2), incubate at 4°C for 30 min, and protect from light; (4) Wash the stained cells with sodium chloride, centrifuge, discard the supernatant and add 1 μL of mouse anti-human anti-hunan-IG1-FC-APC Biolegend 410712-100tests flow cytometry antibody, incubate at 4℃ for 60 min, and protect from light. (5) Wash the cells to be tested with sodium chloride, centrifuge, add flow cytometry buffer, and then test.

[0123] Flow cytometry results showed that FP878, FP879, FP880, FP881, FP882, FP883, and FP884 had a higher percentage of cells specifically binding to B7H3 jurkat. Figures 3-4 Similarly, after co-incubation with Jurakat cells, secondary antibody incubation with mouse anti-human anti-hunan-IG1-FC-APC was performed for 1 hour, followed by flow cytometry analysis. Figure 5 As shown, FP878, FP879, FP880, FP881, FP882, FP883, FP884, and FP885 are not associated with jurkat.

[0124] The above conclusions indicate that FP878, FP879, FP880, FP881, FP882, FP883, FP884 and FP885 specifically bind to B7-H3jurkat.

[0125] Example 7 ELISA Quantitative Detection of Antibody Supernatant The expression levels of fusion antibodies FP878, FP879, FP880, FP881, FP882, FP883, FP884, and FP885 were quantitatively detected using ELISA, as detailed below: Remove the ELISA plate from the aluminum foil bag after equilibration to room temperature for 20 minutes. Set up standard wells and sample wells. Add 50 μL of human IgG Fc fragment standards of different concentrations to each standard well. Add 50 μL of the test samples (fusion antibodies FP878, FP879, FP880, FP881, FP882, FP883, FP884, and FP885) to each sample well (serial dilution, 1:100 / 1:1000 / 1:10000). Do not add any to the blank wells. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each standard and sample well. Seal the reaction wells with sealing film and incubate at 37°C in a water bath or incubator for 60 minutes. Discard the liquid, pat dry on absorbent paper, fill each well with washing buffer (350 μL), let stand for 1 minute, discard the washing buffer, pat dry on absorbent paper, and repeat this washing process 5 times (or a plate washer can be used). Add 50 μL of substrate A and substrate B to each well and incubate at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well and measure the OD value of each well at 450 nm within 15 min.

[0126] The results are shown in Table 5. Among the fusion antibodies, the fusion antibodies FP877 and FP883 showed the strongest expression.

[0127] Table 5 Protein content in each supernatant as determined by ELISA

[0128] Example 8: Detection of binding ability of anti-B7H3 fusion antibody after quantification Cell staining: Take B7H3-jurkat cells and jurkat cells to be stained by flow cytometry, wash with sodium chloride / PBS, and add fusion antibodies at doses of 100 ng, 25 ng, 6.25 ng, 0.391 ng, 0.156 ng, and 0.098 ng, respectively. Incubate at 4°C for 30 min in the dark. Wash the stained cells with sodium chloride, centrifuge, discard the supernatant, add anti-hunan-IG1-FC-APC flow cytometry antibody, and incubate at 4°C for 60 min in the dark. Wash the cells to be tested with sodium chloride, centrifuge, add flow cytometry buffer, and perform flow cytometry analysis.

[0129] Test results as follows Figures 6-9 As shown in Tables 6 and 7, the fusion antibody FP880 has the highest affinity for Anti-B7H3 antibodies.

[0130] Table 6. Quantitative detection and target cell Anti-B7H3 binding capacity (MFI)

[0131] Table 7. Quantitative detection and target cell Anti-B7H3 binding capacity (percentage of positive cells)

[0132] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An anti-B7H3 nanobody, wherein the anti-B7H3 nanobody comprises a complementarity-determining region (CDR), the CDR comprising CDR1, CDR2, and CDR3, wherein, The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the anti-B7H3 nanobody are shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively.

2. The anti-B7H3 nanobody according to claim 1, characterized in that, The amino acid sequence of the anti-B7H3 nanobody is as shown in SEQ ID NO:46, or an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:46 after substitution, deletion, or addition of one or more amino acids.

3. A fusion antibody, comprising a first domain and a second domain; The first structural domain is the anti-B7H3 nanobody as described in claim 1 or 2; The second domain has the effect of prolonging the in vivo half-life and / or binding to effector cells.

4. The fusion antibody according to claim 3, characterized in that, The second structural domain includes: Immunoglobulin Fc region; and / or Serum albumin or fragments thereof, domains that bind to serum albumin, polyethylene glycol, polyethylene glycol-liposome complexes, or combinations thereof; and / or Molecules that have an affinity for T cell surface molecules and / or are able to bind to surface molecules present on T cells; Preferably, the immunoglobulin is selected from one or more combinations of IgG, IgA1, IgA2, IgD, IgE, and IgM; Preferably, the first and second domains are hinged together or connected via a linker peptide.

5. Biomaterials related to the anti-B7H3 nanobody of claim 1 or 2 or the fusion antibody of claim 3 or 4; said biomaterials being any one of a1) to a12): a1) A nucleic acid molecule encoding the anti-B7H3 nanobody of claim 1 or 2 or the fusion antibody of claim 3 or 4; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecules described in a1); a4) A recombinant vector containing the expression cassette described in a2); a5) Recombinant microorganisms containing the nucleic acid molecules described in a1); a6) Recombinant microorganisms containing the expression cassette described in a2); a7) Recombinant microorganisms containing the recombinant vector described in a3); a8) Recombinant microorganisms containing the recombinant vector described in a4); a9) Transgenic cell lines containing the nucleic acid molecules described in a1); a10) Transgenic cell lines containing the expression cassette described in a2); a11) Transgenic cell lines containing the recombinant vector described in a3); a12) Transgenic cell lines containing the recombinant vector described in a4).

6. An immunoconjugate, comprising: (A) The anti-B7H3 nanobody according to claim 1 or 2 or the fusion antibody according to claim 3 or 4; as well as (B) Functional molecules connected to (A).

7. The immunoconjugate according to claim 6, characterized in that, The (B) includes cytotoxins, radioactive isotopes, bioactive proteins, molecules that target tumor surface markers, molecules that inhibit tumors, molecules or detectable markers that target immune cell surface markers, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or combinations thereof. Preferably, the molecule targeting the tumor surface marker is an antibody or ligand that binds to the tumor surface marker; Preferably, the tumor-inhibiting molecule is an anti-tumor cytokine or an anti-tumor toxin.

8. A drug comprising the anti-B7H3 nanobody of claim 1 or 2, the fusion antibody of claim 3 or 4, the biomaterial of claim 5, or the immunoconjugate of claim 6 or 7.

9. The use of the anti-B7H3 nanobody of claim 1 or 2, the fusion antibody of claim 3 or 4, the biomaterial of claim 5, the immunoconjugate of claim 6 or 7, or the drug of claim 8 in any one of (1) to (4): (1) To prepare preparations, kits or boxes for the diagnosis, treatment or prevention of cancer; (2) To prepare preparations or kits for the treatment or prevention of autoimmune diseases; (3) Quantitative / qualitative detection of B7H3 expression level; (4) Prepare reagents / kits for quantitative / qualitative detection of B7H3 expression levels; Preferably, the cancer described in (1) includes: At least one of the following: glioblastoma, neuroblastoma, non-small cell lung cancer, cervical cancer, liver cancer, bladder cancer, prostate cancer, colon cancer, melanoma, ovarian cancer, osteosarcoma, neuroblastoma, hematologic malignancy, and kidney cancer; Preferably, the autoimmune diseases described in (2) include at least one of lupus, sepsis, arthritis, pancreatitis and type I diabetes.

10. A kit or cassette comprising the anti-B7H3 nanobody of claim 1 or 2, the fusion antibody of claim 3 or 4, the biomaterial of claim 5, the immunoconjugate of claim 6 or 7, or the drug of claim 8.

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