CN119823268A

CN119823268A - Anti-B7H 3 nano antibody and application thereof

Info

Publication number: CN119823268A
Application number: CN202411934933.8A
Authority: CN
Inventors: 马欣然; 陈涵; 张雁; 吴雅静
Original assignee: Guangzhou Fanen Biotechnology Co ltd
Current assignee: Guangzhou Fanen Biotechnology Co ltd
Priority date: 2024-12-26
Filing date: 2024-12-26
Publication date: 2025-04-15

Abstract

本发明属于生物医药技术领域，公开了一种抗B7H3纳米抗体及其应用，具体公开了一种抗B7H3纳米抗体，限定了其具体的氨基酸序列。本发明通过免疫羊驼获得的抗B7H3纳米抗体，且亲和力高、特异性强，可用于免疫印记、酶联免疫反应试剂盒、流式细胞检测计数，也可用于肿瘤、自身免疫性疾病等疾病的治疗。The present invention belongs to the field of biomedicine technology, discloses an anti-B7H3 nano antibody and its application, specifically discloses an anti-B7H3 nano antibody, and defines its specific amino acid sequence. The anti-B7H3 nano antibody obtained by immunizing alpacas has high affinity and strong specificity, 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 and autoimmune diseases.

Description

Anti-B7H 3 nano antibody and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an anti-B7H 3 nano antibody and application thereof.

Background

Immune checkpoint blocking therapies represented by targeted PD-1/PD-L1 and CTLA-4 have achieved unusual efficacy in the treatment of solid tumors, but the reality that less than 30% of patients can benefit from it suggests that other immune checkpoint molecule-mediated immunosuppression phenomena also exist. B7H3 (also known as CD 276), a transmembrane glycoprotein, is a member of the B7 family of immunomodulators, expressed on APC antigen presenting cells. Unlike CTLA-4/PD-1, which is predominantly expressed in T cells and mediates immunosuppression or depletion of the latter, B7H3 protein is not only inducibly expressed in immune cells, but is also constitutively and highly expressed in various tumor cells and tumor-associated vasculature, such as glioblastoma (glioblastoma, GBM), neuroblastoma (neuroblastoma, NB), non-small cell lung cancer, cervical cancer, liver cancer, bladder cancer, ovarian cancer, osteosarcoma, hematological tumor, renal cancer, and the like. The B7H3 not only can activate a plurality of signal paths to directly promote malignant phenotype of tumor cells, but also plays an important role in biological processes such as tumor generation, proliferation, migration and the like. Some studies have shown that B7H3 plays an important role in a variety of autoimmune diseases, such as lupus erythematosus, sepsis, arthritis, pancreatitis, and type I diabetes.

The traditional monoclonal antibody is more easily lost by the epitope caused by chemical treatment factors, and compared with the traditional alpaca antibody, the deformation temperature of the alpaca antibody can reach 60-80 ℃. Meanwhile, in order to ensure the correct structural folding, glycosylation and other post-translational modifications in production, the traditional monoclonal antibody is produced by using a mammalian cell line, so that the production cost is high and the time consumption is long. Monoclonal antibodies of non-fully human origin are more immunogenic.

The characteristic that the B7H3 protein is highly expressed in tumor tissues and tumor related vascular tissues and is low expressed or not expressed in normal tissues makes the B7H3 protein become an ideal tumor antigen target designed by CAR-T cells, and the currently-developed clinical test project of the B7H 3-targeted CAR-T cells shows that the B7H 3-targeted CAR-T cells show strong anti-tumor activity in various solid tumors and hematological tumors, and no obvious toxicity is detected, wherein one B7H3-CAR-T cell (TAA-06) has been qualified for FDA treatment of neuroblastoma orphism.

Studies have shown that the combined blockade of B7H3 and PD-L1 can significantly increase the tumor antigen specific CD8 ⁺ T cell anti-tumor effect. Phase I clinical trial results show that enoxaparin combined CTLA-4 antibody or PD-1 antibody treatment is more effective than that of either antibody drug alone, and the combined treatment of the enoxaparin and the PD-1 antibody remarkably improves the intratumoral recruitment and the anti-tumor activity of TIL without dose-dependent toxicity and serious adverse reaction. The total response rate of enoxazumab in combination with palbociclib in patients with primary/metastatic non-small cell lung cancer and recurrent/metastatic head and neck squamous cell carcinoma can reach 36% and 33%, respectively.

Nanobody (VHH) has the same domains as common antibody VH, namely 4 conserved framework regions (FR 1/2/3/4) and 3 complementarity determining regions (CDR 1/2/3). The molecular weight of the nano antibody is only 10% of that of the traditional antibody, and the characteristics of HCAbs complete antigen binding capacity, strong specificity, good affinity and high stability are maintained. Because nanobodies do not have an Fc segment and cannot produce cytotoxicity such as ADCC/CDC as in conventional antibodies, VHH antibodies are often fused to the Fc segment, and Fc-VHH fusion antibodies are constructed to increase ADCC (antibody-dependent cell-mediated cytotoxicity, anti-DEPENDENT CELL-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity, complement Dependent Cytotoxicity) activities. These forms of nanobodies can be widely used in the treatment of various diseases, as compared with the conventional antibodies.

Disclosure of Invention

The object of the first aspect of the present invention is to provide an anti-B7H3 nanobody.

The object of the second aspect of the present invention is to provide a fusion antibody.

It is an object of a third aspect of the present invention to provide a biomaterial associated with the anti-B7H 3 nanobody of the first aspect of the invention or the fusion antibody of the second aspect of the invention.

The object of the fourth aspect of the present invention is to provide an immunoconjugate.

The object of the fifth aspect of the present invention is to provide a medicament.

The sixth aspect of the present invention is directed to the use of the anti-B7H 3 nanobody of the first aspect of the invention, the fusion antibody of the second aspect of the invention, the biomaterial of the third aspect of the invention, the immunoconjugate of the fourth aspect of the invention or the medicament of the fifth aspect of the invention.

The seventh aspect of the present invention aims to provide a kit or a medicine box.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

In a first aspect of the invention there is provided an anti-B7H 3 nanobody comprising complementarity determining region CDRs comprising complementarity determining region CDR1, complementarity determining region CDR2 and complementarity determining region CDR3, wherein,

The amino acid sequence of the CDR1 of the complementarity determining region is shown as SEQ ID NO. 19, SEQ ID NO. 28, SEQ ID NO. 33, SEQ ID NO. 38, SEQ ID NO. 43, SEQ ID NO. 48 and SEQ ID NO. 53;

The amino acid sequence of the complementarity determining region CDR2 is shown in SEQ ID NO:20、SEQ ID NO:24、SEQ ID NO:29、SEQ ID NO:34、SEQ ID NO:39、SEQ ID NO:44、SEQ ID NO:49、SEQ ID NO:54;

The amino acid sequence of the complementarity determining region CDR3 is shown in SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:30、SEQ ID NO:35、SEQ ID NO:40、SEQ ID NO:45、SEQ ID NO:50、SEQ ID NO:55.

In some embodiments of the invention, the CDRs of the anti-B7H 3 nanobody comprise:

CDR1 as shown in SEQ ID NO. 19, CDR2 as shown in SEQ ID NO. 20 and CDR3 as shown in SEQ ID NO. 21, or

CDR1 as shown in SEQ ID NO. 19, CDR2 as shown in SEQ ID NO. 24 and CDR3 as shown in SEQ ID NO. 25, or

CDR1 as shown in SEQ ID NO. 28, CDR2 as shown in SEQ ID NO. 29 and CDR3 as shown in SEQ ID NO. 30, or

CDR1 as shown in SEQ ID NO. 33, CDR2 as shown in SEQ ID NO. 34 and CDR3 as shown in SEQ ID NO. 35, or

CDR1 as shown in SEQ ID NO 38, CDR2 as shown in SEQ ID NO 39 and CDR3 as shown in SEQ ID NO 40, or

CDR1 as shown in SEQ ID NO. 43, CDR2 as shown in SEQ ID NO. 44 and CDR3 as shown in SEQ ID NO. 45, or

CDR1 as shown in SEQ ID NO. 48, CDR2 as shown in SEQ ID NO. 49 and CDR3 as shown in SEQ ID NO. 50, or

CDR1 shown in SEQ ID NO. 53, CDR2 shown in SEQ ID NO. 54 and CDR3 shown in SEQ ID NO. 55.

In some embodiments of the invention, the CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

In some embodiments of the invention, the amino acid sequence of the anti-B7H 3 nanobody is an amino acid sequence which is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:17、SEQ ID NO:22、SEQ ID NO:26、SEQ ID NO:31、SEQ ID NO:36、SEQ ID NO:41、SEQ ID NO:46、SEQ ID NO:51 or SEQ ID NO:17、SEQ ID NO:22、SEQ ID NO:26、SEQ ID NO:31、SEQ ID NO:36、SEQ ID NO:41、SEQ ID NO:46、SEQ ID NO:51, modified by substitution, deletion or addition of one or more amino acids.

In a second aspect of the invention, there is provided a fusion antibody comprising a first domain and a second domain;

the first domain is an anti-B7H3 nanobody of the first aspect of the invention;

The second domain has an in vivo half-life extending effect and/or a binding effect on effector cells.

In some embodiments of the invention, the second domain includes (but is not limited to):

An immunoglobulin Fc region (e.g., a human immunoglobulin Fc region), and/or

Serum albumin (e.g., human HSA) or a fragment thereof, a domain that binds serum albumin (e.g., an anti-serum albumin antibody, including nanobodies), polyethylene glycol-liposome complexes, or combinations thereof, and/or

A molecule having affinity for a T cell surface molecule and/or capable of binding to a surface molecule (e.g., CD 3) present on a T cell.

In some embodiments of the invention, the human immunoglobulin Fc region comprises a mutation therein for altering an Fc-mediated effector function, including one or more of CDC activity, ADCC activity, ADCP activity.

In some embodiments of the invention, the immunoglobulin is a combination of one or more selected from the group consisting of IgG, igA1, igA2, igD, igE, igM.

In some embodiments of the invention, the IgG is selected from a combination of one or more of the IgG1, igG2, igG3, or IgG4 subtypes.

In some embodiments of the invention, the immunoglobulin Fc region has an amino acid sequence as shown in SEQ ID NO. 56, or an amino acid sequence which has the same or similar function as the amino acid sequence shown in SEQ ID NO. 56 after being modified by substitution, deletion or addition of one or more amino acids.

In some embodiments of the invention, the first domain is linked to the second domain at the N-terminus or the C-terminus.

In some embodiments of the invention, the first domain and the second domain are hingedly linked (e.g., anti-B7H 3 nanobody is fused directly to the hinge region of Fc) or are linked by a linker peptide.

In some embodiments of the invention, the connecting peptide is a flexible linker.

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

In some embodiments of the invention, the amino acid sequence of the fusion antibody is shown as SEQ ID NO. 1-8.

In a third aspect of the invention, there is provided a biomaterial associated with the anti-B7H 3 nanobody of the first aspect of the invention or the fusion antibody of the second aspect of the invention, wherein the biomaterial is any one of a 1) to a 12):

a1 A nucleic acid molecule encoding an anti-B7H 3 nanobody of the first aspect of the invention or a fusion antibody of the second aspect of the invention;

a2 An expression cassette comprising a 1) said nucleic acid molecule;

a3 A recombinant vector comprising the nucleic acid molecule of a 1);

a4 A recombinant vector comprising the expression cassette of a 2);

a5 A) a recombinant microorganism comprising a nucleic acid molecule according to a 1);

a6 A) a recombinant microorganism comprising the expression cassette of a 2);

a7 A) a recombinant microorganism comprising the recombinant vector of a 3);

a8 A) a recombinant microorganism comprising the recombinant vector of a 4);

a9 A transgenic cell line comprising a 1) said nucleic acid molecule;

a10 A transgenic cell line comprising the expression cassette of a 2);

a11 A transgenic cell line comprising a 3) the recombinant vector;

a12 A transgenic cell line comprising the recombinant vector of a 4).

In some embodiments of the invention, the nucleic acid molecule encoding the anti-B7H 3 nanobody of the first aspect of the invention is shown as SEQ ID NO:18、SEQ ID NO:23、SEQ ID NO:27、SEQ ID NO:32、SEQ ID NO:37、SEQ ID NO:42、SEQ ID NO:47、SEQ ID NO:52.

In some embodiments of the invention, the nucleic acid molecule encoding the fusion antibody of the second aspect of the invention is shown in SEQ ID NO 9-18.

In some embodiments of the invention, the transgenic animal cell line does not comprise propagation material.

In some embodiments of the invention, the expression cassette comprises a 5' transcriptional control region, an open reading frame encoding a fusion antibody of the first aspect of the invention, a translational control signal, a 3' untranslated region (3 ' utr), and a transcriptional termination signal.

In some embodiments of the invention, the 5' transcriptional control region comprises a promoter (a general purpose promoter such as a viral promoter (SV 40 promoter) or mammalian "housekeeping" promoter may be used), a transcription initiation site, an enhancer, and/or a silencer element.

In some embodiments of the invention, the 3'utr may encode AU-rich elements that ARE common regulators of mRNA stability via the 3' -5 'exosome pathway, and ARE typically located in the 3' utr. The AU-rich element may comprise one or more repetitions of the sequence AUUUA. It may also comprise one or more so-called US2B elements having the sequence AUAUAU.

In some embodiments of the invention, the vector comprises a promoter operably linked to the nucleic acid molecule.

In some embodiments of the invention, the vector is independently selected from the group consisting of a non-pathogenic viral vector and a viral vector.

In some embodiments of the invention, the viral vector comprises at least one of a lentiviral vector, an adenoviral vector, a baculovirus vector, a retroviral vector, a poxviral vector, a sendai viral vector, a herpes simplex viral vector.

In some embodiments of the invention, the non-viral vector comprises at least one of a plasmid vector, a cationic polymer vector, chitosan, polyethylenimine, a nanoparticle vector, a liposome.

In some embodiments of the invention, the vector is a plasmid vector, phagemid, viral vector, cellular vector, phage, cosmid, F cosmid, artificial chromosome.

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

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

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

In some embodiments of the invention, the prokaryotic cells include E.coli, streptomyces, bacillus subtilis, and the like, bacteria known in the art that can be used to express the protein of interest.

In some embodiments of the invention, the eukaryotic cell comprises at least one of a yeast cell, a mammalian cell, a plant cell, and an insect cell.

In a fourth aspect of the invention, there is provided an immunoconjugate comprising:

(A) An anti-PD-L1 nanobody of the first aspect of the invention or a fusion antibody of the second aspect of the invention, and

(B) Functional molecules linked (including but not limited to covalent attachment, coupling, attachment, adsorption) to (a).

In some embodiments of the invention, the (B) comprises a cytotoxin, a radioisotope, a bioactive protein, a tumor surface marker-targeting molecule, a tumor-inhibiting molecule, an immune cell surface marker-targeting molecule or a detectable marker, an extracellular hinge region based on chimeric antigen receptor technology, a transmembrane region (such as the transmembrane region of CD8 or CD 28), and an intracellular signaling region (such as the cd3ζ chain, fcsry tyrosine activation motif, the intracellular signaling region of co-stimulatory signaling molecules CD27, CD28, CD137, CD134, myD88, CD40, etc.), or a combination thereof.

In some embodiments of the invention, the molecule that targets a tumor surface marker is an antibody or ligand that binds a tumor surface marker.

In some embodiments of the invention, the tumor-inhibiting molecule is an anti-tumor cytokine (e.g., IL-12, IL-15, IFN-beta, TNFalpha) or an anti-tumor toxin.

In some embodiments of the invention, the detectable label is selected from the group consisting of a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or any combination thereof.

In some embodiments of the invention, the (B) is selected from fluorescent substances, chemiluminescent labels, colored substances, radioisotopes, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes, chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticle, etc.

In a fifth aspect of the invention, there is provided a medicament comprising an anti-B7H 3 nanobody of the first aspect of the invention, a fusion antibody of the second aspect of the invention, a biomaterial of the third aspect of the invention or an immunoconjugate of the fourth aspect of the invention.

In some embodiments of the invention, the medicament further comprises pharmaceutically acceptable excipients.

In some embodiments of the invention, the pharmaceutically acceptable excipients include at least one of fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, wetting agents, flavoring agents, co-solvents, suspending agents, solvents, slow release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, fragrances, and solvents.

In some embodiments of the invention, the medicament further includes combination medicaments including, but not limited to, immune effector molecules, cells, cytotoxic substances, multi-kinase inhibitors.

In a sixth aspect, the invention provides the use of an anti-B7H 3 nanobody of the first aspect, a fusion antibody of the second aspect, a biomaterial of the third aspect, an immunoconjugate of the fourth aspect or a medicament of the fifth aspect of the invention in any one of (1) to (4):

(1) Preparing a formulation, kit or kit for diagnosing, treating or preventing cancer;

(2) Preparing a preparation or a kit for treating or preventing an autoimmune disease;

(3) Quantitatively/qualitatively detecting the expression level of B7H 3;

(4) Preparing a reagent/kit for quantitatively/qualitatively detecting the expression quantity of B7H 3.

Can also be used for targeted delivery of traditional chemotherapeutic drugs (traditional Chinese medicine monomers, microRNA, etc.).

In some embodiments of the invention, the cancer in (1) comprises 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, hematological tumor, and renal cancer.

In some embodiments of the invention, the autoimmune disease in (2) comprises at least one of lupus erythematosus, sepsis, arthritis, pancreatitis, and type I diabetes.

In some embodiments of the invention, the kit/kit described in (4) comprises reagents for immunoblotting, enzyme-linked immunosorbent assay, flow cytometry.

In a seventh aspect of the invention there is provided a kit or kit comprising an anti-B7H 3 nanobody of the first aspect of the invention, a fusion antibody of the second aspect of the invention, a biomaterial of the third aspect of the invention, an immunoconjugate of the fourth aspect of the invention or a medicament of the fifth aspect of the invention.

In some embodiments of the invention, the kit may be used for immunoblotting, enzyme-linked immunosorbent assay, flow cytometry.

The beneficial effects of the invention are as follows:

The anti-B7H 3 nano antibody obtained by immunizing alpaca has high affinity and strong specificity, can be used for immunoblotting, enzyme-linked immunoreaction kit and flow cytometry detection counting, can also be used for treating diseases such as tumor, autoimmune diseases and chronic infections, and has the potential of being developed as an immune check point inhibitor.

Drawings

FIG. 1 is a schematic structural diagram of a fusion antibody of the present invention.

FIG. 2 is a flow test report (B) of the construction of B7H3 target antigen cell line using an antigen lentiviral vector map (A) and the construction of B7H3 target antigen cell line.

FIG. 3 shows the results of flow-through detection of the affinity of supernatants of fusion antibodies FP878, FP879, FP880, FP881, FP882 and FP883 for target antigen cells.

FIG. 4 shows the results of a flow assay for the affinity of supernatants of fusion antibodies FP884 and FP885 for target antigen cells.

FIG. 5 shows the results of a clear affinity flow assay for non-target antigen cells on each fusion antibody.

FIG. 6 is a flow chart report of quantitative detection of binding capacities of fusion antibodies FP878, FP879, FP880 or FP881 and target cells.

FIG. 7 is a flow chart report of quantitative detection of binding capacity of fusion antibodies FP882, FP883, FP884 or FP885 to target cells.

FIG. 8 is a graph showing quantitative determination of the binding capacity-MFI-of each fusion antibody to target cell Anti-B7H 3.

FIG. 9 is a graph showing quantitative determination of the binding capacity of each fusion antibody to target cells Anti-B7H 3-positive cell proportion.

Detailed Description

The following describes the present invention in further detail by way of specific examples.

It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1 plasmid construction

A plurality of candidate nano antibody sequences are obtained by immunizing alpaca with the B7H3 target antigen amino acid sequence, the VHH region nucleic acid sequence of the nano antibody is connected with the human IgG Fc nucleic acid sequence by utilizing a multi-fragment homologous recombination technology, the connecting region is finger, the sequence analysis is carried out after enzyme digestion and identification of the plasmid subjected to homologous recombination, and the fusion antibody sequence structure diagram is shown in figure 1.8 fusion antibodies of FP878, FP879, FP880, FP881, FP882, FP883, FP884, FP885 and the like are constructed, the amino acid sequence of the fusion antibodies is shown as SEQ ID NO 1-8, and the nucleotide sequence of the fusion antibodies is shown as SEQ ID NO 9-16.

The VHH of fusion antibody FP878 has an amino acid sequence shown as SEQ ID NO. 17, a nucleotide sequence shown as SEQ ID NO. 18, and amino acid sequences of CDR1, CDR2 and CDR3 shown as SEQ ID NO. 19-21 in sequence.

QVQLVESGGGLVQAGGSLRLSCAASGRTSGNYIMGWFRQAPGKEREFVAAINWTPGTTYYANSVKGRFTISGDNAKNTVYLQMNGLRAEDTAVYSCGARLGWNIQNTNYPYWGQGTQVTVS(SEQ ID NO:17);

ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGGACTTCCGGTAACTATATCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCAGCTATTAACTGGACTCCTGGTACTACATACTATGCAAACTCCGTGAAGGGCCGATTCACCATCTCCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACGGCCTGAGAGCTGAGGACACGGCCGTTTATTCCTGTGGAGCTCGGTTGGGTTGGAATATTCAAAATACGAATTATCCCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCC(SEQ ID NO:18);

CDR1:GRTSGNYI(SEQ ID NO:19);CDR2:INWTPGTT(SEQ ID NO:20);CDR3:GARLGWNIQNTNYPY(SEQ ID NO:21)。

The VHH of fusion antibody FP879 has an amino acid sequence shown as SEQ ID NO. 22, a nucleotide sequence shown as SEQ ID NO. 23, and the amino acid sequences of CDR1, CDR2 and CDR3 shown as SEQ ID NO. 24-26 in sequence.

QVQLVESGGGLVQAGGSLRLSCAASGRTSGNYIQGWFRQAPGKEREFVAAINWSAGTTYYADSVKGRFTISRDNAKNTVYLQMTSLRAEDTAVYSCGARPGWNIQNTNYQHWGQGTQVTVS(SEQ ID NO:22);

ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGGACTTCCGGTAACTATATCCAGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCAGCTATTAACTGGAGTGCTGGTACTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGACTAGCCTGAGAGCTGAGGACACGGCCGTTTATTCCTGTGGAGCTCGGCCGGGTTGGAATATTCAAAATACGAATTATCAACACTGGGGCCAGGGGACCCAGGTCACCGTCTCC(SEQ ID NO:23);

CDR1:GRTSGNYI(SEQ ID NO:19);CDR2:INWSAGTT(SEQ ID NO:24);CDR3:GARPGWNIQNTNYQH(SEQ ID NO:25)。

The VHH fusion antibody FP880 has the amino acid sequence shown as SEQ ID NO. 26, the nucleotide sequence shown as SEQ ID NO. 27, and the amino acid sequences of CDR1, CDR2 and CDR3 shown as SEQ ID NO. 28-30.

QVQLVESGGGLVQPGGSLRLSCGASGRTFSRDTMAWFRQAPGKEREFVAGISWLGATTYYADSVKGRFTISRDNAQNTVYLQMNAPKPEDTAEYYCAASSRLDPRVRTTDMDYWGKGTLVTVS(SEQ ID NO:26);

ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGGTCTCTGAGACTCTCCTGTGGAGCCTCTGGACGCACCTTCAGTAGGGATACCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGGTATTAGCTGGCTTGGTGCGACCACATACTATGCAGACTCCGTAAAGGGCCGATTCACCATCTCCAGAGACAACGCCCAGAACACGGTGTATCTGCAAATGAACGCACCGAAACCTGAGGACACGGCCGAATATTACTGTGCAGCAAGTTCCCGGCTAGATCCCCGAGTACGTACTACGGACATGGACTACTGGGGCAAAGGGACCCTGGTCACCGTCTCC(SEQ ID NO:27);

CDR1:GRTFSRDT(SEQ ID NO:28);CDR2:ISWLGATT(SEQ ID NO:29);CDR3:AASSRLDPRVRTTDMDY(SEQ ID NO:30)。

The VHH of fusion antibody FP881 has an amino acid sequence shown as SEQ ID NO. 31, a nucleotide sequence shown as SEQ ID NO. 32, and the amino acid sequences of CDR1, CDR2 and CDR3 shown as SEQ ID NO. 33-35.

QVQLVESGGGLVQAGGSLRLSCAASGATFSSLLLGWFRQAPGKEREFVALRSGIGAITYYADSVEDRFTISRDSTKNTVYLQMNDLKPEDTAVFYCAARPLGGSFTYSRPRSYDYWGQGTQVTVS(SEQ ID NO:31);

ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGCCACCTTCAGCAGTCTTCTCTTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTGGCACTTCGTAGCGGGATTGGTGCAATCACATACTATGCAGACTCCGTGGAGGACCGATTCACCATCTCCAGAGACAGCACCAAGAACACGGTGTATCTGCAAATGAACGACCTGAAACCTGAGGACACGGCCGTTTTTTACTGTGCAGCGCGACCCCTGGGTGGTAGTTTCACATATAGCCGTCCCAGATCTTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCC(SEQ ID NO:32);

CDR1:GATFSSLL(SEQ ID NO:33);CDR2:RSGIGAIT(SEQ ID NO:34);CDR3:AARPLGGSFTYSRPRSYDY(SEQ ID NO:35)。

The VHH fusion antibody FP882 has the amino acid sequence shown in SEQ ID NO. 36, the nucleotide sequence shown in SEQ ID NO. 37, and the amino acid sequences of CDR1, CDR2 and CDR3 shown in SEQ ID NO. 38-40.

QVQLVESGGGLVQAGGSLRLSCAASGRTSGAYVMGWFRQAPGKEREFVAAIQWTAGTTYYADSAEGRFIISRDNAKNTVYLEMNSLRTEDAAVYSCGARPGWNVQSTNYPYWGQGTQVTVS(SEQ ID NO:36);

ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGGACTTCCGGTGCCTATGTCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATTCAGTGGACTGCTGGTACTACATACTATGCAGACTCCGCGGAGGGCCGATTCATCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGGAAATGAACAGCCTGAGAACTGAGGACGCGGCCGTTTATTCCTGTGGAGCTCGGCCGGGTTGGAATGTTCAAAGTACAAATTATCCCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCC(SEQ ID NO:37);

CDR1:GRTSGAYV(SEQ ID NO:38);CDR2:IQWTAGTT(SEQ ID NO:39);CDR3:GARPGWNVQSTNYPY(SEQ ID NO:40)。

The VHH fusion antibody FP883 has an amino acid sequence shown as SEQ ID NO. 41, a nucleotide sequence shown as SEQ ID NO. 42, and amino acid sequences of CDR1, CDR2 and CDR3 shown as SEQ ID NO. 43-45.

QLQLVESGGGLVQSGGSLRLSCTASGYTLNYYAIGWFRQAPGKEREGISCITSIGTGGSTNYADSVKGRFTISRDSAKNTVYLQTYSLKPEDTAVYYCAAAPRFGSSCNRSGGMYDYWGQGTQVTVS(SEQ ID NO:41);

ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGTCTGGGGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATACACTTTGAATTACTACGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGATCTCATGTATCACTAGTATTGGTACCGGTGGTAGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAACACGGTGTATCTCCAAACGTATAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCGGCCGCCCCTCGCTTCGGTAGTAGCTGTAACCGGTCAGGGGGAATGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCC(SEQ ID NO:42);

CDR1:GYTLNYYA(SEQ ID NO:43);CDR2:ITSIGTGGST(SEQ ID NO:44);CDR3:AAAPRFGSSCNRSGGMYDY(SEQ ID NO:45)。

The VHH of fusion antibody FP884 has an amino acid sequence shown as SEQ ID NO. 46, a nucleotide sequence shown as SEQ ID NO. 47, and the amino acid sequences of CDR1, CDR2 and CDR3 shown as SEQ ID NO. 48-50.

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).

The VHH fusion antibody FP885 has an amino acid sequence shown as SEQ ID NO. 51, a nucleotide sequence shown as SEQ ID NO. 52, and amino acid sequences of CDR1, CDR2 and CDR3 shown as SEQ ID NO. 53-55.

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)。

The amino acid sequence of the human IgG Fc is shown as SEQ ID NO. 56, and the nucleotide sequence is shown as SEQ ID NO. 57.

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:56);

GAACCTAAAAGCAGCGACAAGACCCACACCTGCCCCCCTTGTCCTGCTCCTGAGCTGCTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA(SEQ ID NO:57).

The specific experimental procedure for plasmid construction of each antibody was as follows:

(1) Preparation of single fragment amplification reaction system and program

A single fragment amplification reaction system was prepared as shown in Table 1, and the reaction was carried out according to the reaction procedure shown in Table 2. The PCR amplification primers of the VHH region of the Anti-B7H3 nanobody are shown in Table 3, and the amplified fragment size is 408bp.

HIgG1-Fc fragment amplification primer:

F-IGg1:CTGAGCTGCTGGGAGGAccg(SEQ ID NO:58)(70% GC Tm=68);

R-IGg1:AAAAGGCGCAACCCGCTAGCtcatttacccggagacagggagag(SEQ ID NO:59)(54%GC Tm=67);

TABLE 1 Single-segment amplification reaction System

5×Q5 Reaction Buffer	10μL
		10mM dNTPs	1μL
Q5 High-Fidelity DNA Polymerase	0.5μL
		Template plasmid	0.1~2ng
Primer F	1μM
		Primer R	1μM
UltraPure^TM Distilled Water	to 50μL

Table 2 reaction procedure

TABLE 3 Anti-B7H3 nanobody VHH region PCR amplification primers

(2) Multi-fragment homologous recombination preparation system and program

The double fragments and 8044bp of lentiviral vector skeleton recovered by double cleavage of NotI and NheI are subjected to homologous recombination, and the system is prepared as shown in Table 4.

A multi-fragment homologous recombination reaction system was prepared as shown in Table 4, and the multi-fragment recombination reaction was performed at 50℃for 15min, and was cooled down to 4℃or immediately placed on ice.

TABLE 4 Multi-fragment homologous recombination reaction System

(3) Construction of target antigen Gene over-expression lentiviral vector

1) Fragment PCR, constructing B7H3 target antigen amino acid sequence (SEQ ID NO: 63) into lentiviral shuttle plasmid, and PCR amplification primers are as follows:

F-EF1-B7H3:catttcaggtgtcgtgaagcggccgcgccaccatgctgcgtcggcgg(SEQ ID NO:64);

P2A-B7H3:gAAgTTaGTAGCTCCggatccggctatttcttgtccatcatc(SEQ ID NO:65)

a large fragment 7802bp/718bp was recovered by double cleavage with NotI-BamHI, and B7H3 target antigen lentiviral expression vector was constructed with B7H3 target antigen single fragment GIBSON (FIG. 2A).

2) Multi-fragment homologous recombination preparation system and program

A multi-fragment homologous recombination reaction system was prepared as shown in Table 4 and reacted according to the following reaction procedure, multi-fragment recombination reaction was performed at 50℃for 15min, and was reduced to 4℃or immediately cooled on ice.

Example 2 plasmid extraction

(1) Plasmid transformation is carried out on the correct vector through sequence comparison, and the temperature is 37 ℃ and 14h;

(2) Selecting monoclonal colony, culturing with small bacterial liquid, culturing with culture volume of 4mL Amp+LB (100 mg/L Amp+), culturing at 37deg.C for 10h at 220rpm,

(3) The bacterial liquid is cultivated in large quantity, the next day, the seed liquid is inoculated into 250mL of Amp+LB (100 mg/L Amp+), the temperature is 37 ℃ and the cultivation time is 220rpm for 14h,

(4) Plasmid extraction was performed using the Tiangen plasmid large extraction kit.

Example 3 production of antibodies

(1) And observing 293T cells for packaging under a microscope, and screening out cells with cell density of 80% -95%.

(2) Preparing 10% packaging culture medium, newly opening a bottle of 500mL DMEM medium, adding 55mL FBS, adding 550 μL 1mol/L sodium pyruvate solution and 550 μL 25mmol/L chloroquine phosphate solution, and mixing.

(3) The selected cells were removed from the incubator, the old culture medium in the flask was poured into a waste liquid jar, 17ml of 10% packaging medium was added, and then returned to the carbon dioxide incubator for adaptation.

(4) The number of flasks to be transfected was calculated to give the desired plasmid (30. Mu.g per flask, prepared in example 2), and 0.125mol/L of calcium chloride solution to prepare a DNA-CaCl ₂ mixture. Then, an equal volume of 2 XHBS solution was added dropwise to the DNA-CaCl ₂ mixture, while swirling the DNA-CaCl ₂ mixture. And standing at room temperature for 20min after the dripping is completed.

(5) And (3) taking out the cells obtained in the step (3) from the incubator, adding 8mL of calcium phosphate-DNA precipitation complex into each bottle, putting back into the incubator, and replacing the solution with 5% packaging culture medium for 4-6 hours.

Preparation of 5% packaging Medium A500 mL bottle of DMEM medium was opened, factor complex ITX-100X gibco 51500056 and 27.5mL gibco KnockOut ^TM serum substitute (cat. No. 10828028) for promoting cell adhesion were added thereto, and 550. Mu.L of 1mol/L sodium pyruvate solution and 550. Mu.L of 25mmol/L chloroquine phosphate solution were added thereto and mixed well.

(6) And (3) taking out the 293T cells obtained in the step (5) from the incubator, discarding the old culture medium, adding 25mL of 5% packaging culture medium into each bottle, putting back into the incubator for continuous culture, and harvesting the supernatant after culturing for 72 hours to obtain the fusion antibody.

EXAMPLE 4 preparation of target antigen over-expressed lentiviruses

(4) Calculating added expression plasmid, packaging auxiliary plasmid, envelope plasmid and 0.125mol/L calcium chloride solution to prepare DNA-CaCl ₂ mixed solution. Then, an equal volume of 2 XHBS solution was added dropwise to the DNA-CaCl ₂ mixture, while swirling the DNA-CaCl ₂ mixture. After the dripping is completed, the mixture is kept stand at room temperature for 20min until white precipitate is formed.

(5) And (3) taking out the cells obtained in the step (2) from the incubator, adding 8mL of calcium phosphate-DNA precipitation complex into each bottle, marking the virus name and the operation date on the bottle body, and putting back into the incubator for 4-6 hours, and using 5% packaging culture medium to replace the liquid.

(6) A5% packaging medium was prepared, one bottle of 500mL of DMEM medium was filled, 27.5mL of FBS and 27.5mL of serum replacement were added, 550. Mu.L of 1mol/L sodium pyruvate solution and 550. Mu.L of 25mmol/L chloroquine phosphate solution were added, and the mixture was homogenized.

(7) Taking out the 293T cells obtained in the step (5) from the incubator, discarding the old culture medium, adding 25mL of 5% packaging culture medium into each bottle, putting back into the incubator for continuous culture, and sterilizing after culturing for 42-48 h.

EXAMPLE 5 construction of target antigen overexpressing cell lines

Resuscitates the jurkat cell line by taking out the jurkat cells to be resuscitated from a liquid nitrogen tank, transferring the cell suspension to RM1640 culture medium which is put with 10mL of 10% FBS in advance by using a 10mL pipette after the rapid thawing in a 37 ℃ constant-temperature water bath, centrifuging at 1000rpm for 5min, discarding the supernatant after centrifugation, re-suspending the cell sediment in fresh RM1640 with 10mL of 10% FBS uniformly, transferring to T25, culturing in a 37 ℃ CCO ₂ incubator, and carrying out subculture according to the proportion of 1:3 every two days;

Cells after 3 passages of stable culture calculated from the beginning of resuscitation were inoculated into 24-well plates, 0.8XE5/well, the harvested lentiviral supernatant (example 4) was transduced according to 10. Mu.L, 20. Mu.L, 40. Mu.L, and EGFP expression was detected using a flow cytometer for 72H with a positive rate of greater than 85%, the positive rate of B7H3 target antigen overexpressing cells according to the present invention was 86.6% (B in FIG. 2), and cell lines (i.e., B7H3 jurkat) were successfully constructed and could be used for subsequent antibody supernatant affinity property detection.

EXAMPLE 6 antibody supernatant specificity test

(1) Antibody supernatants from 72h of transfection (example 3) were collected 1mL in 1.5mL centrifuge tubes.

(2) Centrifuging at 12000rpm for 5min to remove cell debris, and clarifying with 0.22 μm small filter;

(3) Cell staining, namely taking cells B7H3 jurkat cells (example 5) to be subjected to flow staining and the jurkat cells, washing the cells by using sodium chloride/PBS, adding 50 mu L of the antibody supernatant in the step (2), 200 mu L of the antibody supernatant, and incubating at 4 ℃ for 30min, and keeping away from light;

(4) Washing the stained cells with sodium chloride, centrifuging, removing supernatant, adding mouse anti-human anti-hunan-IG1-FC-APC Biolegend 410712-100tests flow antibody, incubating for 60min at 1 μL and 4 ℃, and keeping away from light;

(5) And (3) washing the cells to be detected by using sodium chloride, centrifuging, adding a flow type on-machine buffer solution, and detecting on-machine.

The flow results showed that FP878, FP879, FP880, FP881, FP882, FP883, FP884 specifically bound to a higher percentage of cells of B7H3jurkat (fig. 3-4 TCR). Similarly, flow assays were performed after incubation with jurkat cells for 1h using murine anti-human anti-hunan-IG1-FC-APC after incubation, as shown in FIG. 5, with no binding of FP878, FP879, FP880, FP881, FP882, FP883, FP884, and FP885 to jurkat.

The above conclusions indicate that FP878, FP879, FP880, FP881, FP882, FP883, FP884 and FP885 are specific binding for the binding of B7-H3 jurkat.

EXAMPLE 7 ELISA quantitative detection of antibody supernatants

The expression ability of fusion antibodies FP878, FP879, FP880, FP881, FP882, FP883, FP884 and FP885 was quantitatively detected by ELISA, concretely as follows:

The ELISA well plates were removed from the aluminum foil bags after 20min equilibration at room temperature. Setting a standard substance hole and a sample hole, wherein 50 mu L of human IgG Fc fragment standard substances with different concentrations are respectively added into the standard substance hole, 50 mu L (gradient dilution, 1:100/1:1000/1:10000) of samples (fusion antibodies FP878, FP879, FP880, FP881, FP882, FP883, FP884 and FP 885) to be detected are added into the sample hole, and blank holes are not added. In addition to the blank wells, 100 μl of horseradish peroxidase (HRP) -labeled detection antibody was added to each of the standard wells and sample wells, and the reaction wells were sealed with a sealing plate membrane and incubated for 60min in a 37 ℃ water bath or incubator. The liquid is discarded, the water-absorbing paper is beaten to dryness, each hole is filled with the washing liquid (350 mu L), the mixture is stood for 1min, the washing liquid is thrown off, the water-absorbing paper is beaten to dryness, and the plate is repeatedly washed for 5 times (the plate can be washed by a plate washer). Substrate A, B. Mu.L each was added to each well and incubated at 37℃for 15min in the absence of light. Adding stop solution 50 μl for 15min into each hole

In this, the OD of each well was measured at a wavelength of 450 nm.

As a result, as shown in Table 5, the fusion antibodies FP877 and FP883 were the most expressible.

TABLE 5 Elisa detection of protein content in respective supernatants

Example 8 detection of binding Capacity after quantification of anti-B7H3 fusion antibodies

Cell staining, namely taking cells B7H3-jurkat and jurkat to be subjected to flow staining, washing with sodium chloride/PBS, adding each fusion antibody according to 100ng, 25ng, 6.25ng, 0.391ng, 0.156ng and 0.098ng, incubating for 30min at 4 ℃ and keeping away from light, washing the stained cells with sodium chloride, centrifuging, adding anti-hunan-IG1-FC-APC flow antibody after supernatant discarding, incubating for 60min at 4 ℃ and keeping away from light, washing the cells to be detected with sodium chloride, centrifuging, adding flow type on-machine buffer solution, and on-machine detecting.

The detection results are shown in FIG. 6-FIG. 9 and Table 6-Table 7, and the fusion antibody FP880 has the highest affinity for the Anti-B7H3 antibody.

Table 6 quantitative determination of binding Capacity to target cells Anti-B7H3 (MFI)

TABLE 7 quantitative determination of binding Capacity to target cells Anti-B7H3 (positive cell proportion)

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims

1. An anti-B7H3 nanobody, comprising a complementary determining region CDR, wherein the complementary determining region CDR comprises a complementary determining region CDR1, a complementary determining region CDR2 and a complementary determining region CDR3, wherein:

The amino acid sequence of the complementary determining region CDR1 is shown in SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:48, and SEQ ID NO:53;

The amino acid sequence of the complementary determining region CDR2 is shown in SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:44, SEQ ID NO:49, and SEQ ID NO:54; the amino acid sequence of the complementary determining region CDR3 is shown in SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:45, SEQ ID NO:50, and SEQ ID NO:55.

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:17, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:51, or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids with the amino acid sequence shown in SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:51 and having the same or similar function.

3. A fusion antibody comprising a first domain and a second domain;

The first domain is the anti-B7H3 nanobody according to claim 1 or 2;

The second domain has the effect of extending the half-life in vivo and/or has a binding effect on effector cells.

4. The fusion antibody according to claim 3, characterized in that the second domain comprises:

Immunoglobulin Fc region; and/or

Serum albumin or a fragment thereof, a domain that binds serum albumin, polyethylene glycol, a polyethylene glycol-liposome complex, or a combination thereof; and/or

a molecule having affinity for a T cell surface molecule and/or capable of binding to a surface molecule present on a T cell;

Preferably, the immunoglobulin is selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, and IgM;

Preferably, the first domain and the second domain are connected by a hinge or by a connecting peptide.

5. A biological material related to the anti-B7H3 nanobody according to claim 1 or 2 or the fusion antibody according to claim 3 or 4; the biological material is any one of a1) to a12):

a1) a nucleic acid molecule encoding the anti-B7H3 Nanobody according to claim 1 or 2 or the fusion antibody according to claim 3 or 4;

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) a recombinant microorganism containing the nucleic acid molecule described in a1);

a6) a recombinant microorganism containing the expression cassette described in a2);

a7) a recombinant microorganism containing the recombinant vector described in a3);

a8) a recombinant microorganism containing the recombinant vector described in a4);

a9) a transgenic cell line containing the nucleic acid molecule described in a1);

a10) a transgenic cell line containing the expression cassette described in a2);

a11) a transgenic cell line containing the recombinant vector described in a3);

a12) A transgenic cell line 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; and

(B) Functional molecule linked to (A).

7. The immunoconjugate according to claim 6, characterized in that (B) comprises cytotoxins, radioactive isotopes, biologically active proteins, molecules targeting tumor surface markers, tumor-suppressing molecules, molecules or detectable markers targeting surface markers of immune cells, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or a combination thereof;

Preferably, the molecule targeting a tumor surface marker is an antibody or a ligand that binds to a 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 according to claim 1 or 2, the fusion antibody according to claim 3 or 4, the biomaterial according to claim 5, or the immunoconjugate according to claim 6 or 7.

9. Use of the anti-B7H3 nanobody according to claim 1 or 2, the fusion antibody according to claim 3 or 4, the biomaterial according to claim 5, the immunoconjugate according to claim 6 or 7, or the drug according to claim 8 in any one of (1) to (4):

(1) Preparation of preparations, test kits or pharmaceutical kits for diagnosing, treating or preventing cancer;

(2) preparing a preparation or kit for treating or preventing an autoimmune disease;

(3) Quantitative/qualitative detection of B7H3 expression;

(4) preparing a reagent/kit for quantitative/qualitative detection of B7H3 expression;

Preferably, the cancer in (1) comprises 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, hematological tumor and renal cancer;

Preferably, the autoimmune disease in (2) comprises at least one of lupus erythematosus, sepsis, arthritis, pancreatitis and type I diabetes.

10. A test kit or a medicine kit comprising the anti-B7H3 nanobody according to claim 1 or 2, the fusion antibody according to claim 3 or 4, the biomaterial according to claim 5, the immunoconjugate according to claim 6 or 7, or the drug according to claim 8.