CN118406150A - Anti-B7-H3 antibody and application thereof - Google Patents

Abstract

The invention discloses an anti-B7-H3 antibody and application thereof, and relates to the technical field of biological medicines, wherein the anti-B7-H3 antibody or antigen binding fragment thereof comprises HCDR1 with an amino acid sequence shown as SEQ ID NO. 10, HCDR2 with an amino acid sequence shown as SEQ ID NO. 11, HCDR3 with an amino acid sequence shown as SEQ ID NO. 12, LCDR1 with an amino acid sequence shown as SEQ ID NO. 14, LCDR2 with an amino acid sequence shown as SEQ ID NO. 15 and LCDR3 with an amino acid sequence shown as SEQ ID NO. 16. According to the invention, through an in-vitro killing experiment, the anti-tumor effect of the B7-H3-CD3 bispecific antibody is verified, and the result shows that the B7-H3-CD3 bispecific antibody has a remarkable killing effect on A375-lucf tumor cells. The invention can be applied to the preparation of medicaments for diagnosing, preventing and treating human tumors.

Description

An anti-B7-H3 antibody and its application

This invention is based on the divisional application of the invention patent with the application date of December 26, 2022, the priority date of August 26, 2022, the application number CN202211676252.7, and the invention name of "An anti-B7-H3 antibody and its application".

Technical Field

The present invention belongs to the field of biomedicine technology and relates to an anti-B7-H3 antibody and its application, in particular to an antibody or its functional fragment that can specifically bind to human B7-H3 and its application in tumor treatment.

Background technique

As an innovative treatment method, tumor immunotherapy has become a hot topic in the field of tumor treatment research and is expected to bring new treatment options to patients. By blocking the PD-L1/PD-1 pathway or the CTLA-4 signaling pathway, the immunosuppressive state of T cells can be reversed, and the ability of the patient's own immune system to inhibit or kill tumors can be improved. However, currently, blockers of the PD-L1/PD-1 pathway or the CTLA-4 pathway are only effective for a small number of patients in clinical practice, and new immunotherapy targets or new immunotherapy strategies still need to be explored.

B7-H3 (CD276) is a member of the B7 family and is located on human chromosome 15. Studies have shown that mouse B7-H3 is a negative regulator of T cell activation (Prasad DV, et al., Murine B7-H3 is anegative regulator of T cells. J Immunol, 2004, 173: 2500-2506). Compared with normal mice, B7-H3 knockout mice have more severe respiratory inflammation and earlier experimental autoimmune encephalomyelitis (Suh WK, et al., The B7 family member B7-H3 preferentially down-regulates T helper type 1-mediated immune responses. Nat Immunol, 2003, 4: 899-906). In humans, B7-H3 was initially reported as a T cell co-stimulatory molecule (Chapoval AI, et al., B7-H3: a costimulatory molecule for T cell activation and IFN-gamma production. Nat Immunol, 2001, 2: 269-274). However, recent studies have shown that human B7-H3 can inhibit the production of IFN-γ, IL-2, IL-10 and IL-13 during T cell activation, and has an inhibitory effect on T cell activation (Leitner J, et al., B7-H3 is a potent inhibitor of human T-cell activation: No evidence for B7-H3 and TREML2 interaction. Eur J Immunol, 2009, 39: 1754-1764). To date, the receptor or ligand of B7-H3 has not been identified.

By detecting various clinical samples, many studies have shown that B7-H3 is lowly expressed in most normal tissues, but highly expressed in a variety of tumor tissues, and its expression level is associated with poor prognosis, such as lung cancer (Zhang G, et al., Diagnosis value of serum B7-H3expression in non-small cell lung cancer. Lung Cancer, 2009, 66: 245-249), colorectal cancer (Sun J, et al., Clinical significance and regulation of the costimulation molecule B7-H3 in human colorectal carcinoma. Cancer Immunol Immunother, 2010, 59: 1163-1171), prostate cancer (Zang X, et al., B7-H3 and B7x are highly expressed in human prostate cancer and associated with disease spread and poor outcome. Proc Natl Acad Sci U SA, 2007, 104: 19458-19463), bladder cancer (Xu Z, et al., High expression of B7-H3and CD163 in cancer tissues indicates malignant clinicopathological status and poor prognosis of patients with urothelial cell carcinoma of the bladder. Oncol Lett, 2018, 15: 6519-6526), and medulloblastoma (Purvis IJ, et al., Role of MYC-miR-29-B7-H3 in Medulloblastoma Growth and Angiogenesis. J Clin Med, 2019, 8, pii: E1158). In addition, B7-H3 has also been reported to be highly expressed in many other tumors, such as gastric cancer, ovarian cancer, glioblastoma, craniopharyngioma, cervical cancer, osteosarcoma, hematological tumors, head and neck tumors, pancreatic cancer, skin cancer, renal cancer, meningioma and breast cancer.

In addition, relevant studies have shown that B7-H3 plays a role in the pathogenesis of a variety of autoimmune diseases and has potential diagnostic and therapeutic value for a variety of autoimmune diseases, such as lupus erythematosus, sepsis, arthritis, pancreatitis and type 1 diabetes.

In summary, since B7-H3 is widely and highly expressed in various tumor tissues and lowly expressed in normal tissues, it is considered to be a highly potential target for tumor treatment.

Summary of the invention

One aspect of the present invention is to provide an anti-B7-H3 antibody or a functional fragment thereof that can specifically bind to B7-H3 (CD276).

The present invention provides an antibody or a functional fragment thereof, comprising a heavy chain CDR selected from the following amino acid sequences or variants of any of the sequences: SEQ ID NO: 8n+2, SEQ ID NO: 8n+3 and SEQ ID NO: 8n+4; and/or a light chain CDR selected from the following amino acid sequences or variants of any of the sequences: SEQ ID NO: 8n+6, SEQ ID NO: 8n+7 and SEQID NO: 8n+8, wherein each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In some specific embodiments, the present invention provides an anti-B7-H3 antibody or an antigen-binding fragment thereof, wherein the heavy chain CDR comprises the following three complementarity determining regions:

CDR1 shown in SEQ ID NO: 8n+2,

CDR2 shown in SEQ ID NO: 8n+3, and

CDR3 shown in SEQ ID NO: 8n+4;

The light chain CDR includes the following three complementarity determining regions:

CDR1 shown in SEQ ID NO: 8n+6,

CDR2 shown in SEQ ID NO: 8n+7, and

CDR3 shown in SEQ ID NO: 8n+8;

wherein each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In some specific embodiments, the present invention provides an anti-B7-H3 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 8n+1 or any variant of the sequence, and/or a light chain variable region selected from the amino acid sequence SEQ ID NO: 8n+5 or any variant of the sequence, wherein each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In some specific embodiments, the present invention provides an anti-B7-H3 antibody or an antigen-binding fragment thereof, wherein the antibody or the functional fragment thereof is a chimeric antibody, a single-chain antibody or a bispecific antibody.

In some specific embodiments, the present invention provides an anti-B7-H3 antibody or an antigen-binding fragment thereof, wherein the structural representation of the single-chain antibody is: VH-(G4S)3-VL-huIgG1Fc, wherein VH is the heavy chain variable region, VL is the light chain variable region, (G4S)3 is a peptide linker, and huIgG1Fc is the constant region of human IgG1 antibody. The structural representation of the bispecific antibody (B7-H3-CD3) is: VL1-(G4S)3-VH1-G4S-VH2-(G4S)3-VL2, wherein VH1 is the B7-H3 heavy chain variable region, VL1 is the B7-H3 light chain variable region, VH2 is the CD3 heavy chain variable region, VL2 is the CD3 light chain variable region, G4S and (G4S)3 are peptide linkers, and VH2-(G4S)3-VL2 is CD3scFv, which uses the amino acid sequence of OKT3 (as shown below):

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEW IGYINPSRGYTNYNQKFKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYY DDHYCLDYWGQGTTLTVSS(VH2)GGGGSGGGGSGGGGS((G4S)3)DIQLTQS PAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGT SPKRWIYDTSKVASGVP YRFSGGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(VL2).

In some specific embodiments, the present invention provides an anti-B7-H3 antibody or an antigen-binding fragment thereof, wherein the functional fragment includes Fab, Fv, sFv, F(ab')2, single-chain antibody, nanobody, domain antibody and multispecific antibody.

Another aspect of the present invention provides an expression vector comprising the antibody and a host cell comprising the expression vector.

Another aspect of the present invention provides a pharmaceutical composition comprising the antibody of the present invention or a functional fragment thereof, or comprising the nucleic acid molecule and an expression vector or a host cell, or any combination thereof, and a pharmaceutically acceptable carrier.

Another aspect of the present invention provides an immunoconjugate comprising an antibody of the present invention or a functional fragment thereof conjugated to a therapeutic agent. In some embodiments, the therapeutic agent is a toxin, a radioisotope, a drug, or a cytotoxic agent. The immunoconjugate includes a fusion protein of an antibody or a functional fragment and a factor, and a conjugate of an antibody or a functional fragment and a toxin, a reflective isotope, a drug, or a cytotoxic agent.

Another aspect of the present invention provides a method for preparing an anti-B7-H3 antibody or a functional fragment thereof, comprising: culturing the host cell of the present invention under conditions allowing the production (expression) of the antibody or the functional fragment thereof, and recovering the produced antibody or the functional fragment thereof from the host cell.

Another aspect of the present invention provides a method for diagnosing, preventing or treating a disease or condition by targeting B7-H3 therapy, which comprises administering a therapeutically effective amount of an antibody or a functional fragment thereof, an expression vector, a host cell, a pharmaceutical composition or an immunoconjugate of the present invention to a subject in need thereof. Wherein, the disease is selected from tumors or inflammatory diseases, and the tumor preferably includes glioma, neuroblastoma, medulloblastoma, meningioma, lung cancer, esophageal cancer, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, bladder cancer, ureteral cancer, prostate cancer, skin cancer, melanoma, ovarian cancer, endometrial cancer, cervical cancer, soft tissue sarcoma, acute and chronic leukemia, Hodgkin's and non-Hodgkin's lymphoma, gastric cancer and head and neck tumors; the inflammatory disease includes lupus erythematosus, ankylosing spondylitis, multiple sclerosis, psoriasis, antiphospholipid antibody syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, autoimmune hepatitis, arthritis, rheumatoid arthritis, pemphigus, Guillain-Barre syndrome, Crohn's disease, vasculitis and autoimmune diabetes.

The present invention has the following beneficial effects:

The present invention prepares a specific monoclonal antibody of B7-H3 by traditional hybridoma technology, and constructs a single-chain antibody (scFv) and a bispecific antibody (B7-H3-CD3) thereof, and verifies the anti-tumor effect of the B7-H3-CD3 bispecific antibody of the present invention by an in vitro killing experiment, and the results show that the B7-H3-CD3 bispecific antibody has a significant killing effect on A375-lucf tumor cells. The present invention can be applied to the preparation of drugs for preventing, diagnosing and treating human tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1 is an immunofluorescence analysis of the binding results of recombinant mouse monoclonal antibodies 1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, and 15D7 to B7-H3 molecules overexpressed in cells;

FIG2 is a FACS analysis of the binding results of single-chain antibodies 1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, and 15D7 to endogenous B7-H3 in cells (2a) and knockout endogenous B7-H3 in cells (2b);

FIG3 shows the in vitro tumor killing results of bispecific antibodies 1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, and 15D7 in series with CD3scfv, respectively;

Figure 4 shows the results of in vivo anti-tumor analysis of bispecific antibodies 1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, and 15D7 in tandem with CD3scfv, respectively, using the NSG mouse model.

Detailed ways

In the following description, for illustration rather than for limitation, specific details such as specific system structure, technology and the like are proposed, so as to thoroughly understand the embodiments of the present invention. However, it should be clear to those skilled in the art that the present invention can also be implemented in other embodiments without these specific details. Unless there is a specific indication, all scientific and technological terms used in the present invention are the same meanings understood by those skilled in the art. The abbreviation of amino acid adopts the standard 3 letters or 1 letter code of one of 20 kinds of commonly used amino acids in this area, as described in J.biol.chem, 243, p3558 (1968).

Those skilled in the art can replace, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention, and replace amino acids with similar properties in the variable region without substantially affecting the binding activity of the antibody, thereby obtaining variants of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention.

the term

The “functional fragment” mentioned in the present invention mainly refers to the functional fragment of an antibody that can specifically bind to an antigen, such as scFv (single-chain antibody), Fv, Fab, F(ab’)2, Fab’, scFv-Fc, single-domain antibody, antibody-like antibody, bispecific antibody, multispecific antibody, or any antibody fragment that has been chemically modified to increase the half-life.

The "pharmaceutical composition" of the present invention refers to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient that are combined together to achieve a specific purpose. In certain embodiments, the pharmaceutical composition includes a combination separated in time and/or space, as long as they can work together to achieve the purpose of the present invention. Some pharmaceutical compositions are achieved by co-administering some pharmaceutically acceptable ingredients or compounds to enhance the biological efficacy of the present invention or reduce the side effects of the drug (for example, it can be used in combination with other anti-tumor drugs to enhance the anti-tumor effect). The purpose of other pharmaceutical compositions is to promote administration to an organism, facilitate the absorption of active ingredients, enhance stability or targeting, prolong the half-life, and thus better exert the biological efficacy of the present invention.

The "host cell" described in the present invention includes prokaryotic host cells, eukaryotic host cells and bacteriophages. The prokaryotic host cells may be Escherichia coli, Streptomyces or Bacillus subtilis, etc. The eukaryotic host cells may be 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, Per6, Saccharomyces cerevisiae, Pichia pastoris, Hansen yeast, Candida, some insect cells and plant cells. 293 series cells, Per6 cells and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins, and are well known to those of ordinary skill in the art.

The "chimeric antibody" described in the present invention is an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, a hybridoma that secretes mouse-specific monoclonal antibodies must first be established, and then the variable region gene must be cloned from the mouse hybridoma cells, and then the constant region gene of the human antibody must be cloned as needed, and the mouse variable region gene and the human constant region gene must be connected into a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule must be expressed in a eukaryotic or prokaryotic industrial system.

The "expression vector" of the present invention refers to any recombinant polynucleotide construct, which can directly or indirectly (such as packaged into a virus) introduce the target DNA fragment into the host cell by transformation, transfection or transduction to express the target gene. One type of vector is a plasmid, i.e. a circular double-stranded DNA molecule, which can connect the target DNA fragment to the plasmid ring. Another type of vector is a viral vector, which can connect and package the target DNA fragment into a viral genome (such as adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, the target gene can be expressed.

A person skilled in the art can also transcribe the nucleic acid sequence of the present invention into RNA by in vitro transcription, and further transfect, transduce or transform the RNA into a host cell to express the antibody of the present invention or its functional fragment to exert the biological efficacy of the present invention.

The "effective amount" of the present invention refers to a dose sufficient to show its benefit to the object to which it is administered. The actual amount administered, as well as the rate and time course of administration will depend on the individual condition and severity of the person being treated. The prescription of treatment is ultimately determined by the doctor, who usually takes into account the individual condition of the patient, the delivery site, the method of administration, the severity of the disease, and other conventional factors for the doctor.

The "subject" described in the present invention refers to mammals, such as humans, but can also be other animals, such as wild animals, livestock or experimental animals.

The present invention provides an anti-B7-H3 antibody and a functional fragment thereof that can specifically bind to B7-H3. The antibody or the functional fragment thereof of the present invention can specifically bind to human B7-H3 but not to mouse B7-H3.

The antibodies of the invention may be full length (eg, IgG1 or IgG4 antibodies) or may comprise only the antigen binding portion (eg, Fab, F(ab')2 or scFv fragments), which may be functional fragments of single chain antibodies (scFv) and bispecific antibodies.

The present invention provides an application method that can be used for tumor treatment. The method includes cell therapy products (such as chimeric antigen receptor T cells), bispecific antibodies (such as B7-H3/CD3 bispecific antibodies), immunoconjugates (such as antibodies coupled to maytansine), and pharmaceutical compositions comprising the antibodies and their functional fragments of the present invention and pharmaceutically acceptable carriers.

The present invention can be used for qualitative or quantitative detection of B7-H3 antigen, for example, the antibody of the present invention or its functional fragment can be applied to ELISA, immunohistochemistry, immunofluorescence and flow cytometry. It can also be used for antigen tracing of cells, tissues or living bodies, for example, the antibody of the present invention or its functional fragment can be fluorescently or isotopically labeled to perform antigen tracing.

The following examples are specifically provided to demonstrate and further explain some preferred embodiments and aspects of the present invention, but should not be construed as limiting the scope thereof. The experimental methods in the examples of the present invention that do not specify specific conditions are generally performed under conventional conditions, such as the Cold Spring Harbor Antibody Technology Laboratory Manual, the Molecular Cloning Manual; or under the conditions recommended by the raw material or commodity manufacturer. Reagents that do not specify specific sources are conventional reagents purchased from the market.

Example 1

(1) Preparation of B7-H3 recombinant protein: The nucleic acid sequence encoding human B7-H3 (NM_001024736.2) was synthesized by Anhui General Biotechnology Co., Ltd. PCR amplification and subcloning into the pcDNA3.1 expression vector (Invitrogen). Then, the extracellular domain of B7-H3 was subcloned into the pcDNA3.1 expression vector carrying an Fc or His tag at the C-terminus. The Fc tag includes human Fc (hFc) and mouse Fc (mFc). 293FT was transiently transfected and cultured in a shake flask using FreeStyleTM serum-free medium (Life Technologies) for 5-7 days. The supernatant was collected, subjected to centrifugal ultrafiltration, and then purified by ProteinA/G or nickel column affinity chromatography and molecular sieve chromatography.

(2) Preparation of stable cell lines expressing human B7-H3 antigen: The full-length sequence encoding human B7-H3 was constructed into the pcDNA 3.1 expression vector carrying IRES-EGFP. CHO-S (ATCC) cells were cultured in CD-CHO medium (Life Technologies); Hela cells were cultured in DMEM containing 10% fetal bovine serum. Cells were transfected using Lipofectamine LTX (Life Technologies) transfection reagent, flow sorted 48 hours later, cultured in 96-well plates, and monoclonal stable cell lines were screened and identified. CHO (CHO.B7-H3-EGFP) and Hela (Hela.B7-H3-EGFP) cells stably expressing B7-H3 were maintained.

Example 2

1. Preparation of anti-B7-H3 monoclonal antibody:

(1) Animal immunization

Balb/c female mice aged 5-6 weeks were used as immunized animals, and the immunization dose was 100μg/mouse. For the first immunization, 100μl of Freund's complete adjuvant (Sigma) was mixed with an equal volume of recombinant B7-H3 protein, and multiple subcutaneous injections were performed after sufficient emulsification. Every 2 weeks, an equal volume of Freund's incomplete adjuvant (Sigma) was mixed with the recombinant protein, and multiple subcutaneous injections were performed after sufficient emulsification. The booster immunization was performed 4 times in total. On the 10th day after the last booster immunization, the mouse antibody titer was detected by cutting the tail and collecting blood. 3 days before cell fusion, 100μg of recombinant protein was injected into the abdominal cavity once.

(2) Cell fusion and hybridoma screening

Under sterile conditions, the spleen of mice was taken to prepare a suspension rich in B cells, and the cells were fused with SP2/0 according to the classic PEG (Sigma) method. The fused cells were resuspended in HAT medium for culture. Fresh HAT medium was used for half-medium replacement culture on the 5th and 10th days after fusion. ELISA, immunofluorescence and flow cytometry were performed on the 11th to 15th day after fusion to screen positive clones.

ELISA screening was performed using a 96-well plate. Briefly, the B7-H3 recombinant protein was coated at 100 ng/well at 4 degrees overnight on the bottom of the well plate, and HRP-conjugated anti-mouse IgG antibody and chemiluminescent reagent (Biyuntian Biotechnology) were used for color development, and the values were read on a microplate reader at a wavelength of 450 nm. Immunofluorescence staining used Hela cell lines that stably expressed B7-H3. Briefly, the cell lines were cultured on 96-well plates, 50 μl of hybridoma supernatant was added as the primary antibody, incubated at 4 degrees for 2 hours, washed 3 times with PBS, and Cy3-labeled Goat Anti-Mouse IgG (Proteintech) was used as the secondary antibody, incubated at room temperature for 1 hour, washed 3 times with PBS, and images were collected using a fluorescence microscope. Flow cytometry analysis was performed using CHO cells stably expressing B7-H3. Briefly, cells were collected by centrifugation and resuspended in PBS buffer containing 50 μl of hybridoma supernatant, incubated at 4°C for 2 h, washed three times with PBS, and CY3-labeled Goat Anti-Mouse IgG (Beyotime Biotech) was used as a secondary antibody. The cells were incubated at room temperature for 1 h, washed three times with PBS, and analyzed on a flow cytometer (Agilent-Novosampler Pro).

Based on the above ELISA analysis, immunofluorescence analysis and flow cytometry analysis results, eleven optimal hybridoma clones (named 1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, and 15D7) were finally determined for subsequent experiments such as sequence cloning and affinity analysis.

Example 3

Hybridoma antibody variable region sequence cloning: Hybridoma cells in the logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen) and reverse transcribed (PrimeScriptTM Reverse Transcriptase, Takara). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen) and then sequenced, and finally the heavy chain and light chain variable region sequences of eleven monoclonal antibodies were obtained. The variable region CDR sequences of the heavy chain and light chain are shown in Table 1.

Table 1. CDR sequences contained in the heavy and light chain variable regions of mouse monoclonal antibodies

Example 4

Binding analysis of recombinant chimeric antibodies to HeLa cervical cancer cells overexpressing B7-H3

The human (IgG1) and mouse (IgG2a) heavy chain constant regions, as well as the human/mouse light chain constant regions, were cloned into the pcDNA3.1 (Invitrogen) plasmid vector, and then the VH and VL gene fragments of the hybridoma clones 1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, and 15D7 were respectively constructed into the gene recombination vectors containing the human IgG1 heavy chain constant region and the human IgGκ light chain constant region to obtain the recombinant chimeric antibody heavy chain expression vector and light chain expression vector. The recombinant chimeric antibody was transiently transfected into 293FT and cultured in a shake flask using FreeStyleTM serum-free medium (Life Technologies) for 5-7 days. The supernatant was collected, subjected to centrifugal ultrafiltration, and then purified by Protein A/G affinity chromatography and molecular sieve chromatography columns to obtain the corresponding type of anti-B7-H3 recombinant monoclonal antibodies. Hela.B7-H3-EGFP cells were plated in a 24-well cell culture dish, and the next day, recombinant chimeric antibodies 1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, and 15D7 were used as primary antibodies, and CY3-labeled Goat Anti-Mouse IgG (Biyuntian Biotechnology Co., Ltd.) was used as secondary antibodies, and fluorescence confocal microscopy was used to observe and photograph them. As shown in Figure 1, the results in Figure 1 show that recombinant chimeric antibodies 1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, and 15D7 can specifically bind to cells Hela.B7-H3-EGFP.

Example 5

Binding analysis of single-chain antibody to endogenous B7-H3

Human skin cancer cells A375 with positive expression of B7-H3 and A375 cells with targeted knockout of B7-H3 by CRISPR/Cas9 were selected and flow cytometry analysis was performed according to the method described in Example 2.

Among them, the preparation of A375 cells for targeted knockout of B7-H3 adopted the classic CRISPR/Cas9 gene editing method. The gRNA targeting B7-H3 was designed using CHOPCHOP (http://chopchop.cbu.uib.no) software. The gRNA was then subcloned into the lentiCRISPR V2 lentiviral vector (Addgene: #52961). The lentiviral vector and packaging plasmid were co-transfected into HEK293T cells, and the viral supernatant was harvested. After centrifugation and concentration, A375 cells were infected, and 1 μg/ml of puromycin was added for screening after 48 hours. After 3-5 days of screening, the cells were collected for flow cytometry analysis, and the experimental results are shown in Figure 2.

The results of Figure 2a show that B7-H3 positive A375 cells can be stained almost 100% positive by 1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, 15D7 mouse single chain antibodies, among which 1H9S, 2H2, 5A3, 5H5, 7H7, 8F3, G10H mouse single chain antibodies show stronger staining. The results of Figure 2b show that B7-H3 targeted knockout A375 cells are stained negative. This shows that 1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, 15D7 mouse single chain antibodies can recognize B7-H3 molecules endogenously expressed in cells.

Example 6

Binding analysis of hybridoma antibodies to mouse B7-H3

The cDNA clone of mouse B7-H3 was purchased from Sino Biological, and then the stable expression cell line CHO-mB7-H3 of mouse B7-H3 was constructed according to Example 2. Then the supernatant of hybridomas 1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, and 15D7 was taken and flow cytometry analysis was performed according to Example 2. The results showed that the monoclonal antibody could not bind to mouse B7-H3.

Example 7

In vitro binding affinity and kinetics experiments

This example uses the surface plasmon resonance (SPR) method for determination, and uses the GE Biacore 8K instrument for analysis. Using the kit provided by Biacore, the B7-H3-His recombinant protein was covalently linked to the CM5 (GE) chip using the standard amino coupling method, and then the single-chain antibody to be tested was diluted in the same buffer according to different concentration gradients and injected. After injection, it was regenerated with the regeneration reagent in the kit. The data was analyzed and collected using the Biacore 8K supporting analysis software. The results are shown in Table 2 below.

Table 2. Antibody-antigen in vitro binding affinity and kinetic analysis

Example 8

In vitro killing experiment of B7-H3-CD3 bispecific antibody

Preparation of effector cells and target cells: Peripheral blood was drawn from healthy donors, PBMC was separated, and T cells were separated using a T cell isolation kit (Miltenyi T Cell Isolation Kit). The separated T cells were cultured in X-VIVO (LONZA) medium containing 5% AB serum. The 6-well plate coated with TC was pre-incubated with 1 ml of coating solution containing 50 ng/ml anti-human CD3 antibody (PeproTech) and 50 ng/ml CD28 antibody (PeproTech) at 37°C for 2 hours, and the coating solution was removed before use. The cells were inoculated into the 6-well plate coated with antibodies at 1 ml/well. After stimulation and culture for 48 hours, IL-2 (100 U/mL) and IL-15 (10 ng/mL) were added as activation factors to continue the culture. The target cells were selected from human skin cancer cells A375 with high expression of B7-H3 and cultured in DMEM high-glucose culture medium supplemented with 10% fetal bovine serum (Gibco).

Luciferase reporter gene refers to a reporter system that uses luciferin as a substrate to detect the activity of firefly luciferase. Luciferase can catalyze the oxidation of luciferin to oxyluciferin, and during the oxidation process of luciferin, bioluminescence is emitted. The lentiviral plasmid carrying the luciferase gene (Addgene: #72486) and the packaging plasmid were co-transfected into HEK293T cells, and the viral supernatant was harvested. After centrifugation and concentration, A375 cells were infected and flow sorted 48 hours later, and finally A375-luciferase cells (A375-lucf) were obtained for subsequent co-culture experiments.

Cell co-culture experiment: A375-lucf tumor cells in the logarithmic growth phase were added to a 96-well plate. After overnight culture, 0 ng/ml (Control) or 10 ng/ml of bispecific antibody was added. T cells were added at the same time according to the effector-target ratio of T cells to tumor cells of 1:1, 2:1, 4:1 and 8:1. After 24 hours, luciferin was added and the cell survival rate was measured by reading the bioluminescence value. The formula is as follows:

Cell survival rate % = bioluminescence value of experimental group/bioluminescence value of negative control group × 100%

The negative control group refers to the A375-lucf group with normal growth without addition of bispecific antibody and T cells. The data shown are mean ± s.d. P values were obtained using unpaired two-tailed Student's t-test. The experiment was repeated three times using T cells from different donors and similar results were obtained. Representative results are shown in Figure 3.

As can be seen from the results in Figure 3, compared with the group without adding bispecific antibodies (Control), eleven B7-H3-CD3 bispecific antibodies (1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, 15D7) had a significant killing effect on A375-lucf tumor cells (*P<0.05, **P<0.01, ***P<0.001).

Embodiment 9

Xenograft mouse model anti-tumor experiment

This example uses a xenograft mouse model to evaluate the in vivo antitumor activity of B7-H3-targeted bispecific antibodies (1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, 15D7). An immunodeficient mouse model was used for evaluation.

NCG severe immunodeficiency mouse model: NCG severe immunodeficiency mice were purchased from the Model Animal Institute of Nanjing University. 2×10 ⁶ A375 cells in the logarithmic growth phase were inoculated subcutaneously on the right back of the NCG mice. After about 6 days when the tumor grew to 200 mm ³ , the mice with uniform tumor volume were randomly divided into groups, with 5 mice in each group. A control group was set up with a saline solution of the same volume as the drug administration. The bispecific antibody was administered intraperitoneally, 30 μg/mouse, once every 3 days, for a total of 4 times. The mice were weighed and the tumor size was measured every 3 days. The average volume of the transplanted tumor was calculated according to the formula V=1/2(L×W ² ), where L represents the length of the tumor and W represents the width of the tumor. When the mouse tumor volume reached 2000 mm3 or the tumor surface showed obvious ulceration, the mouse was killed and the animal experiment was terminated. All data are mean ± sdP values obtained using an unpaired two-tailed Student's t test. The experimental results are shown in Figure 4.

As shown in the results of Figure 4, compared with the control group without drug administration, the eleven B7-H3-CD3 bispecific antibodies (1H9S, 2H2, 3HS, 5A3, 5H5, 7H7, 8F3, 8HS, G10H, 10HS, 15D7) all had significant inhibitory effects on the growth of A375 tumor cells, among which the 3HS/8HS/10HS/15D7-CD3 group **P<0.01, the 1H9S/7H7/8F3-CD3 group ***P<0.001, and the 2H2/5A3/5H5/G10H-CD3 group had the best inhibitory effect ****P<0.0001.

The following is the sequence involved in the present invention:

The above specific embodiments are merely explanations of the present invention, and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims (10)

1. An anti-B7-H3 antibody or an antigen-binding fragment thereof, characterized in that the antibody or the antigen-binding fragment thereof comprises an amino acid sequence such as HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 11, and HCDR3 shown in SEQ ID NO: 12, and an amino acid sequence such as LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 15, and LCDR3 shown in SEQ ID NO: 16. 2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 9 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 13. 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that the antibody or antigen-binding fragment thereof is a chimeric antibody, a single-chain antibody or a bispecific antibody. 4. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3. 5. An expression vector comprising the nucleic acid molecule of claim 4. A host cell comprising the expression vector of claim 5 . 7. A pharmaceutical composition, characterized in that it comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, or any combination thereof, and a pharmaceutically acceptable carrier. 8. An immunoconjugate, characterized in that it comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 conjugated to a therapeutic agent; the therapeutic agent is a toxin, a drug, a radioactive isotope or a cytotoxic agent. 9. A method for preparing the anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising the steps of expressing the antibody or antigen-binding fragment thereof in the host cell according to claim 6, and recovering and isolating the antibody or antigen-binding fragment thereof from the host cell. 10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the nucleic acid molecule according to claim 4, or the expression vector according to claim 5, or the host cell according to claim 6, the pharmaceutical composition according to claim 7, or the immunoconjugate according to claim 8 in the preparation of an agent or drug for diagnosing or treating inflammatory diseases or tumors, characterized in that the tumors include melanoma and cervical cancer.