Monoclonal antibody against immune checkpoint molecule CD276 and application thereof

Abstract

This invention belongs to the field of biomedicine and tumor immunotherapy technology, specifically relating to a monoclonal antibody targeting the immune checkpoint molecule CD276 (B7-H3) and its applications. The applicant conducted immunization and screening work around the human CD276 antigen, selecting two representative monoclonal antibody clones, 2B2-1 and 5G4-2, from multiple candidate antibodies. The nucleotide and amino acid sequences of their heavy and light chain variable regions were determined, and their performance in flow cytometry, T-cell killing assays, immunohistochemistry, ELISA, and Biacore affinity assays was systematically evaluated. Experimental results show that the above antibodies can specifically recognize and bind to CD276, efficiently distinguish between CD276-high and low-expressing cells, and enhance the killing activity of T cells against CD276-high expressing tumor cells in an in vitro co-culture system. This provides an excellent antibody backbone for the subsequent construction of diagnostic kits, ADC drugs, or bispecific antibodies.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and tumor immunotherapy technology, specifically relating to a monoclonal antibody targeting the immune checkpoint molecule CD276 (B7-H3) and its applications, including the antibody amino acid / nucleotide sequence, diagnostic reagents for tumor cell detection, and pharmaceutical compositions for tumor immunotherapy. Background Technology

[0002] Tumor immunotherapy has made groundbreaking progress in recent years, with immune checkpoint inhibitors, such as PD-1 / PD-L1 and CTLA-4, approved for use in various malignant tumors. However, numerous clinical and cohort studies have shown that only a subset of patients benefit from existing immune checkpoint therapies, and some patients with initial responses develop acquired resistance, suggesting the continued need to explore new immune regulatory targets and develop more precise antibody drugs. B7-H3 (also known as CD276), as a novel immune checkpoint molecule in the B7 family, is widely and highly expressed in various solid tumors and hematological malignancies, and has been considered a highly promising "next-generation" tumor immunotherapy target in recent years.

[0003] B7-H3 / CD276 is a type I transmembrane glycoprotein with an extracellular region composed of immunoglobulin V-like and C-like domains. In humans, it primarily exists in two splicing isoforms: 2IgB7-H3 and 4IgB7-H3, and its gene is located in the human 15q24 region. Early studies suggested that B7-H3 had a co-stimulatory effect on T cells, but subsequent extensive evidence indicates that B7-H3 primarily functions as a co-inhibitory molecule in the tumor microenvironment, inhibiting T cell activation and proliferation, and reducing the secretion of cytokines such as IFN-γ and TNF-α, thereby participating in tumor immune escape. Unlike traditional B7 family molecules, B7-H3 expression levels are low in most normal tissues, but significantly upregulated in various tumor tissues, including lung cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, and glioma. Its high expression is often significantly associated with tumor stage progression, lymph node metastasis, and poor prognosis, thus it is considered a tumor-associated antigen with both diagnostic and therapeutic value.

[0004] Besides tumor cells themselves, B7-H3 is also widely expressed in tumor stroma and vascular-associated components, including tumor-associated endothelial cells, cancer-associated fibroblasts (CAFs), and some myeloid immune cells. In tumors such as ovarian cancer, colorectal cancer, and breast cancer, high expression of B7-H3 is often closely related to suppressed tumor-infiltrating lymphocyte function, an immune-rejecting microenvironment, and the upregulation of other inhibitory checkpoint molecules (such as PD-1 and TIM-3). This suggests that B7-H3 is not only a single tumor cell surface antigen, but also a crucial hub for reshaping the tumor immune microenvironment and driving the immunosuppressive network.

[0005] Recent multi-omics and functional studies have further revealed that B7-H3 also has significant non-immune functions in tumor progression. B7-H3 can promote tumor cell proliferation, epithelial-mesenchymal transition (EMT), invasion, and metastasis by activating multiple signaling pathways, including JAK2 / STAT3, PI3K / AKT, and ERK / MAPK. It also participates in multiple processes such as tumor angiogenesis, cell metabolic reprogramming, radiotherapy and chemotherapy tolerance, and tumor stemness maintenance. Some studies have also found that the synergistic high expression of B7-H3 in tumor stem-like cells and tumor vascular endothelium helps tumors gain a growth advantage at both the immune and non-immune levels. Therefore, treatment targeting B7-H3 holds promise for simultaneously combating tumor immune escape and the "seed-soil" system of solid tumors.

[0006] Based on the aforementioned biological characteristics, targeting strategies around B7-H3 / CD276 have developed rapidly in recent years, encompassing various forms such as therapeutic monoclonal antibodies, antibody-drug conjugates (ADCs), bispecific antibodies, CAR-T / CAR-NK cells, and radioimmunoconjugates. Taking anti-B7-H3 ADCs as an example, drugs such as MGC018 and DS-7300a (ifinatamab deruxtecan) have demonstrated considerable objective response rates and manageable safety in various clinical trials for solid tumors with high B7-H3 expression. Furthermore, new-generation B7-H3 ADCs such as YL201 have achieved positive phase I / Phase Ib results in patients with advanced small cell lung cancer and nasopharyngeal carcinoma. Simultaneously, B7-H3-CAR-T therapy has shown promising early anti-tumor activity in refractory diseases such as pediatric solid tumors and brain tumors. However, no B7-H3 targeted drugs have yet been approved for marketing, and indications, patient stratification, safety, and combination therapy strategies are still under investigation.

[0007] Despite the increasing prominence of B7-H3 as a dual target for both tumor immune and non-immune processes, existing anti-B7-H3 antibodies still have several limitations: First, most drugs in clinical trials are based on proprietary humanized or fully human monoclonal antibody backbones developed by individual companies. Publicly available information mainly focuses on clinical results and some pharmacological parameters, and the specific sequence of the antibody variable region is often not fully disclosed, which is not conducive to researchers' replication and derivation design in basic research. Second, most of the research-grade B7-H3 antibodies commonly used in the literature are murine or rabbit monoclonal or polyclonal antibodies. Although they can be used for immunohistochemistry or flow cytometry, different clones vary greatly in terms of epitopes, affinity, and cross-species reactivity. Furthermore, there is a lack of standardized antibodies that have been systematically validated and are suitable for flow cytometry (FACS), immunohistochemistry (IHC), ELISA, and functional cell experiments. Third, there is still a relative scarcity of candidate antibodies that can significantly enhance T cell-mediated tumor cell killing while detecting B7-H3 expression and are easy to further modify into ADCs, bispecific antibodies, or CAR vectors.

[0008] Therefore, developing a new generation of anti-B7-H3 monoclonal antibodies with fully defined sequences, high affinity and specificity, stable and reliable performance on multiple detection platforms, and the ability to enhance T-cell killing and / or combine with immunotherapy is of great significance for promoting the research and development of CD276-targeted diagnostic reagents and therapeutic drugs. Summary of the Invention

[0009] The overall objective of this invention is to provide a class of monoclonal antibodies against the human CD276 antigen, comprising at least two representative clones, 2B2-1 and 5G4-2, with clearly defined nucleotide and amino acid sequences of their heavy and light chain variable regions; and further to provide the application of said antibodies in tumor cell detection (such as flow cytometry, immunohistochemistry, ELISA) and in T cell-mediated tumor killing systems, thereby providing new antibody tools and drug candidates for CD276-targeted diagnosis and treatment.

[0010] This invention is achieved through the following technical solution: A monoclonal antibody targeting the immune checkpoint molecule CD276, wherein the monoclonal antibody specifically binds to the human CD276 antigen, the amino acid sequence of the heavy chain variable region (containing the signal peptide and the FR1–FR4 segment) of the monoclonal antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region (containing the signal peptide and the FR1–FR4 segment) is shown in SEQ ID NO:4 (representative clone 2B2-1).

[0011] A nucleic acid molecule encoding the heavy chain variable region and / or light chain variable region of the above-mentioned monoclonal antibody, the nucleotide sequence of the nucleic acid molecule being shown in SEQ ID NO:1 and / or SEQ ID NO:3.

[0012] A monoclonal antibody targeting the immune checkpoint molecule CD276, wherein the monoclonal antibody specifically binds to the human CD276 antigen, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8 (representative clone 5G4-2).

[0013] A nucleic acid molecule encoding the heavy chain variable region and / or light chain variable region of the aforementioned monoclonal antibody, the nucleotide sequence of the nucleic acid molecule being shown in SEQ ID NO:5 and / or SEQ ID NO:7.

[0014] An expression vector comprising the aforementioned nucleic acid molecule.

[0015] A CD276 detection reagent or kit, wherein the detection reagent or kit contains the monoclonal antibody as a specific recognition component, for qualitative or quantitative detection of CD276 in flow cytometry, immunohistochemical staining or enzyme-linked immunosorbent assay.

[0016] A pharmaceutical composition comprising the monoclonal antibody and a pharmaceutically acceptable carrier.

[0017] The use of the monoclonal antibody in the preparation of a drug for treating tumors, wherein the tumor is a tumor that highly expresses CD276.

[0018] Furthermore, the monoclonal antibody can be used as a single agent or in combination with PD-1 / PD-L1 antibodies, chemotherapy drugs, radiotherapy agents, or targeted drugs.

[0019] An antibody derivative, wherein the antibody derivative is obtained by modifying the monoclonal antibody, and the derivative is selected from antibody-drug conjugates (ADCs), bispecific antibodies, Fc engineered antibodies, Fab fragments, scFv fragments, B7-H3-targeted CAR-T receptors or CAR-NK receptors.

[0020] The present invention has the following advantages over the prior art: This invention focuses on immunization and screening of the human CD276 antigen. Two representative monoclonal antibody clones, 2B2-1 and 5G4-2, were selected from multiple candidate antibodies. The nucleotide and amino acid sequences of their heavy and light chain variable regions were determined, and their performance in flow cytometry, T-cell killing assays, immunohistochemistry, ELISA, and Biacore affinity assays was systematically evaluated. Experimental results show that these antibodies can specifically recognize and bind to CD276, efficiently distinguish between CD276-high and low-expressing cells, and enhance the killing activity of T cells against CD276-high expressing tumor cells in an in vitro co-culture system. This provides an excellent antibody backbone for the subsequent construction of diagnostic kits, ADC drugs, or bispecific antibodies. Its main advantages are: (1) The sequences are well-defined and highly reproducible: The heavy chain and light chain variable region nucleotide and amino acid sequences of the 2B2-1 and 5G4-2 antibodies provided by this invention have been clearly given, which facilitates patent protection, sequence listing submission, and subsequent recombinant expression and engineering modification, overcoming the limitation that some existing B7-H3 antibody sequences are not publicly available or are limited to commercial reagents.

[0021] (2) It has good specificity and high affinity for CD276: According to the biophysical detection results of Biacore and other existing biophysical assays, the antibody of the present invention can bind stably and specifically to the extracellular region of human CD276, with a low dissociation rate and an affinity that reaches the high affinity level commonly found in monoclonal antibody drugs.

[0022] (3) Applicable to multiple detection platforms: The applicant has verified in flow cytometry, immunohistochemical staining and ELISA that the antibody of this invention has low background signal and high signal-to-noise ratio when detecting CD276 expression, and can effectively distinguish between cell or tissue samples with high and low CD276 expression, making it suitable as a standardized detection tool.

[0023] (4) It has the potential to enhance T cell killing: In the in vitro T cell killing co-culture system, after adding the antibody of the present invention, the proportion of CD276-expressing tumor cells that die is significantly increased compared with the control IgG group, indicating that this type of antibody can not only be used for expression detection, but also has a certain immune effect function, providing a functional basis for further development of ADC, bispecific or CAR-T, etc.

[0024] (5) Possesses broad engineering and translation potential: Since the antibody variable region sequence is known, Fc engineering, humanization, affinity maturation and different isotype switching can be carried out without changing its specific epitope recognition. It can also be used to construct drug forms adapted to different clinical needs (such as long-acting antibodies, Fc-silent antibodies or high ADCC antibodies, etc.). Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the structures of the anti-CD276 antibodies 2B2-1 and 5G4-2 of the present invention; Figure 2 The results of flow cytometry staining of the antibody of this invention on different cell lines; Figure 3 Immunohistochemical staining of the antibody of this invention in tumor tissue; Figure 4 These are the results of a T-cell killing experiment; Figure 5 This is the ELISA binding curve of the antibody of this invention with CD276; Figure 6 The curve for determining Biacore affinity. Detailed Implementation

[0026] To further explain the present invention, the following specific embodiments are described.

[0027] Antibody sequence information Multiple candidate antibodies were obtained through immunization and screening against the human CD276 antigen. After comprehensive flow cytometry, affinity assays, and functional verification, two representative clones, 2B2-1 and 5G4-2, were ultimately selected. The screening process employed at least one or more of the following criteria: First, in ELISA and / or surface plasmon resonance (SPR) assays using recombinant human CD276 protein as the coating antigen, the candidate antibodies exhibited high binding capacity and low dissociation rate, with affinity at a superior level within the candidate library. Second, in flow cytometry, they demonstrated a significant difference in average fluorescence intensity between CD276-high expression cell lines and low-expression / negative cell lines, with low background signal and high signal-to-noise ratio. Third, in in vitro T-cell co-culture killing experiments, they significantly enhanced the killing effect of T cells on CD276-high expression tumor cells. Fourth, they maintained good expression levels and antigen-binding activity after multiple batches of expression and freeze-thaw storage. Based on the above comprehensive indicators, clones 2B2-1 and 5G4-2 were selected as the representative antibodies of the present invention, and their variable region nucleotide and amino acid sequences are shown in SEQ ID NO:1–8, respectively.

[0028] SEQ ID NO:1 (2B2-1 heavy chain DNA sequence) ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTAATTTTAAAAGGTGTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGGCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCA ACCATTAGTGGTGGTGGTCGTTACACCTACTTTCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCTTGTATTACTGTGCAAGACATGATGGTAACCGTCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO:2 (2B2-1 heavy chain amino acid sequence) MNFGLSLIFLVLILKGVQCEVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVATISGGGRYTYFPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARHDGNRLDYWGQGTTLTVSS SEQ ID NO:3 (2B2-1 light chain DNA sequence) ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGTGACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAACAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTATTGTCAACATCATTATGGTACTCCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA SEQ ID NO:4 (2B2-1 light chain amino acid sequence) MSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPPYTFGGGTKLEIK SEQ ID NO:5 (5G4-2 heavy chain DNA sequence) ATGGGCTGGTCCTGCATCATCCTGTTCCTGGTGGCCACCGCCACAGGCGTTCATAGCAAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATACACCGTACACTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAAGTGTTAATCCTGGCGATGGTGGTACTTACTACAACCAGAAATTCAGGGGCAGGGCCTCATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTGCAAGACATGCCTACGGTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA SEQ ID NO:6 (5G4-2 heavy chain amino acid sequence) MGWSCIILFLVATATGVHSKVQLQQSGPELVKPGASVKISCKTSGYTFTEYTVHWVKQSHGKSLEWIGSVNPGDGGTYYNQKFRGRASLTVDKSSSTAYMELRSLTSEDSAVYYCARHAYGAMDYWGQGTSVTVSS SEQ ID NO:7 (5G4-2 light chain DNA sequence) ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGTGACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAACAGAAACAGGGAAAATCTCCT CAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTATTGTCAACATCATTATGGTACTCCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAAAAA SEQ ID NO:8 (5G4-2 light chain amino acid sequence) MSVPTQVLGLLLLWLTGARCDIQMTQSPASLSSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPPYTFGGGTKLEIK Figure 1 The diagram shows the structural composition of the anti-CD276 antibodies 2B2-1 and 5G4-2 of the present invention, including the heavy and light chain domains of the two antibodies, including a signal peptide, a variable region (FR / CDR), and a constant region. The SEQ ID numbers corresponding to the heavy and light chain variable region sequences are marked.

[0029] Example 1: Antibody Preparation and Expression The antibodies of this invention are obtained in the following manner: (1) Prepare recombinant proteins containing the extracellular region of human CD276, or construct cell lines that stably and highly express human CD276 for repeated immunization of mice. The immunization regimen may include basic immunization and 2-4 booster immunizations to induce a high-titer antibody response against CD276.

[0030] (2) After the last immunization, spleen cells of immunized animals are taken and fused with myeloma cells to prepare hybridomas, or a phage display antibody library is constructed from immunized spleen cells.

[0031] (3) Double screening was performed using ELISA coated with recombinant CD276 protein and flow cytometry targeting CD276 high-expression / low-expression cells to enrich positive clones that specifically recognize CD276.

[0032] (4) The positive clones were subcloned by limiting dilution to obtain stable cell lines that secrete a single specific antibody. The supernatant was collected for subsequent functional verification.

[0033] (5) Total RNA was extracted from the above cell lines, and the heavy and light chain variable region sequences were amplified by RT-PCR using universal primers targeting the variable region of mouse immunoglobulins, followed by sequencing. The primers can be the Mouse IgG Library Primer Set or heavy / light primer mix commonly used in the art, and can be combined with 3′ primers targeting the constant region (CH1 region) of the mouse IgG heavy chain and the constant region (Cκ) of the κ light chain, respectively, to obtain complete VH and VL fragments. This invention is not limited to specific primer sequences; those skilled in the art can design or select other equivalent primers based on known conserved framework region sequences of immunoglobulins, as long as the correct variable region sequence can be amplified, it falls within the scope of this invention.

[0034] (6) The measured heavy and light chain variable region sequences were cloned into recombinant expression vectors containing the human IgG heavy chain and κ chain constant regions, respectively. These vectors were then transiently or stably co-transfected into mammalian cells such as HEK293 or CHO. The cell supernatant was collected and purified using Protein A / G affinity chromatography and gel filtration to obtain the antibody of this invention. If necessary, antibody purity can be detected by SDS-PAGE and SEC-HPLC, and the molecular weight and sequence correctness of the heavy and light chains can be confirmed by mass spectrometry or N-terminal sequencing.

[0035] Example 2: Flow cytometry verification of CD276 binding specificity Tumor cell lines with high CD276 expression and those with low CD276 expression were selected as controls. Cells in the logarithmic growth phase were digested, centrifuged, and resuspended in PBS containing 1% fetal bovine serum or 1% BSA, adjusting the concentration to approximately 1×10⁻⁶. 6 Add antibody 2B2-1 or 5G4-2 of this invention to a final concentration of 0.1-10 μg / mL, incubate at 4°C in the dark for 30 min, and set up an isotype control IgG at an equal concentration as a negative control. After incubation, wash 2-3 times with PBS, add fluorescently labeled anti-human IgG secondary antibody (such as FITC or PE labeling), and continue incubation at 4°C in the dark for 20-30 min. After washing, collect no less than 1×10⁻⁶ cells / mL using a flow cytometer. 4Each cell event was analyzed, and geometric mean fluorescence intensity (MFI) and positivity rate were recorded. The specificity and detection sensitivity of different antibody clones were evaluated by comparing the MFI differences and signal-to-noise ratios between CD276-high expression cells and low expression / negative cells. Figure 2 The flow cytometry staining results of the antibodies of this invention on different cell lines are shown. The horizontal axis represents CD276 staining intensity, and the vertical axis represents cell count. The peak shape differences between CD276 high-expression cell lines and low-expression cell lines under 2B2-1, 5G4-2, and isotype control staining are compared to demonstrate the ability of the antibodies to distinguish between high and low expression. The results show that on CD276 high-expression cells, both antibodies can obtain significantly higher specific positive signals than the isotype control, while the background signal is lower on CD276 low-expression or negative cells. Both antibodies can clearly distinguish between CD276 high-expression and low-expression cells, verifying their good specificity and detection sensitivity.

[0036] Example 3: Evaluation of Immunohistochemical (IHC) Staining Performance Tumor tissue microarrays or surgically removed specimens fixed in 10% neutral formaldehyde and embedded in paraffin were selected, with a section thickness of 3-4 μm. After dewaxing and hydration, antigen retrieval was performed by high-pressure or microwave in citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) for 10-20 min. After natural cooling, endogenous peroxidase was blocked with 3% hydrogen peroxide. The sections were then blocked with 5% normal goat serum for 20 min, and the antibodies 2B2-1 or 5G4-2 of this invention (e.g., diluted 1:100-1:400) were added. The sections were incubated overnight at 4°C. The next day, the sections were washed with PBS, and HRP-labeled secondary antibody was added. The sections were incubated at room temperature for 30 min. After DAB staining, hematoxylin counterstaining, dehydration, and mounting, the sections were observed under an optical microscope. The results are as follows: Figure 3 As shown. The CD276 staining level was evaluated using a semi-quantitative integral score (staining intensity × proportion of positive cells) and compared with negative control sections from the same batch.

[0037] Example 4: T-cell killing experiment (functional verification) CD276-overexpressing tumor cells were seeded into 96-well plates. After stable adhesion, human peripheral blood T cells pre-activated with anti-CD3 / CD28 microbeads were added. The effector cell to target cell ratio (E:T) could be set to multiple gradients such as 1:1, 5:1, and 10:1. Antibodies 2B2-1 or 5G4-2 and an equal amount of isotype control IgG were added to the experimental wells. If necessary, cross-linked secondary antibodies or bispecific bridging structures were added to enhance the effect. After co-culturing for 24–72 h, the proportion of tumor cell death was detected by flow cytometry combined with live / dead staining or lactate dehydrogenase (LDH) release assay. Figure 4The results of the T-cell killing experiment are shown in bar charts or line graphs, illustrating the changes in tumor cell mortality or survival rates under different antibody treatment conditions. Comparisons were made between the control IgG group and the 2B2-1 and 5G4-2 treatment groups, with statistical significance indicated based on the actual data. The results show that, at the same E:T ratio, the target cell mortality rate in the antibody treatment group was significantly higher than that in the isotype control group, suggesting that the antibody of this invention can enhance the T-cell-mediated killing effect on CD276-overexpressing tumor cells.

[0038] Example 5: ELISA Binding Activity Detection Recombinant human CD276 protein was coated at concentrations of 1–5 μg / mL onto 96-well high-binding-strength microplates (100 μL per well) and incubated overnight at 4°C. After discarding the coating solution, the plates were blocked with 1% BSA-containing PBS for 1 h, and then serially diluted antibodies of this invention (e.g., from 0.01 μg / mL to 10 μg / mL) were added and incubated at room temperature for 1 h. After washing, HRP-labeled anti-human IgG secondary antibody was added, and the plates were incubated at room temperature for 30 min. After TMB colorimetry, OD values ​​were read at 450 nm, and concentration-response curves were plotted. The half-maximum effect concentration (EC50) was calculated to compare the binding activity of different clones to CD276. Figure 5 ).

[0039] Example 6: Biacore Affinity Measurement Recombinant human CD276 protein was immobilized on the CM5 chip surface via amine coupling in an acetate buffer at pH 4.5, with the coupling level controlled at approximately 500–1500 RU. Using a buffer containing 10 mmol / L HEPES, 150 mmol / L NaCl, and 0.005% Tween-20 (pH 7.4) as the mobile phase, different concentrations (e.g., 0.5–32 nM) of the antibody of this invention were sequentially injected into the flow path. Binding and dissociation curves were recorded, and the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (Kd) were obtained by fitting the data using a 1:1 Langmuir kinetic model. Figure 6 The Biacore affinity assay curves show the binding and dissociation sensing curves of 2B2 (Anti-CD276-2) and 5G4-2 (Anti-CD276-5) with immobilized CD276 protein at different concentrations, highlighting their high affinity characteristics. Experimental results show that the binding curve of the antibody of this invention to CD276 fits well, the dissociation rate is slow, and the affinity reaches the high affinity level commonly seen in therapeutic monoclonal antibodies.

[0040] Example 7: Construction of derived molecules based on antibodies of the present invention Based on the understanding of the variable region sequence of the antibody of this invention, those skilled in the art can further construct various derivative molecules, such as: 1. B7-H3-targeting ADCs are prepared by conjugating full-length IgG of 2B2-1 or 5G4-2 with cytotoxic small molecule drugs (such as topoisomerase I inhibitors, microtubule inhibitors, etc.) via cleavable linkers.

[0041] 2. The scFv fragment of the antibody of the present invention is linked with the scFv fragment of the anti-CD3 antibody to construct a B7-H3×CD3 bispecific T cell connector, so as to further amplify the T cell-mediated killing effect.

[0042] 3. The scFv sequence of the antibody of the present invention is embedded into the CAR receptor framework to construct B7-H3-targeting CAR-T or CAR-NK cells for adoptive cell therapy of refractory and relapsed solid tumors. The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A monoclonal antibody targeting the immune checkpoint molecule CD276, characterized in that, The monoclonal antibody specifically binds to the human CD276 antigen. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

2. A monoclonal antibody targeting the immune checkpoint molecule CD276, characterized in that, The monoclonal antibody specifically binds to the human CD276 antigen. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the heavy chain variable region and / or light chain variable region of the monoclonal antibody of claim 1, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:1 and / or SEQ ID NO:

3.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the heavy chain variable region and / or light chain variable region of the monoclonal antibody of claim 2, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:5 and / or SEQ ID NO:

7.

5. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 3 or 4.

6. A CD276 detection reagent or kit, characterized in that, The detection reagent or kit contains the monoclonal antibody as described in claim 1 or 2 as a specific recognition component, for qualitative or quantitative detection of CD276 in flow cytometry, immunohistochemical staining or enzyme-linked immunosorbent assay.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the monoclonal antibody as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

8. The use of the monoclonal antibody according to claim 1 or 2 in the preparation of a medicament for treating tumors, characterized in that, The tumor is a tumor that highly expresses CD276.

9. The use according to claim 8, characterized in that, The monoclonal antibody can be used as a single agent or in combination with PD-1 / PD-L1 antibodies, chemotherapy drugs, radiotherapy agents, or targeted drugs.

10. An antibody derivative, characterized in that, The antibody derivative is obtained by modifying the monoclonal antibody according to claim 1 or 2, and the derivative is selected from antibody-drug conjugates (ADCs), bispecific antibodies, Fc engineered antibodies, Fab fragments, scFv fragments, B7-H3-targeted CAR-T receptors or CAR-NK receptors.

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