B7-H3 specific binding molecule and application thereof

Abstract

The invention belongs to the field of medicines, and discloses a B7-H3 specific binding molecule and application thereof. The inventor obtains a B7-H3 monoclonal antibody Ab-82 through screening, the VH-CDR of the B7-H3 monoclonal antibody Ab-82 is as shown in SEQ ID NO.3-5 and SEQ ID NO.9, and the VL-CDR of the B7-H3 monoclonal antibody Ab-82 is as shown in SEQ ID NO.6-8. The Ab-82 antibody and the humanized antibody thereof can be highly specifically combined with a B7-H3 antigen and preferentially recognize N91 / 104 / 309 / 322 glycosylation sites of the B7-H3 antigen, have human, mouse and monkey cross activity, and have better B7-H3 internalization mediating ability than that of an Ifinatamab antibody used by a B7-H3 ADC drug DS-7300 with the most advanced clinical test progress at present. When the compound is independently used, tumor growth can be effectively inhibited in vivo, T cells are promoted to infiltrate into tumor tissues, and a potent B7-H3 ADC drug is expected to be developed on the basis of the compound, so that the compound is beneficial to vast tumor patients expressing B7-H3.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a B7-H3 specific binding molecule and its applications. Background Technology

[0002] B7 homolog 3 protein (B7-H3), also known as CD276 or B7RP-2, belongs to the B7 family, the same family as PD-L1 (i.e., B7-H1). It is an important immune checkpoint molecule closely related to the growth, metastasis, and immune escape of malignant tumors, making it a popular target for tumor immunotherapy. Compared to its low expression in normal tissues, B7-H3 is abnormally highly expressed in many human tumor tissues, such as lung cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, osteosarcoma, oral cancer, and hematologic malignancies. It is highly expressed on the surface of tumor cells, tumor stem cells, and matrix fibroblasts, vascular endothelial cells, and antigen-presenting cells in the tumor microenvironment, making it a broad-spectrum tumor marker. Initially considered a co-stimulatory molecule, B7-H3 is increasingly defined as a co-inhibitory molecule, promoting tumor growth and invasion by inhibiting the body's anti-tumor immune response. Multiple studies have shown that B7-H3 inhibits CD4 in tumor tissues. + and CD8 + B7-H3 inhibits the activation and proliferation of T cells while reducing the secretion of active factors such as IL-2, IFN-γ, and TNF-α. Furthermore, it can suppress the activity of immune cells such as NK cells, dendritic cells, macrophages, and neutrophils, reduce the production of perforin and Granzyme B, and create an immunosuppressive tumor microenvironment by promoting the release of TGF-β and IL-10. The expression characteristics and functional features of B7-H3 make it a promising target for research and development. Targeting B7-H3 to relieve immunosuppression and restore the anti-tumor activity of immune cells is an important therapeutic strategy for achieving immune normalization. For many years, drug research and clinical trials targeting B7-H3 have been ongoing. Antibody-drug conjugates (ADCs) based on B7-H3 have demonstrated significant clinical value with the maturation of ADC technology—B7-H3 is stably and highly expressed in most malignant tumors, making it an ideal target for ADC development.

[0003] Antibody-drug conjugates (ADCs) link monoclonal antibodies to a payload via linkers. Using the monoclonal antibody as a carrier, and leveraging its high specificity for the target antigen, the payload (usually a small-molecule cytotoxic drug) is accurately delivered to the surface of tumor cells. The payload then enters the tumor cells via receptor-mediated internalization, releasing its biologically active form and killing the tumor cells. ADCs are a treatment method that effectively improves efficacy while reducing the toxic side effects of drug molecules. The advent of monoclonal antibodies in 1975 and the maturity of protein recombinant engineering technology have made ADCs a hot area in antibody drug development. With the continuous improvement of antibody modification technology, the ongoing advancement of site-specific conjugation technology, and the large-scale development of potent small-molecule toxins, ADC drug research has entered a golden age of rapid development. As of November 2023, 15 ADC drugs have been approved worldwide for the treatment of solid or hematologic malignancies, and 244 ADC drugs are undergoing different phases of clinical trials. Ideally, an ADC drug should be stably transported in plasma, should not release its payload prematurely before reaching tumor tissue, and should achieve highly efficient killing once it enters tumor cells. B7-H3 is a good biomarker for most malignant tumors. Its expression characteristics and functional features are highly compatible with the target antigen selection requirements of ADCs (antibody-drug conjugates). Therefore, the development of ADC drugs targeting B7-H3 has broad prospects and significant clinical application value. Monoclonal antibodies, as the guidance component of ADCs, determine the selectivity of ADCs for tumor cells and are a crucial core framework. An ideal monoclonal antibody framework should have good antigen affinity and effectively mediate the internalization of antigen molecules to achieve precise targeting of tumor cells. Summary of the Invention

[0004] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a B7-H3 specific binding molecule and its application.

[0005] Through in-depth research and creative work, the inventors expressed recombinant B7-H3 using a mammalian cell expression system as an antigen to immunize Japanese white rabbits. Using single-B cell antibody preparation technology, they obtained a monoclonal antibody that specifically binds to B7-H3. By screening a large number of monoclonal antibodies, the inventors obtained a specific monoclonal antibody (named Ab-82) that specifically binds to B7-H3 and preferentially recognizes its N91 / 104 / 309 / 322 glycosylation sites. This monoclonal antibody can mediate B7-H3 internalization very efficiently.

[0006] Furthermore, the inventors obtained Ab-82 through artificial expression and creatively obtained humanized antibodies against B7-H3 (named Hu-Ab-82-Ab5, Hu-Ab-82-Ab14, and Hu-Ab-82-Ab16, respectively).

[0007] The inventors have also surprisingly discovered that the antibodies Ab-82 and Hu-Ab-82-Ab5 of this invention can effectively inhibit tumor growth in vivo and promote T cell infiltration into tumor tissue; they have the potential to be used in the preparation of drugs for the prevention and treatment of osteosarcoma, sarcoma, nephroblastoma, melanoma, mesothelioma, adrenocortical carcinoma, uterine carcinoma, head and neck cancer, endometrioid carcinoma, lung cancer, cholangiocarcinoma, prostate cancer, glioma, neuroblastoma, breast cancer, pancreatic cancer, bladder cancer, testicular cancer, ovarian cancer, esophageal cancer, colorectal cancer, kidney cancer, gastric cancer, cervical cancer, liver cancer, leukemia, lymphoma and other tumors and hematological diseases.

[0008] This leads to the following invention: A first aspect of the invention provides: a B7-H3 specific binding molecule comprising a heavy chain variable region (V H ) and light chain variable region (V L The amino acid sequences of CDR1 to CDR3 of the complementarity-determining region (CDR) of the heavy chain variable region are as shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.9, SEQ ID NO.5, respectively (i.e., the amino acid sequences of CDR1 to CDR3 are as shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, or as shown in SEQ ID NO.3, SEQ ID NO.9, SEQ ID NO.5), and / or the sequences obtained after affinity maturation of the CDRs shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.9, SEQ ID NO.5; The amino acid sequences of CDR1 to CDR3 of the complementarity-determining region (CDR) of the light chain variable region are shown in SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively, and / or the sequences obtained after affinity maturation of the CDRs shown in SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8.

[0009] According to any one of the B7-H3 specific binding molecules of the present invention, the amino acid sequence of the heavy chain variable region of the B7-H3 specific binding molecule is as shown in SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.15, and / or the sequence obtained after affinity maturation of the variable region shown in SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.15.

[0010] According to any one of the B7-H3 specific binding molecules of the present invention, the amino acid sequence of the light chain variable region of the B7-H3 specific binding molecule is as shown in SEQ ID NO.2, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.16, and / or the sequence obtained after affinity maturation of the variable region as shown in SEQ ID NO.2, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.16.

[0011] According to any one of the B7-H3 specific binding molecules of the present invention, the B7-H3 specific binding molecule comprises: (1) The heavy chain variable region as shown in SEQ ID NO.1 and the light chain variable region (Ab-82) as shown in SEQ ID NO.2; (2) The heavy chain variable region as shown in SEQ ID NO.10 and the light chain variable region (Ab-82-Ab5) as shown in SEQ ID NO.11; (3) The heavy chain variable region as shown in SEQ ID NO.12 and the light chain variable region (Ab-82-Ab14) as shown in SEQ ID NO.13. (4) The heavy chain variable region as shown in SEQ ID NO.14 and the light chain variable region (Ab-82-Ab16) as shown in SEQ ID NO.13.

[0012] The amino acid sequences of the three CDR regions of the variable region of the B7-H3 specific binding molecule Ab-82 heavy chain of the present invention are as follows: VH-CDR1:GFSFSSNYWIC (SEQ ID NO.3) VH-CDR2: CIYAGGDGNTYYASWAKG (SEQ ID NO.4) VH-CDR3:DYAGDAYVYFNL (SEQ ID NO.5); The amino acid sequences of the three CDR regions of its light chain variable region are as follows: VL-CDR1: QASENIYSGLA (SEQ ID NO.6) VL-CDR2: LASTLES (SEQ ID NO.7) VL-CDR3: QSVYYSSSDLA (SEQ ID NO. 8).

[0013] The B7-H3 specific binding molecules Hu-Ab-82-Ab5, Hu-Ab-82-Ab14, and Hu-Ab-82-Ab16 of the present invention have the same CDR region, and the amino acid sequences of the three CDR regions of their heavy chain variable region are as follows: VH-CDR1:GFSFSSNYWIC (SEQ ID NO.3) VH-CDR2: CIYAGGEGNTYYASWAKG (SEQ ID NO.9) VH-CDR3:DYAGDAYVYFNL (SEQ ID NO.5); The amino acid sequences of the three CDR regions of its light chain variable region are as follows: VL-CDR1: QASENIYSGLA (SEQ ID NO.6) VL-CDR2: LASTLES (SEQ ID NO.7) VL-CDR3: QSVYYSSSDLA (SEQ ID NO. 8).

[0014] According to any one of the B7-H3 specific binding molecules of the present invention, the main molecule is its Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, chimeric antibody, humanized antibody, single-domain antibody fused with Fc fragment, bivalent or multivalent single-domain antibody, dual-target antibody or multi-target antibody.

[0015] According to any one of the B7-H3 specific binding molecules of the present invention, the Fc fragment is directly or through a linker element attached to the C-terminus or N-terminus of the B7-H3 specific binding molecule; the Fc fragment includes the Fc segment of human IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM, IgE or its variants or modified forms.

[0016] According to any one of the B7-H3 specific binding molecules of the present invention, the B7-H3 specific binding molecule includes a non-CDR region, and the non-CDR region is derived from a species other than rabbits, such as from human antibodies.

[0017] These features can be combined arbitrarily as long as they do not conflict with each other.

[0018] The second aspect of the present invention relates to a conjugate comprising the B7-H3 specific binding molecule described in the first aspect of the present invention and a conjugation portion, wherein the conjugation portion comprises a drug molecule and a detectable label, preferably, the drug molecule comprising a chemical drug, a biological product, or a traditional Chinese medicine monomer, and the detectable label comprising a radioactive substance, a fluorescent substance, a luminescent substance, a colored substance, an enzyme, or biotin.

[0019] A third aspect of the present invention relates to a nucleic acid sequence molecule comprising a nucleic acid sequence capable of encoding the variable region of the heavy chain of the B7-H3 specific binding molecule described in the first aspect of the present invention, wherein the variable region of the heavy chain of the B7-H3 specific binding molecule comprises a CDR with amino acid sequences of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.9, and SEQ ID NO.5; preferably, the heavy chain of the B7-H3 specific binding molecule has the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, and SEQ ID NO.15.

[0020] A fourth aspect of the present invention relates to a nucleic acid sequence molecule comprising a nucleic acid sequence capable of encoding a variable region of the light chain of the B7-H3 specific binding molecule described in the first aspect of the present invention, wherein the variable region of the light chain of the B7-H3 specific binding molecule comprises a CDR with amino acid sequences of SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8; preferably, the light chain of the B7-H3 specific binding molecule has amino acid sequences shown in SEQ ID NO. 2, SEQ ID NO. 11, SEQ ID NO. 13, and SEQ ID NO. 16.

[0021] The fifth aspect of the present invention relates to a vector comprising the nucleic acid sequence molecule described in the third and / or fourth aspects of the present invention.

[0022] Another aspect of the invention relates to a host cell comprising the nucleic acid sequence molecule described in the third and / or fourth aspects of the invention, or the vector described in the fifth aspect of the invention.

[0023] Another aspect of the present invention relates to an expression method comprising the steps of preparing an expression system for the B7-H3 specific binding molecule described in the first aspect of the present invention, transferring the expression system into a host cell, culturing the host cell, and recovering the B7-H3 specific binding molecule from the cell culture.

[0024] Another aspect of the present invention relates to a kit comprising the B7-H3 specific binding molecule as described in any of the first aspects of the present invention, or comprising the conjugate as described in any of the second aspects of the present invention; preferably, the kit further comprises a second antibody that specifically recognizes the B7-H3 specific binding molecule as described in the first aspect of the present invention; optionally, the second antibody further comprises a detectable label, such as a radioactive substance, a fluorescent substance, a luminescent substance, a colored substance, an enzyme, or biotin.

[0025] Another aspect of the invention relates to the use of the B7-H3 specific binding molecule according to any one of the first aspects of the invention or the conjugate according to any one of the second aspects of the invention in the preparation of a kit for detecting the presence or level of B7-H3 in a sample.

[0026] Another aspect of the invention relates to a pharmaceutical composition comprising a B7-H3 specific binding molecule as described in any of the first aspects of the invention or a conjugate as described in any of the second aspects of the invention; optionally, it further comprises a pharmaceutically acceptable carrier and / or excipient.

[0027] Another aspect of the present invention relates to the use of a B7-H3 specific binding molecule as described in any of the first aspects of the present invention or a conjugate as described in any of the second aspects of the present invention in the preparation of medicaments for treating and / or preventing and / or diagnosing tumors or hematologic disorders; preferably, the tumor is selected from osteosarcoma, sarcoma, nephroblastoma, melanoma, mesothelioma, adrenocortical carcinoma, uterine carcinosarcoma, head and neck cancer, endometrioid carcinoma, lung cancer, cholangiocarcinoma, prostate cancer, glioma, neuroblastoma, breast cancer, pancreatic cancer, bladder cancer, testicular cancer, ovarian cancer, esophageal cancer, colorectal cancer, kidney cancer, gastric cancer, cervical cancer, liver cancer, leukemia, and lymphoma.

[0028] Another aspect of the present invention relates to the use of a B7-H3 specific binding molecule as described in any of the first aspects of the present invention or a conjugate as described in any of the second aspects of the present invention in the preparation of the following pharmaceutical products: Drugs that block the binding of B7-H3 to its receptor; Drugs that regulate (e.g., downregulate) the activity or level of B7-H3; Drugs that relieve the immunosuppression caused by B7-H3; Drugs that regulate (e.g., enhance) the activity of immune cells; Compounds, biologics, or cell therapy products that target B7-H3.

[0029] Another aspect of the invention relates to an in vivo or in vitro method comprising the step of applying to cells an effective amount of the B7-H3 specific binding molecule according to any one of the first aspects of the invention or the conjugate according to any one of the second aspects of the invention, said method being selected from the following: Methods to block the binding of B7-H3 to its receptor; Methods for regulating (e.g., downregulating) the activity or level of B7-H3; Methods to relieve the immunosuppression caused by B7-H3; Methods for regulating (e.g., enhancing) the activity of immune cells; Methods to target (e.g., kill) B7-H3 expressing cells.

[0030] In one specific embodiment of the present invention, the in vitro method is for non-therapeutic or diagnostic purposes.

[0031] Another aspect of the present invention relates to a method for treating and / or preventing and / or diagnosing tumors or hematologic disorders, comprising the step of administering to a subject an effective amount of any of the B7-H3 specific binding molecules or conjugates of the present invention; preferably, the tumor is selected from osteosarcoma, sarcoma, nephroblastoma, melanoma, mesothelioma, adrenocortical carcinoma, uterine carcinosarcoma, head and neck cancer, endometrioid carcinoma, lung cancer, cholangiocarcinoma, prostate cancer, glioma, neuroblastoma, breast cancer, pancreatic cancer, bladder cancer, testicular cancer, ovarian cancer, esophageal cancer, colorectal cancer, kidney cancer, gastric cancer, cervical cancer, liver cancer, leukemia, and lymphoma.

[0032] Dosage is determined by many factors, such as the severity of the condition being treated, the sex, age, weight, and individual response of the patient or animal, as well as the patient's condition and medical history. Common practice in the art is to start with a dose below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0033] The B7-H3 specific binding molecule or conjugate of any one of the present invention is used for the treatment and / or prevention and / or diagnosis of tumors or hematologic disorders; preferably, the tumor is selected from osteosarcoma, sarcoma, nephroblastoma, melanoma, mesothelioma, adrenocortical carcinoma, uterine carcinosarcoma, head and neck cancer, endometrioid carcinoma, lung cancer, cholangiocarcinoma, prostate cancer, glioma, neuroblastoma, breast cancer, pancreatic cancer, bladder cancer, testicular cancer, ovarian cancer, esophageal cancer, colorectal cancer, kidney cancer, gastric cancer, cervical cancer, liver cancer, leukemia, and lymphoma.

[0034] The B7-H3 specific binding molecule according to any one of the first aspects of the present invention or the conjugate according to any one of the second aspects of the present invention is used for: Blocking the binding of B7-H3 to its receptor; Regulate (e.g., downregulate) the activity or level of B7-H3; To relieve the immunosuppression caused by B7-H3; Regulating (e.g., enhancing) the activity of immune cells; Target (e.g., kill) B7-H3 expressing cells.

[0035] The use of the B7-H3 specific binding molecule according to any one of the first aspects of the present invention or the conjugate according to any one of the second aspects of the present invention for use in the treatment of tumors, simultaneously, separately, or sequentially in combination with one or more antitumor agents. The tumors described are osteosarcoma, sarcoma, nephroblastoma, melanoma, mesothelioma, adrenocortical carcinoma, uterine carcinosarcoma, head and neck cancer, endometrioid carcinoma, lung cancer, cholangiocarcinoma, prostate cancer, glioma, neuroblastoma, breast cancer, pancreatic cancer, bladder cancer, testicular cancer, ovarian cancer, esophageal cancer, colorectal cancer, kidney cancer, gastric cancer, cervical cancer, liver cancer, leukemia, and lymphoma.

[0036] The beneficial effects of this invention are as follows: The B7-H3 monoclonal antibody Ab-82 and its humanized antibody can bind to the B7-H3 antigen with high specificity and preferentially recognize its N91 / 104 / 309 / 322 glycosylation sites. It has cross-activity with humans, mice, and monkeys, and its ability to mediate B7-H3 internalization is superior to that of the Ifinatamab antibody used in DS-7300, the B7-H3 ADC drug with the most advanced clinical trial progress. When used alone, it can also effectively inhibit tumor growth and promote T cell infiltration into tumor tissue in vivo. It is expected that based on this, a potent B7-H3 ADC drug can be developed to benefit a large number of tumor patients expressing B7-H3. Attached Figure Description

[0037] Figure 1 This is the preparation process for B7-H3 monoclonal antibodies.

[0038] Figure 2 This is the ELISA-specific screening result of the obtained B cell supernatant.

[0039] Figure 3 This is the flow cytometry-specific screening result of the supernatant of positive B cells obtained from the initial ELISA screening.

[0040] Figure 4 This is the result of flow cytometry screening of the internalization capacity of positive B cell supernatants obtained through flow cytometry-specific screening.

[0041] Figure 5 This is the in vitro recombinant expression and purification process for Ab-82 antibody.

[0042] Figure 6 This is the result of in vitro recombinant expression and purification of the Ab-82 antibody.

[0043] Figure 7 This is the result of an ELISA test showing the binding of the Ab-82 antibody to the human B7-H3 fusion protein.

[0044] Figure 8 This is the result of a Dot blot analysis of the binding of the Ab-82 antibody to the human B7-H3 fusion protein.

[0045] Figure 9 This is the result of flow cytometry analysis showing that the Ab-82 antibody recognizes B7-H3 on the surface of living cells.

[0046] Figure 10 This is the SPR test result of the affinity of serially diluted Ab-82 antibody for human B7-H3 fusion protein.

[0047] Figure 11 These are the flow cytometry results of the binding ability of the Ab-82 antibody to B7-H3 proteins from different species.

[0048] Figure 12 This is the result of a Dot blot analysis of the binding of the Ab-82 antibody to the glycosylated and deglycosylated forms of the B7-H3 fusion protein.

[0049] Figure 13 The results are Western blot analysis of the binding of Ab-82 antibody to wild-type B7-H3 (WT), fully deglycosylated B7-H3 (8NQ), glycosylated B7-H3 at N91 / 104 / 309 / 322 sites, and deglycosylated B7-H3 at N91 / 104 / 309 / 322 sites.

[0050] Figure 14 The results are flow cytometry analysis of the binding of Ab-82 antibody to wild-type B7-H3 (WT), fully deglycosylated B7-H3 (8NQ), glycosylated B7-H3 at N91 / 104 / 309 / 322 sites, and deglycosylated B7-H3 at N91 / 104 / 309 / 322 sites.

[0051] Figure 15 The results are from a flow cytometry model for temperature-induced internalization and flow cytometry analysis of Ab-82 antibody-mediated internalization of B7-H3 protein on the cell membrane surface.

[0052] Figure 16 It is a flow cytometry detection model of internalization and recirculation to the cell membrane surface, and the flow cytometry analysis results of the recirculation of B7-H3 protein on the cell membrane surface mediated by Ab-82 antibody.

[0053] Figure 17 This is a detection model for pH probe-indicated internalization and the results of immunofluorescence analysis of Ab-82 antibody internalization into cells.

[0054] Figure 18 This is a comparison of the efficiency of Ab-82 antibody and Ifinatamab antibody in mediating B7-H3 protein internalization in different tumor cell lines.

[0055] Figure 19This is a comparison of the efficiency of Ab-82 antibody-mediated internalization of wild-type B7-H3 (WT), completely deglycosylated B7-H3 (8NQ), B7-H3 glycosylated at N91 / 104 / 309 / 322 sites, and B7-H3 deglycosylated at N91 / 104 / 309 / 322 sites.

[0056] Figure 20 These are the flow cytometry and Western blot results of the degradation of B7-H3 on the cell membrane surface promoted by Ab-82 antibody.

[0057] Figure 21 These are the in vivo antitumor efficacy and liver and kidney toxicity test results of the Ab-82 antibody.

[0058] Figure 22 This is the result of immunohistochemical detection showing that Ab-82 antibody promotes the infiltration of immune cells into tumor tissue.

[0059] Figure 23 This is the humanization process for the Ab-82 antibody.

[0060] Figure 24 The results are SPR assays of the affinity of humanized Ab-82 antibodies Hu-Ab-82-Ab5, Hu-Ab-82-Ab14, and Hu-Ab-82-Ab16 for the human B7-H3 fusion protein.

[0061] Figure 25 This is a comparison of the efficiency of Hu-Ab-82-Ab5 antibody and Ifinatamab antibody in mediating B7-H3 protein internalization.

[0062] Figure 26 These are the in vivo antitumor efficacy and liver and kidney toxicity test results of the Hu-Ab-82-Ab5 antibody.

[0063] Figure 27 This is the immunohistochemical result of Hu-Ab-82-Ab5 antibody promoting the infiltration of immune cells into tumor tissue. Detailed Implementation

[0064] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0065] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). The variable regions (V) of the light and heavy chains determine antigen binding. Each chain's variable region contains three hypervariable regions called complementarity-determining regions (CDRs). The CDRs of the heavy chain (H) include VH-CDR1, VH-CDR2, and VH-CDR3, and the CDRs of the light chain (L) include VL-CDR1, VL-CDR2, and VL-CDR3; these are named by Kabat et al., see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1–3, NIH Publication 91-3242, Bethesda Md. The amino acid sequence of the antibody's CDR regions is analyzed using techniques well known to those skilled in the art, such as through the VBASE2 database. Antibody light chains can be classified into κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by a “J” region of approximately 12 or more amino acids, and the heavy chain also contains a “D” region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly denatured regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form antibody-binding sites. The allocation of amino acids to each region or domain follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0066] In this document, unless the context clearly indicates otherwise, when referring to the term “antibody,” it includes not only the complete antibody but also the antigen-binding fragment of the antibody. The term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the complete antibody. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody antibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0067] As used herein, the term "Fd fragment" refers to an antibody fragment consisting of the VH and CH1 domains; the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody; the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains; and the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region. The term "Fab' fragment" refers to a monovalent fragment resulting from further cleavage of the F(ab')2 fragment by removing one arm, comprising one arm of the antibody and the hinge region.

[0068] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), where the VL and VH domains pair to form a monovalent molecule by enabling them to generate linkers that are single polypeptide chains (see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such scFv molecules can have a general structure: NH2- V L -Connector-V H -COOH or NH2-V H -Connector-V L -COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

[0069] In some cases, the antigen-binding fragment of an antibody is a biantibody, i.e., a bivalent antibody, in which the VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).

[0070] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the monoclonal antibodies Ab-82, Hu-Ab-82-Ab5, Hu-Ab-82-Ab14, Hu-Ab-82-Ab16 provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0071] As used herein, the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are usually obtained using hybridoma techniques first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see USP 4,816,567).

[0072] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain is derived from a portion of an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855(1984)).

[0073] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, see, for example, Jones et al., Nature, 321: 522 525 (1986); Reichmann et al., Nature, 332: 323 329 (1988); Presta, Curr. Op. Struct. Biol., 2: 593 596 (1992); and Clark, Immunol. Today 21: 397 402 (2000).

[0074] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0075] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as B cells, fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0076] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to the antigen.

[0077] As used in this article, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies (e.g., the monoclonal antibodies Ab-82, Hu-Ab-82-Ab5, Hu-Ab-82-Ab14, Hu-Ab-82-Ab16 of the present invention) have a dissociation equilibrium constant of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D The antigen (e.g., B7-H3 protein) is bound to the antigen, for example, as determined in a BIACORE instrument using surface plasmon resonance (SPR).

[0078] As used in this article, the term "affinity maturation" refers to the process of introducing mutations into the variable region of an antibody gene through random or directed mutagenesis, followed by screening antibody variants with higher affinity from a large number of mutants using affinity screening techniques (e.g., phage display, yeast display, ribosome display, etc.), thereby optimizing the antibody's affinity, specificity, and stability.

[0079] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; and the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0080] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0081] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when administered to the body along with or before an antigen, can enhance the body's immune response to the antigen or alter the type of immune response. There are many types of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, and cytokines. Freund's adjuvant is currently the most commonly used adjuvant in animal studies. Aluminum hydroxide adjuvant is more frequently used in clinical trials.

[0082] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0083] The term "subject" can refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the composition of the present invention to treat, prevent, reduce and / or alleviate the disease or condition described in the present invention.

[0084] The term "disease and / or symptom" refers to a physical condition of the subject that is related to the disease and / or symptom described in this invention.

[0085] The technical solution of the present invention will be further explained below with reference to experiments.

[0086] Preparation and screening of B7-H3 monoclonal antibodies Using purchased human B7-H3 fusion protein as the antigenic peptide, Japanese white rabbits were subcutaneously immunized multiple times. After successful animal immunization, over 700 B cell monoclonal antibodies were obtained using single-cell B cell antibody preparation technology. Preliminary screening using ELISA identified 192 positive clones that specifically recognize the human B7-H3 fusion protein. Figure 1-2 Subsequently, using a commercially available B7-H3 flow cytometry antibody (eBioscience, 17-2769-42) as a control, 114 positive clones that specifically bind to B7-H3 on the surface of live cells were screened using flow cytometry analysis. Figure 3 Finally, using a commercially available B7-H3 internalization antibody (Santa Cruz, sc-376769) as a control, the B7-H3 monoclonal antibody Ab-82 with the strongest internalization ability was screened based on a flow cytometry model induced by temperature change. Figure 4 ).

[0087] The monoclonal antibody Ab-82 was externally sequenced to determine its amino acid sequence, which is as follows: Heavy chain variable region: QSLEESGGDLVKPGASLTLTCTAS GFSFSSNYWIC WVRQAPGKGLEWIA CIYAGGDGNTYYASWAKG RFTISKTSSTTVTLQMTSLTGADTATYFCAR DYAGDAYVYFNL WGPGTLVTVSS (SEQ ID NO.1); Light chain variable region: ALVMTQTPSSVSAAVGGTVTINC QASENIYSGLA WYQQKPGQRPKLLIY LASTLES GVPSRFKGSGSGTEFTLTISDLECADAATYYC QSVYYSSSDLA FGGGTEVVVK (SEQ ID NO. 2).

[0088] Further analysis determined the amino acid sequences of the complementarity-determining regions (CDRs) of the heavy chain and light chain variable regions of the monoclonal antibody Ab-82 as follows:

[0089] The inventors further used the HEK293F eukaryotic expression system and affinity chromatography purification technology to perform in vitro recombinant expression and purification of the Ab-82 antibody. Figure 5 The results showed that clear heavy and light chains were visible in the antibody elution buffer of Ab-82, and no antibody bands were observed in the culture supernatant and impurity wash buffer after affinity chromatography. Figure 6 This indicates that the Ab-82 antibody expression and purification were successful.

[0090] Assay for the binding activity of Ab-82 antibody to human B7-H3 protein First, the microplates were coated with human B7-H3 fusion protein. Serially diluted Ab-82 antibody was used as the primary antibody, and the binding activity of Ab-82 antibody to the human B7-H3 fusion protein was detected by ELISA. The results showed that Ab-82 antibody bound well to the human B7-H3 fusion protein. Figure 7 Subsequently, serially diluted human B7-H3 fusion protein was spotted onto a PVDF membrane. A commercially available B7-H3 immunoblotting antibody (R&D, AF1027) was used as a positive control, and Ab-82 antibody at the same concentration was used as the primary antibody. The recognition ability of Ab-82 antibody for human B7-H3 fusion protein was semi-quantitatively detected by Dot blot. The results showed that Ab-82 antibody can effectively bind to human B7-H3 fusion protein (…). Figure 8 Subsequently, using serially diluted Ab-82 antibody as the primary antibody, flow cytometry was used to observe its ability to recognize B7-H3 on the surface of live cells. The results showed that the Ab-82 antibody could specifically recognize the B7-H3 protein on the surface of various cancer cells. Figure 9 Finally, the affinity of serially diluted Ab-82 antibody for the human B7-H3 fusion protein was observed using SPR detection technology. The results showed that Ab-82 has extremely high affinity for the B7-H3 protein, with an affinity constant K0. D The value is 3×10 -9 M ( Figure 10 ).

[0091] Assay for the binding activity of Ab-82 antibody to human / mouse / monkey B7-H3 protein Human, mouse, and rhesus monkey-derived B7-H3-Flag molecules were overexpressed in HEK293T-B7-H3-KO cell line. Ab-82 antibody was used as the primary antibody for flow cytometry staining, while a commercially available Flag antibody (CST, 14793S) was used as a positive control. Flow cytometry analysis was used to detect the binding ability of the antibodies to B7-H3 proteins from different species. The results showed that, compared with the untransfected control group, Ab-82 antibody effectively recognized B7-H3 proteins from humans, mice, and rhesus monkeys, and its recognition ability for human and rhesus monkey-derived B7-H3 proteins was stronger. Figure 11 ).

[0092] Assay for the binding activity of Ab-82 antibody to glycosylated B7-H3 protein Glycosylation modification of human B7-H3 fusion protein was removed using PNGase. Then, serially diluted glycosylated and deglycosylated human B7-H3 fusion proteins were spotted onto PVDF membranes. A commercially available B7-H3 immunoblotting antibody (R&D, AF1027) was used as a positive control, and Ab-82 antibody at the same concentration was used as the primary antibody. The recognition ability of Ab-82 antibody for glycosylated B7-H3 fusion protein was semi-quantitatively detected by dot blot. The results showed that Ab-82 antibody specifically recognized glycosylated B7-H3 but not deglycosylated B7-H3. Figure 12 Subsequently, different glycosylated forms of B7-H3-Flag protein were expressed in MDA-MB-231-B7-H3-KO and A549-B7-H3-KO cell lines, and enriched by immunoprecipitation. A commercially available anti-Flag antibody (CST, 14793S) was used as a positive control, and Ab-82 antibody at the same concentration was used as the primary antibody. The recognition ability of Ab-82 antibody for different glycosylated forms of B7-H3 protein was detected by Western blot. The results showed that the Ab-82 antibody specifically recognized fully glycosylated B7-H3 (WT) and B7-H3 glycosylated at N91 / 104 / 309 / 322 sites (N91 / 104 / 309 / 322), but did not recognize fully deglycosylated B7-H3 (8NQ) and B7-H3 deglycosylated at N91 / 104 / 309 / 322 sites (N91 / 104 / 309 / 322Q). Figure 13 Furthermore, using a commercially available B7-H3 flow cytometry detection antibody (eBioscience, 17-2769-42) as a positive control, the recognition ability of the Ab-82 antibody for different glycosylated forms of B7-H3 on the surface of live cells was detected by flow cytometry analysis. The results showed that the Ab-82 antibody preferentially recognized WT-B7-H3 and N91 / 104 / 309 / 322-B7-H3 (…). Figure 14 ).

[0093] Detection of the ability of Ab-82 antibody to mediate the internalization of B7-H3 on the cell membrane Based on a flow cytometry model for temperature-induced internalization, using serially diluted Ab-82 antibody as the primary antibody, the ability of Ab-82 to mediate the internalization of B7-H3 protein on the cell membrane surface was detected. The results showed that with increasing Ab-82 antibody concentration, the amount of B7-H3 protein internalized on the cell membrane surface increased (…). Figure 15Simultaneously, Primaquine was used to block the recirculation of internalized proteins to the cell membrane surface, and flow cytometry was used to detect whether B7-H3 protein recirculated to the cell membrane after internalization. The results showed that Primaquine application could not further reduce the level of B7-H3 at the membrane surface; therefore, Ab-82 antibody-mediated B7-H3 internalization no longer circulated to the cell membrane. Figure 16 Subsequently, based on an analytical model that uses pH probes to indicate internalization, the Ifinatamab antibody used in DS-7300, the B7-H3 ADC drug currently in the most advanced clinical trials, was compared with the efficiency of Ab-82 antibody in mediating B7-H3 protein internalization. Immunofluorescence assay results showed that both pH probe-labeled Ab-82 antibody and Ifinatamab antibody could internalize into tumor cells. Figure 17 Flow cytometry analysis on various tumor cells showed that the internalization of B7-H3 protein increased with increasing incubation time, and the efficiency of B7-H3 internalization mediated by Ab-82 antibody was superior to that mediated by Ifinatamab. Figure 18 Simultaneously, the inventors also tested the ability of the Ab-82 antibody to mediate the internalization of B7-H3 in different glycosylation forms. Flow cytometry analysis showed that the Ab-82 antibody preferentially mediated the internalization of WT-B7-H3 and N91 / 104 / 309 / 322-B7-H3. Figure 19 Furthermore, tumor cells were co-incubated with Ab-82 antibody for 24 to 48 hours, and the degradation of B7-H3 after internalization was detected by flow cytometry and Western blot. The results showed that Ab-82 antibody promoted a decrease in the level of B7-H3 on the cell membrane surface and the total intracellular B7-H3 protein content. Figure 20 ).

[0094] In vivo antitumor efficacy assay of Ab-82 antibody B7-H3 (mB7-H3) in murine 4T1 cells was knocked out, and then human B7-H3 (hB7-H3) was reintroduced to construct the 4T1-hB7-H3 cell line. This line was then inoculated into BALB / c mice, and after tumor formation, the mice were treated with Ab-82 antibody at doses of 0 / 0.25 / 0.5 / 1 mg / kg, respectively. Results showed that Ab-82 antibody effectively inhibited tumor growth in vivo in a dose-dependent manner, with no significant hepatotoxicity or renal toxicity. Figure 21 Simultaneously, immunohistochemical analysis of T cell infiltration in tumor tissue showed that Ab-82 antibody treatment could promote CD4 cell proliferation. + T cells and CD8 + T cells infiltrate tumor tissue and promote the release of granzyme B (GZMB). Figure 22 ).

[0095] Affinity and internalization ability of humanized Ab-82 antibody detection To reduce the immunogenicity of the Ab-82 antibody, the inventors humanized it. Figure 23 We obtained Hu-Ab-82-Ab5, Hu-Ab-82-Ab14, and Hu-Ab-82-Ab16 antibodies with a humanization level of over 95%. These three humanized antibodies share the same complementarity-determining region (CDR), and their amino acid sequences are as follows:

[0096] The amino acid sequences of the heavy chain variable region and light chain variable region of Hu-Ab-82-Ab5, Hu-Ab-82-Ab14, and Hu-Ab-82-Ab16 antibodies are as follows, with the CDR region marked by underline in the sequence: Hu-Ab-82-Ab5-VH: QVQLVESGGGVVQPGRSLRLSCTAS GFSFSSNYWIC WVRQAPGKGLEWIA CIYAGGEGNTYYASWAKG RFTISRDNSTNTVFLQMNSLRAEDTAVYFCAR DYAGDAYVYFNL WGQGTLVTVSS (SEQ ID NO. 10).

[0097] Hu-Ab-82-Ab5-VL: DIQMTQSPSSLSASVGDRVTITC QASENIYSGLA WYQQKPGKVPKLLIY LASTLES GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QSVYYSSSDLA FGGGTKVEIK (SEQ ID NO. 11).

[0098] Hu-Ab-82-Ab14-VH: QVQLVESGGGVVQPGRSLRLSCTAS GFSFSSNYWIC WVRQAPGKGLEWIA CIYAGGEGNTYYASWAKG RFTISKDNSTTVFLQMNSLRAEDTAVYFCAR DYAGDAYVYFNL WGQGTLVTVSS (SEQ ID NO. 12).

[0099] Hu-Ab-82-Ab14-VL: ALVMTQSPSSLSASVGDRVTITC QASENIYSGLA WYQQKPGKRPKLLIY LASTLES GVPSRFSGSGSGTEFTLTISSLQPEDVATYYC QSVYYSSSDLA FGGGTKVEIK (SEQ ID NO. 13).

[0100] Hu-Ab-82-Ab16-VH: QSLVESGGGVVQPGRSLRLSCTAS GFSFSSNYWIC WVRQAPGKGLEWIA CIYAGGEGNTYYASWAKG RFTISKDNSTTVFLQMNSLRAEDTAVYFCAR DYAGDAYVYFNL WGQGTLVTVSS (SEQ ID NO. 14).

[0101] Hu-Ab-82-Ab16-VL: (SEQ ID NO. 13).

[0102] The amino acid sequence of the humanized Ab-82 antibody variant is as follows: Humanized Ab-82-VH variant: QVQLVESGGGVVQPGRSLRLSCTAS GFX1X2SSX3YWIC WVRQAPGKGLEWIA CIX4AGGX5GX6TYY ASWAKG RFTISRDNSTNTVFLQMNSLRAEDTAVYFCA X7 DX8AGX9AX10VYFNL WGQGTLVTVSS (X1 can be S or T; X2 can be F or Y; X3 can be Q or N; X4 can be F or Y; X5 can be D or E; X6 can be Q or N; X7 can be R or K; X8 can be F or Y; X9 can be D or E; X10 can be F or Y) (SEQ ID NO.15).

[0103] Humanized Ab-82-VL variant: DIQMTQSPSSLSASVGDRVTITC QASEX11IX12SX13LA WYQQKPGKVPKLLIY X14ASTLX15S GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QSX16YX17SSX18X19LA FGGGTKVEIK (X11 can be Q or N; X12 can be F or Y; X13 can be G or S or T or A; X14 can be L or M or I or V; X15 can be D or E; X16 can be L or I or V; X17 can be F or Y; X18 can be S or T or A; X19 can be D or E) (SEQ ID NO.16).

[0104] The affinity constant K of the Hu-Ab-82-Ab5 antibody was determined by SPR affinity assay. D The value is 3.2 × 10 -9 The affinity constant K of M and Hu-Ab-82-Ab14 antibodies D The value is 1.17 × 10 -8 The affinity constant K of M and Hu-Ab-82-Ab16 antibodies D The value is 7.85 × 10 -9 M ( Figure 24 It still exhibits extremely high affinity for the B7-H3 antigen. Subsequently, based on an analytical model where pH probes indicate internalization, the efficiency of Ifinatamab in mediating B7-H3 protein internalization was compared with that of the antibody with the highest affinity, Hu-Ab-82-Ab5. Flow cytometry analysis on various tumor cells showed that with increasing incubation time, the amount of internalized B7-H3 protein increased, and the efficiency of Hu-Ab-82-Ab5 antibody in mediating B7-H3 internalization was superior to that of Ifinatamab. Figure 25 ).

[0105] In vivo antitumor efficacy assay of humanized Hu-Ab-82-Ab5 antibody Human peripheral blood mononuclear cells (huPBMCs) were reinfused into NOG-MHC I / II-2 KO mice to reconstruct their immunity. A549 cells were then inoculated into this humanized mouse model. After tumor formation, the mice were treated with 5 mg / kg of isotype control IgG antibody, 2.5 mg / kg, and 5 mg / kg of Hu-Ab-82-Ab5 antibody, respectively. Results showed that 5 mg / kg of Hu-Ab-82-Ab5 antibody significantly inhibited tumor growth in vivo without significant hepatotoxicity or nephrotoxicity. Figure 26 Simultaneously, immunohistochemical observation of T cell infiltration showed that 5 mg / kg Hu-Ab-82-Ab5 treatment could promote CD8 cell growth. + T cells infiltrate tumor tissue and promote the release of granzyme B (GZMB). Figure 27 ).

[0106] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A B7-H3 specific binding molecule, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of CDR1 of the complementarity-determining region (CDR) of the heavy chain variable region is shown in SEQ ID NO.3, the amino acid sequence of CDR2 is shown in SEQ ID NO.4 or SEQ ID NO.9, the amino acid sequence of CDR3 is shown in SEQ ID NO.5, and / or the sequence obtained after affinity maturation of the CDRs shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.9, and SEQ ID NO.5; The amino acid sequences of CDR1 to CDR3 of the complementarity-determining region (CDR) of the light chain variable region are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively, and / or the sequences obtained after affinity maturation of the CDRs shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.

8.

2. The B7-H3 specific binding molecule according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the B7-H3 specific binding molecule is shown in SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, and SEQ ID NO.15, and / or the sequence obtained after affinity maturation of the variable region shown in SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, and SEQ ID NO.15; The amino acid sequence of the light chain variable region of the B7-H3 specific binding molecule is shown in SEQ ID NO.2, SEQ ID NO.11, SEQ ID NO.13, and SEQ ID NO.16, and / or the sequence obtained after affinity maturation of the variable region shown in SEQ ID NO.2, SEQ ID NO.11, SEQ ID NO.13, and SEQ ID NO.

16.

3. The B7-H3 specific binding molecule according to any one of claims 1 to 2, characterized in that, Its main molecules are Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies, chimeric antibodies, humanized antibodies, single-domain antibodies fused with Fc fragments, bivalent or multivalent single-domain antibodies, dual-target antibodies or multi-target antibodies.

4. The B7-H3 specific binding molecule according to claim 3, characterized in that, The Fc fragment is directly or via a linker element attached to the C-terminus or N-terminus of the B7-H3 specific binding molecule; the Fc fragment includes the Fc fragment of human IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM, IgE or its variants or modifications.

5. The B7-H3 specific binding molecule according to any one of claims 1 to 2, characterized in that, The B7-H3 specific binding molecule includes a non-CDR region, and the non-CDR region is derived from a species other than rabbits, such as from human antibodies.

6. A coupling agent, characterized in that, It includes the B7-H3 specific binding molecule and the coupling portion according to any one of claims 1 to 5; preferably, the coupling portion includes a drug molecule and a detectable label; preferably, the drug molecule includes a chemical drug, a biological product, or a traditional Chinese medicine monomer, and the detectable label includes a radioactive substance, a fluorescent substance, a luminescent substance, a colored substance, an enzyme, or biotin.

7. A nucleic acid sequence molecule, characterized in that, It at least encodes the heavy chain variable region of the B7-H3 specific binding molecule according to any one of claims 1 to 5, wherein the heavy chain variable region of the B7-H3 specific binding molecule comprises: a CDR with the amino acid sequence SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.9, and SEQ ID NO.5; preferably, the heavy chain of the B7-H3 specific binding molecule has the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, and SEQ ID NO.

15.

8. A nucleic acid sequence molecule, characterized in that, It at least encodes the light chain variable region of the B7-H3 specific binding molecule according to any one of claims 1 to 5, wherein the light chain variable region of the B7-H3 specific binding molecule comprises: a CDR with amino acid sequences of SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8; preferably, the light chain of the B7-H3 specific binding molecule has the amino acid sequences shown in SEQ ID NO. 2, SEQ ID NO. 11, SEQ ID NO. 13, and SEQ ID NO.

16.

9. A carrier, characterized in that, It comprises the nucleic acid sequence molecule as described in claims 7 and / or 8.

10. A host cell, characterized in that, It comprises the nucleic acid sequence molecule as described in claim 7 and / or 8, or the vector as described in claim 9.

11. A method of expression, characterized in that, include: Prepare an expression system for the B7-H3 specific binding molecule as described in any one of claims 1 to 5; The expression system is transferred into host cells; Cultivating the host cells, and The step of recovering the B7-H3 specific binding molecule from cell culture.

12. A reagent kit, characterized in that, It includes the B7-H3 specific binding molecule as described in any one of claims 1 to 5, or the conjugate as described in claim 6; Preferably, the kit further includes a second antibody that specifically recognizes the B7-H3-specific binding molecule; optionally, the second antibody further includes a detectable marker, preferably selected from radioactive substances, fluorescent substances, luminescent substances, colored substances, enzymes, or biotin.

13. Use of the B7-H3 specific binding molecule of any one of claims 1 to 5 or the conjugate of claim 6 in the preparation of a kit for detecting the presence or level of B7-H3 in a sample.

14. A pharmaceutical composition, characterized in that, It comprises the B7-H3 specific binding molecule as described in any one of claims 1 to 5 or the conjugate as described in claim 6; optionally, it further comprises a pharmaceutically acceptable carrier and / or excipient.

15. Use of the B7-H3 specific binding molecule according to any one of claims 1 to 5 or the conjugate according to claim 6 in the preparation of the following pharmaceutical products: Drugs that block the binding of B7-H3 to its receptor. Drugs that regulate the activity or level of B7-H3 Drugs that relieve the immunosuppression caused by B7-H3. Drugs that regulate the activity of immune cells. Biologics or cell therapy products that target B7-H3.

16. Use of the B7-H3 specific binding molecule according to any one of claims 1 to 5 or the conjugate according to claim 6 in the preparation of a medicament for treating and / or preventing and / or diagnosing tumors or hematological diseases; Preferably, the tumor is selected from osteosarcoma, sarcoma, nephroblastoma, melanoma, mesothelioma, adrenocortical carcinoma, uterine carcinosarcoma, head and neck cancer, endometrioid carcinoma, lung cancer, bile duct cancer, prostate cancer, glioma, neuroblastoma, breast cancer, pancreatic cancer, bladder cancer, testicular cancer, ovarian cancer, esophageal cancer, colorectal cancer, kidney cancer, gastric cancer, cervical cancer, liver cancer, leukemia, and lymphoma.

17. An in vivo or in vitro method, characterized in that, The method includes the step of applying to cells in an effective amount of the B7-H3 specific binding molecule of any one of claims 1 to 5 or the conjugate of claim 6, wherein the method is selected from the following: Methods to block the binding of B7-H3 to its receptor. Methods for regulating B7-H3 activity or levels Methods to relieve the immunosuppression caused by B7-H3 Methods for regulating immune cell activity A method for targeting B7-H3 expressing cells.

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