B7H3 monoclonal antibody group and its pharmaceutical uses

Abstract

Anti-B7H3 receptor monoclonal antibodies and humanized antibodies thereof are provided. [Solution] The antibodies obtained by hybridoma technology have B7H3 affinity, cell endocytosis function, and antibody-dependent cellular toxicity, or can effectively block the immunosuppressive effect of B7H3, and can be used to suppress cancer cells, regulate the effect and level of B7H3, and prepare related drugs used to improve the body's immunity.

Description

[Technical field]

[0001] This application claims priority to a Chinese patent application filed with the China Patent Office on June 9, 2021, bearing application number 202110639996.0 and entitled "B7H3 monoclonal antibody group and its medical use", the entire contents of which are incorporated herein by reference.

[0002] The present invention belongs to the field of tumor antibody therapy and molecular immunotherapy, and relates to B7H3 antibodies and their uses. Specifically, the present invention relates to various B7H3 monoclonal antibodies. [Background technology]

[0003] B7H3, also known as CD276, is a type I transmembrane protein (Chapoval A.I., et al., (2001), Nat. Immunol. 2:269). In mice, B7H3 is encoded on chromosome 9, whereas in humans, it is encoded on chromosome 15. B7H3 shares 20%-27% amino acid identity with other B7 family ligands (Sun M., et al., (2002), J. Immunol. 168:6294; Loos M., (2010), Clin. Dev. Immunol. 2010:683875). In terms of protein composition and structure, mouse B7H3 has only two IgG7H3 types (consisting of one pair of extracellular IgV and IgC), while human B7H3 has four IgG7H3 types, which are almost completely identical IgV-IgC tandem repeats (Sun M., et al., (2002), J. Immunol. 168:6294; Steinberger P., et al., (2004), J. Immunol. 172:2352). B7H3 mRNA is present in some normal tissues, such as liver, small intestine, pancreas, testis, heart and colon, but B7H3 protein is rarely found in normal tissues (Greenwald R. J., et al., (2005), Annu. Rev. Immunol. 23:515-48). Such differences between B7H3 mRNA and protein may reflect the existence of a post-transcriptional tight regulatory mechanism (Hofmeyer K.A., et al., (2008), Proc. Natl. Acad. Sci. 105:10277; Calabro L., et al., (2011), J. Cell Physiol. 226:2595). Overall, B7H3 is present only in some non-immune fibroblasts, endothelial cells and osteoblasts, and in some immune cells, such as B cells, T cells, monocytes, dendritic cells or NK cells.B7H3 expression can be produced by CSF or lipopolysaccharide stimulation induction of granulocyte-macrophages (Suh W.K., et al., (2003), Nat. Immunol. 4:899; Chapoval A.I., et al., (2001), Nat. Immunol. 2:269; Greenwald R.J. et al., (2005), Annu. Rev. Immunol. 23:515).

[0004] B7H3 is an important immune checkpoint protein. Through preliminary research, it has been concluded that B7H3 has a stimulating effect on T cells, and can promote the proliferation of CD4+ and CD8+ T cells, enhance the cytotoxicity of T lymphocytes, and stimulate the production of interferon-γ (IFN-γ) to a certain extent (Chapoval A.I., et al., (2001), Nat.Immunol.2:269), and the deletion of B7H3 can alleviate the chronic repulsive effect of allogeneic transplantation (Wang L.et al., (2005), Eur.J.Immunol.35:428). However, more and more studies have shown that B7H3 has a significant immunosuppressive effect. For example, B7H3 can significantly suppress the activation effect of CD3 antibody or allogeneic DC cells on T cells, but B7H3 blocking antibody can effectively reverse such suppressive effect (Prass D. VR, et al., (2004), J. Immunol. 173: 2500). Structural information of B7H3 protein indicates that the FG loop in the B7H3 IgV domain may play an important role in suppressing T cells (Vigdorovich V., et al., (2013), Structure 21: 707). In addition to T cells, B7H3 may also have an inhibitory effect on natural killer cells (NK) (Castriconi R., et al., (2004), Proc. Natl. Acad. Sci. 101: 12640). One possible mechanism is related to the suppression of stimulatory activity of dendritic cells (DCs) on T lymphocytes by inducing DC B7H3 expression by regulatory T cells (Tregs) (Mahnke K., et al., (2007), Eur. J. Immunol. 37:2117). In mouse models, mice lacking B7H3 suffer from severe airway inflammation and earlier onset of several autoimmune diseases, such as experimental autoimmune strain encephalomyelitis (Suh W. K., et al., (2003), Nat. Immunol. 4:899).

[0005] The expression level of B7H3 is very high in many malignant tumors such as melanoma (Wang J., et al., (2013), J. Invest. Dermatol. 133: 2050), leukemia (Hu Y., et al., (2015), Hemmelo 20: 187; Sun J., et al., (2014), OncoTargets Ther. 7: 1979), prostate cancer (Zang X., et al., (2007), Proc. Natl. Acad. Sci. 104: 19458), ovarian cancer (Zang X., et al., (2010), Mod. Pathol. 23: 1104) and pancreatic adenocarcinoma (Chen Y., et al., (2014), Onco. Targets Ther. 7: 1465-72). Although B7H3 mRNA is present in both normal and tumor tissues, B7H3 protein is expressed at high levels only in tumor cells. Such differences may be related to miRNA-29 regulatory mechanisms. For example, a study found that the levels of miRNA-29 and B7H3 protein are inversely correlated in normal or tumor tissues and cancer cell lines (Xu H., et al., (2009), Cancer Res. 69:6275). In tumor patients, abnormal expression of B7H3 is closely associated with poor prognosis, increased tumor fractionation and metastasis, treatment resistance, and reduced overall survival rate of tumor patients (Picarda E., et al., (2016), Clin. Cancer Res. 22:3425). The inhibitory effect of B7H3 on T cells, NK cells and DC cells significantly promotes the occurrence of immune escape of tumor cells. B7H3 also plays an important role in tumor cell proliferation, metastasis, invasion, angiogenesis, epithelial-mesenchymal transition (EMT), cancer stemness and Warburg effect, as well as drug resistance of tumor cells.

[0006] Increased B7H3 can promote the expression of Bcl-2 and Bcl-xl and enhance the anti-apoptotic activity of tumor cells by activating the JAK2-STAT3 signaling pathway (Zhang T., et al., (2015), World J. Gastroenterol. 21:1804). At the same time, activation of AKT, ERK, and JAK2 / STAT3 routes can induce the expression of protein factors involved in tumor cell metastasis (including MMP2, MMP9, CXCR4, etc.), thereby enhancing the metastatic and invasive abilities of tumor cells (Tekle C., et al., (2013), Int. JCancer 130: 2282; Li Y., et al., (2017), Oncotarget 8: 71725; Wang L., et al., (2013), PLoSOne 8: e70689; Liu F., et al., (2015), Mol. Med. Rep. 12: 5455).

[0007] Many studies have found that B7H3 in tumor endothelial cells can also induce the activation of NF-κB through a TLR4-dependent mechanism, significantly increasing the expression levels of VEGF and IL-8 (Tekle C., et al., (2012), Int. J. Cancer 130: 2282), and further promoting tumor invasion and angiogenesis (Ferrara N., et al., (2002), Nat. Rev. Cancer 2: 795). Meanwhile, B7H3 can also function to regulate tumor cell EMT and tumor stem cell activity by reducing E-calcitonin and increasing the expression of N-calcitonin, volucrin, CD133 and CD44 (Jiang B., et al., (2016), Oncotarget 731755). B7H3 can activate the transduction of STAT3 signaling and increase the expression of hexokinase 2. Therefore, B7H3 also plays a role in promoting aerobic glycolysis in tumor cells (Shi T., et al., (2019), CellDeathDis.10:308). At the same time, B7H3 increases the levels of reactive oxygen species (ROS) and HIFI1a by suppressing the activity of the transcription factor NRF2, further enhancing the aerobic glycolysis of tumor cells and promoting tumor cell growth (Lim S., et al., (2016), CancerRes.76:2231). In addition, more and more studies have found that suppressing or reducing the expression of B7H3 can increase the response of tumor cells to some drugs, such as drugs that inhibit DNA replication (including alkylating drugs) and drugs that inhibit PI3K / Akt / mTOR and Ras / Raf / MEK signaling proteins (Flem-Karlsen K., et al., (2017), Pigment Cell Melanoma Res. 30: 467; Kasten B. B., et al., (2017), Nucl. Med. Biol. 47: 23; Liu H., et al., (2011), Mol. Cancer Ther. 10: 960).

[0008] By transplanting tumor cells into B7H3-deficient mice or treating tumor mice with B7H3 antibodies, tumor growth was clearly suppressed (Cai D., et al., (2020), Cell. Mol. Immunol. 17: 227; Lee Y. H., et al., (2017), Cell Res. 27: 1034), indicating that signaling blocking B7H3 may be used for tumor treatment. In addition, by combining with anti-PD-1 antibodies, it exerts a dual blocking function against B7H3 and PD-1, resulting in a synergistic anti-tumor effect (Lee Y. H., et al., (2017), Cell Res. 27: 1034). Due to the significant difference in the expression level of B7H3 between normal and tumor tissues, the ADCC effect or toxin coupling of B7H3 antibodies can effectively kill tumor cells without significantly affecting normal tissues (Koenig S., et al., (2014), Medicographia 36:285). In summary, based on current research information, B7H3 is considered to be a promising target for cancer therapy. Summary of the Invention

[0009] The present invention provides a group of anti-B7H3 monoclonal antibodies using hybridoma technology, which have high affinity for B7H3, prominent endocytosis function (can selectively kill or inhibit tumors by toxin coupling), prominent antibody-dependent cellular cytotoxicity (ADCC), and can effectively block immunosuppressive effects caused by B7H3. The present invention has successfully humanized six of the antibody candidates. The above-mentioned antibodies are considered promising for a wide range of applications in the areas of suppressing cancer cells, regulating the effects and levels of B7H3, and preparing drugs for improving biological immunity, especially for the preparation of drugs for cancer treatment.

[0010] The murine or humanized B7H3 antibody or functional fragment provided by the present invention includes a heavy chain sequence and a light chain sequence. The amino acid sequence information of the anti-human B7H3 mouse antibody, the heavy chain variable region, and the light chain variable region is as follows: The amino acid sequences of the 7F5 heavy chain variable region and light chain variable region are SEQ ID NOs: 1 and 2, respectively; the amino acid sequences of the 9C8 heavy chain variable region and light chain variable region are SEQ ID NOs: 3 and 4, respectively; the amino acid sequences of the 5B6 heavy chain variable region and light chain variable region are SEQ ID NOs: 5 and 6, respectively; the amino acid sequences of the 7C9 heavy chain variable region and light chain variable region are SEQ ID NOs: 7 and 8, respectively; the amino acid sequences of the 2A9 heavy chain variable region and light chain variable region are SEQ ID NOs: 9 and 10, respectively; the amino acid sequences of the 4F11 heavy chain variable region and light chain variable region are SEQ ID NOs: 11 and 12, respectively; the amino acid sequences of the 15A2 heavy chain variable region and light chain variable region are SEQ ID NOs: 13 and 14, respectively; and the amino acid sequences of the 7B7 heavy chain variable region and light chain variable region are SEQ ID NOs: The amino acid sequences of the 7E6 heavy chain variable region and light chain variable region are SEQ ID NOs: 17 and 18, respectively, the amino acid sequences of the 2E10 heavy chain variable region and light chain variable region are SEQ ID NOs: 19 and 20, respectively, the amino acid sequences of the 2F12 heavy chain variable region and light chain variable region are SEQ ID NOs: 21 and 22, respectively, the amino acid sequences of the 2F7 heavy chain variable region and light chain variable region are SEQ ID NOs: 23 and 24, respectively, the amino acid sequences of the 13A2 heavy chain variable region and light chain variable region are SEQ ID NOs: 25 and 26, respectively, and the amino acid sequences of the 14B3 heavy chain variable region and light chain variable region are SEQ ID NOs: 27 and 28, respectively.

[0011] The amino acid sequence information of the heavy chain and light chain CDR1, CDR2, and CDR3 of the above antibodies is as follows: the amino acid sequences of the 7F5 heavy chain CDR1, CDR2, and CDR3 are SEQ ID NOs: 29, 30, and 31, respectively, and the amino acid sequences of the light chain CDR1, CDR2, and CDR3 are SEQ ID NOs: 32, 33, and 34, respectively; the amino acid sequences of the 9C8 heavy chain CDR1, CDR2, and CDR3 are SEQ ID NOs: 35, 36, and 37, respectively, and the amino acid sequences of the light chain CDR1, CDR2, and CDR3 are SEQ ID NOs: 38, 39, and 40, respectively; the amino acid sequences of the 5B6 heavy chain CDR1, CDR2, and CDR3 are SEQ ID NOs: 41, 42, and 43, respectively, and the amino acid sequences of the light chain CDR1, CDR2, and CDR3 are SEQ ID NOs: 41, 42, and 43, respectively. The amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of 7C9 are SEQ ID NOs: 47, 48 and 49, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 of 2A9 are SEQ ID NOs: 53, 54 and 55, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 of 2A9 are SEQ ID NOs: 56, 57 and 58, respectively; the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of 4F11 are SEQ ID NOs: 59, 60 and 61, respectively, and the amino acid sequences of the light .... The amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 of 15A2 are SEQ ID NOs:65, 66, and 67, respectively, and the amino acid sequences of the light chain CDR1, CDR2, and CDR3 of 15A2 are SEQ ID NOs:68, 69, and 70, respectively; the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 of 7B7 are SEQ ID NOs:71, 72, and 73, respectively, and the amino acid sequences of the light chain CDR1, CDR2, and CDR3 of 7B7 are SEQ ID NOs:74, 75, and 76, respectively; and the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 of 7E6 are SEQ ID NOs:77, 78, and 79, respectively, and the amino acid sequences of the light chain CDR1, CDR2, and CDR3 of 7E6 are SEQ ID NOs:79, 70, and 71, respectively.The amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of 2E10 are SEQ ID NOs: 83, 84 and 85, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 of 2E10 are SEQ ID NOs: 86, 87 and 88, respectively. The amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of 2F12 are SEQ ID NOs: 89, 90 and 91, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 of 2F12 are SEQ ID NOs: 92, 93 and 94, respectively. The amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of 2F7 are SEQ ID NOs: 95, 96 and 97, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 of 2F7 are SEQ ID NOs: 96, 97 and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 of 2F7 are SEQ ID NOs: 97, 98 and 99, respectively. The amino acid sequences of the 13A2 heavy chain CDR1, CDR2, and CDR3 are SEQ ID NOs: 101, 102, and 103, respectively, and the amino acid sequences of the light chain CDR1, CDR2, and CDR3 are SEQ ID NOs: 104, 105, and 106. The amino acid sequences of the 14B3 heavy chain CDR1, CDR2, and CDR3 are SEQ ID NOs: 107, 108, and 109, respectively, and the amino acid sequences of the light chain CDR1, CDR2, and CDR3 are SEQ ID NOs: 110, 111, and 112, respectively.

[0012] Furthermore, the anti-human B7H3 antibody or fragment may be transformed to a humanized antibody.

[0013] The amino acid sequence of the 2A9 heavy chain variable region of the anti-human B2H3 humanized antibody is SEQ ID NO:113, the amino acid sequence of the light chain variable region of the humanized antibody is SEQ ID NO:114, the amino acid sequence of the 4F11 heavy chain variable region of the humanized antibody is SEQ ID NO:115, the amino acid sequence of the light chain variable region of the humanized antibody is SEQ ID NO:116, the amino acid sequence of the 9C8 heavy chain variable region of the humanized antibody is SEQ ID NO:117, the amino acid sequence of the light chain variable region of the humanized antibody is SEQ ID NO:118, the amino acid sequence of the 15A2 heavy chain variable region of the humanized antibody is SEQ ID NO:119, the amino acid sequence of the light chain variable region of the humanized antibody is SEQ ID NO:120, and the amino acid sequence of the 5B6 heavy chain variable region of the humanized antibody is SEQ ID NO:121, the amino acid sequence of the light chain variable region of the humanized antibody is SEQ ID NO:122. NO:122, the amino acid sequence of the 7B7 heavy chain variable region of the humanized antibody is SEQ ID NO:123, and the amino acid sequence of its light chain variable region is SEQ ID NO:124.

[0014] The expression vector comprises the antibody nucleic acid molecule.

[0015] The pharmaceutical composition comprises the above-mentioned antibody or a functional fragment thereof and a pharmaceutical vector. The above-mentioned antibodies or functional fragments thereof, nucleic acid molecules, expression vectors, host cells, and pharmaceutical compositions are used for the preparation of B7H3 immunologically functional drugs.

[0016] In this patent, a mammalian cell expression system is used to prepare recombinant B7H3 receptor protein as an antigen, and after immunizing mice, the mouse spleen cells are fused with myeloma cells to obtain hybridoma cells. A large number of hybridoma cells are cloned and screened multiple times to obtain multiple monoclonal antibody doma cell lines. These hybridoma cell lines can secrete and produce monoclonal antibodies that specifically bind to B7H3 receptors (Figures 1 and 2). Among these monoclonal antibodies, some have obvious endocytosis function by cells (Figures 3 and 4), some have the function of promoting the secretion of cellular factors, such as IFN-γ, by human immune cells (Figure 5), and some have obvious ADCC participation function (Figures 6, 7, and 8), where some monoclonal antibodies may be used in the future development of ADC, ADCC, and B7H3 antagonists. Furthermore, the genes of the light chain variable region and the heavy chain variable region of the antibody are cloned and coded by RT-PCR (Reverse Transcription-Polymerase Chain Reaction), and a humanized antibody is constructed by complementary arity-determining region grafting (CDR-graft). In vitro functional experiments have shown that the humanized B7H3 antibody can specifically bind to the B7H3 receptor protein (Figures 9 and 10), and has a clear endocytosis function by cells (Figure 11). After coupling with MMAE toxin, it has a clear poisoning function against Calu-6 tumor cells (Figure 12), an ADCC participation function (Figure 13), an ADCC poisoning function against Jurkat-B7H3 and PC9 cells via NK92MI-hCD16 cells (Figure 14), and a function of promoting the production and secretion of cellular factors, such as IFN-γ, by human immune cells (Figure 15). The positive control antibodies used in this patent are derived from patents US8802091 and WO2017180813Al. [Brief description of the drawings]

[0017] In order to more clearly describe the specific embodiments of the present invention or the technical solutions of the prior art, the following briefly introduces the accompanying drawings necessary for describing the specific embodiments or the prior art. It is obvious that the accompanying drawings described below are only some examples of the present invention, and those skilled in the art can obtain other accompanying drawings based on these accompanying drawings without creative efforts.

[0018] [Figure 1] 1 shows that the binding characteristics of the B7H3 hybridoma monoclonal antibody and the B7H3-hFc protein are measured by ELISA. [Diagram 2] 1 shows that the binding characteristics of B7H3 hybridoma monoclonal antibody to PC9 cells are measured by the FACS method. [Diagram 3] 1 shows that the activity of the B7H3 hybridoma antibody to be endocytosed by Jurkat-B7H3 cells is measured by the FACS method. [Figure 4] FIG. 1 shows that the activity of the B7H3 hybridoma antibody to be endocytosed by PC9 cells is measured by the FACS method. [Diagram 5] 1 shows the promoting effect of the B7H3 hybridoma antibody on the secretion of IFN-γ from human PBMS cells. [Figure 6] 1 shows that the ADCC function of B7H3 hybridoma or chimeric antibody against Jurkat-B7H3 cells is measured by the reporter gene method. [Figure 7] We show that the reporter gene method is used to measure the ADCC function against PC9 tumor cells involving several B7H3 chimeric antibodies. [Figure 8] We show that the reporter gene method is used to measure the ADCC function against DLD1 tumor cells involving several B7H3 chimeric antibodies. [Figure 9] 1 shows that the binding characteristics of humanized B7H3 monoclonal antibody and B7H3-His protein are measured by ELISA. [Figure 10]1 shows that the binding characteristics of a humanized B7H3 monoclonal antibody to the B7H3 protein in PC9 cells are measured by FACS. [Figure 11] 1 shows that the humanized B7H3 monoclonal antibody is endocytosed by PC9 cells as measured by FACS. [Figure 12] This shows the toxic effect of humanized B7H3 antibody ADC on Calu-6 tumor cells. [Figure 13] 1 shows that the ADCC function of humanized B7H3 antibody against Jurkat-B7H3 cells and PC9 cells is measured by the reporter gene method. [Figure 14] 1 shows the ADCC killing function of NK92MI-hCD16 cells in relation to Jurkat-B7H3 cells and PC9 cells in which humanized B7H3 antibody is involved. [Figure 15] 1 shows the promoting effect of humanized B7H3 monoclonal antibody on the production and secretion of IFN-γ from human PBMS cells. Specific embodiments

[0019] The term "antibody" as used herein is used in the broadest sense to refer to a protein or polypeptide that includes immunoglobulins or other types of molecules that contain one or more antigen-binding domains that specifically bind to an antigen and that exhibit binding specificity to a particular antigen. Specific examples of antibodies include complete antibodies (e.g., conventional four-chain antibody molecules), single-chain antibodies, single-domain antibodies, bispecific antibodies, and multispecific antibodies. Conventional antibody molecules are usually tetramers that consist of two identical heavy chains and two identical light chains linked together by disulfide bonds. Due to conservative differences in amino acid sequence, heavy and light chains are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxyl terminus. The variable region is responsible for recognizing and binding to antigens, and the constant region (e.g., Fc fragment) is responsible for downstream effects such as antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy and light chain variable regions each have three local regions of amino acid composition, with a higher degree of variation in sequence, which are important positions for antibody-antigen binding. Therefore, they are also called complementarity determining regions (CDRs). The amino acid sequences of CDRs can be easily identified by numbering schemes recognized in the art, such as Kabat, Chothia, IMGT, AbM, or Contact.

[0020] An "antigen-binding fragment" of an antibody refers to an amino acid fragment of an antibody molecule that is involved in specific binding to an antigen, and may be, for example, one of F(ab')2, Fab, and scFv.

[0021] EC50 (concentration for 50% of maximal effect) refers to the concentration that produces 50% of the maximum effect. In enzyme-linked immunosorbent assay (ELISA), when expressing the binding ability of an antibody molecule to a corresponding antigen, it may refer to the antibody molecule concentration that produces half of the maximum detection signal (e.g., colorimetric intensity or fluorescent intensity). The lower the EC50 value, the greater the binding affinity with the antigen.

[0022] The present invention relates to a B7H3 monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain and a light chain, wherein the heavy chain CDR1 amino acid sequence is selected from one of SEQ ID NOs: 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107; the heavy chain CDR2 amino acid sequence is selected from one of SEQ ID NOs: 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108; the heavy chain CDR3 amino acid sequence is selected from one of SEQ ID NOs: 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109; and the light chain CDR1 amino acid sequence is selected from one of SEQ ID NOs: the light chain CDR2 amino acid sequence is selected from one of SEQ ID NOs: 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, and the light chain CDR3 amino acid sequence is selected from one of SEQ ID NOs: 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, wherein the heavy chain and light chain in the antigen-binding fragment comprise the amino acid sequences spanning CDR1 to CDR3 of the heavy chain and light chain of the antibody, respectively. The B7H3 antibody or antigen-binding fragment thereof provided by the present invention comprises heavy chain CDRs and light chain CDRs, and the heavy chain CDRs and light chain CDRs have one or more pairs selected from the CDR combinations shown in a to n.

[0023] [Table 10]

[0024] In some embodiments, the amino acid sequence in the heavy chain variable region is selected from one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and the amino acid sequence in the light chain variable region is selected from one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28.

[0025] In some embodiments, the B7H3 monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region being selected from one or more sets of variable region combinations shown in A to N.

[0026] [Table 11]

[0027] As will be generally understood by those skilled in the art, functional variants of antibodies or antigen-binding fragments thereof are also within the scope of the present invention, and the term "functional variant" as used herein refers to a variant molecule obtained by introducing one or more amino acid insertions, deletions or substitutions based on a parent protein molecule (e.g., a naturally occurring protein molecule), while still retaining at least some of the functions of the parent protein molecule (particularly functions of interest, e.g., the ability to bind to a corresponding antigen). Regarding the CDR region, the functional variant in the present invention includes a heavy chain complementarity determining region and a light chain complementarity determining region, and includes up to three amino acid mutations (e.g., substitution, deletion or addition of one, two or three amino acids, or any combination thereof) compared to any one of the combinations of complementarity determining regions shown in a to n. Preferably, the mutations are conservative mutations.

[0028] Regarding the variable region, in some embodiments, the functional variant of the present invention comprises a heavy chain variable region, and the amino acid sequence in the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any of the heavy chain variable region sequences shown in combinations A to N. In some embodiments, the functional variant of the present invention comprises a light chain variable region, and the amino acid sequence in the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any of the light chain variable region sequences shown in combinations A to N. The term "identity" refers to the degree of match between two amino acid or nucleotide sequences (e.g., between a query sequence and a reference sequence), and is generally expressed as a percentage. Usually, before calculating the identity percentage between two amino acid or nucleotide sequences, sequence alignment is first performed and gaps (if any) are introduced. If the amino acid residues or bases in the two sequences are the same at a certain comparison position, the two sequences are considered to be identical or matched at that position. If the amino acid residues or bases in the two sequences are different, the two sequences are considered to be non-identical or non-matched at that position. In some algorithms, the identity of the sequences is obtained by dividing the number of matched positions by the total number of positions in the comparison window. In some other algorithms, the number of gaps and / or the length of the gaps are also taken into account. General-purpose sequence comparison algorithms or software include DANMAN, CLUSTALW, MAFFT, BLAST, MULCLE, etc. For the purpose of the present invention, the public comparison software BLAST (available from https: / / www.ncbi.nlm.nih.gov / ) is used, and the default settings may be used to obtain optimal sequence comparison and calculate the sequence identity between two amino acid or nucleotide sequences.

[0029] A functional variant has the ability to specifically bind to B7H3. A person skilled in the art can identify suitable variants of the antigen-binding molecules described herein using known techniques. In some embodiments, a person skilled in the art can identify regions of an antibody or antigen-binding fragment thereof that are not important for the activity of specifically binding to B7H3 without destroying the relevant activated regions.

[0030] In some embodiments, the B7H3 monoclonal antibody or antigen-binding fragment thereof is murine or humanized. In some embodiments, the heavy chain variable region and the light chain variable region are humanized.

[0031] In some embodiments, the heavy chain and the light chain are humanized. The term "humanized antibody", also called CDR-grafted antibody, refers to an antibody produced by grafting CDR sequences from a first animal onto a human antibody variable region framework, i.e., onto a different type of human antibody framework sequence. Chimeric antibodies can avoid the induction of xenogeneic reactions by containing a large amount of mouse protein components. Such architectural sequences can be obtained from a common DNA database or published references that contain species-specific antibody gene sequences. Species-specific DNA sequences, such as human heavy and light chain variable region genes, can be obtained from the "VBase" species-specific sequence database (www.mrccpe.com.ac.uk / vbase) and can also be found in Kabat, EA et al., "Sequences of Proteins of Immunological Interest" (5th ed., 1991). To avoid a reduction in immunogenicity and a concomitant reduction in activity, the activity may be maintained by performing minimal reverse mutations or back mutations on the above-mentioned human antibody variable region framework sequences. The humanized antibody of the present invention further includes a humanized antibody in which the CDRs are affinity matured by phage display. In a preferred embodiment of the present invention, the first animal origin is mouse origin.

[0032] In some embodiments, the heavy chain and the light chain are humanized, and the amino acid sequence of the humanized heavy chain variable region is selected from one of SEQ ID NOs: 113, 115, 117, 119, 121, 123, and the amino acid sequence of the humanized light chain variable region is selected from one of SEQ ID NOs: 114, 116, 118, 120, 122, 124.

[0033] In some embodiments, the humanized heavy chain variable region and the humanized light chain variable region are selected from one or more sets of variable region combinations shown in 1 to 6.

[0034] [Table 12]

[0035] In some embodiments, the heavy chain and the light chain are humanized, and the amino acid sequence of the humanized heavy chain variable region is SEQ ID NO:113 and the amino acid sequence of the humanized light chain variable region is SEQ ID NO:114.

[0036] In some embodiments, the B7H3 monoclonal antibody or antigen-binding fragment thereof has a human IgG1 constant region.

[0037] The present invention also relates to the use of a B7H3 monoclonal antibody or an antigen-binding fragment thereof in the preparation of a drug that modulates B7H3 activity or B7H3 levels, has significant endocytosis function (the ability to selectively kill or inhibit tumors by toxin coupling), or has significant antibody-dependent cellular cytotoxicity (ADCC), or improves the body's immunity by blocking the immunosuppressive action of B7H3, promotes T lymphocytes, or improves the production of cellular factors in T lymphocytes, such as IFN-γ. The present invention also relates to a monoclonal antibody coupling agent comprising a monoclonal antibody and a coupling moiety, wherein the monoclonal antibody is a set of B7H3 monoclonal antibodies or antigen-binding fragments thereof as described above, and the coupling moiety is selected from one or more of a radionuclide, a drug, a toxin, a cellular factor, a cellular factor receptor fragment, an enzyme, fluorescein, and biotin.

[0038] The present invention also relates to the use of the monoclonal antibody coupling agents as described above in the preparation of drugs that modulate B7H3 activity or levels, have significant endocytosis function (the ability to selectively kill or inhibit tumors by toxin coupling), have significant antibody-dependent cellular cytotoxicity (ADCC), improve the body's immunity by blocking the immunosuppressive action of B7H3, stimulate T lymphocytes, or improve the production of cellular factors in T lymphocytes, such as IFN-γ.

[0039] The present invention also relates to the use of a monoclonal antibody coupling agent as described above in the preparation of a medicament for the prevention and / or treatment and / or adjuvant treatment of tumors.

[0040] The present invention also relates to expression vectors comprising the above-mentioned antibody nucleic acid molecules. The present invention also relates to an expression vector comprising the above-mentioned B7H3 monoclonal antibody or an antigen-binding fragment thereof.

[0041] The term "vector" refers to a nucleic acid delivery tool into which a polynucleotide may be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, it is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transgenesis to allow the host cell to express the genetic material element carried by the vector. Vectors are known by those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)), phages (e.g., lambda phage or M13 phage), and animal viruses. Animal viruses that may be used as vectors include, but are not limited to, reverse transcriptase viruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), vaccinia viruses, baculoviruses, papilloma viruses, and papilloma viruses (e.g., SV40). In some embodiments, the vectors described herein contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, polynucleotide sequences, etc.).

[0042] In the present invention, the vector may be a composition, for example it may be a mixture of multiple plasmids or it may be part of different plasmids loaded antibodies or antigen-binding fragments thereof.

[0043] The present invention also relates to a pharmaceutical composition comprising (at least one of) the above-mentioned antibody or functional fragment thereof and a pharmaceutical vector.

[0044] As used herein, a "pharmaceutical vector" includes any material that, when combined with an active ingredient, retains the biological activity of the ingredient and does not react with the subject's immune system. Illustrative examples include, but are not limited to, standard pharmaceutical vectors (e.g., phosphate buffered saline, water, emulsions (such as oil / water emulsions), and any of a variety of wetting agents. Compositions containing such vectors are prepared by known and common methods (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, edited by A. Gennaro, Mack Publishing Co., Eason, PA, 1990, and Remington, The Science and Practice of Pharmacy, 21st Edition, Mack Publishing, 2005).

[0045] The present invention also relates to the use of the above-mentioned antibody or functional fragment thereof, nucleic acid molecule, expression vector, host cell, and pharmaceutical composition for the preparation of B7H3 immunologically functional drugs.

[0046] The following describes in detail the embodiments of the present invention in conjunction with examples. Example 1

[0047] Immunization and cell fusion of mice producing anti-B7H3 antibodies The extracellular domain of humanized B7H3 (NCBI Reference Sequence: NM_001024736.2) and mFc fusion protein (B7H3-ECD-mFc) are used as antigens, and are thoroughly emulsified with isomeric complete Freund's adjuvant (Sigma, Cat. No.: F5581), and then subcutaneous immunization is performed on 6-8 week-old Balb / c mice (purchased from Joen (Suzhou) New Drug Research Center Co., Ltd.), with the antigen immunization dose being 50 μg / mouse. After that, the same dose of antigen is thoroughly emulsified with incomplete Freund's adjuvant (Sigma, Cat. No.: F5506) every two weeks, and then subcutaneous immunization is performed on the mice three times. After three immunizations, the serum titers of the mice are measured, and one booster immunization is performed by intraperitoneal injection three days before the fusion. Using PEG Hybri-Max (Sigma, Cat. No.: 7181) as a fusion agent, mouse spleen cells were mixed with SP2 / 0 cells at a ratio of 4:1, and the fused cells were placed in a 96-well plate (1 × 10 5 Each well contains 0.1 mL of 1xHAT (Invitrogen, Cat. No.: 21060-017) medium. On the third day, add 0.1 mL of HT (Invitrogen, Cat. No.: 11067-030) medium. On the seventh day, aspirate the medium in the 96-well plate and add 0.2 mL of fresh HT medium. On the ninth day, collect the supernatant for various screening and testing. Example 2

[0048] Antigen binding by hybridoma antibodies and subcloning Testing of hybridoma antibody binding capacity includes ELISA, FACS and subcloning using limited dilution methods.

[0049] 1) Screen B7H3-binding positive clones by ELISA. 50 μL of B7H3-hFc (final concentration: 2 μg / mL) is used to wrap a 96-well ELISA plate (Corning, Cat. No.: 9018) and left at room temperature overnight. After washing three times with washing buffer (PBS + 0.05% Tween 20), add blocking buffer (PBS + 2% BSA (Sigma, Cat. No.: V90093)) and incubate at room temperature for 1 hour, and wash the ELISA plate three times with washing buffer. Add hybridoma supernatant, incubate at room temperature for 1 hour, and wash three times. Add 100 μL of 10000-fold diluted HRP-coupled goat anti-mouse IgG secondary antibody (Thermo, Cat. No.: 31432) to each well, incubate at room temperature for 1 hour protected from light, and wash three times. Add 100 μL TMB (Beijing Baiwo Sports, Cat. No.: ES-002) to each well and incubate at room temperature for 2 minutes to allow color development. Add 100 μL / well of stopping solution (2NH2SO4) to terminate the color development reaction, and read the OD450 value of each well using a microplate reader (Tecan Spark).

[0050] 2) Screening for B7H3-binding positive clones by FACS. 50 μL of hybridoma supernatant or purified hybridoma antibody that was positive in the above detection was taken and mixed with 50 μL of 293T-B7H3 cells (2×10 5 pcs / well) into a 96-well U-bottom cell plate and incubate at 4oC for 1 hour, wash twice with FACS buffer (PBS+3%FCS) and centrifuge, add 400-fold diluted PE-labeled goat anti-mouse antibody (Biolegend, Cat. No.: 405307), incubate at 4oC for 30 minutes in the dark, wash twice with FACS buffer and centrifuge, and then use a BDAccuiC6 flow cytometer to detect the signal value of the cells in the PE path.

[0051] 3) Subcloning: Subcloning is performed by the finite dilution method. That is, subcloning is performed on the polyclonal hybridoma cells that are positive in the above-mentioned ELISA and FACS detection, and then the detection screening by the ELISA and FACS methods is repeated to obtain positive hybridoma monoclones.

[0052] Purification of antibodies, measurement of concentration and measurement of endotoxin Positive monoclonal hybridoma cells were placed in 50mL of serum-free medium (Invitrogen, Cat. No.: 12045-076) and cultured for 8-9 days, then centrifuged to collect the supernatant. The monoclonal antibodies were purified by Protein A affinity chromatography, and the purified antibody samples were subjected to liquid exchange and concentration using ultrafiltration centrifuge tubes (Millipore, Cat. No.: ACS500024). The protein concentration was then measured by the BCA method, and the endotoxin content of the purified antibody samples was detected using Limulus Reagent (Xiamen Limulus Reagent Biological Science Co., Ltd.).

[0053] The binding ability of the purified antibody samples to B7H3 was detected by ELISA and FACS, and the results are shown in Figure 1 (a-b), Figure 2 (a-b) and Table 1. All of the selected hybridoma antibodies have high affinity.

[0054] Table 1. Binding strength of B7H3 hybridoma monoclonal antibody and B7H3 antigen EC 50 Value of [Table 1] Example 3

[0055] Testing the endocytosis and B7H3 blocking functions of hybridoma monoclonal antibodies Assay for antibody endocytosis function: In a 96-well plate, add 50 μL of 2 × 10 6 1000μL / ml PC9 cells and 50μL of labeled B7H3 antibody were added, and the 96-well cell culture plate was incubated at 4℃ for 1 hour. After washing twice with FACS buffer, Goat anti-mFc (Jackson, Cat. No.: 115-005-071) secondary antibody labeled with pH-dependent fluorescent dye CypHer5E (GE, Cat. No.: PA15401) was added, and then incubated at 4℃ for 0.5 hours. After washing twice, the plate was placed in medium (1640+10% FBS) and left in an incubator for 3 hours. The medium was then centrifuged to remove the medium, and the plate was resuspended in PBS at pH 9.0. The CypHer5E signal of PC9 cells was detected by BD C6 flow cytometer, and the efficiency of B7H3 antibody entering the cells was calculated. As shown in Figures 3(a-d) and 4(a-d), Jurkat-B7H3 and PC9 cells have significant endocytosis activity toward B7H3 antibodies (including 15A2, 5B6, 7C9, 2F7, 7B7, 2E10, 4F11, etc.).

[0056] Testing the effect of B7H3 hybridoma antibody on the secretion of cellular factors by human PBMC cells: In a 96-well plate (Corning, Cat. No.: 3799), PBMC cells (TPCS, Cat. No.: PB025C) resuspended in complete medium (RPMI1640 + 10% FCS) were added, followed by 40ng / mL OKT3 (eBioscience, Cat. No.: 16-0037-85) and incubated at 37°C for 72 hours. Activated PBMC cells were counted and then resuspended in complete medium (2.5 x 10 5In a 96-well plate, 100 μL of PBMC cells and 50 μL of different concentrations of B7H3 antibodies (starting concentration 20 μg / mL, diluted 10-fold) were added to each well, and the 96-well cell culture plate (Corning, Cat. No.: 3599) was incubated at 37°C in a 5% CO2 incubator for 48 hours, and the supernatant was collected. The concentration of cellular factors was detected by IFN-γ ELISA kit (R&D Systems, Cat. No.: DY285). As shown in Figure 5, B7H3 antibodies, including 5B6, 2F12, 2F7, 15A2, 13A2, 14B3, 4F11, 2A9, etc., can significantly promote the secretion of IFN-γ by PBMC cells. Example 4

[0057] Cloning and sequence determination of the variable region genes of B7H3 antibody B7H3 monoclonal hybridoma cell line is digested with TRIzon (Cwbiotech, Cat. No.: CW0580) and total RNA of hybridoma cells is extracted. RNA of hybridoma cells is reverse transcribed to cDNA using HiFi Script cDNA synthesis kit (Cwbiotech, Cat. No.: CW2569). Using cDNA as a template and simple primers, variable region genes of heavy and light chains of antibodies are amplified by PCR (Kettleborough et al., (1993), Eur J Immunology 23: 206-211; Strebe, et al., (2010), Antibody Engineering 1: 3-14). After PCR amplification products are ligated into T / A vector, DH5a competent cells are transformed, plated and cultured at 37℃ overnight. After picking up monoclones from the culture plate and expanding and culturing them, the plasmid is extracted and the antibody gene sequence is measured. Based on the antibody gene sequence, its complementarity determining cluster (CDR) and skeleton region are analyzed. The sequence number of the B7H3 antibody is shown in Table 2, and the specific sequence information can be found in the sequence table.

[0058] Table 2. Description of the sequence numbers of the B7H3 antibody [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] Example 5

[0059] ADCC activity test of hybridoma monoclonal antibodies The ADCC activity of the antibody is tested by the reporter gene method. 293T-B7H3, PC9 or DLD1 tumor cells are used as target cells, and Jurkat-mCD16.2-NF-kB or Jurkat-hCD16-NF-kB cells are used as effector cells, and ADCC-positive clones are screened using the activation signal of the transcription factor NF-kB mediated by mCD16.2 or hCD16. 50 μL of the hybridoma antibody that is positive in the above-mentioned ELISA and FACS detection is taken and mixed with 25 μL of 293T-B7H3, PC9 or DLD1 tumor cells (7.5 × 10 4 cells / well) and added to a 96-well plate, followed by 25 μL of Jurkat-mCD6.2-NF-kB cells (2.5 × 10 4Add 100 μL of pre-warmed Bright-Glo solution (Promega, Cat. No.: E2620) to each well, mix thoroughly, and incubate for 4 hours at 37° C. Add 25 μL of pre-warmed Bright-Glo solution (Promega, Cat. No.: E2620) to each well, and leave the wells at room temperature for 3 minutes in the dark. Then measure the cold luminescence signal value of each sample using a microplate reader (Tecan Spark).

[0060] Among the hybridoma antibodies, mouse IgG1 antibodies have no ADCC activity. Therefore, gene sequence measurement revealed that, of the 14 hybridoma antibodies, 9 were based on mouse IgG1, and 5 were based on IgG2b or IgG2a (see Table 3). Figure 6a shows the ADCC reporter gene test results of the hybridoma antibodies with Jurkat-B7H3 as the target cells. As expected, the 5 hybridoma antibodies based on IgG2b or IgG2a have ADCC activity, but the 9 hybridoma antibodies based on mouse IgG1 do not. Therefore, the 9 hybridoma antibodies based on mouse IgG1 were replaced with Fc, and the antibodies were made into chimeric antibodies based on human IgG1, as shown in Figure 6b. Figures 7 and 8 show the ADCC reporter gene test results of the PC9 or DLD1 tumor cells as the target cells, and the antibody form is a chimeric antibody based on human IgG1. Looking at the ADCC reporter gene test results in general, the antibodies with relatively strong ADCC activity include 9C8, 5B6, 7C9, F5, 7E6, 2A9, and 4F11.

[0061] Table 3. Fc type of B7H3 hybridoma antibody [Table 3] Example 6

[0062] Humanized B7H3 antibodies 2A9, 4F11, 9C8, 15A2, 5B6 and 7B7 After comprehensively considering the characteristics of each aspect of the hybridoma antibodies, six B7H3 antibodies, including 2A9, 4F11, 9C8, 15A2, 5B6 and 7B7, were selected for humanization transformation.

[0063] Humanization transformation of the B7H3 antibody is carried out by the complementary determinant cluster grafting method. First, the IMGT database is searched for human germline antibody sequences with the highest homology to the light chain variable region sequences and heavy chain variable region sequences of mouse 2A9, 4F11, 9C8, 15A2, 5B6 and 7B7 antibodies, respectively. For humanization of the light chain variable region of the 2A9 antibody, germline IGKV3-11*01 is selected, and for humanization of the heavy chain variable region, germline IGHV1-2*02 is selected. For humanization of the light chain variable region of the 4F11 antibody, germline IGKV3-11*01 is selected, and for humanization of the heavy chain variable region, germline IGHV1-2*02 is selected. For humanization of the light chain variable region of the 9C8 antibody, the embryonic system IGKV3-11*01 is selected, and for humanization of the heavy chain variable region, the embryonic system IGHV1-46*01 is selected. For humanization of the light chain variable region of the 15A2 antibody, the embryonic system IGKV3-11*01 is selected, and for humanization of the heavy chain variable region, the embryonic system IGHV1-8*01 is selected. For humanization of the light chain variable region of the 5B6 antibody, the embryonic system IGKV3-39*01 is selected, and for humanization of the heavy chain variable region, the embryonic system IGHV1-69*02 is selected. For humanization of the light chain variable region of the 7B7 antibody, the embryonic system IGKV3-39*01 is selected, and for humanization of the heavy chain variable region, the embryonic system IGHV1-21*01 is selected. The CDR regions of the mouse antibody are left, and the framework region sequences of the mouse antibody are replaced with the framework region sequences of a human embryonic antibody. Establish a structural model of the mouse antibody, and compare the amino acid of the humanized antibody with each site in the corresponding mouse antibody framework region. If the human amino acid sequence is used at a site in the framework region without destroying or changing the spatial structure of the CDR region, the human amino acid sequence is applied to that site, otherwise the corresponding mouse sequence is applied to that site (i.e., the back mutation is the mouse sequence).

[0064] In the structural simulation, Ala at position 24 in the humanized heavy chain of the 2A9 antibody is backmutated to Thr, Met at position 48 to Ile, Val at position 67 to Ala, Met at position 69 to Leu, Arg at position 71 to Val, and Thr at position 73 to Lys. Leu at position 46 in the humanized light chain of the 2A9 antibody is backmutated to Arg, Leu at position 47 to Trp, Ile at position 48 to Val, and Phe at position 71 to Tyr. Ala at position 24 in the humanized heavy chain of the 4F11 antibody is backmutated to Thr, Met at position 48 to Ile, Val at position 67 to Ala, Met at position 69 to Leu, Arg at position 71 to Val, and Thr at position 73 to Lys. In the humanized light chain of the 4F11 antibody, Leu at position 46 is backmutated to Arg, Leu at position 47 is backmutated to Trp, Ile at position 48 is backmutated to Val, and Phe at position 71 is backmutated to Tyr. In the humanized heavy chain of the 9C8 antibody, Met at position 48 is backmutated to Ile, Val at position 67 is backmutated to Ala, Met at position 69 is backmutated to Leu, Arg at position 71 is backmutated to Val, and Thr at position 73 is backmutated to Lys. In the humanized light chain of the 9C8 antibody, Ile at position 2 is backmutated to Thr, Leu at position 46 is backmutated to Arg, Leu at position 47 is backmutated to Trp, and Phe at position 71 is backmutated to Tyr. In the humanized heavy chain of the 15A2 antibody, Met at position 48 is backmutated to Ile, Val at position 67 is backmutated to Ala, Met at position 69 is backmutated to Leu, Arg at position 71 is backmutated to Ala, and Thr at position 73 is backmutated to Lys. In the humanized light chain of the 15A2 antibody, Tyr at position 36 is backmutated to Phe, Leu at position 47 is backmutated to Trp, Tyr at position 49 is backmutated to His, Ile at position 58 is backmutated to Phe, and Phe at position 71 is backmutated to Tyr. In the humanized heavy chain of the 5B6 antibody, Gly at position 27 is backmutated to Tyr, Ser at position 30 is backmutated to Ile, Met at position 48 is backmutated to Ile, Val at position 67 is backmutated to Ala, and Ile at position 69 is backmutated to Leu. In the humanized light chain of the 5B6 antibody, Leu at position 46 is backmutated to Arg, Leu at position 47 is backmutated to Pro, Gly at position 66 is backmutated to Ala, Phe at position 71 is backmutated to Tyr, and Phe at position 98 is backmutated to Ile.In the humanized heavy chain of the 7B7 antibody, Ser at position 49 is backmutated to Ala. In the humanized light chain of the 7B7 antibody, Ile at position 2 is backmutated to Ser and Ile at position 48 is backmutated to Val.

[0065] The amino acid sequence numbers for the heavy and light chain variable regions of the humanized antibody are SEQ ID NO:113 and SEQ ID NO:114, respectively. The amino acid sequence numbers for the heavy and light chain variable regions of the 4F11 humanized antibody are SEQ ID NO:115 and SEQ ID NO:116, respectively. The amino acid sequence numbers for the heavy and light chain variable regions of the 9C8 humanized antibody are SEQ ID NO:117 and SEQ ID NO:118, respectively. The amino acid sequence numbers for the heavy and light chain variable regions of the 15A2 humanized antibody are SEQ ID NO:119 and SEQ ID NO:120, respectively. The amino acid sequence numbers for the heavy and light chain variable regions of the 5B6 humanized antibody are SEQ ID NO:121 and SEQ ID NO:122, respectively. The amino acid sequence numbers of the heavy and light chain variable regions of the 7B7 humanized antibody are SEQ ID NO:123 and SEQ ID NO:124, respectively. The above humanized antibodies are constructed as IgG1 subtype. The amino acid sequence information of the heavy and light chain variable regions of the humanized antibodies is shown in Table 4.

[0066] The nucleic acid sequences for the light and heavy chains of the humanized antibodies numbered 2A9, 4F11, 9C8, 15A2, 5B6, and 7B7 are synthesized and inserted into the expression vector pcDNA3.1. 0.1 mg of the antibody light chain and 0.1 mg of the antibody heavy chain expression plasmid are used to inoculate 200 mL of 293 cells (cell density 1×10 6 1000μg / mL) and cultured in a 37oC shaker for 6 days, then centrifuged to collect the supernatant, purified the humanized antibody with Protein A, and detected the activity against the purified humanized antibody.

[0067] Table 4. Sequence listing of B7H3 humanized antibodies [Table 4] Example 7

[0068] Binding activity of humanized B7H3 antibodies to B7H3 The binding activity of the humanized B7H3 antibody sample with the B7H3 protein was detected by ELISA and FACS, and the specific method is described in Example 2. The measurement results for the humanized antibodies hu2A9, hu4F11, hu5B6, hu9C8, hu15A2 and hu7B7 are shown in Tables 5-6 and Figures 9-10. Overall, the antibodies of the present invention can retain the high antigen affinity effect before humanization after humanization. In particular, hu15A2, hu7B7 and hu5B6 are included.

[0069] Table 5. Detection of binding between humanized B7H3 antibody and B7H3-mFc protein by ELSIA [Table 5]

[0070] Table 6. Measurement of binding activity of humanized B7H3 antibody to cell surface B7H3 by FACS [Table 6] Example 8

[0071] Detection of endocytic function of humanized B7H3 antibody For specific measurement methods, please refer to Example 3. As a result, as shown in Table 7 and Figure 11, it was found that PC9 cells have significant endocytosis function for humanized B7H3 antibody. Compared with positive controls (MGA018 by MacroGenics and 8H9 by MSKCC), the endocytosis function of the antibody in the present invention is much higher than that of 8H9. In addition, hu15A2 and hu7B7 are also higher than MGC018.

[0072] Table 7 Endocytosis function of humanized B7H3 antibody by PC9 tumor cells [Table 7] Example 9

[0073] Toxic killing of Calu-6 tumor cells by humanized B7H3 antibody coupled to toxins (B7H3-ADC) Vc-MMAE is coupled to B7H3 antibody by chemical coupling method. B7H3 antibody is reduced with TCEP in a 3:1 molar ratio, incubated at 37°C for 45 minutes, and TCEP is removed by ultrafiltration. The antibody is resuspended in 0.5ml PBS, and 6 times more molar Vc-MMAE than B7H3 antibody is added, incubated at 4°C for 120 minutes, and uncoupled Vc-MMAE is removed by ultrafiltration, and the antibody concentration is detected for use. 100μl of PC9 (5000 cells per well) and 100μl of gradient diluted B7H3-ADC are added to a 96-well plate. After incubation at 37°C for 5 days, xμl of CellTiter-Glo is added to each well, and the fluorescence intensity is detected to calculate the ability of the antibody to inhibit tumor cell proliferation. As a result, as shown in FIG. 12 and Table 8, it was found that the humanized B7H3 antibody had significant cytotoxicity against Calu-6 tumor cells after coupling to MMAE toxin, and had stronger activity than MacroGene's positive control (MGC018-MMAE).

[0074] Table 8. The cytotoxicity of Calu-6 tumor cells by the coupling agent of humanized B7H3 antibody and MMAE [Table 8] Example 10

[0075] Detection of ADCC function by humanized B7H3 antibody For specific measurement methods, please refer to Example 5. As a result, as shown in Table 9 and Figure 13, it was revealed that the humanized B7H3 antibody has significant ADCC activity. MGA271 (MG-Ab) is an antibody from Macrogenics, and compared with it, the humanized 9C8, 5B6, 2A9 and 4F11 antibodies have smaller EC50 values ​​and exhibit better ADCC activity.

[0076] Table 9. ADCC function of humanized B7H3 antibody [Table 9] Example 11

[0077] ADCC killing function of humanized B7H3 antibody-mediated NK92MI-hCD16 cells against Jurkat-B7H3 and PC9 cells NK92MI-hCD16 cells were used to simulate NK cells to evaluate the tumor killing activity of the B7H3 antibody. 25 μl of CFSE-stained PC9 (0.5 × 10 cells per well) was added to each well. 5 cells) and 25 μl of NK92MI-hCD16 (1 × 10 596-well plate) and add 50μl of gradient diluted B7H3 antibody. After 4 hours of incubation at 37℃, add 5μl of 7-AAD to each well, incubate at room temperature for 10 minutes, centrifuge the 96-well plate to resuspend the supernatant, and then use a flow cytometer to detect the proportion of CFSE and 7-AAD double positive cells in CFSE positive cells, and obtain the NK cell killing activity of B7H3 antibody intervention. As a result, as shown in Figure 14, humanized B7H3 antibodies 9C8, 5B6, 2A9 and 4F11 can obviously trigger the ADCC killing function of NK92MI-hCD16 cells against PC9 tumor cells. Example 12

[0078] Promoting function of humanized B7H3 antibody on secretion of cellular factors by human PBMC cells For specific measurement methods, see Example 3. As a result, as shown in Figure 15, the humanized B7H3 antibodies 5B6, 2A9 and 4F11 have the function of significantly promoting the secretion of IFN-γ by human PBMC cells. The above is merely a preferred embodiment of the present invention, and does not limit the scope of protection of the present invention. That is, any simple equivalent changes and amendments based on the claims and utility model of the present invention will fall within the scope of protection of the patent application of the present invention.

Claims

1. A B7H3 monoclonal antibody or an antigen-binding fragment thereof comprising heavy chain CDRs and light chain CDRs, the heavy chain CDRs and the light chain CDRs having one or more pairs selected from the CDR combinations shown in a to n. 【Table 10】

2. The B7H3 monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region being selected from one or more sets of variable region combinations shown in A to N. 【Table 11】

3. The B7H3 monoclonal antibody or antigen-binding fragment thereof of claim 2, wherein the heavy chain variable region and the light chain variable region are humanized.

4. The B7H3 monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein the humanized heavy chain variable region and the humanized light chain variable region are selected from one or more sets of variable region combinations shown in 1 to 6. 【Table 12】

5. The B7H3 monoclonal antibody or antigen-binding fragment thereof of claim 4, having a human IgG1 constant region.

6. Use of the B7H3 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 in the preparation of a drug that modulates B7H3 activity or B7H3 levels, has a significant endocytosis function (a function that can selectively kill or inhibit tumors by toxin coupling), or has a significant antibody-dependent cellular cytotoxicity (ADCC), or improves the body's immunity by blocking the immunosuppressive action of B7H3, promotes T lymphocytes, or improves the production of cellular factors in T lymphocytes such as IFN-γ.

7. 6. A monoclonal antibody coupling agent comprising a monoclonal antibody and a coupling moiety, wherein the monoclonal antibody is a B7H3 monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, and the coupling moiety is selected from one or more of a radionuclide, a drug, a toxin, a cellular factor, a cellular factor receptor fragment, an enzyme, fluorescein, and biotin.

8. Use of the monoclonal antibody coupling agent of claim 7 in the preparation of a drug that modulates B7H3 activity or B7H3 levels, has a significant endocytosis function (a function that can selectively kill or inhibit tumors by toxin coupling), has a significant antibody-dependent cellular cytotoxicity (ADCC), improves the body's immunity by blocking the immunosuppressive action of B7H3, promotes T lymphocytes, or improves the production of cellular factors in T lymphocytes such as IFN-γ.

9. 10. Use of the monoclonal antibody coupling agent according to claim 7 in the preparation of a drug for the prevention and / or treatment and / or adjuvant treatment of a tumor.

10. An expression vector comprising a nucleic acid molecule of the B7H3 monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 5.

11. A pharmaceutical composition comprising the B7H3 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and a pharmaceutical vector.

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