CN116554325A - anti-B7H 3 antibodies and uses thereof - Google Patents

Abstract

The present application provides murine anti-B7H 3 (CD 276) antibodies, chimeric and humanized forms thereof. The application also provides a pharmaceutical composition comprising the antibody, a vaccine, a nucleotide molecule encoding the antibody, an expression vector comprising the nucleotide molecule, a host cell comprising the expression vector and the application of the antibody in treating B7H3 related diseases.

Description

Anti-B7H3 antibody and its application

technical field

The present application relates to the field of antibodies, more specifically, the present application relates to anti-B7H3 antibodies and applications thereof.

Background technique

In the cellular immune response, the proliferation and activation of T cells not only requires T cell receptor TCR to recognize the first signal presented by APC or MHC on the surface of tumor cells, but also requires the second signal provided by co-stimulatory molecules (Hodge J W, Greiner J W, Tsang KY et al., 2006). The B7-CD28 superfamily is one of the costimulatory molecular families discovered so far, and belongs to the immunoglobulin superfamily. B7 family molecules can provide stimulating signals to enhance and maintain T cell immune responses, and can also produce inhibitory signals to limit and weaken T cell immune responses. Therefore, this family plays an important role in tumor diseases, organ transplantation and autoimmune diseases.

B7H3 is a member of the B7 family. According to function, the B7 family can be divided into 3 classes (Seliger B, Marincola FM, Ferrone S et al., 2008). Class I: B7-1 (CD80) and B7-2 (CD86); Class II: B7-H1 (PD-L1) and B7-DC (PD-L2); Class III: B7H3 (CD276) and B7 -H4(B7x). Receptors for class III members have not been identified, but are thought to be involved in co-stimulatory and co-inhibitory pathways.

The immune checkpoint PD1 antibody O drug (Opdivo) and K drug (Kyytrudo) against B7-H1 (also known as PD-L1) were launched in December and April 2014, respectively. Thanks to this, the development of antibodies against B7H1 It has developed rapidly this year. Roche's Tecentriq was also approved to go on the market two years later in October, making the B7 family gain more attention and enthusiasm for research and development in the field of tumor immunotherapy.

In addition, CTLA-4, which is the B7-1 and B7-2 receptors of the first member of the B7 family, has been the research focus of many pharmaceutical companies. Ipilimumab, which was first released, has a good curative effect in the treatment of advanced melanoma, but its severe side effects have added many uncertainties, so more mechanism research and more effective and low-toxicity therapy are needed.

For the first two types of B7 family, there are relatively many developments and researches on them, while the third type, B7H3, develops slowly. This is mainly due to the fact that its ligands are still unknown and its mechanism of action is still unclear, resulting in many difficulties in the selection of its anti-tumor aspects.

B7H3, also known as CD276, is a type I transmembrane protein. The extracellular region contains one or two pairs of the same immunoglobulin variable region and constant region. The intracellular region is very short and has no clear signal motif, thus forming two The main molecular forms 2IgB7H3 and 4IgB7H3, and the free form. The human 4IgB7H3 gene is located on chromosome 15, while the mouse B7H3 gene is located on chromosome 9. B7H3 is one of the most evolutionarily conserved members of the B7 family, as it is ubiquitously expressed in species ranging from teleosts to mammals. In addition, both forms of B7H3 are expressed in a variety of non-human primates, but only one form of 2IgB7H3 is included in animals such as mice.

Transcripts of B7H3 are widely expressed in tissues such as heart, liver, placenta, prostate, testis, uterus, pancreas, small intestine and colon. Whereas the expression of B7H3 protein is more restricted to the cell surface, such as activated dendritic cells, monocytes, T cells, B cells and NK cells (Wang L, Kang F B, Shan B E. 2014).

Many studies have revealed that B7H3 is abnormally highly expressed in various cancer cells or tissues, including gastric cancer, lung cancer, prostate cancer, kidney cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, liver cancer, colorectal cancer, oral cancer, Bladder cancer, osteosarcoma and malignant diseases of the blood system, its expression level is closely related to the poor prognosis and clinical outcome of patients, and it is speculated that it is involved in the immune evasion of tumors. B7H3 may play a dual role in the immune system. On the one hand, B7H3, as a co-stimulatory molecule, has co-stimulatory effects on CD4+ and CD8+ cells, induces cellular immunity, and selectively enhances interferon-γ (IFNG) production under T cell receptor signaling.

On the other hand, experiments in humans and mice have demonstrated that B7H3 also has a co-suppressive effect (Veenstra R G, Flynn R, Kreymborg K et al., 2015), which can suppress Treg cells, thereby allowing tumors to escape immune responses (Hofmeyer KA, Ray A, Zang X. 2008). This may be related to NFAT, NF-κB and AP-1 factors. Although its molecular mechanism is still unclear, it may be due to the existence of different forms of molecules that cause different functions, but as a possible immune checkpoint molecule, it is a promising target for tumor immunotherapy.

It has been proved by literature that B7H3 plays an important role in tumor immune escape by inhibiting T cells. In addition, it also plays an important role in the non-immune system. Studies have shown that B7H3 is involved in the regulation of the metastatic potential of cancer cells, and may also affect biological characteristics such as tumor proliferation and invasion.

The high expression of B7H3 in bladder cancer, prostate cancer, melanoma and other tumor cell lines has been proved to promote the invasion and metastasis of tumor cells. In melanoma cells, silencing of B7H3 reduced the cells' matrigel invasive ability. This may be because B7H3 silencing reduces the expression of metastasis-associated proteins, such as matrix metalloproteinase (MMP)-2, signal transducer and activator of transcription 3 (STAT3), and secreted interleukin 8 (IL-8).

In addition, it has been found in numerous Chinese and foreign literatures that B7H3 is highly expressed on the vascular endothelium in the tumor microenvironment, which may also suggest that B7H3 has a certain role in inhibiting or promoting angiogenesis, and has a role in inhibiting tumors and treating age-related macular degeneration (AMD) and other diseases. Certain potential.

For the B7H3 molecule, currently the fastest research progress on the market is Enoblituzumab (MGA271) from MacroGenics, whose anti-tumor effect is carried out through antibody-dependent cell-mediated cytotoxicity (ADCC). The antibody has entered Phase II clinical trials and has shown encouraging preliminary results. Antibody-drug conjugates (ADCs) can act as bio-missiles. The radiolabeled monoclonal antibody 8H9 targeting B7H3 has been successfully used in the clinical treatment of metastatic neuroblastoma, and the radiolabeled humanized 8H9 antibody has been used in Carry out clinical trials on various tumors such as peritoneal cancer, glioma, and central nervous system tumors. The development of bispecific antibodies, chimeric antibodies, receptor T cells (CAR-T), and small molecule inhibitors has enriched the strategies of tumor immunotherapy, and combined applications can promote synergistic effects. Further research on their receptors and mechanisms provides a basis for designing more Effective therapeutic drugs lay the foundation.

Contents of the invention

In order to solve the above technical problems, the present application provides an anti-B7H3 antibody and its application. Specifically, the application provides the following technical solutions.

In a first aspect, the application provides an antibody or an antigen-binding portion thereof that specifically binds B7H3, comprising a heavy chain variable region comprising any one or more of HCDR1, HCDR2 and HCDR3 sequences item, wherein the HCDR1 sequence is GDSITSNY (SEQ ID NO: 1); the HCDR2 sequence is ISNSGST (SEQ ID NO: 2); and the HCDR3 sequence is ARGEGRYGFGAY (SEQ ID NO: 3).

In some embodiments, the antibody or antigen-binding portion thereof further comprises a light chain variable region comprising any one or more of LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1 sequence comprising the amino acid sequence KSLLHGNGNTY (SEQ ID NO: 4); the LCDR2 sequence comprising the amino acid sequence RMS (SEQ ID NO: 5); and the LCDR3 sequence comprising the amino acid sequence MQHLEYPFT (SEQ ID NO: 6).

In some embodiments, the antibody is a murine antibody. Preferably, the heavy chain variable region of the murine antibody comprises the amino acid sequence of SEQ ID NO:7, and/or the light chain variable region of the murine antibody comprises the amino acid sequence of SEQ ID NO:8.

In some embodiments, the antibody is a chimeric antibody. Preferably, the chimeric antibody comprises a heavy chain and/or a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:9, and/or the light chain comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody is a humanized antibody. Preferably, the heavy chain variable region of the humanized antibody comprises the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, and/or the light chain variable region of the humanized antibody The region comprises the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:15.

In the second aspect, the present application provides a pharmaceutical composition, which comprises the antibody or antigen-binding portion thereof that specifically binds to B7H3 described in the first aspect and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition further comprises one or more other active ingredients. In some embodiments, the active ingredient is a chemotherapeutic agent, a PD-1 binding antagonist, a 4-1BB binding agonist, and the like.

In a third aspect, the present application provides a vaccine comprising the antibody or antigen-binding portion thereof described in the first aspect, and optionally an immune adjuvant.

In the fourth aspect, the present application provides a nucleotide molecule encoding the antibody or antigen-binding portion thereof that specifically binds to B7H3 described in the first aspect.

In the fifth aspect, the present application provides an expression vector comprising the nucleotide molecule described in the fourth aspect.

In the sixth aspect, the present application provides a host cell comprising the nucleotide molecule described in the fourth aspect or the expression vector described in the fifth aspect.

In the seventh aspect, the present application provides the antibody specifically binding to B7H3 or its antigen-binding portion described in the first aspect, the pharmaceutical composition described in the second aspect, the vaccine described in the third aspect, and the vaccine described in the fourth aspect. The nucleotide molecule of the fifth aspect, the expression vector of the fifth aspect or the host cell of the sixth aspect is used for inhibiting or promoting angiogenesis, inhibiting Treg function, promoting T cell proliferation, triggering T cell mediated response, Use in drugs for improving the function of effector T cells, improving the function of memory T cells, inhibiting or promoting angiogenesis, treating age-related macular degeneration (AMD), and/or effectively inhibiting tumor growth.

In the eighth aspect, the present application provides the antibody specifically binding to B7H3 or its antigen-binding portion described in the first aspect, the pharmaceutical composition described in the second aspect, the vaccine described in the third aspect, the vaccine described in the third aspect, Use of the nucleotide molecule of the fourth aspect, the expression vector of the fifth aspect or the host cell of the sixth aspect in the preparation of a medicament for preventing and/or treating B7H3-related diseases.

In the ninth aspect, the present application provides a detection reagent or kit, which comprises the antibody or antigen-binding portion thereof that specifically binds to B7H3 described in the first aspect.

In a tenth aspect, the present application provides a method for preventing and/or treating a B7H3-related disease, which comprises administering the antibody or antigen-binding portion thereof specifically binding to B7H3 described in the first aspect, the second aspect, to an individual in need The pharmaceutical composition of the third aspect, the vaccine of the third aspect, the nucleotide molecule of the fourth aspect, the expression vector of the fifth aspect or the host cell of the sixth aspect.

In some embodiments, the method further includes coadministering other therapeutic agents, such as chemotherapeutic agents, PD-1 binding antagonists, 4-1BB binding agonists, and the like.

The antibody specifically binding to B7H3 or its antigen-binding portion of the present application can specifically bind to B7H3, and has one or more of the following effects: it has the function of a B7H3 inhibitor, stimulates the proliferation and activation of T cells, and induces B7H3-mediated Anti-tumor immune response, inhibit or promote angiogenesis, treat eye diseases such as age-related macular degeneration (AMD), and/or inhibit tumor growth, etc.

Brief description of the drawings

Figure 1 shows the experimental results of ELISA detection of the combination of hybridoma supernatant and human 4IgB7H3-his after the cell fusion experiment, in which wells 1C and 7A are negative controls, wells 4H and 5H are positive controls, and bold fonts are screened out positive hole.

Figure 2-1 shows the experimental results of ELISA detection of the combination of hybridoma supernatant and mouse mB7H3-his, in which the wells of 12A, 12B, 12C and 12D are negative controls, and the wells of 3F, 3H, 7C, 7G, 8F, 12G and 12H Wells are positive controls, underlined wells are screened positive wells.

Figure 2-2 shows the clone numbers corresponding to the ELISA test results in Figure 2-1, where the 8 wells marked are the clone numbers corresponding to the positive wells screened out in Figure 2-1.

Fig. 3 shows the binding experiment of three positive hybridoma antibodies 2#, 48#, and 248# screened by three ELISA strains and 293T cells by flow cytometry.

Fig. 4 shows the binding experiment of 7 subcloned antibodies including A4, A5, B8, D2, D4, G7 and G9 of clone 384# detected by flow cytometry and 293T cells.

Figure 5 shows the binding experiments of different concentrations of B7H3 chimeric antibody B7H3-Chi and HCC827 cells detected by flow cytometry.

Figure 6 shows the binding experiments of B7H3 humanized antibodies (B7H3-1, B7H3-2, B7H3-3) and chimeric antibody B7H3-Chi to CT26-B7H3 cells detected by flow cytometry.

Figure 7 shows the detection of the binding of B7H3-1 to HCC827 cells by laser confocal microscopy (magnification 40X).

Detailed description of the invention

The following definitions and methods are provided to better define this application and to guide those of ordinary skill in the art in the practice of this application. Unless otherwise specified, terms in this application are to be understood according to conventional usage by those of ordinary skill in the relevant art.

definition

To facilitate understanding of this application, certain terms used herein are first defined.

As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof ( such as IgM). Each heavy chain is comprised of a heavy chain variable region (abbreviated VH) and a heavy chain constant region (abbreviated CH). The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated VL) and a light chain constant region (abbreviated CL). The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with conserved regions called framework regions (FRs). In some embodiments, from N-terminus to C-terminus, both the light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

As used herein, the term "antigen-binding portion" of an antibody refers to a portion or segment of an intact antibody molecule that is responsible for binding antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Antigen-binding fragments of antibodies can be prepared from intact antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, among others. Non-limiting examples of antigen binding portions include: Fab fragments, F(ab') ₂ fragments, Fd fragments, Fv fragments, single chain Fv (scFv) molecules, single domain antibodies, dAb fragments, and amino acids derived from hypervariable regions of mimicking antibodies. The smallest unit of recognition composed of residues (eg, isolated CDRs). The term "antigen-binding moiety" also includes other engineered molecules such as diabodies, triabodies, tetrabodies, and minibodies. For example, the Fd fragment described herein refers to an antibody fragment composed of VH and CH1 domains; Fv fragment consists of VL and VH domains in a single arm of an antibody; dAb fragment (Ward et al., Nature 1989; 341:544-546) Consists of VH domains.

It is well known to those skilled in the art that complementarity determining regions (CDRs, usually CDR1, CDR2 and CDR3) are regions in the variable region that have the greatest influence on the affinity and specificity of antibodies. There are two common ways to define the CDR sequence of VH or VL, namely Kabat definition and Chothia definition, for example, see Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, MD. (1991); Al- Lazikani et al., J Mol Biol 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). For the variable region sequence of a given antibody, the CDR region sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In an embodiment of the present application, Kabat is used to define CDR sequences. Herein, CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively; CDR1, CDR2 and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2 and LCDR3, respectively.

For the variable region sequence of a given antibody, the CDR region sequence in the variable region sequence can be analyzed in various ways, for example, it can be determined using the online software Abysis (http://www.abysis.org/).

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope, such as an antibody with an affinity at least two times greater than its affinity for a non-specific antigen. The ability to bind to specific antigens with double the affinity. It is to be understood, however, that an antibody is capable of specifically binding to two or more antigens related to its sequence. For example, the antibodies of the present application can specifically bind to human and non-human (eg, mouse or non-human primate) B7H3.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for the possibility of naturally occurring mutations in a small number of individuals. The monoclonal antibodies described herein specifically include "chimeric" antibodies, in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the heavy chain and/or the remainder of the light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, so long as they exhibit the desired (See, US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

As used herein, the term "murine antibody" refers to any antibody in which all constant domain sequences are mouse sequences. Such antibodies can be produced by hybridomas.

As used herein, the term "chimeric antibody" refers to an antibody comprising segments from two or more different antibodies. In some embodiments, one or more CDRs are derived from a mouse anti-B7H3 antibody. In other embodiments, all CDRs are derived from mouse anti-B7H3 antibodies. In some embodiments, CDRs from more than one mouse anti-B7H3 antibody are combined in a chimeric antibody. For example, a chimeric antibody can comprise CDR1 from a light chain in a first mouse anti-B7H3 antibody, CDR2 from a light chain in a second mouse anti-B7H3 antibody, and a combination of CDR1 from a light chain in a third mouse anti-B7H3 antibody. The CDR3 of , and the CDRs from the heavy chain can be derived from one or more other anti-B7H3 antibodies. Furthermore, the framework regions can be from the same anti-B7H3 antibody or from one or more different individuals. The chimeric antibody of the present application may comprise variable regions of murine antibodies (including heavy chain variable region VH and/or light chain variable region VL) and constant regions of human antibodies.

As used herein, the term "humanized antibody" refers to a CDR-grafted antibody, specifically an antibody produced by grafting a mouse CDR region sequence into a human antibody variable region framework. The purpose is to overcome the strong immune side effects induced by chimeric antibodies in humans due to carrying a large number of protein components from other species such as mice.

As used herein, the term "nucleotide molecule" can refer to DNA molecules as well as RNA molecules, which can be single-stranded or double-stranded. Nucleotide molecules can also be cDNA.

As used herein, the term "B7H3-associated disease" includes diseases and/or disorders associated with the B7H3 signaling pathway. Exemplary B7H3-related diseases or conditions include cancers, such as colon cancer, melanoma, mesothelioma, renal cell carcinoma, lymphoma, advanced solid tumors and the above-mentioned metastatic tumors, and ocular lesions such as AMD.

As used herein, the term "EC50" refers to the concentration for 50% of maximal effect (EC50), which refers to the concentration that can cause 50% of the maximal effect.

As used herein, the term "immune response" refers to a biological response in a vertebrate to a foreign agent that protects the organism against such agent and the disease caused thereby. The immune response is composed of cells of the immune system (eg, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and by Either of these cells or mediated by the action of soluble macromolecules produced by the liver, including antibodies, cytokines, and complement, which result in selective targeting, binding, damage, destruction, and/or elimination from the vertebrate body Invasion of pathogens, pathogen-infected cells or tissues, cancerous or otherwise abnormal cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation. The immune response includes the activation or suppression of T cells (eg effector T cells) or Th cells (eg CD4 ⁺ or CD8 ⁺ T cells) or suppression of Treg cells.

As used herein, the term "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Dysregulated cell division can form malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream.

As used herein, the term "treatment" refers to any type of intervention or method performed on a subject or the administration of an active agent thereto for the purpose of reversing, alleviating, ameliorating, inhibiting or alleviating or preventing a symptom, complication, condition or Disease-related progression, development, severity, or recurrence.

As used herein, the term "prophylaxis" refers to administration to a subject who does not have a disease in order to prevent the occurrence or minimize the effects, if any, of the disease.

As used herein, the term "angiogenesis" refers to the process of neovascularization and vascular maturation in a subject's normal body tissue or tumor microenvironment.

As used herein, the term "eye disease" refers to age-related macular degeneration (AMD), wet phase of age-related macular degeneration/wet macular degeneration (wAMD), diabetic macular edema (DME), choroidal neovascularization (CNV), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), pathological myopia (PM) and one or more diseases.

Detailed ways

The present application provides novel anti-B7H3 antibodies or antigen-binding portions thereof that specifically bind B7H3. The antibody of the present application or its antigen-binding part can bind to the B7H3 molecule on the surface of the target cell, and can bind to two molecular structures of 2IgB7H3 and 4IgB7H3.

The present inventors screened and obtained some hybridoma cells, and the antibodies in the supernatants could bind to mouse B7H3 and human or monkey 2IgB7H3 and 4IgB7H3 molecular structures.

The present inventors have also prepared chimeric and humanized antibody forms from mouse-derived anti-B7H3 antibodies by means of genetic engineering. These antibodies can also bind to human or monkey B7H3, and can block the binding of ligands to B7H3, thereby Effectively induce the immune response mediated by B7H3, and play a role in preventing or treating B7H3-related diseases.

The present application also provides a nucleotide molecule encoding the antibody or its antigen-binding fragment, an expression vector comprising the nucleotide molecule, a host cell comprising the nucleotide molecule or expression vector, and methods for preparing and purifying the antibody. Methods and medical and biological applications of the antibodies or antigen-binding fragments thereof, such as the prevention or treatment of B7H3-associated diseases or disorders. The present application also covers methods for detecting B7H3 and regulating B7H3 activity using the antibody or antigen-binding fragment thereof, as well as related detection reagents or kits.

Suitable techniques that may additionally be used in the antibody method include B7H3-based affinity purification, native gel purification, HPLC or RP-HPLC, purification on a protein A column, or any combination of these techniques. B7H3 antibody isotypes can be determined using ELISA assays, for example human Ig can be identified using mouse Ig adsorbed anti-human Ig.

B7H3 suitable for antibody production can be produced by any of a number of standard protein purification or recombinant expression techniques known in the art. Forms of B7H3 suitable for generating an immune response include B7H3 subsequences (eg, immunogenic fragments). Additional forms of B7H3 include B7H3 expressing cells, B7H3 containing preparations or cell extracts or fractions, partially purified B7H3.

In the first aspect, the present application provides an antibody specifically binding to B7H3 or an antigen-binding portion thereof, which comprises a heavy chain variable region comprising any one or more of HCDR1, HCDR2 and HCDR3 sequences , wherein the HCDR1 sequence comprises GDSITSNY (SEQ ID NO: 1); the HCDR2 sequence comprises ISNSGST (SEQ ID NO: 2); and the HCDR3 sequence comprises ARGEGRYGFGAY (SEQ ID NO: 3).

In a preferred embodiment, the heavy chain variable region of the antibody or antigen-binding portion thereof described herein comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR2 shown in SEQ ID NO: 3 HCDR3 sequence.

In some embodiments, an antibody or antigen-binding portion thereof described herein further comprises a light chain variable region comprising any one or more of LCDR1, LCDR2, and LCDR3 sequences, wherein said The LCDR1 sequence comprises KSLLHGNGNTY (SEQ ID NO: 4); the LCDR2 sequence comprises RMS (SEQ ID NO: 5); and the LCDR3 sequence comprises MQHLEYPFT (SEQ ID NO: 6).

In a preferred embodiment, the light chain variable region of the antibody or antigen-binding portion thereof described herein comprises LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR2 shown in SEQ ID NO: 6 LCDR3 sequence.

In a preferred embodiment, an antibody described herein, or an antigen-binding portion thereof, comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, SEQ ID NO: 1 HCDR2 shown in ID NO: 2 and HCDR3 sequence shown in SEQ ID NO: 3, and the light chain variable region includes LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, SEQ ID NO: LCDR3 sequence shown in 6.

In some more specific embodiments, the antibodies disclosed herein may be anti-human B7H3 monoclonal antibodies. Anti-B7H3 antibody type and subtype can be determined by any means known in the art. Typically, antibody classes and subtypes can be determined using antibodies specific for particular antibody classes and subtypes. Anti-B7H3 antibody isotypes can be determined using ELISA assays, for example human Ig can be identified using mouse Ig adsorbed anti-human Ig.

In some embodiments, the antibody described herein is a murine antibody, preferably, the heavy chain variable region of the murine antibody comprises the amino acid sequence of SEQ ID NO: 7, and/or the light chain of the murine antibody The variable region comprises an amino acid sequence such as SEQ ID NO:8.

In a preferred embodiment, the murine antibody described herein comprises a heavy chain variable region represented by SEQ ID NO:7 and a light chain variable region represented by SEQ ID NO:8.

In some embodiments, the antibodies described herein are chimeric antibodies. The chimeric antibodies described herein comprise the variable region of a murine antibody (including the heavy chain variable region VH and/or the light chain variable region VL) and the constant region of a human antibody.

In preferred embodiments, the chimeric antibodies described herein comprise the variable regions of murine antibodies (including heavy chain variable regions and light chain variable regions) and the constant regions of human antibodies.

Preferably, the chimeric antibody comprises the heavy chain shown in SEQ ID NO:9, and/or the light chain shown in SEQ ID NO:10.

In a preferred embodiment, the chimeric antibody described herein comprises a heavy chain set forth in SEQ ID NO:9 and a light chain set forth in SEQ ID NO:10.

In some embodiments, the antibodies described herein are humanized antibodies. The humanized antibodies described herein comprise the CDR regions of murine antibodies (including any one or more of HCDR1, HCDR2 and HCDR3 and/or any one or more of LCDR1, LCDR2 and LCDR3), human antibody The framework regions of the variable regions (including any one or more of FR1, FR2, FR3 and FR4), and optionally the constant regions of a human antibody.

In a preferred embodiment, the humanized antibodies described herein comprise the CDR regions of murine antibodies (including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3), the framework regions of human antibody variable regions (including FR1, FR2 , FR3 and FR4), and optionally the constant region of a human antibody.

Preferably, the amino acid sequence of the heavy chain variable region of the humanized antibody is shown in SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, and/or the light chain of the humanized antibody is variable The amino acid sequence of the region is SEQ ID NO:14 or SEQ ID NO:15.

In a preferred embodiment, the humanized antibody described herein comprises the heavy chain variable region shown in SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 and SEQ ID NO: 14 or SEQ ID NO: The variable region of the light chain shown in 15.

The antibodies described herein may also comprise murine or human antibody constant regions. The murine antibody constant region includes the heavy chain constant region of murine IgG1, IgG2a, IgG2b or IgG3 and the light chain constant region of the κ or λ type, etc. The human antibody constant region includes the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and the light chain constant region of κ or λ type, etc.

In some embodiments, the B7H3 described herein is a rodent B7H3. Preferably, the rodent B7H3 described herein is selected from mouse or rat B7H3.

In some embodiments, the B7H3 described herein is a primate B7H3. Preferably, the primate B7H3 described herein is selected from human B7H3 or monkey B7H3.

In some embodiments, the antigen binding moieties described herein are selected from the group consisting of: Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fv fragments, scFv fragments, Fd fragments, or single domain antibodies.

The term "Fab fragment" as used herein comprises the light chain as well as the CH1 and variable regions of the heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The term "Fab'fragment" as used herein contains the light chain as well as a part or fragment of the heavy chain containing the VH domain and the CH1 domain and the region between the CH1 and CH2 domains such that in two Fab An interchain disulfide bond can be formed between the two heavy chains of the 'fragment to form an F(ab') ₂ molecule.

The term "F(ab') ₂ fragment" as used herein contains two light chains and two heavy chains containing part of the constant region between the CH1 and CH2 domains such that between the two heavy chains form interchain disulfide bonds. The F(ab') ₂ fragment thus consists of two Fab' fragments linked together by a disulfide bond between the two heavy chains.

The term "Fv fragment" as used herein comprises the variable regions from the heavy and light chains, but lacks the constant regions.

The term "single-chain Fv" or "scFv" as used herein refers to an antibody fragment comprising the VH domain and VL domain of an antibody, wherein these domains exist as a single polypeptide chain. Typically, the Fv polypeptide also comprises a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding.

The term "single domain antibody" as used herein refers to an antigen-binding portion comprising a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. It was originally found in the peripheral blood of alpacas as an antibody that naturally lacks light chains. Although this antibody only contains a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, it is not like the artificially modified Single-chain antibody fragments (scFv) are as easy to stick to each other and even aggregate into blocks. More importantly, the VHH structure cloned and expressed separately has the same structural stability and antigen-binding activity as the original heavy chain antibody, and is the smallest known unit that can bind the target antigen. VHH crystal molecular weight is only 15KDa, so it is also called nanobody (Nanobody, Nb).

In the second aspect, the present application provides a pharmaceutical composition, which comprises the antibody or antigen-binding portion thereof described in the first aspect and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its Derivatives such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; ( 7) Lubricants such as magnesium stearate, sodium lauryl sulfate, and talc; (8) Excipients such as cocoa butter and suppository waxes; (9) Oils such as peanut oil, cottonseed oil, safflower oil, sesame oil , olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as oil (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; ( 18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solution; (21) polyester, polycarbonate and/or polyanhydride; (22) fillers such as polypeptides and amino acids; (23) Serum components such as serum albumin, HDL and LDL; (22) _C2 - _C12 alcohols such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical preparations.

In some embodiments, the pharmaceutical compositions described herein further comprise one or more other active ingredients, such as agents for treating B7H3-related diseases such as tumors.

In a third aspect, the present application provides a vaccine comprising the antibody or antigen-binding portion thereof described in the first aspect, and optionally an immune adjuvant.

Generally, the vaccines are prepared as injectables, eg, as liquid solutions or suspensions, or in solid forms suitable for resuspension in liquid prior to injection. The preparation may also be emulsified. The active immunogenic component is usually mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, etc. or combinations thereof. In addition, the vaccine may also contain a small amount of auxiliary materials as required, such as wetting agents or emulsifying agents, pH buffering agents or adjuvants to improve the effect of the vaccine.

A vaccine is administered in a manner compatible with its dosage form, and in an amount that is therapeutically effective and immunogenic. The amount administered depends on the subject being treated, including, for example, the ability of the individual's immune system to mount an immune response, the route of administration, etc., and the specific amount is at the discretion of the physician. Applicable treatment regimens may also vary for initial administration and booster injections. In some embodiments, the vaccine is delivered intravenously, or directly to the tumor or site of infection, or other traditional methods of vaccine administration.

In a fourth aspect, the present application provides a nucleotide molecule encoding the antibody or antigen-binding portion thereof of the first aspect.

In preferred embodiments, the nucleotide molecules described herein may be codon-optimized nucleotide molecules suitable for expression in a host cell. For example according to the degeneracy of the codons it still encodes the same protein. Methods for codon optimization depending on the host cell used are well known to those skilled in the art.

In the fifth aspect, the present application provides an expression vector comprising the nucleotide molecule described in the third aspect.

Any suitable expression vector can be used. For example, prokaryotic cloning vectors include plasmids from E. coli such as colE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNA such as M13 and other filamentous single-stranded DNA phages. An example of a vector that can be used in yeast is the 2μ plasmid. Suitable vectors for expression in mammalian cells include the following well-known derivatives: SV-40, adenovirus, retrovirus-derived DNA sequences as well as those derived from functional mammalian vectors (such as those described above) and functional plasmids and Combinatorial shuttle vectors for phage DNA.

Additional eukaryotic expression vectors are known in the art (e.g., P J. Southern & P. Berg, J. Mol. Appl. Genet, 1:327-341 (1982); Subramani et al., Mol. Cell. Biol, 1 : 854-864 (1981); Kaufinann & Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene," J.Mol.Biol, 159:601-621 (1982); Kaufhiann & Sharp, Mol.Cell.Biol, 159 : 601-664 (1982); Scahill et al., "Expression And Characterization Of The Product Of A Human Immune InterferonDNA Gene In Chinese Hamster Ovary Cells," Proc. Nat'l Acad. Sci USA, 80: 4654-4659 (1983) ; Urlaub & Chasin, Proc. Nat'l Acad. Sci USA, 77:4216-4220, (1980), which is hereby incorporated by reference in its entirety).

Expression vectors useful in this application contain at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. Control sequences are inserted into the vector to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, the major operator and promoter region of bacteriophage lambda, the control region of the fd coat protein, the glycolytic promoter of yeast, e.g. 3-phosphate Promoters for glycerate kinase, yeast acid phosphatase, e.g. Pho5, yeast alpha-mating factor, and promoters from polyoma, adenovirus, retrovirus, and simian virus, e.g., SV40 Early and late promoters and other sequences known to control gene expression in prokaryotic or eukaryotic cells and their viruses or combinations thereof.

In the sixth aspect, the present application provides a host cell comprising the nucleotide molecule described in the fourth aspect or the expression vector described in the fifth aspect.

In some embodiments, the host cells described herein are mammalian cells. Mammalian cells may include, but are not limited to, CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, and PER.C6 cells. Those skilled in the art can select suitable host cells as needed.

The preparation method of the anti-B7H3 monoclonal antibody disclosed herein may include: culturing host cells under expression conditions, thereby expressing the anti-B7H3 monoclonal antibody; isolating and purifying the expressed anti-B7H3 monoclonal antibody. Using the above method, a crude anti-B7H3 monoclonal antibody can be obtained. Anti-B7H3 monoclonal antibodies are then purified by purification methods including B7H3-based affinity purification, native gel purification, HPLC or RP-HPLC, size exclusion, purification on protein A columns, or any combination of these techniques It is a substantially uniform substance, such as a single band on SDS-PAGE electrophoresis.

In the seventh aspect, the present application provides the antibody specifically binding to B7H3 or its antigen-binding portion described in the first aspect, the pharmaceutical composition described in the second aspect, the vaccine described in the third aspect, and the vaccine described in the fourth aspect. The nucleotide molecule of the fifth aspect, the expression vector of the fifth aspect or the host cell of the sixth aspect is used for inhibiting or promoting angiogenesis, inhibiting Treg function, promoting T cell proliferation, triggering T cell mediated response, Use in drugs for improving the function of effector T cells, improving the function of memory T cells, inhibiting or promoting angiogenesis, treating age-related macular degeneration (AMD), and/or effectively inhibiting tumor growth.

In the eighth aspect, the present application provides the antibody specifically binding to B7H3 or its antigen-binding portion described in the first aspect, the pharmaceutical composition described in the second aspect, the vaccine described in the third aspect, the vaccine described in the third aspect, Use of the nucleotide molecule of the fourth aspect, the expression vector of the fifth aspect or the host cell of the sixth aspect in the preparation of a medicament for preventing and/or treating B7H3-related diseases.

In the ninth aspect, the present application provides a detection reagent or kit, which comprises the antibody or antigen-binding portion thereof that specifically binds to B7H3 described in the first aspect.

An antibody, or antigen-binding portion thereof, described herein can be bound to a detectable moiety. Exemplary detectable moieties include, but are not limited to, radioactive isotopes such as iodine-125, iodine-131, cesium-137, iridium-192, and cobalt-60, horseradish peroxidase, fluorescein isothiocyanate, biotin, alkaline phosphate Enzymes, chemiluminescent agents such as luminol, etc. Those skilled in the art can select a suitable detectable moiety to bind to the antibody of the present application or its antigen-binding portion according to needs, so as to achieve different detection purposes.

In a tenth aspect, the present application provides a method for preventing and/or treating a B7H3-related disease, which comprises administering the antibody or antigen-binding portion thereof specifically binding to B7H3 described in the first aspect, the second aspect, to an individual in need The pharmaceutical composition of the third aspect, the vaccine of the third aspect, the nucleotide molecule of the fourth aspect, the expression vector of the fifth aspect or the host cell of the sixth aspect.

In some embodiments, the method further comprises administering a second agent for preventing and/or treating a B7H3-associated disease such as a tumor.

The term "individual" as used herein refers to mammals, including but not limited to primates, cows, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. Preferably, the mammal is a non-human primate or a human. A particularly preferred mammal is a human. As used herein, "individual" and "subject" are used interchangeably.

"Treatment" refers to both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent or slow down (lessen) the targeted pathological state or disorder. Those in need of treatment include those already with the disorder as well as those who will develop the disorder or those in which the disorder is to be prevented. Accordingly, the individual to be treated herein has been diagnosed with the disorder or is predisposed or susceptible to the disorder.

The "therapeutically effective dose" used herein can be determined according to specific conditions, and those of ordinary skill in the art can easily grasp according to the actual required dose, for example, it can be determined according to the patient's weight, age and disease condition.

In any one embodiment of the eighth and tenth aspects, the B7H3-related disease is a tumor.

In any one embodiment of the eighth and tenth aspects, the B7H3-related disease is an eye disease.

In some embodiments, a tumor described herein is a primary cancer or a metastatic cancer. In specific embodiments, the tumor is selected from lung cancer such as non-small cell lung cancer, colorectal cancer, bladder cancer, hematopoietic cancer such as leukemia, breast cancer, gastric cancer, esophageal cancer, B-lymphocyte non-Hodgkin's lymphoma, Hodgkin's lymphoma, Chickkin's Lymphoma, Anaplastic Large Cell Lymphoma, Head and Neck Cancers such as Head and Neck Squamous Cell Carcinoma, Glioblastoma, Renal Cancer, Melanoma, Prostate Cancer, Bone Cancer, Giant Cell Tumor of Bone, Pancreatic Cancer, Ovarian Cancer , sarcoma, liver cancer, squamous cell carcinoma of the skin, thyroid cancer, cervical cancer, nasopharyngeal cancer, endometrial cancer, or metastatic cancer of the above tumors.

In some embodiments, angiogenesis as described herein refers to the process of neovascularization and vascular maturation in a subject's normal body tissue or tumor microenvironment.

In some embodiments, the eye diseases described herein refer to age-related macular degeneration (AMD), wet phase of age-related macular degeneration/wet macular degeneration (wAMD), diabetic macular edema (DME), choroidal neovascularization (CNV), retinal One or more diseases such as vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), pathological myopia (PM), etc.

In embodiments of either aspect, the methods, uses and pharmaceutical compositions described herein may further comprise administering to the individual a second agent and/or treatment, for example as part of a combination therapy. Non-limiting examples of second agents and/or treatments may include radiation therapy, surgery, gemcitabine, cilastatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, Rapamycin, chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine ; vindesine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate ); daunomycin; aminopterin; xeloda; PKC-α, Raf, H-Ras, EGFR (eg, erlotinib) that reduce cell proliferation ) and an inhibitor of VEGF-A and any pharmaceutically acceptable salt, acid or derivative thereof.

In the present description and claims, the words "comprising", "comprising" and "containing" mean "including but not limited to", and are not intended to exclude other parts, additives, components, or steps.

It should be understood that features, characteristics, components or steps described in one particular aspect, embodiment or example of the present application can be applied to any other aspect, embodiment or example described herein unless incompatible therewith.

The invention discloses an antibody specifically binding to B7H3 of mammals (humans, primates, etc.), and the invention provides applications of such proteins in treatment, screening and detection, such as in cancer treatment. Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included within the scope of the present invention. Those skilled in the art can implement and apply the present invention by making changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention. In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with examples.

Example

The following examples are used to illustrate the present application, but not to limit the scope of the present application. Without departing from the spirit and essence of the present application, any modifications or replacements made to the methods, steps or conditions of the present application belong to the scope of the present application.

Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, insert genes encoding proteins into vectors and plasmids, or introduce plasmids into host cells. Such methods are well known to those skilled in the art and are described in numerous publications, see for example Sambrook, J., Fritsch, EF. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, Cold spring Harbor Laboratory Press.

Example 1. Preparation of anti-B7H3 antibody hybridoma

The preparation method is as follows:

1. Take 6-8 week-old Balb/c mice and subcutaneously immunize them with 4IgB7H3-his (Beijing Baipusaisi Biotechnology Co., Ltd., Cat: B73 H52E7) protein (100 μg/dose/mouse) on the back. Immunization once every 2 weeks, a total of 3 times of immunization. The mice were injected with 4IgB7H3-his protein for 3 consecutive days before fusion.

2. Two days before the fusion, the intraperitoneal macrophages of common Kunming mice (KM) were taken as a feeder layer, inoculated in 20 96-well cell culture plates, and then cultured in a 37°C 5% CO ₂ cell incubator.

3. The lymphocytes from the spleen and lymph nodes of the immunized mice were fused with the non-secretory myeloma SP2/0 cell line, and the cells were added to a 96-well plate with a feeder layer in advance, and the fused cells were fused. HAT selection (Galfre and Milstein, Methods Enzymol 1981; 73:3-46).

4. Recovering a group of hybridoma cells all secreting anti-4IgB7H3 specific antibody. The primary screening is to use enzyme-linked immunoassay (ELISA) to determine the titer of anti-4IgB7H3 antibody secreted by the hybridoma.

Example 2. Construction of human 4IgB7H3 high expression cell line

Construction of human 4IgB7H3 high expression cell line

The constructed cell line with high expression of human 4IgB7H3 is a stably transformed CT26-B7H3 cell line (purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd., product name: CT26 CD276 (B7H3) (KC-1364), Lot No: 20190822) . The amino acid sequence of human 4IgB7H3 is shown in SEQ ID NO:16.

SEQ ID NO: 16: NP_001019907.1 CD276 antigen isoform a precursor [Homo sapiens]

Example 3. Screening of hybridoma antibodies

First, when the hybridoma cells were cultured for 7-10 days, the growth of the hybridoma clones in the 96-well cell culture plate was observed under a microscope, and the blank wells and the corresponding number of clones in the wells with long clones were marked on the plate. Then the murine antibody against 4IgB7H3 was screened by the following method.

Method 1: ELISA method to screen hybridoma clones producing affinity antibodies Prepare 4IgB7H3-his protein at a concentration of 1 μg/ml, coat 96-well ELISA plates with 100 μl/well, and detect the antibody titer of hybridomas.

Take 50 μl of hybridoma supernatant, add 50 μl of PBS, and detect the antibody titer in the supernatant by ELISA. The positive control is 1:1k diluted serum from pre-fusion mice. Figure 1 shows the ELISA results of antibodies secreted by hybridoma cells in one of the 96-well plates, wherein the wells of 1C and 7A are negative controls, the wells of 4H and 5H are positive controls, and the bold fonts are the screened positive wells.

The wells with OD450 greater than or close to the OD value of the positive control were selected as positive clones, and positive clones were screened out according to this rule, and further ELISA screening was performed on them.

Method 2: ELISA method to screen mouse cross-positive clones

Prepare mB7H3-his protein (Shenzhou Science and Technology Co., Ltd., Cat: 50973-M08H) at a concentration of 1 μg/ml, coat 96-well ELISA plate with 100 μl/well, and detect hybridoma cell supernatants screened by method 1.

Take 50 μl of hybridoma supernatant, add 50 μl of PBS, and detect the antibody titer in the supernatant by ELISA. The positive control is 1:1k diluted serum from pre-fusion mice. Figure 2-1 shows the results of one of the ELISA screens for cross-reactivity with mouse B7H3 protein. Wells 12A, 12B, 12C and 12D are negative controls, and wells 3F, 3H, 7C, 7G, 8F, 12G and 12H are negative controls. Positive control, underlined wells are screened positive wells. Figure 2-2 is the clone number corresponding to Figure 2-1, and the 8 wells marked are the clone numbers corresponding to the positive wells screened in Figure 2-1.

Select wells with OD450 greater than or close to the OD value of the positive control as positive clones, and screen out positive clones according to this rule, and use them for flow screening.

Method 3: Screening hybridoma clones producing affinity antibodies by flow cytometry

The B7H3 positive detection antibody used was anti-B7H3-Ab-APC (CD276 Ab APC Mouse mAb, Sino Biological Technology Co., Ltd., Cat: 11188-MM06-A); the secondary antibody used was anti-mouse IgG APC (Biolegend, Cat: 405308).

Collect 293T cells in a T75 cell culture flask, wash once with FACS buffer, resuspend and count with FACS buffer, and adjust the cell concentration to 2×10 ⁶ /ml. Add 293T cells to the flow tube at a volume of 50 μL/tube, then add 50 μL of the hybridoma supernatant of positive clones screened by ELISA to each well, and use anti-B7H3-Ab-APC as a positive control, and then incubate for 0.5 hours .

Add 3ml FACS buffer to each well to wash twice, add APC goat anti-mouse IgG Fc secondary antibody to the hybridoma supernatant sample, incubate for 1 hour, and then detect with flow cytometer.

The screening rule is that the Geom. average value of the flow cytometry index of the sample is significantly higher than the Geom. average value of the secondary antibody negative control.

Finally, after the screening of methods 1, 2, and 3, a total of 17 hybridoma cells with positive clones were obtained, and compared with the original wells, the statistics are as follows:

Table 1. Statistical table of positive clones obtained by hybridoma screening

Example 4. Subcloning Screening for Anti-B7H3 Antibodies

Almost none of the 17 positive clones obtained in Example 3 are single clones, so no matter which clone is selected as a candidate, subcloning is required to continue screening.

In order to optimize and narrow the range of positive hybridomas selected for subcloning, 17 strains of hybridomas were cultured in a 6-well cell culture plate until the supernatant turned yellow and the cells basically died. source hybridoma antibodies. Then detect the combination of hybridoma antibody and 293T cells, the method is shown in the third method of Example 3, and calculate EC50. Table 2 and Figure 3 show the EC50 detection results of the binding experiments of the three positive mouse antibodies selected at random.

Table 2. EC50 of positive hybridoma antibodies binding to 293T cells

The 348# clone was then selected for subclonal screening. According to 0.8 cells/well, a total of five 96-well plates were laid, and the medium was 10% FBS+1640. When the cell clones in the wells grew to more than 1/4, flow cytometry screening was carried out. The method was as in Example 3. method three. The test results are shown in Table 3 and Figure 4.

Table 3. EC50 results of 384# subcloned antibody binding experiment

Subsequently, the affinities of the supernatants of the seven subclones to 4IgB7H3-his were detected by Fortebio, and the results are shown in Table 4.

Example 5. Preparation of anti-B7H3 chimeric antibody and verification of binding activity

From Example 4, it can be found that all the subcloned antibodies of 384# clone have an affinity close to that of 4IgB7H3-his. Subsequently, RNA was extracted from the 7 subcloned cells, and the cDNA of the antibody was obtained by reverse transcription. Afterwards, the antibody primers designed by our laboratory were amplified and sequenced by PCR, and finally the amino acid sequences of the murine anti-B7H3 antibody encoding antibody variable regions VH and VL were obtained (the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 ). Then VH and VL are respectively constructed on the plasmid pQKX1 (hIgG1 CH) and pQKX2 (hIgG1 CL) vectors to obtain heavy chain expression vector pQK B7H3-CH (heavy chain sequence shown in SEQID NO: 9) and light chain expression vector pQK B7H3-CL (light chain sequence shown in SEQ ID NO: 10).

293Fv cells (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were diluted to 1.5×10 ⁶ cells/mL, with a final volume of 200 ml, and cultured on a shaker at 37°C for 24 hours. Plasmids pQKB7H3-CH and pQKB7H3-CL were diluted with fresh medium at 10% of the transfection volume, calculated according to the transfected cell volume, to a concentration of 1 μg/mL. Add 200 μl of 3 mg/mL PEI to the diluted plasmid according to 1/1000 of the cell volume, vortex and shake immediately for 10 seconds, and let it stand at room temperature for 15 minutes, add the plasmid/PEI mixture dropwise to the cell culture medium, and gently Gently shake the culture bottle, put it into a shaker for culture, collect the culture supernatant after 7 days, filter through a 0.4 μm filter membrane, use Mab sure Lx 5ml purification column to capture the antibody from the culture supernatant, set the flow rate to 3ml/min, use Equilibrate the purification column with 5 column volumes of 20mM PB+150mM Nacl, pH 7.4 balance solution, load the sample after the column is stable, choose 20mM PB+150mM Nacl, pH 7.4 for rinsing at the end of the rinsing, and use 50mM citric acid PH3 at the end of the rinsing .0 eluent was eluted, and collected with 1M Tris-Hcl, pH9.0 neutralized the eluted antibody (the amino acid sequences of its heavy chain and light chain variable regions are respectively as SEQ ID NO: 7 and SEQ ID NO: 8), named B7H3-Chi.

Binding Activity Assay:

Method 1: Fortebio detected the affinity between B7H3-Chi and 4IgB7H3-his and mB7H3-his, and the results are shown in Table 5. B7H3-Chi has good affinity with 4IgB7H3-his and mB7H3-his.

Method 2: Detection by flow cytometry

Collect HCC827 cells in a T75 cell culture flask (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), wash once with FACS buffer, resuspend and count with FACS buffer, adjust the cell concentration to 2×10 ⁶ cells/ml; antibody gradient dilution. Take 10 μL of the diluted antibodies of each concentration and add them to 100 μL of the above-mentioned adjusted cell samples, mix well, and incubate at room temperature for 1 hour.

Add 2ml FACS buffer to each well to wash twice, add APC anti-human IgG Fc secondary antibody (Biolegend, Cat409306), incubate for 1 hour, and perform flow cytometry detection. The results are shown in Table 6 and Figure 5. The results in Table 6 show that the chimeric antibody B7H3-Chi specifically binds to HCC827 cells.

Table 6. EC50 results of the binding experiment between B7H3-Chi and HCC827

Example 6. Preparation of anti-B7H3 humanized antibody and detection of binding experiments

1. Preparation of anti-B7H3 humanized antibody

The amino acid sequence of the VH of the anti-B7H3 humanized antibody is shown in SEQ ID NO: 11, 12 or 13. The amino acid sequence of the VL of the anti-B7H3 humanized antibody is shown in SEQ ID NO: 14 or 15.

The VH and VL coding nucleic acid sequences of the anti-B7H3 humanized antibody were constructed into vector plasmids pQKX1 and pQKX2 by total synthesis to obtain heavy chain expression vectors pQK B7H3-H1, pQKB7H3-H2, pQKB7H3-H3 and light chain expression vectors pQKB7H3-L1, pQKB7H3-L2.

The 293Fv cells were diluted to 1.5×10 ⁶ cells/mL, the final volume was 200ml, and cultured on a shaker at 37°C for 24h. The above 8 plasmids were diluted with fresh medium with 10% transfection volume, calculated according to the volume of transfected cells, to a concentration of 1 μg/mL. Add 200 μl of 3 mg/mL PEI to the diluted plasmid according to 1/1000 of the cell volume, vortex and shake immediately for 10 seconds, and let it stand at room temperature for 15 minutes, add the plasmid/PEI mixture dropwise to the cell culture medium, and gently Gently shake the culture bottle, put it into a shaker for culture, collect the culture supernatant after 7 days, filter through a 0.4 μm filter membrane, use Mab sure Lx 5ml purification column to capture the antibody from the culture supernatant, set the flow rate to 3ml/min, use Equilibrate the purification column with 5 column volumes of 20mM PB+150mM Nacl, pH 7.4 equilibrium liquid, load the sample after the column is stable, choose 20mM PB+150mM Nacl, pH 7.4 for rinsing at the end of the rinsing, and use 50mM citric acid at the end of the rinsing The pH 3.0 eluent was eluted, and the antibodies neutralized and eluted with 1M Tris-Hcl, pH 9.0 were collected and named as B7H3-1, B7H3-2, and B7H3-3, as shown in Table 7.

Table 7. Heavy chain variable region and light chain variable region of anti-B7H3 humanized antibody

2. Binding experiment of anti-B7H3 humanized antibody

Method 1: Fortebio detects the binding of anti-B7H3 humanized antibody to 2IgB7H3-his (Beijing Baipusaisi Biotechnology Co., Ltd., Cat: B73 H52E2) and mB7H3-his.

The detection results are shown in Table 8. The humanized antibodies B7H3-1, B7H3-2, and B7H3-3 all bind to human and mouse B7H3.

Method 2: B7H3 humanized antibody and chimeric antibody B7H3-Chi binding experiment to CT26-B7H3 cells detected by flow cytometry

For the method, refer to Method 2 of Example 5, and the result is shown in FIG. 6 .

Example 7. Laser Confocal Microscopy Detection of the Binding of B7H3 Humanized Antibody to Tumor Cells

HCC827 cells were taken and diluted according to 2e4 cells/mL after trypsinization. Spread the circular glass slides in a 24-well plate, slowly add the cell suspension into the 24-well plate at 1 mL per well, and place the culture plate in a 37°C/5% CO ₂ incubator for overnight culture until the cells are fully attached. wall.

The next day, take the slides of the cell slides in the culture plate, wash them three times in PBS, and then add PKH67 (Sigma, MKCN7808) at a volume of 150 μL per slide for cell membrane staining: first incubate at 37°C for 5 minutes, then place Incubate at 4°C for 15 minutes. After the staining was finished, the staining was terminated with serum-supplemented medium and washed three times in PBS. PE (Sigma, GR3392101-1) directly labeled B7H3 humanized antibody (B7H3-1, concentration: 50 μg/mL) was added dropwise on the slide, first placed at 4 °C for 30 min, and then incubated at 37 °C for 2 h.

After the antibody incubation time point, slides were washed three times in PBS. The slides were fixed in 4% paraformaldehyde for 20 min. After washing three times in PBS, stain with DAPI for 3-5 minutes; after washing three times in PBS, take the mounting medium and place it on a glass slide for mounting.

Wait for the mounting medium to dry at room temperature, and finally perform laser confocal fluorescence photography to observe the localization of the antibody in the cells. The result is shown in Figure 7.

It can be understood that although the present application describes the related inventions in the above specific forms, these inventions are not limited to the specific content described in these specific forms. It is obvious to those skilled in the art that without departing from the spirit of the invention described in this application, various equivalent changes can also be made to the technical features contained in the invention involved, and these changes should all belong to the invention within range.

sequence listing

<110> Beijing Immunity Ark Pharmaceutical Technology Co., Ltd.

<120> Anti-B7H3 antibody and application thereof

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 1

Gly Asp Ser Ile Thr Ser Asn Tyr

1 5

<210> 2

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 2

Ile Ser Asn Ser Gly Ser Thr

1 5

<210> 3

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 3

Ala Arg Gly Glu Gly Arg Tyr Gly Phe Gly Ala Tyr

1 5 10

<210> 4

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 4

Lys Ser Leu Leu His Gly Asn Gly Asn Thr Tyr

1 5 10

<210> 5

<211> 3

<212> PRT

<213> Artificial Sequence

<400> 5

Arg Met Ser

1

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 6

Met Gln His Leu Glu Tyr Pro Phe Thr

1 5

<210> 7

<211> 118

<212> PRT

<213> Artificial Sequence

<400> 7

Gln Val Gln Leu Lys Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Asn

20 25 30

Tyr Trp Asn Trp Leu Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met

35 40 45

Gly Tyr Ile Ser Asn Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu

65 70 75 80

Gln Leu Ile Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Gly Arg Tyr Gly Phe Gly Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 8

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Asp Ile Val Met Thr Gln Ala Ala Pro Ser Ile Pro Val Ala Pro Gly

1 5 10 15

Ala Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Gly

20 25 30

Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Leu Arg Pro Ser Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 9

<211> 448

<212> PRT

<213> Artificial Sequence

<400> 9

Gln Val Gln Leu Lys Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Asn

20 25 30

Tyr Trp Asn Trp Leu Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met

35 40 45

Gly Tyr Ile Ser Asn Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu

65 70 75 80

Gln Leu Ile Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Gly Arg Tyr Gly Phe Gly Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 10

<211> 219

<212> PRT

<213> Artificial Sequence

<400> 10

Asp Ile Val Met Thr Gln Ala Ala Pro Ser Ile Pro Val Ala Pro Gly

1 5 10 15

Ala Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Gly

20 25 30

Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Leu Arg Pro Ser Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 11

<211> 118

<212> PRT

<213> Artificial Sequence

<400> 11

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Asn

20 25 30

Tyr Trp Asn Trp Leu Arg Gln Pro Pro Gly Asn Lys Leu Glu Tyr Met

35 40 45

Gly Tyr Ile Ser Asn Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Gly Arg Tyr Gly Phe Gly Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 12

<211> 118

<212> PRT

<213> Artificial Sequence

<400> 12

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Asn

20 25 30

Tyr Trp Asn Trp Leu Arg Gln Pro Pro Gly Asn Lys Leu Glu Tyr Met

35 40 45

Gly Tyr Ile Ser Asn Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Lys

50 55 60

Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Gly Arg Tyr Gly Phe Gly Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 13

<211> 118

<212> PRT

<213> Artificial Sequence

<400> 13

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Asn

20 25 30

Tyr Trp Asn Trp Leu Arg Gln Pro Pro Gly Asn Lys Leu Glu Tyr Met

35 40 45

Gly Tyr Ile Ser Asn Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Gly Arg Tyr Gly Phe Gly Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 14

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 14

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Thr Cys Arg Ser Ser Lys Ser Leu Leu His Gly

20 25 30

Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 15

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 15

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Thr Cys Arg Ser Ser Lys Ser Leu Leu His Gly

20 25 30

Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 16

<211> 534

<212> PRT

<213> Homo sapiens (homo sapiens)

<400> 16

Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala

1 5 10 15

Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln

20 25 30

Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu

35 40 45

Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn

50 55 60

Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala

65 70 75 80

Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe

85 90 95

Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val

100 105 110

Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp

115 120 125

Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys

130 135 140

Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr

145 150 155 160

Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val

165 170 175

Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr

180 185 190

Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Ile Leu

195 200 205

Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn

210 215 220

Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Ile Thr Pro Gln

225 230 235 240

Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val

245 250 255

Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro

260 265 270

Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr

275 280 285

Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly

290 295 300

Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln

305 310 315 320

Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly

325 330 335

Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val

340 345 350

Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu

355 360 365

Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser

370 375 380

Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln

385 390 395 400

Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu

405 410 415

Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu Gly Ala

420 425 430

Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp

435 440 445

Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro

450 455 460

Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu

465 470 475 480

Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys

485 490 495

Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly

500 505 510

Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu Asp

515 520 525

Asp Gly Gln Glu Ile Ala

530

Claims (15)

1. An antibody or antigen-binding portion thereof that specifically binds B7H3 comprising a heavy chain variable region comprising any one or more of HCDR1, HCDR2 and HCDR3 sequences wherein the HCDR1 sequence comprises the amino acid sequence GDSITSNY (SEQ ID NO: 1); the HCDR2 sequence comprises the amino acid sequence ISNSGST (SEQ ID NO: 2); and the HCDR3 sequence comprises amino acid sequence ARGEGRYGFGAY (SEQ ID NO: 3), optionally the antigen binding portion is selected from the group consisting of: fab fragment, fab 'fragment, F (ab') ₂ Fragments, fv fragments, scFv fragments, fd fragments or single domain antibodies.

2. The antibody or antigen-binding portion thereof of claim 1, further comprising a light chain variable region comprising any one or more of LCDR1, LCDR2, and LCDR3 sequences wherein the LCDR1 sequence comprises amino acid sequence KSLLHGNGNTY (SEQ ID NO: 4); the LCDR2 sequence comprises the amino acid sequence RMS (SEQ ID NO: 5); and the LCDR3 sequence comprises amino acid sequence MQHLEYPFT (SEQ ID NO: 6).

3. The antibody or antigen binding portion thereof of claim 1 or 2, wherein the antibody is a murine antibody, optionally the heavy chain variable region comprises amino acid sequence SEQ ID No. 7, and/or the light chain variable region comprises amino acid sequence SEQ ID No. 8, optionally wherein the antibody is a chimeric antibody, optionally the chimeric antibody comprises a heavy chain and/or a light chain, the heavy chain comprises amino acid sequence SEQ ID No. 9, and the light chain comprises amino acid sequence SEQ ID No. 10, optionally wherein the antibody is a humanized antibody, optionally the heavy chain variable region comprises amino acid sequence SEQ ID No. 11, SEQ ID No. 12 or SEQ ID No. 13, and/or the light chain variable region comprises amino acid sequences SEQ ID No. 14, SEQ ID No. 15.

4. The antibody or antigen-binding portion thereof of any one of claims 1 to 3, wherein the antibody or antigen-binding portion thereof specifically binds primate and rodent B7H3; preferably, the primate and rodent B7H3 are selected from the group consisting of mouse B7H3, human 2IgB H3, human 4IgB H3, monkey 2IgB7H3 and monkey 4IgB H3.

5. The antibody or antigen-binding portion thereof of any one of the preceding claims, wherein the antibody or antigen-binding portion thereof is at 1 x 10 ^-10 Up to 1X 10 ^-9 KD of M specifically binds to mouse mB7H3, human 2IgB7H3, human 4IgB7H3, monkey 2IgB7H3, and/or monkey 4IgB7H3.

6. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof of any one of claims 1-5, and a pharmaceutically acceptable carrier;

optionally, the pharmaceutical composition further comprises one or more additional active ingredients, such as a chemotherapeutic agent, a PD-1 binding antagonist, a 4-1BB binding agonist, and the like.

7. A vaccine comprising the antibody or antigen-binding portion thereof of any one of claims 1-5, and optionally an immunoadjuvant.

8. A nucleotide molecule encoding the antibody or antigen binding portion thereof of any one of claims 1-5.

9. An expression vector comprising the nucleotide molecule of claim 8.

10. A host cell comprising the nucleotide molecule of claim 8 or the expression vector of claim 9.

11. Use of the antibody or antigen binding portion thereof of any one of claims 1-5, the pharmaceutical composition of claim 6, the vaccine of claim 7, the nucleotide molecule of claim 8, the expression vector of claim 9, or the host cell of claim 10 in the manufacture of a medicament for blocking/activating ligand binding to B7H3, promoting/inhibiting T cell activation, eliciting a T cell mediated response, increasing, inhibiting the function of effector T cells, inhibiting or promoting angiogenesis, treating age-related macular degeneration (AMD), and/or inhibiting tumor growth.

12. Use of the antibody or antigen binding portion thereof of any one of claims 1-5, the pharmaceutical composition of claim 6, the vaccine of claim 7, the nucleotide molecule of claim 8, the expression vector of claim 9 or the host cell of claim 10 in the manufacture of a medicament for the prevention and/or treatment of a B7H 3-related disease.

13. The use of claim 12, wherein the B7H 3-related disease is a tumor;

optionally, the tumor is selected from one or more of the following: colon cancer, melanoma, mesothelioma, renal cell carcinoma, lymphoma, advanced solid tumors, and metastases as described above.

14. The use of claim 12, wherein the B7H 3-related disease is an ocular disease;

optionally, the ocular disease is selected from one or more of the following: age-related macular degeneration (AMD), age-related macular degeneration wet stage/wet macular degeneration (wtmd), diabetic Macular Edema (DME), choroidal Neovascularization (CNV), retinal Vein Occlusion (RVO), central Retinal Vein Occlusion (CRVO), branch Retinal Vein Occlusion (BRVO), pathological Myopia (PM).

15. A detection reagent or kit comprising the antibody or antigen-binding portion thereof of any one of claims 1-5.