Antibody cm0735 and uses thereof

Abstract

The application discloses an antibody CM0735 and application thereof, the antibody CM0735 is an antibody targeting B7-H3 protein, HCDR1-3 in the heavy chain variable region are respectively shown as SEQ ID NO:1-3, LCDR1-3 in the light chain variable region are respectively shown as SEQ ID NO:5-7, the antibody has higher binding activity to B7-H3, can be used in the detection and treatment of B7-H3 abnormal expression diseases, and has important significance for preventing and controlling B7-H3 abnormal expression related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to antibody CM0735 and its applications. Background Technology

[0002] B7-H3 is a member of the B7 family of costimulatory regulatory proteins. It was first discovered in 2001 in a cDNA library derived from human dendritic cells and was later named CD276, belonging to the immunoglobulin superfamily. B7-H3 has two isoforms: the two-region 2IgB7-H3 and the four-region 4IgB7-H3 (IgV-IgC-IgV-IgC). Both are expressed in human tissues, with the latter being predominant. 4IgB7-H3 is only found in humans; only 2IgB7-H3 is found in mouse tissues. Currently, no functional difference has been found between the two. B7-H3 is widely expressed in the biological world, not only in mammals such as humans but also in lower vertebrates such as fish, and its sequence is highly similar across different species.

[0003] B7-H3 is widely expressed in many human tissues. Researchers have found its protein expression in various organs, including the circulatory, reproductive, digestive, endocrine, and musculoskeletal systems. However, B7-H3 protein expression is limited and maintained at low levels. Under normal stable conditions, B7-H3 protein is expressed at low levels only in tissues such as the liver, lungs, breast, prostate, testes, and placenta. With further research into B7-H3, researchers have found high expression of the protein in cancerous tissues from various cancer patients, such as osteosarcoma, prostate cancer, colorectal cancer, and cervical cancer. Immunohistochemistry and other detection methods have shown that B7-H3 is highly expressed on the surface and in the cytoplasm of tumor cells. Multiple studies have indicated that B7-H3 may play a role in promoting tumorigenesis. Therefore, the development and research of antibodies targeting B7-H3 has significant clinical and scientific value. Currently, there is an urgent need in this field to develop an antibody product with good affinity and specificity for B7-H3. Summary of the Invention

[0004] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0005] The first aspect of the present invention provides a B7-H3 antibody or an antigen-binding fragment thereof, wherein the B7-H3 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3.

[0006] The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:1, 2 and 3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:5, 6 and 7, respectively.

[0007] Furthermore, the heavy chain variable region of the B7-H3 antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 75% identity with SEQ ID NO:4, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with SEQ ID NO:8.

[0008] In this invention, the B7-H3 antibody or its antigen-binding fragment may be a monoclonal antibody, a domain antibody, a single-chain (scFv), a Fab fragment, an F(ab')2 fragment multispecific antibody, a single-domain heavy chain antibody, or a single-domain light chain antibody. In some embodiments, such antibodies binding to the B7-H3 protein or their antigen-binding fragments are mouse, other rodent, chimeric, humanized, or fully human monoclonal antibodies.

[0009] The "complementarity-determining region" (CDR) or "hypervariant region" is a region in the antibody's variable region that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes.

[0010] Based on the variable region amino acid sequence contained in the given B7-H3 antibody or its antigen-binding fragment according to this invention, those skilled in the art can routinely determine the CDR contained therein. For example, the Kabat, AbM, Chothia, or Contact protocols can be used to define the CDR in the variable region amino acid sequence.

[0011] When referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0012] The boundaries of the CDR of the antibody of the present invention can be determined artificially according to any method or combination thereof in the art. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers the CDR sequence determined in any of the foregoing methods.

[0013] In some embodiments, functional variants of the B7-H3 antibody or its antigen-binding fragment described herein are also included within the scope of protection of this invention. A "functional variant" refers to a protein that has significant or marked sequence identity or similarity to the parent antibody, and that retains the biological activity of the parent antibody. Functional variants encompass, for example, the following variants of the B7-H3 antibody or its antigen-binding fragment (parent antibody) described herein, which retain the ability to recognize target cells to a similar, equal, or greater extent than the parent antibody. Referring to the parent antibody, the functional variant may, for example, have at least about 75%, 80%, 85%, 90%, 95%, or higher identity with the parent antibody in terms of amino acid sequence.

[0014] In some embodiments, the functional variant may, for example, comprise the amino acid sequence of a parent antibody having at least one conserved amino acid substitution. Alternatively or supplementally, the functional variant may comprise the amino acid sequence of a parent antibody having at least one non-conserved amino acid substitution. In this case, the non-conserved amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution can enhance the biological activity of the functional variant, resulting in increased biological activity of the functional variant compared to the parent antibody.

[0015] In some embodiments, conservative amino acid substitution is known in the art and includes the substitution of one amino acid having a particular physical and / or chemical property with another amino acid having the same or similar chemical or physical property. For example, conservative amino acid substitutions can be: replacing one acidic / negatively charged polar amino acid with another (e.g., Asp or Glu); replacing one amino acid with a nonpolar side chain with another (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.); replacing one basic / positively charged polar amino acid with another (e.g., Lys, His, Arg, etc.); replacing one uncharged amino acid with a polar side chain with another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.); replacing one amino acid with a β-branched side chain with another (e.g., He, Thr, and Val); and replacing one amino acid with an aromatic side chain with another (e.g., His, Phe, Trp, and Tyr).

[0016] A second aspect of the present invention provides a bispecific antibody comprising the B7-H3 antibody or its antigen-binding fragment described in the first aspect of the present invention.

[0017] Furthermore, the bispecific antibody also includes a second antibody that specifically binds to other antigens.

[0018] In some implementations, the second antibody that specifically binds to other antigens is not particularly limited; it can be any antibody targeting B7-H3 or any antigen other than B7-H3. Those skilled in the art can make conventional selections according to actual needs.

[0019] A third aspect of the present invention provides a polynucleotide molecule or a carrier comprising the polynucleotide molecule, said polynucleotide molecule encoding the B7-H3 antibody or its antigen-binding fragment as described in the first aspect of the present invention or the bispecific antibody as described in the second aspect of the present invention.

[0020] In this invention, the polynucleotide molecule may comprise natural, non-natural, or modified nucleotides; and it may comprise natural, non-natural, or modified internucleotide linkages, such as aminophosphate linkages or thiophosphate linkages, instead of phosphodiester linkages present between unmodified oligonucleotide nucleotides. In some embodiments, the nucleotides do not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, it may be suitable for a nucleotide to contain one or more insertions, deletions, inversions, and / or substitutions, and therefore, nucleotides formed by these insertions, deletions, inversions, and / or substitutions are also within the scope of this invention.

[0021] Those skilled in the art can readily mutate the nucleotide sequence corresponding to the antibody described in this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 75% or more identity with the nucleotide sequence corresponding to the B7-H3 antibody or its antigen-binding fragment described in this invention, as long as they encode the aforementioned B7-H3 antibody or its antigen-binding fragment, are all derived from and equivalent to the nucleotide sequence of this invention, and are also included within the scope of protection of this invention.

[0022] When applied to polynucleotide molecules, the term "encoding" refers to a polynucleotide that, if in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce an mRNA containing a polypeptide and / or fragments thereof, is called "encoding" the polypeptide. The antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced from it.

[0023] In some embodiments, the vector may contain expression regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host cell into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), depending on whether the vector is DNA-based or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0024] In some embodiments, the vector may also contain one or more marker genes that allow selection of host cells for transformation or transfection. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.); and prototrophic complementation in auxotrophic hosts. Suitable marker genes for the expression vector of this invention include neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, and puromycin resistance genes.

[0025] Various vectors known in the art can be used, such as commercially available vectors, and then a polynucleotide encoding the antibody or its antigen-binding fragment can be operatively linked to the expression regulatory sequence to form an expression vector. In some embodiments, the vector includes, but is not limited to, plasmids, phage particles, granules, artificial chromosomes, and virus-derived vectors.

[0026] A fourth aspect of the present invention provides a modified host cell or a population of host cells comprising thereto, said modified host cell comprising the polynucleotide molecule described in the third aspect of the present invention or a vector comprising thereto.

[0027] Furthermore, the host cell population also includes host cells other than the modified host cells.

[0028] Furthermore, the modified host cells include prokaryotic cells and eukaryotic cells.

[0029] In some implementations, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsia.

[0030] In some implementations, the bacteria include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Staphylococcus.

[0031] In some implementations, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0032] In some embodiments, the modified host cells are prepared by introducing the polynucleotide molecules or vectors containing them as described above into the host cells. The methods of introduction include, but are not limited to, physical, chemical, and biological methods. The physical methods include, but are not limited to, microinjection, electroporation, calcium phosphate precipitation, lipid transfection, and particle bombardment. The chemical methods include, but are not limited to, colloidal dispersion systems and lipid-based systems. The colloidal dispersion systems include, but are not limited to, macromolecular complexes, nanocapsules, microspheres, and beads. The lipid-based systems include, but are not limited to, oil-in-water emulsions, micelles, mixed micelles, and liposomes. The biological methods include, but are not limited to, DNA vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, and adeno-associated virus vectors.

[0033] The fifth aspect of the present invention provides an antibody derivative of a B7-H3 antibody or an antigen-binding fragment thereof, wherein the antibody derivative comprises a complex formed by direct or indirect conjugation of the B7-H3 antibody or an antigen-binding fragment thereof described in the first aspect of the present invention or a bispecific antibody described in the second aspect of the present invention to a detectable marker or therapeutic agent.

[0034] Furthermore, the detectable markers include fluorescent dyes, enzymes, chemiluminescent markers, radioactive isotopes, electron-dense reagents, colored particles, biotin, or digoxin.

[0035] In some implementations, the detectable marker often generates a measurable signal, such as radioactivity, fluorescence, color, or enzyme activity. Examples of suitable fluorescent dyes include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine, fluorescein, dansyl chloride, and phycoerythrin; examples of suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; chemiluminescent markers include, but are not limited to, luminol and its derivatives, isoluminol and its derivatives, acridine esters and their derivatives, adamantane, rare earth elements, and ruthenium bipyridine complexes; radioactive isotopes include, but are not limited to, those that... 68 Ga、 86 Y、 110 In、 111 In、 177 Lu、 18 F, 52 Fe、 62 Cu、 64 Cu、 11 C 67 Cu、 94 Tc, 99 mTc, 120 I, 123 I,124 I, 15 O. The aforementioned detectable markers can be directly linked or conjugated with antibodies or indirectly via intermediates such as linkers known in the art using techniques known in the art.

[0036] Furthermore, the therapeutic agents include cytotoxic agents, hormonal preparations, targeted small molecule preparations, proteasome inhibitors, chemotherapeutic agents, oncolytic drugs, cytokines, activators of co-stimulatory molecules, or inhibitors of inhibitory molecules.

[0037] The sixth aspect of the present invention provides a detection reagent or detection product, the detection reagent or detection product comprising the B7-H3 antibody or its antigen-binding fragment as described in the first aspect of the present invention, the bispecific antibody as described in the second aspect of the present invention, or the antibody derivative as described in the fifth aspect of the present invention.

[0038] Furthermore, the product includes reagent kits, chips, and test strips.

[0039] Furthermore, the kits include, but are not limited to, ELISA kits, immunofluorescence kits, flow cytometry kits, and immunohistochemistry kits.

[0040] In some embodiments, the kit may include a container, instructions, buffers, etc. In other embodiments, the kit may also include a lysis medium for dissolving the sample to be tested, universal reagents and buffers required for detection, such as various buffer solutions, detection labels, detection substrates, etc. This test kit can be an in vitro diagnostic device.

[0041] The seventh aspect of the present invention provides a pharmaceutical composition for treating diseases or conditions with abnormal B7-H3 expression, the pharmaceutical composition comprising the B7-H3 antibody or its antigen-binding fragment as described in the first aspect of the present invention, the bispecific antibody as described in the second aspect of the present invention, or the antibody derivative as described in the fifth aspect of the present invention.

[0042] In this invention, the treatment refers to the suppression, containment, relief, improvement, slowing, cessation, delay, or reversal of the course of a disease in order to prevent and reduce the occurrence or development of the disease. Various indicators of maintaining and / or using the medication during the course of the disease, disorder, or pathological state include the reduction or elimination of symptoms or complications, or the cure or elimination of the disease, disorder, or condition.

[0043] In this invention, prevention refers to various means or measures, including medical, physical or chemical methods, used to prevent the occurrence or development of a disease before it is recognized as a disease by clinical standards, in order to prevent and reduce the occurrence or development of various symptoms of the disease.

[0044] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. Excipients refer to additives in a pharmaceutical preparation other than the active pharmaceutical ingredient (API), also known as excipients. General requirements for excipients include: stability, no incompatibility with the API, no side effects, no impact on efficacy, resistance to deformation, cracking, mold, and insect infestation at room temperature, harmlessness to humans, no physiological effects, no chemical or physical reactions with the API, and no interference with the determination of the API's content.

[0045] In some embodiments, the excipients include binders, fillers, disintegrants, lubricants, ointments, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solvents, osmotic pressure regulators, and colorants.

[0046] In some embodiments, the above-described pharmaceutical composition may be administered via any suitable route known in the art, including but not limited to: oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and / or cerebrospinal fluid administration.

[0047] Furthermore, the diseases or conditions in which B7-H3 is abnormally expressed include osteosarcoma, glioma, melanoma, hematologic malignancies, prostate cancer, esophageal cancer, cervical cancer, ovarian cancer, colorectal cancer, breast cancer, or lung cancer.

[0048] In some implementations, the term "diseases with abnormal B7-H3 expression" is a general term for a class of diseases related to abnormal B7-H3 expression, and is not limited to the specific disease types listed above. Any disease related to abnormal B7-H3 expression will fall within the protection scope of this invention.

[0049] The eighth aspect of the present invention provides any of the following methods:

[0050] 1) A method for preparing the B7-H3 antibody or its antigen-binding fragment as described in the first aspect of the present invention or the bispecific antibody as described in the second aspect of the present invention, the method comprising the following steps: culturing the modified host cells or host cell populations containing the modified host cells as described in the fourth aspect of the present invention, and isolating the B7-H3 antibody or its antigen-binding fragment or bispecific antibody from the culture.

[0051] 2) A method for detecting B7-H3 or a fragment thereof in a test sample, the method comprising the following steps: contacting the test sample with the B7-H3 antibody or its antigen-binding fragment as described in the first aspect of the present invention, the bispecific antibody as described in the second aspect of the present invention, the antibody derivative as described in the fifth aspect of the present invention, or the detection reagent or detection product as described in the sixth aspect of the present invention, and detecting the formation of the corresponding antibody-antigen complex.

[0052] 3) A method for preparing the modified host cell or host cell population containing the present invention according to the fourth aspect of the present invention, the method comprising the following steps: introducing the polynucleotide molecule or vector containing the present invention according to the third aspect of the present invention into the host cell.

[0053] 4) A method for inhibiting B7-H3 activity in a sample in vitro, the method comprising: contacting the sample with the B7-H3 antibody or its antigen-binding fragment as described in the first aspect of the present invention or the bispecific antibody as described in the second aspect of the present invention.

[0054] In some implementations, the sample or test sample may be selected from blood, serum, plasma, urine, saliva, ascites, brain tissue, cerebrospinal fluid, non-tissue-associated cells, tissues, histological preparations, etc., from the test subject. The present invention does not have any particular limitation on the specific type of the sample or test sample, and any sample that may contain B7-H3 may be used as the sample or test sample.

[0055] The ninth aspect of the present invention provides any of the following applications:

[0056] 1) The use of the B7-H3 antibody or its antigen-binding fragment as described in the first aspect of the present invention, the bispecific antibody as described in the second aspect of the present invention, the polynucleotide molecule or a carrier containing the thereof as described in the third aspect of the present invention, the modified host cell or a host cell population containing the thereof as described in the fourth aspect of the present invention, or the antibody derivative as described in the fifth aspect of the present invention in the preparation of a detection reagent or detection product for detecting B7-H3.

[0057] 2) The use of the B7-H3 antibody or its antigen-binding fragment as described in the first aspect of the present invention, the bispecific antibody as described in the second aspect of the present invention, the polynucleotide molecule or a carrier containing the thereof as described in the third aspect of the present invention, the modified host cell or a host cell population containing the thereof as described in the fourth aspect of the present invention, the antibody derivative as described in the fifth aspect of the present invention, or the detection reagent or detection product as described in the sixth aspect of the present invention in the preparation of diagnostic products for diagnosing or assisting in the diagnosis of diseases with abnormal B7-H3 expression.

[0058] 3) The use of the B7-H3 antibody or its antigen-binding fragment as described in the first aspect of the present invention, the bispecific antibody as described in the second aspect of the present invention, the polynucleotide molecule or a carrier containing the thereof as described in the third aspect of the present invention, the modified host cell or a population of host cells containing the thereof as described in the fourth aspect of the present invention, or the antibody derivative as described in the fifth aspect of the present invention in the preparation of a medicament for treating and / or preventing diseases with abnormal B7-H3 expression.

[0059] 4) The use of the B7-H3 antibody or its antigen-binding fragment as described in the first aspect of the present invention, the bispecific antibody as described in the second aspect of the present invention, the polynucleotide molecule or a carrier containing the thereof as described in the third aspect of the present invention, the modified host cell or a host cell population containing the thereof as described in the fourth aspect of the present invention, the antibody derivative as described in the fifth aspect of the present invention, or the detection reagent or detection product as described in the sixth aspect of the present invention in the detection of B7-H3 for non-diagnostic and non-therapeutic purposes.

[0060] Advantages and beneficial effects of the present invention:

[0061] This invention discloses antibody CM0735 and its applications, wherein antibody CM0735 is an antibody targeting the B7-H3 protein. The B7-H3 antibody or its antigen-binding fragment of this invention can bind to the B7-H3 protein with high activity and specificity. This invention also relates to polynucleotide molecules encoding B7-H3 antibodies or their antigen-binding fragments, vectors, host cells, and antibody derivatives or other products derived from the B7-H3 antibody or its antigen-binding fragment according to this invention. Attached Figure Description

[0062] Figure 1 To verify the binding ability of antibody CM0735 to B7-H3; where A is immunoprecipitation (IP); B is immunohistochemistry (IHC) of frozen sections; and C is immunohistochemistry (IHC) of paraffin blocks.

[0063] Figure 2 The purpose of this study was to verify the binding affinity, specificity, and affinity of antibody CM0735 for B7-H3. Specifically, A was an ELISA assay to verify the binding affinity and specificity of antibody CM0735 for B7-H3 antigen, and B was a flow cytometry assay to verify the binding affinity of antibody CM0735 for B7-H3 antigen on the surface of 143B cells. Detailed Implementation

[0064] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent changes to the technical content disclosed herein. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the present invention fall within the protection scope of the present invention.

[0065] Example 1: Preparation of antibody CM0735

[0066] Antibody CM0735 was prepared by ABclonal (Urisys (Wuhan) Biotechnology Co., Ltd.). The following is a brief description of the experimental procedure:

[0067] 1. Preparation and processing of immunogens

[0068] The immunogen was Recombinant Human B7-H3 / CD276 Protein, C-hFc&His (Abclonal, cat: #RP01020).

[0069] The original test result was Recombinant Human B7-H3 / CD276 Protein, C-His (Abclonal, cat:#RP01020).

[0070] 2. Animal immunization

[0071] Five healthy Balb / C mice were selected as immunization animals. All mice were in a normal physiological state before and during immunization. Recombinant human B7-H3 / CD276 protein (with a His tag at the C-terminus, hereinafter referred to as "immunogen") was used as the immunogen. Mice were systemically immunized according to the time points and immunization regimens shown in Table 1.

[0072] Table 1 Animal Immunization Experiment Protocol

[0073]

[0074] After the fourth and shock immunizations, blood was collected from the mice, with approximately 15–30 μl of peripheral blood collected per mouse each time, for the preparation and detection of subsequent polyclonal antibody serum.

[0075] Antibody titers were measured in serum samples collected after the fourth and fifth pulse immunizations using enzyme-linked immunosorbent assay (ELISA). The binding activity of specific polyclonal antibodies in serum was assessed using the same immunogen (Human B7-H3 / CD276 Protein, C-His) as the coating antigen.

[0076] 3. Cell fusion

[0077] Two mice with high binding activity to the specific polyclonal antibody were selected (one for further antibody development, and one as a backup). Mouse spleens were fused with the mouse osteosarcoma cell line (SP2 / 0), resulting in 20 96-well cell plates. The cells were first screened using HAT medium, and the selector was then screened a second time. The fusion supernatant that specifically recognized the selector was selected using ELISA. ELISA-positive clones (ODs selected by ELISA screening) were then chosen. 450 (The value was greater than or equal to 5 times the background value), and a total of 80 positive clones were selected.

[0078] 4. Subcloning screening

[0079] Twenty ELISA-positive fusion cells were selected for subcloning based on the ELISA binding results.

[0080] 5. Sequence Fishing

[0081] Subcloned cells were collected, lysed, and RNA was extracted. Using the extracted RNA as a template, cDNA was obtained in two steps using the Yiqiao Technology reverse transcription kit. The cDNA obtained through reverse transcription was then used as a template for PCR amplification. The amplified fragment was inserted into an expression vector or a commercial vector, and sequencing was performed to obtain plasmids containing the correct sequence.

[0082] 6. Experimental Results

[0083] The sequence information of the prepared antibody CM0735 is shown in Table 2.

[0084] Table 2 Sequence information of antibody CM0735

[0085]

[0086] Example 2: Functional Study of Antibodies Targeting B7-H3

[0087] 1. Verification of the binding ability of antibodies targeting B7-H3

[0088] (1) Immunoprecipitation (IP) was used to verify the binding ability of antibody CM0735 to B7-H3.

[0089] The human osteosarcoma cell line 143B used in the experiments was purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number TCHU264. Cells were cultured in DMEM (Gibco, C11995500BT) complete medium containing 10% fetal bovine serum (Wisent, 085-150) at 37°C under humid conditions of 5% CO2. Immunoprecipitation (IP) experiments were performed using an immunoprecipitation kit (Protein A+G agarose gel method) (Beyotime, P2197S). Cell lysis, antibody binding (50 μg / ml), immunoprecipitation, and elution were performed according to the kit's instructions. Western blotting was then used for verification.

[0090] (2) Immunohistochemistry (IHC) was used to verify the binding ability of antibody CM0735 to B7-H3.

[0091] All paraffin sections were obtained from the specimen bank of the Department of Bone Tumors, Peking University People's Hospital, and were 4 μm in size, stored at -20℃. Immunohistochemical staining was performed on a Bond RX staining machine, including dewaxing, antigen recapture, blocking of endogenous peroxidase, washing, primary antibody incubation (CM0730 concentration 1:100), secondary antibody incubation (DS9800), DAB staining, counterstaining, dehydration, clearing, and mounting.

[0092] Fresh tissue is immediately immersed in a tissue fixative or a specific fixative for at least 24 hours before being stored and transported at room temperature. The tissue is removed from the fixative and trimmed smooth using a scalpel. The tissue portion to be frozen sectioned should be no more than 3 mm thick and no more than 2 cm in length and width, and the corresponding tube should be electronically numbered. The dehydrated tissue is removed, slightly blotted with filter paper, and placed cut-side up on a sample holder. OCT embedding medium (Sakura, 4583) is applied around the tissue. The sample holder is then rapidly frozen and embedded on a quick-freezing stage. Once the OCT medium turns white and hardens, it is ready for sectioning. The sample holder is fixed to the cryostat head. A coarse trimming of 40 μm is performed initially, followed by a fine trimming of 8 μm at the target mounting position. The cut surface is then smoothed before sectioning. The standard section thickness is 8 μm. Sections removed from the cryostat are placed in pre-chilled acetone and fixed at 4°C for 10 minutes. After acetone fixation, remove the slides and air-dry them at room temperature for 10-20 minutes. Then, immerse them in PBS for 5-10 minutes to hydrate the tissue and wash away the fixative. Add 3% hydrogen peroxide (H2O2) solution to cover the tissue. After incubation, rinse three times with PBS for 2 minutes each time. Block with blocking solution (normal goat serum, Servicebio, G1208) for about 30 minutes, then add CM0730 diluted antibody (concentration 1:100), incubate at room temperature for 2 hours, add MicroStacker™ RX polyHRP-labeled secondary antibody polymer (CELNOVTE, CSM1003), incubate at room temperature for 30 minutes, rinse three times with PBS, add DAB (diaminobenzidine) chromogenic solution (CELNOVTE, CSK5001), and counterstain with hematoxylin. Dehydrate by passing through 75%, 85%, 95%, and 100% alcohol sequentially, and clear with xylene. Mount with neutral resin.

[0093] (3) Flow cytometry was used to verify the binding ability of antibody CM0735 to B7-H3 on the surface of 143B cells.

[0094] The human osteosarcoma cell line 143B used in the experiment was purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number TCHU264. The cells were cultured in DMEM (Gibco, C11995500BT) complete medium containing 10% fetal bovine serum (Wisent, 085-150) at 37°C in a humid environment with 5% CO2.

[0095] After discarding the supernatant, wash twice with PBS (Gibco, C10010500BT), digest with trypsin (Gibco, 25200-072) for 2 min, then terminate digestion with complete culture medium. Take 20 μl for counting, retaining 2×10⁻⁶. 5 1 cell / tube, 2 tubes in total (negative, experimental group), centrifuge cells at 1200 rpm for 5 min, discard supernatant.

[0096] Resuspend cells twice in pre-chilled PBS at 4°C, and centrifuge as before. Add 100 μL PBS for the negative control and 5 μL CCM0735 + 95 μL PBS for the positive control, incubate at 4°C for 1 h, and repeat PBS washing and centrifugation three times. Add 100 μL of secondary antibody (YSFluor™488 Goat Anti-Mouse IgG (H+L), 33206ES60) at a 1:400 ratio to each tube, incubate at 4°C for 1 h, and repeat PBS washing and centrifugation three times. After resuspending cells in 100 μL PBS, analyze using a BD Accri@ C6 flow cytometer.

[0097] 2. Validation of the affinity and specificity of antibodies targeting B7-H3

[0098] ELISA was used to verify the affinity and specificity of the antibodies: 100 μl of 0.2 μg / ml antigen (Recombinant Human B7-H3 / CD276 Protein (Abclonal, cat: #RP01020); Recombinant Human B7-H1 / PD-L1 / CD274 Protein (Abclonal, cat: #RP00068)) was added to each well of a 96-well ELISA plate (LABSELECT, 31121) and incubated at 4°C for 16 h; the plates were washed 3 times with PBST (PBS + 0.05% Tween), and 300 μl / well of blocking buffer (SW3015) was added, and the plates were blocked at 37°C for 1.5 h; the plates were washed 3 times; 0.5 μg / ml antibody CM0735 was diluted 4-fold and incubated at 37°C at 600 rpm for 1 h; the plates were washed 5 times, and enzyme-labeled secondary antibody HRP-IgG (H+L) was added. Incubate the plate with a 1:250 solution of Mouse / human (A0350 / A0201) at 37℃ and 60 rpm for 1 h. Wash the plate 5 times. Add 100 μl of TMB (Solarbio, PR1200) per well and incubate at room temperature in the dark for 30 s. Add 50 μl of stop solution (Solarbio, C1058) per well and measure the OD values ​​at 450 / 620 nm using a microplate reader.

[0099] 3. Experimental Results

[0100] Immunoprecipitation (IP) and immunohistochemistry (IHC) results showed that antibody CM0735 could effectively bind to the B7-H3 antigen ( Figure 1 A, Figure 1 B. Figure 1 C). Subsequently, we demonstrated by flow cytometry that antibody CM0735 has a strong specific binding ability to the B7-H3 antigen on the surface of 143B cells. Figure 2 B). ELISA results showed that antibody CM0735 had a high affinity for the B7-H3 antigen, with an EC50% (half-maximal effective concentration). 50 The value was 0.001351 mg / ml. Figure 2 A), we then used the B7-H1 antigen, which has a similar structure to the B7-H3 antigen, to verify the antibody's specificity. We found that CM0735 had no binding activity against B7-H1, proving its binding specificity. Figure 2 A).

[0101] The above results collectively demonstrate that the antibody CM0735 prepared in this invention has a strong binding ability to the B7-H3 antigen, can detect the expression of B7-H3 in the sample, and that the binding of antibody CM0735 to B7-H3 is specific.

[0102] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A B7-H3 antibody or its antigen-binding fragment, characterized in that, The B7-H3 antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the light chain variable region includes LCDR1, LCDR2 and LCDR3. The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:1, 2 and 3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:5, 6 and 7, respectively.

2. The B7-H3 antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region of the B7-H3 antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 75% identity with SEQ ID NO:4, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with SEQ ID NO:

8.

3. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the B7-H3 antibody or its antigen-binding fragment as described in any one of claims 1 or 2.

4. A carrier, characterized in that, The carrier comprises the polynucleotide molecule of claim 3.

5. A modified host cell, characterized in that, The modified host cell comprises the polynucleotide molecule of claim 3 or the vector of claim 4.

6. The modified host cell according to claim 5, characterized in that, The modified host cells include prokaryotic cells and eukaryotic cells.

7. A modified host cell population, characterized in that, The modified host cell population includes the modified host cells as described in any one of claims 5 or 6, as well as other host cells.

8. A B7-H3 antibody or an antibody derivative thereof containing an antigen-binding fragment, characterized in that, The antibody derivative comprises a complex formed by direct or indirect coupling of the B7-H3 antibody or its antigen-binding fragment to a detectable marker as described in any one of claims 1 or 2.

9. The antibody derivative according to claim 8, characterized in that, The detectable markers include fluorescent dyes, enzymes, chemiluminescent markers, electron-dense reagents, colored particles, biotin, or digoxigenin.

10. A testing reagent or testing product, characterized in that, The detection reagent or detection product includes the B7-H3 antibody or its antigen-binding fragment as described in any one of claims 1 or 2, or the antibody derivative as described in any one of claims 8 or 9.

11. The detection reagent or detection product according to claim 10, characterized in that, The products include reagent kits, chips, and test strips.

12. A pharmaceutical composition for treating diseases or conditions with abnormal B7-H3 expression, characterized in that, The pharmaceutical composition comprises the B7-H3 antibody or its antigen-binding fragment as described in any one of claims 1 or 2, or the antibody derivative as described in any one of claims 8 or 9.

13. The pharmaceutical composition according to claim 12, characterized in that, The diseases or conditions associated with abnormal B7-H3 expression include osteosarcoma, glioma, melanoma, hematologic malignancies, prostate cancer, esophageal cancer, cervical cancer, ovarian cancer, colorectal cancer, breast cancer, or lung cancer.

14. Any of the following methods: 1) A method for preparing the B7-H3 antibody or its antigen-binding fragment as described in any one of claims 1 or 2, the method comprising the following steps: culturing the modified host cells as described in any one of claims 5 or 6 or the modified host cell population as described in claim 7, and isolating the B7-H3 antibody or its antigen-binding fragment from the culture; 2) A method for detecting B7-H3 or its fragments in a test sample for non-diagnostic, non-therapeutic purposes, the method comprising the following steps: contacting the test sample with the B7-H3 antibody or its antigen-binding fragment as described in any one of claims 1 or 2, the antibody derivative as described in any one of claims 8 or 9, or the detection reagent or detection product as described in any one of claims 10 or 11, and detecting the formation of the corresponding antibody-antigen complex; 3) A method for preparing the modified host cell as described in any one of claims 5 or 6 or the modified host cell population as described in claim 7, the method comprising the steps of: introducing the polynucleotide molecule as described in claim 3 or the vector as described in claim 4 into the host cell; 4) A method for inhibiting B7-H3 activity in a sample in vitro, the method comprising: The sample is contacted with the B7-H3 antibody or its antigen-binding fragment as described in any one of claims 1 or 2.

15. Any of the following applications: 1) The use of the B7-H3 antibody or antigen-binding fragment thereof as described in any one of claims 1 or 2, the polynucleotide molecule as described in claim 3, the vector as described in claim 4, the modified host cell as described in any one of claims 5 or 6, the modified host cell population as described in claim 7, or the antibody derivative as described in any one of claims 8 or 9 in the preparation of a detection reagent or detection product for detecting B7-H3; 2) The use of the B7-H3 antibody or its antigen-binding fragment as described in any one of claims 1 or 2, the polynucleotide molecule as described in claim 3, the vector as described in claim 4, the modified host cell as described in any one of claims 5 or 6, the modified host cell population as described in claim 7, the antibody derivative as described in any one of claims 8 or 9, or the detection reagent or detection product as described in any one of claims 10 or 11 in the preparation of diagnostic products for the diagnosis or auxiliary diagnosis of diseases with abnormal B7-H3 expression; 3) The use of the B7-H3 antibody or its antigen-binding fragment as described in any one of claims 1 or 2, the polynucleotide molecule as described in claim 3, the vector as described in claim 4, the modified host cell as described in any one of claims 5 or 6, the modified host cell population as described in claim 7, the antibody derivative as described in any one of claims 8 or 9, or the detection reagent or detection product as described in any one of claims 10 or 11 in the detection of B7-H3 for non-diagnostic, non-therapeutic purposes.

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