Antibodies targeting b7h3 and uses thereof

Abstract

The application discloses an antibody targeting B7H3 and application thereof. The application provides a brand-new antibody targeting B7H3 for the field, and the antibody can specifically bind to B7H3, has good affinity and specificity. The application lays a foundation for developing B7H3 related detection and / or auxiliary detection products for the field, and is expected to provide more diagnostic tools and treatment strategies for diagnosis and / or treatment of B7H3 related diseases, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antibody targeting B7H3 and its application. Background Technology

[0002] B7H3 (also known as CD276) is a type I membrane protein with a sequence similar to the extracellular domain of PD-L1 (also known as B7H1). B7H3 RNA is widely expressed in both lymphoid and non-lymphoid organs and can act as a co-stimulatory molecule against CD4. + and CD8 + T cell proliferation plays a crucial role. B7H3 signaling induces cellular immunity, promoting T cell activation and IFN-γ proliferation. B7H3 is aberrantly expressed in various cancers, including lung cancer, prostate cancer, esophageal cancer, ovarian cancer, cervical cancer, colorectal cancer, breast cancer, and osteosarcoma. Numerous studies have confirmed the important role of B7H3-mediated non-immune responses in influencing host cell biology.

[0003] B7H3 antibodies have wide applications in scientific research and medicine. In research, they can be used to identify and sort cells expressing B7H3 to study cell function and disease mechanisms. In medicine, they can serve as diagnostic markers to aid in the diagnosis of diseases such as breast cancer and osteosarcoma. Furthermore, B7H3 antibodies hold promise for development into therapeutic drugs, restoring the body's anti-tumor immune response by blocking the immunosuppressive function of B7H3. Therefore, the development and research of antibodies targeting B7H3 has significant clinical and research value. Currently, there is still an urgent need in this field to develop an antibody product with good affinity and specificity for B7H3. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the art with a novel antibody that specifically binds to B7H3, as well as related products and uses.

[0005] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0006] The first aspect of the present invention provides a B7H3 antibody, wherein the heavy chain variable region CDR1 is shown in SEQ ID NO:1; CDR2 is shown in SEQ ID NO:2; and CDR3 is shown in SEQ ID NO:3; and the light chain variable region CDR1 is shown in SEQ ID NO:5; CDR2 is shown in SEQ ID NO:6; and CDR3 is shown in SEQ ID NO:7.

[0007] Alternatively, the heavy chain variable region CDR1 of the antibody is shown as SEQ ID NO:9; CDR2 is shown as SEQ ID NO:2; CDR3 is shown as SEQ ID NO:3; the light chain variable region CDR1 of the antibody is shown as SEQ ID NO:5; CDR2 is shown as SEQ ID NO:6; CDR3 is shown as SEQ ID NO:7.

[0008] Alternatively, the heavy chain variable region CDR1 of the antibody is shown as SEQ ID NO:12; CDR2 is shown as SEQ ID NO:2; CDR3 is shown as SEQ ID NO:3; the light chain variable region CDR1 of the antibody is shown as SEQ ID NO:14; CDR2 is shown as SEQ ID NO:6; CDR3 is shown as SEQ ID NO:7.

[0009] Furthermore, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 75% homology with SEQ ID NO:4; 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% homology with SEQ ID NO:8.

[0010] Alternatively, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 75% homology with SEQ ID NO:10; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 75% homology with SEQ ID NO:11.

[0011] Alternatively, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 75% homology with SEQ ID NO:13; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 75% homology with SEQ ID NO:15.

[0012] In some embodiments, CDR1, CDR2, and CDR3 can be defined using any one or any combination of two or more of the following numbering schemes: IMGT, Chothia, Kabat, Martin (enhanced Chothia), AbM, Aho (or other existing CDR numbering schemes or new CDR numbering schemes that may be generated in the future) to define the amino acid sequences corresponding to the light or heavy chains of the antibody as described above. The sequences corresponding to CDR1, CDR2, and CDR3 defined by the above methods also fall within the protection scope of this invention.

[0013] In some embodiments, functional variants of the B7H3 antibody provided in the first aspect of the present invention are also included within the scope of protection of the present invention.

[0014] In some embodiments, the functional variant refers to a protein that has significant or marked homology or similarity to the parent antibody (the B7H3 antibody described in the first aspect of the invention), and the functional variant retains the biological activity of the parent antibody.

[0015] Functional variants encompass the following variants of, for example, the B7H3 antibody (parental 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 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or higher homology in the amino acid sequence of the parent antibody.

[0016] 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 supplementarily, 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. Non-conserved amino acid substitution can enhance the biological activity of the functional variant, resulting in increased biological activity compared to the parent antibody. Such functional variants obtained from a parent antibody through conserved or non-conserved amino acid substitutions also fall within the scope of protection of this invention.

[0017] In some embodiments, conservative amino acid substitution is known in the art, including replacing one of the amino acids having specific physical and / or chemical properties with another amino acid having the same or similar chemical or physical properties. 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).

[0018] In some embodiments, homology refers to sequence similarity to a target amino acid sequence or nucleotide sequence. Homology includes amino acid sequences having 75% or higher, 85% or higher, 90% or higher, or 95% or higher homology to the amino acid sequence of the antibody provided by the present invention. Homology can be evaluated visually or by computer software. When using computer software, the homology between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences. The 75% or higher homology can be 75%, 80%, 85%, 90%, or 95% or higher homology.

[0019] In some embodiments, modifications to one or more amino acids in a protein typically do not affect the protein's function. Those skilled in the art will recognize that altering a single amino acid or a small percentage of amino acids, or individual additions, deletions, insertions, or substitutions of amino acid sequences, are conservative modifications, where the protein alteration produces a protein with similar function. Providing tables of conservative substitutions for functionally similar amino acids is well known in the art. Such amino acid sequences obtained through conservative substitutions are also included within the scope of this invention.

[0020] The final conjugate or variant can be achieved using substitution, deletion, insertion, or any combination thereof. Typically, these changes are made on a few amino acids to minimize alterations to the molecule, particularly the immunogenicity and specificity of the antigen-binding protein. However, larger changes can be tolerated in some cases. Amino acid substitutions are usually single-base substitutions; insertions are typically on the order of about one to about twenty amino acid residues, although significantly larger insertions may be tolerated. Deletions range from about one to about twenty amino acid residues, although in some cases, deletions can be much larger.

[0021] A second aspect of the present invention provides a bispecific antibody comprising the antibody described in the first aspect of the present invention.

[0022] Furthermore, the bispecific antibody also includes a second antibody that specifically binds to other antigens.

[0023] In some implementations, the second antibody that specifically binds to other antigens is not particularly limited; it can be any antibody targeting B7H3 or any antigen other than B7H3. Those skilled in the art can make conventional selections according to actual needs.

[0024] A third aspect of the present invention provides a nucleic acid molecule that encodes the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention.

[0025] Those skilled in the art can easily use known methods, such as directed evolution and point mutation, to mutate the nucleotide sequence corresponding to the antibody described in this invention. As long as the antibody encoding the first aspect of this invention or the bispecific antibody encoding the second aspect of this invention is derived from the nucleotide sequence of this invention and is equivalent to the sequence of this invention, it also falls within the protection scope of this invention.

[0026] In some embodiments, the nucleic acid molecule is isolated or purified. The sequence of the nucleic acid molecule can be obtained using conventional techniques or hybridoma techniques. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. Alternatively, the relevant sequence can be synthesized artificially, especially when the fragment length is short. Generally, longer fragments can be obtained by first synthesizing multiple small fragments and then ligating them.

[0027] A fourth aspect of the present invention provides an expression vector comprising the nucleic acid molecule described in the third aspect of the present invention.

[0028] In some embodiments, the vector includes, but is not limited to, plasmids, phage particles, granules, artificial chromosomes, and virus-derived vectors. The virus-derived vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, herpesvirus vectors, and baculovirus vectors.

[0029] In some implementations, the expression vector can be constructed using methods well known to those skilled in the art. These methods include, but are not limited to, recombinant DNA technology, DNA synthesis technology, etc. The DNA encoding the antibody can be effectively ligated to a multiple cloning site in the vector to guide mRNA synthesis and thus protein expression, or for homologous recombination.

[0030] A fifth aspect of the present invention provides a recombinant host cell comprising the expression vector described in the fourth aspect of the present invention.

[0031] Furthermore, the recombinant host cells include prokaryotic cells and eukaryotic cells.

[0032] In some implementations, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsia.

[0033] In some implementations, the bacteria include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Staphylococcus.

[0034] In some implementations, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0035] In some embodiments, the recombinant host cells are prepared by introducing the nucleic acid molecules or expression vectors of the present invention 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.

[0036] The sixth aspect of the present invention provides any of the following products:

[0037] 1) An antibody conjugate, wherein the antibody conjugate is a complex formed by directly or indirectly conjugating the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention to a detectable marker.

[0038] 2) A detection reagent comprising the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, or the antibody conjugate thereof.

[0039] 3) A detection product comprising the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the antibody conjugate, or the detection reagent.

[0040] 4) A pharmaceutical composition comprising the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention.

[0041] 5) A biological agent comprising the pharmaceutical composition.

[0042] Furthermore, the detectable markers include bioluminescent agents, chemiluminescent agents, enzymes, photosensitizing diagnostic agents, paramagnetic ions, or radionuclides.

[0043] In some embodiments, the bioluminescent agent includes, but is not limited to: luciferin, luciferase, and jellyfish luminescent protein; the chemiluminescent agent includes, but is not limited to: imidazole, luminol, isoluminol, aromatic acridine esters, acridine salts, and oxalates; the enzyme includes, but is not limited to: catalase, alkaline phosphatase, urease, horseradish peroxidase, glucose oxidase, β-D-galactosidase, or glucosyl amylase; the photosensitizing diagnostic agent includes, but is not limited to: protoporphyrin, hematoporphyrin, methylene blue, dihydroxysilylphthalocyanine, and photoporphyrin; the paramagnetic ion includes, but is not limited to: iron(II), cobalt(II), chromium(III), samarium(III), ytterbium(III), manganese(II), iron(III), nickel(II), copper(II), neodymium(III), gadolinium(III), and vanadium(II); the radionuclide includes, but is not limited to: 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.

[0044] Furthermore, the testing products include reagent kits, chips, and test strips.

[0045] In some embodiments, the pharmaceutical composition further comprises other therapeutic compounds that can be administered simultaneously with, or even simultaneously with, the main active ingredient in the pharmaceutical composition of the present invention. Other therapeutic compounds can also be administered alone as a separate composition or in a dosage form different from that of the main active ingredient. A portion of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered alone. During treatment, the dosage of the pharmaceutical composition of the present invention can be adjusted according to the severity of symptoms, the frequency of relapses, and the physiological response to the treatment regimen.

[0046] In some embodiments, the biopharmaceutical may further comprise a pharmaceutically acceptable carrier and / or excipient, which is described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). The present invention does not impose any particular limitation on the pharmaceutically acceptable carriers and / or excipients contained in the biopharmaceutical.

[0047] In some embodiments, the dosage forms of the biological agent include, but are not limited to: solutions, emulsions, suspensions, tablets, pills, granules, capsules, suspensions, syrups, powders, sterile aqueous solutions, non-aqueous solutions, lyophilized preparations, or suppositories.

[0048] In some embodiments, the administration methods of the pharmaceutical composition or biological agent include, but are not limited to: subcutaneous, intradermal, intravenous, intramuscular, intrapulmonary, intraperitoneal, oral, local, intranasal, and rectal administration. When administered orally, it may be formulated with a coating that protects the active ingredient in the pharmaceutical composition or biological agent from degradation in the stomach. Furthermore, the active ingredient may be administered via any device capable of delivery to the target tissue.

[0049] In some embodiments, the pharmaceutical compositions or biological agents provided by the present invention can be formulated into various dosage forms as needed, and can be administered by clinicians based on factors such as the subject's type, age, weight, general disease condition, and route of administration to determine the beneficial dosage for the patient. The route of administration can be, for example, injection or any other suitable route of administration known to those skilled in the art.

[0050] The seventh aspect of the present invention provides any of the following methods:

[0051] 1) A method for preparing recombinant host cells according to the fifth aspect of the present invention, the method comprising: introducing the expression vector according to the fourth aspect of the present invention into host cells to obtain the recombinant host cells according to the fifth aspect of the present invention.

[0052] 2) A method for producing an antibody according to the first aspect of the present invention or a bispecific antibody according to the second aspect of the present invention, the method comprising: culturing a recombinant host cell according to the fifth aspect of the present invention, and isolating the antibody or bispecific antibody from the culture product of the recombinant host cell.

[0053] 3) A method for detecting B7H3 in a test sample for non-diagnostic and non-therapeutic purposes, the method comprising: contacting the test sample with the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the antibody-drug conjugate described in the sixth aspect of the present invention, a detection reagent or a detection product, and detecting the formation of B7H3 protein-antibody immune complex.

[0054] 4) A method for inhibiting B7H3 activity in a sample in vitro, the method comprising: contacting the sample with the antibody described in the first aspect of the present invention and the bispecific antibody described in the second aspect of the present invention.

[0055] 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 B7H3 may be used as the sample or test sample.

[0056] The eighth aspect of the present invention provides any of the following applications:

[0057] 1) The use of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, or the recombinant host cell described in the fifth aspect of the present invention in the preparation of antibody-drug conjugates for detecting B7H3.

[0058] 2) The use of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, or the antibody conjugate described in the sixth aspect of the present invention in the preparation of a detection reagent for detecting B7H3.

[0059] 3) The use of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, the antibody conjugate or detection reagent described in the sixth aspect of the present invention in the preparation of detection products for detecting B7H3.

[0060] 4) The use of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, the antibody conjugate described in the sixth aspect of the present invention, the detection reagent or detection product in non-diagnostic, non-therapeutic target detection of B7H3.

[0061] 5) The use of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, the antibody conjugate described in the sixth aspect of the present invention, the detection reagent or detection product in the preparation of diagnostic products for diagnosing or assisting in the diagnosis of diseases with abnormal B7H3 expression.

[0062] 6) The use of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, or the recombinant host cell described in the fifth aspect of the present invention in the preparation of a medicament for treating and / or preventing diseases with abnormal B7H3 expression.

[0063] 7) The use of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention in the preparation of a biological agent for the treatment and / or prevention of diseases with abnormal B7H3 expression.

[0064] Furthermore, the diseases characterized by abnormal B7H3 expression include osteosarcoma, glioma, melanoma, hematologic malignancies, prostate cancer, esophageal cancer, cervical cancer, ovarian cancer, colorectal cancer, breast cancer, or lung cancer.

[0065] In some implementations, the term "B7H3 abnormal expression disease" is a general term for a class of diseases related to B7H3 abnormal expression, and is not limited to the specific disease types listed above. Any disease related to B7H3 abnormal expression will fall within the protection scope of this invention.

[0066] Advantages and beneficial effects of the present invention:

[0067] This invention provides a novel antibody targeting B7H3, which specifically binds to B7H3 with good affinity and specificity. This invention lays the foundation for the development of B7H3-related detection and / or auxiliary detection products in this field. Furthermore, this invention is expected to provide more diagnostic tools and treatment strategies for the diagnosis and / or treatment of B7H3-related diseases, demonstrating promising application prospects. Attached Figure Description

[0068] Figure 1 To verify the binding ability of the murine antibody CM0730 to B7H3; where A is immunoprecipitation (IP); B is immunohistochemistry (IHC) of frozen sections; and C is immunohistochemistry (IHC) of paraffin blocks.

[0069] Figure 2 The binding ability, affinity, and specificity of human and murine antibodies to B7H3 were verified. Specifically, A was the flow cytometry verification of the binding ability of the three antibodies to the B7H3 antigen on the surface of 143B cells; B was the ELISA verification of the binding affinity and specificity of the three antibodies to the B7H3 antigen; and C was the fluorescent probe verification of the in vivo binding ability of antibody 4KQ3 to the B7H3 antigen. Detailed Implementation

[0070] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents.

[0071] The materials and equipment used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions are typically performed under conventional conditions or as recommended by the manufacturer. Specifically, the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0072] Example 1: Preparation of an antibody targeting B7H3

[0073] Antibody CM0730 was prepared by ABclonal (Urisys (Wuhan) Biotechnology Co., Ltd.). The following is a brief description of the experimental procedure:

[0074] 1. Preparation and processing of immunogens

[0075] The immunogen was Recombinant Human B7-H3 / CD276 Protein, C-hFc&His (Abclonal, cat: #RP01020).

[0076] The original test result was Recombinant Human B7-H3 / CD276 Protein, C-His (Abclonal, cat:#RP01020).

[0077] 2. Animal immunization

[0078] 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 (C-terminal His tag, hereinafter referred to as "immunogen") was used as the immunogen. Mice were systemically immunized according to the following time points and immunization regimen:

[0079] Table 1 Animal Immunization Experiment Protocol

[0080]

[0081] 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.

[0082] 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.

[0083] 3. Cell fusion

[0084] 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.

[0085] 4. Subcloning screening

[0086] Twenty ELISA-positive fusion cells were selected for subcloning based on the ELISA binding results.

[0087] 5. Sequence Fishing

[0088] 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.

[0089] 6. Modification and preparation of humanized antibodies 4KQ3 and 3QRG

[0090] The following humanization method was used: Complementarity-Determining Region Grafting (CDR Grafting): The CDR region of the parental antibody CM0730 was grafted onto the human frame region (FR). The specific steps are as follows:

[0091] (1) Identify CDR regions: Upload the VH and VL sequences of the parental antibody to IMGT / V-QUEST. Extract the sequences of CDR1, CDR2, and CDR3 from the results (as defined by IMGT numbers).

[0092] (2) Selection of human frame region (FR): IgBLAST was used to align the FR region of the parental antibody with the human antibody database. The human FR with the highest homology (IGHV3-23*01) was selected. The sequence of the human FR was recorded.

[0093] (3) The parental CDR region is transplanted onto the human FR to form a humanized sequence.

[0094] (4) Construction and expression of humanized antibodies: Synthesize humanized VH and VL genes and clone them into expression vectors (such as pcDNA3.4). Transiently or stably express humanized antibodies in CHO cells and purify them for subsequent experiments.

[0095] 7. Experimental Results

[0096] The sequence information of the prepared antibodies targeting B7H3 is shown in Table 2.

[0097] Table 2 Sequence information of antibodies targeting B7H3

[0098]

[0099]

[0100] Example 2: Functional Study of Antibodies Targeting B7H3

[0101] 1. Verification of the binding ability of antibodies targeting B7H3

[0102] (1) Immunoprecipitation (IP) was used to verify the binding ability of antibody CM0730 to B7H3.

[0103] 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.

[0104] (2) Immunohistochemistry (IHC) was used to verify the binding ability of antibody CM0730 to B7H3.

[0105] 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.

[0106] Fresh tissue is immediately immersed in a tissue fixative or a specific fixative for the tissue and fixed for at least 24 hours before transport and storage 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. After dehydration, the tissue is removed and slightly blotted with filter paper. It is then placed cut-side up on a sample holder, and 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.

[0107] (3) Flow cytometry was used to verify the binding ability of the antibody to B7H3 on the surface of 143B cells.

[0108] 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.

[0109] 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. 51 cell / tube, 4 tubes in total (negative, CM0730, 4KQ3, 3QRG), centrifuge cells at 1200 rpm for 5 min, discard supernatant.

[0110] Resuspend cells twice in pre-chilled PBS at 4°C, and centrifuge as before. Add 100 μL PBS for negative control and 5 μL (CM0730, 4KQ3, 3QRG) + 95 μL PBS for 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) diluted 1:400 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.

[0111] (4) Fluorescent probes were used to verify the in vivo B7H3 binding ability of antibody 4KQ3.

[0112] The synthesis procedure for the 4KQ3--IRDye800CW fluorescent probe is detailed in the patent—Optical Molecular Imaging Probe for Osteosarcoma Detection and its Preparation Method and Application (Authorization Announcement No.: CN117982685B). BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the 143B cell line was used in this experiment. For the subcutaneous OS model, 20 μL of 143B cell suspension (5 × 10⁶ cells / mL) was used. 7 (4KQ3-IRDye800CW group) was subcutaneously injected into the right posterior region of mice. After the tumor grew to 1 cm, it was administered via the tail vein at a dose of 1 μg / g body weight. At 2, 4, 8, 12, 24, 48 and 72 hours after administration of the probe, mice were first anesthetized with 2.5% isoflurane (2.5 L / min), and then NIR-I (850 nm) and NIR-II (1000 nm) fluorescence images were captured.

[0113] 2. Validation of the affinity and specificity of antibodies targeting B7H3

[0114] 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; the antibody concentrations (CM0730, 4KQ3, 3QRG) were diluted 4-fold at 0.5 μg / ml, and the plates were incubated at 37°C at 600 rpm for 1 h; the plates were washed 5 times, and the 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.

[0115] 3. Experimental Results

[0116] Immunoprecipitation (IP) and immunohistochemistry (IHC) results showed that the murine antibody CM0730 could effectively bind to the B7H3 antigen ( Figure 1 A, Figure 1 B. Figure 1 C). Subsequently, we demonstrated by flow cytometry that the murine antibody CM0730 and the humanized antibodies 4KQ3 and 3QRG had a strong specific binding ability to the B7H3 antigen on the surface of 143B cells, with the two humanized antibodies showing stronger binding ability to the 143B surface antigen. Figure 2 A). ELISA results showed that all three antibodies had high affinity for the B7H3 antigen, with an EC50% (half-maximal effective concentration). 50 The values ​​were CM0730: 0.01020 mg / ml; 4KQ3: 0.008758 mg / ml; 3QRG: 0.05768 mg / ml. Figure 2 (B) We then used the B7H1 antigen, which has a similar structure to the B7H3 antigen, to verify the specificity of the antibody. We found that CM0730 and 4KQ3 had no binding activity against B7H1, proving their binding specificity. Figure 2B). Next, we used the humanized antibody 4KQ3 to verify the in vivo binding of B7H3 with a fluorescent probe. The results showed that the 4KQ3 antibody could effectively bind to the B7H3 antigen on the surface of 143B cells in mice. Figure 2 C).

[0117] The above results collectively demonstrate that the mouse antibody and its corresponding two humanized antibodies prepared in this invention have strong binding ability to the B7H3 antigen and can detect the expression of B7H3 in in vivo and in vitro samples. Among them, the mouse antibody CM0730 and the humanized antibody 4KQ3 have specific binding to B7H3.

[0118] 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 B7H3 antibody, characterized in that, The heavy chain variable region CDR1 of the antibody is shown in SEQ ID NO:1; CDR2 is shown in SEQ ID NO:2; CDR3 is shown in SEQ ID NO:3; the light chain variable region CDR1 of the antibody is shown in SEQ ID NO:5; CDR2 is shown in SEQ ID NO:6; CDR3 is shown in SEQ ID NO:

7. Alternatively, the heavy chain variable region CDR1 of the antibody is shown as SEQ ID NO:9; CDR2 is shown as SEQ ID NO:2; CDR3 is shown as SEQ ID NO:3; the light chain variable region CDR1 of the antibody is shown as SEQ ID NO:5; CDR2 is shown as SEQ ID NO:6; CDR3 is shown as SEQ ID NO:

7. Alternatively, the heavy chain variable region CDR1 of the antibody is shown as SEQ ID NO:12; CDR2 is shown as SEQ ID NO:2; CDR3 is shown as SEQ ID NO:3; the light chain variable region CDR1 of the antibody is shown as SEQ ID NO:14; CDR2 is shown as SEQ ID NO:6; CDR3 is shown as SEQ ID NO:

7.

2. The antibody according to claim 1, characterized in that, The heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 75% homology with SEQ ID NO:4; 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% homology with SEQ ID NO:

8. Alternatively, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 75% homology with SEQ ID NO:10; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 75% homology with SEQ ID NO:

11. Alternatively, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 75% homology with SEQ ID NO:13; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 75% homology with SEQ ID NO:

15.

3. A bispecific antibody, characterized in that, The bispecific antibody comprises the antibody as described in any one of claims 1 or 2.

4. The bispecific antibody according to claim 3, characterized in that, The bispecific antibody also includes a second antibody that specifically binds to other antigens.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody as described in any one of claims 1 or 2, or the bispecific antibody as described in any one of claims 3 or 4.

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

7. A recombinant host cell, characterized in that, The recombinant host cell comprises the expression vector of claim 6.

8. The recombinant host cell according to claim 7, characterized in that, The recombinant host cells include prokaryotic cells and eukaryotic cells.

9. Any of the following products, characterized in that: The products include: 1) An antibody conjugate, wherein the antibody conjugate is a complex formed by directly or indirectly conjugating the antibody of any one of claims 1 or 2 or the bispecific antibody of any one of claims 3 or 4 to a detectable marker; 2) A detection reagent comprising the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in any one of claims 3 or 4, or the antibody conjugate thereof; 3) A detection product comprising the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in any one of claims 3 or 4, the antibody-drug conjugate, or the detection reagent; 4) A pharmaceutical composition comprising the antibody as described in any one of claims 1 or 2 or the bispecific antibody as described in any one of claims 3 or 4; 5) A biological agent comprising the pharmaceutical composition.

10. The product according to claim 9, characterized in that, The detectable markers include bioluminescent agents, chemiluminescent agents, enzymes, photosensitizing diagnostic agents, paramagnetic ions, or radionuclides.

11. The product according to claim 9, characterized in that, The testing products include reagent kits, chips, and test strips.

12. Any of the following methods, characterized in that, The method includes: 1) A method for preparing the recombinant host cell according to claim 7, the method comprising: introducing the expression vector according to claim 6 into a host cell to obtain the recombinant host cell according to claim 7; 2) A method for producing an antibody as described in any one of claims 1 or 2, or a bispecific antibody as described in any one of claims 3 or 4, the method comprising: culturing the recombinant host cell as described in claim 7, and isolating the antibody or bispecific antibody from the culture product of the recombinant host cell; 3) A method for detecting B7H3 in a test sample for non-diagnostic and non-therapeutic purposes, the method comprising: contacting the test sample with the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in any one of claims 3 or 4, the antibody conjugate as described in claim 9, a detection reagent or a detection product, and detecting the formation of an immune complex between the B7H3 protein and the antibody; 4) A method for inhibiting B7H3 activity in a sample in vitro for non-diagnostic, non-therapeutic purposes, the method comprising: contacting the sample with an antibody as described in any one of claims 1 or 2 or a bispecific antibody as described in any one of claims 3 or 4.

13. The following application, characterized in that, The applications include: 1) The use of the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in any one of claims 3 or 4, the nucleic acid molecule as described in claim 5, the expression vector as described in claim 6, or the recombinant host cell as described in claim 7 in the preparation of an antibody-drug conjugate for the detection of B7H3; 2) The use of the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in any one of claims 3 or 4, the nucleic acid molecule as described in claim 5, the expression vector as described in claim 6, the recombinant host cell as described in claim 7, or the antibody conjugate as described in claim 9 in the preparation of a detection reagent for detecting B7H3; 3) The use of the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in any one of claims 3 or 4, the nucleic acid molecule as described in claim 5, the expression vector as described in claim 6, the recombinant host cell as described in claim 7, the antibody conjugate or detection reagent as described in claim 9 in the preparation of a detection product for detecting B7H3; 4) The use of the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in any one of claims 3 or 4, the nucleic acid molecule as described in claim 5, the expression vector as described in claim 6, the recombinant host cell as described in claim 7, the antibody conjugate as described in claim 9, the detection reagent or detection product in non-diagnostic, non-therapeutic target detection of B7H3; 5) The use of the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in any one of claims 3 or 4, the nucleic acid molecule as described in claim 5, the expression vector as described in claim 6, the recombinant host cell as described in claim 7, the antibody conjugate as described in claim 9, the detection reagent or detection product in the preparation of diagnostic products for the diagnosis or auxiliary diagnosis of diseases with abnormal B7H3 expression; 6) The use of the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in any one of claims 3 or 4, the nucleic acid molecule as described in claim 5, the expression vector as described in claim 6, or the recombinant host cell as described in claim 7 in the preparation of a medicament for the treatment of osteosarcoma; 7) The use of the antibody of any one of claims 1 or 2, the bispecific antibody of any one of claims 3 or 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the recombinant host cell of claim 7, or the pharmaceutical composition of claim 9 in the preparation of a biological agent for the treatment of osteosarcoma.

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