B7H3 targeting antibody and application thereof

Abstract

The invention discloses an antibody targeting B7H3 and an application of the antibody. The invention provides a brand new B7H3 targeting antibody for the field, and the antibody can specifically bind to B7H3 and has good affinity and specificity. A foundation is laid for developing detection and / or auxiliary detection products related to B7H3 in the field, meanwhile, more diagnostic tools and treatment strategies are expected to be provided for diagnosis and / or treatment of B7H3 related diseases, and good application prospects are achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an antibody targeting B7H3 and application thereof. BACKGROUND

[0002] B7H3 (also known as CD276) is a type I membrane protein, and its sequence is similar to the extracellular domain of PD-L1 (also known as B7H1). The RNA of B7H3 is widely expressed in lymphoid and non-lymphoid organs, and can act as a costimulatory molecule to CD4 + and CD8 + T cell proliferation. B7H3 signaling induces cellular immunity, which can promote T cell activation and IFN-gamma proliferation. B7H3 is abnormally expressed in various cancers, including lung cancer, prostate cancer, esophageal cancer, ovarian cancer, cervical cancer, colorectal cancer, breast cancer, osteosarcoma, etc. A large number of studies have confirmed that B7H3-mediated non-immune response plays an important role in affecting host cell biology.

[0003] B7H3 antibodies are widely used in scientific research and medical fields. In scientific research, they can be used to identify and sort cells expressing B7H3, and to study cell function and disease mechanisms. In medicine, they can be used as diagnostic markers to assist in the diagnosis of breast cancer, osteosarcoma and other diseases. Moreover, B7H3 antibodies are expected to be developed as therapeutic drugs to restore the body's anti-tumor immune response by blocking the immune suppression function of B7H3. Therefore, the development and research of antibodies targeting B7H3 have important clinical and scientific research value. At present, there is an urgent need in the field to develop an antibody product with good affinity and specificity to B7H3. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a new antibody specifically binding to B7H3 and related products and uses thereof in the field.

[0005] The technical scheme adopted by the present application to achieve the above-mentioned purpose of the application is as follows:

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

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

[0008] or, the CDR1 of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 9; the CDR2 is as shown in SEQ ID NO: 2; the CDR3 is as shown in SEQ ID NO: 3; the CDR1 of the light chain variable region of the antibody is as shown in SEQ ID NO: 5; the CDR2 is as shown in SEQ ID NO: 6; the CDR3 is as shown in SEQ ID NO: 7;

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

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

[0011] or, the heavy chain variable region of the antibody contains an amino acid sequence as 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 an amino acid sequence as 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, the CDR1, CDR2, CDR3 can be defined by any one or any combination (two or more) of IMGT numbering scheme, Chothia numbering scheme, Kabat numbering scheme, Martin (enhanced Chothia) numbering scheme, AbM numbering scheme, Aho numbering scheme (or other existing CDR numbering schemes or new CDR numbering schemes that can be generated in the future) on the corresponding amino acid sequences of the light chain or heavy chain of the antibody of the present application as described above, and the CDR1, CDR2, CDR3 defined by the above definition methods also fall within the scope of the present application.

[0013] In some embodiments, functional variants of the B7H3 antibodies provided by the first aspect of the present application are also encompassed by the scope of the present application.

[0014] In some embodiments, the functional variant refers to a protein having substantial or significant homology or similarity to the parent antibody (the B7H3 antibody described in the first aspect of the present application), which retains the biological activity of the parent antibody.

[0015] The functional variant encompasses, for example, a variant of the B7H3 antibody (parent antibody) described herein, which retains the ability to recognize the target cell to a similar degree, to the same degree, or to a higher degree as compared to the parent antibody. With reference to the parent antibody, the functional variant may, for example, have at least about 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or more homology in the amino acid sequence with the parent antibody.

[0016] In some embodiments, the functional variant may, for example, comprise the amino acid sequence of the parent antibody with at least one conservative amino acid substitution. Alternatively or additionally, the functional variant may comprise the amino acid sequence of the parent antibody with at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is improved as compared to the parent antibody. Such a functional variant obtained by conservative amino acid substitution or non-conservative amino acid substitution based on the parent antibody is also within the scope of the present application.

[0017] In some embodiments, conservative amino acid substitutions are known in the art and include the substitution of one amino acid for another amino acid having similar chemical or physical properties. For example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituted for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituted for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side chain substituted for another amino acid with a beta-branched side chain (e.g., He, Thr, and Val), an amino acid with an aromatic side chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr).

[0018] In some embodiments, the homology refers to sequence similarity to the target amino acid sequence or nucleotide sequence. Homology includes an amino acid sequence that has 75% or more, or 85% or more, or 90% or more, or 95% or more homology to the amino acid sequence of the antibody provided herein. Homology can be assessed by eye or by computer software. When computer software is used, the homology between two or more sequences can be expressed as a percentage (%) that can be used to assess the homology between related sequences. The 75% or more homology can be 75%, 80%, 85%, 90%, or 95% or more homology.

[0019] In some embodiments, modification of one or more amino acids in a protein will not typically affect the function of the protein. Those skilled in the art will recognize that changing a single amino acid or a small percentage of amino acids or making individual additions, deletions, insertions, substitutions to an amino acid sequence are conservative modifications in which the altered protein produces a protein with similar function. Tables of conservative substitution of amino acids that provide functionally similar amino acids are well known in the art. Such amino acid sequences that result from conservative substitutions are also included within the scope of the present application.

[0020] Substitutions, deletions, insertions or any combination thereof can be made to arrive at a final conjugate or variant. Generally these changes are made in a few amino acids to minimize changes to the molecule, particularly to the immunogenicity and specificity of the antigen binding protein. However, in some cases larger changes can be tolerated. Amino acid substitutions are typically of single residues; insertions are typically of the order of about one to about twenty amino acid residues, although substantially larger insertions can be tolerated. Deletions range from about one to about twenty amino acid residues, although in some cases much larger deletions can be tolerated.

[0021] The second aspect of the present application provides a bispecific antibody comprising the antibody of the first aspect of the present application.

[0022] Further, the bispecific antibody further comprises a second antibody which specifically binds to another antigen.

[0023] In some embodiments, the second antibody which specifically binds to another antigen is not particularly limited, which can be another antibody targeting B7H3 or an antibody targeting any antigen other than B7H3, and can be routinely selected by those skilled in the art according to actual needs.

[0024] The third aspect of the present application provides a nucleic acid molecule encoding the antibody of the first aspect of the present application or the bispecific antibody of the second aspect of the present application.

[0025] Those skilled in the art can easily mutate the nucleotide sequence corresponding to the antibody of the present application using known methods, for example, methods of directed evolution and point mutation, as long as the nucleotide sequence encoding the antibody of the first aspect of the present application or the bispecific antibody of the second aspect of the present application is derived from the nucleotide sequence of the present application and is equivalent to the sequence of the present application, which also falls within the scope of the present application.

[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 using hybridoma technology. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities using recombinant methods. This is usually by cloning into a vector, transferring into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. In addition, the relevant sequence can be synthesized using artificial synthesis methods, especially when the length of the fragment is short. Generally, longer fragments of the sequence can be obtained by first synthesizing a plurality of small fragments and then connecting them.

[0027] The fourth aspect of the present application provides an expression vector comprising the nucleic acid molecule of the third aspect of the present application.

[0028] In some embodiments, the vector includes, but is not limited to, plasmid, phagemid, cosmid, artificial chromosome, virus-derived vector. The virus-derived vector includes, but is not limited to, lentivirus vector, retrovirus vector, adenovirus vector, adeno-associated virus vector, poxvirus vector, herpesvirus vector, baculovirus vector, etc.

[0029] In some embodiments, the expression vector can be constructed by 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 linked to a multiple cloning site in the vector to direct mRNA synthesis and thus express the protein, or for homologous recombination.

[0030] The fifth aspect of the present application provides a recombinant host cell comprising the expression vector of the fourth aspect of the present application.

[0031] Further, the recombinant host cell includes prokaryotic cell, eukaryotic cell.

[0032] In some embodiments, the prokaryotic cell includes bacteria, actinomycete, cyanobacterium, mycoplasma, chlamydia, rickettsia.

[0033] In some embodiments, the bacteria includes Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, Staphylococcus.

[0034] In some embodiments, the eukaryotic cell includes mammalian cell, insect cell, plant cell, yeast cell.

[0035] In some embodiments, the recombinant host cell is prepared by introducing the nucleic acid molecule or expression vector of the present application as described above into a host cell, and the method of introduction includes, but is not limited to, physical method, chemical method, biological method. The physical method includes, but is not limited to, microinjection, electroporation, calcium phosphate precipitation, lipofection, particle bombardment; the chemical method includes, but is not limited to, colloidal dispersion system, lipid-based system; the colloidal dispersion system includes, but is not limited to, macromolecular complex, nanocapsule, microsphere, bead; the lipid-based system includes, but is not limited to, oil-in-water emulsion, micelle, mixed micelle, liposome; the biological method includes, but is not limited to, DNA vector, lentivirus vector, poxvirus vector, herpes simplex virus vector, adenovirus vector, adeno-associated virus vector.

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

[0037] 1) An antibody conjugate which is a complex formed by direct or indirect conjugation of the antibody of the first aspect of the application or the bispecific antibody of the second aspect of the application to a detectable label.

[0038] 2) A detection reagent comprising the antibody of the first aspect of the application, the bispecific antibody of the second aspect of the application or the antibody conjugate.

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

[0040] 4) A pharmaceutical composition comprising the antibody of the first aspect of the application or the bispecific antibody of the second aspect of the application.

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

[0042] Further, the detectable label includes a bioluminescent agent, a chemiluminescent agent, an enzyme, a photosensitive diagnostic agent, a paramagnetic ion or a radionuclide.

[0043] In some embodiments, the bioluminescent agent includes, but is not limited to, a luciferin, a luciferase, an aequorin; the chemiluminescent agent includes, but is not limited to, an imidazole, a luminol, an isoluminol, an aromatic acridinium ester, an acridinium salt, an oxalate ester; the enzyme includes, but is not limited to, a catalase, an alkaline phosphatase, a urease, a horseradish peroxidase, a glucose oxidase, a beta-D-galactosidase, or a glucoamylase; the photosensitive diagnostic agent includes, but is not limited to, a protoporphyrin, a hematoporphyrin, a methylene blue, a dihydroxy silicon phthalocyanine, a photofrin; 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), 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] Further, the detection products include kits, chips, test strips.

[0045] In some embodiments, the pharmaceutical composition further comprises other therapeutic compounds, which can be administered simultaneously with the primary active ingredient in the pharmaceutical composition of the present application, even in the same composition. The other therapeutic compounds can also be administered separately in a separate composition or in a different dosage form from the primary active ingredient. Part of the dosage of the primary ingredient can be administered simultaneously with the other therapeutic compounds, while other dosages can be administered separately. During the course of treatment, the dosage of the pharmaceutical composition of the present application can be adjusted according to the severity of the symptoms, the frequency of recurrence, and the physiological response of the treatment regimen.

[0046] In some embodiments, the biological agent can further comprise a pharmaceutically acceptable carrier and / or excipient, which is described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995), and the present application does not have a particular limitation on the pharmaceutically acceptable carrier and / or excipient contained in the biological agent.

[0047] In some embodiments, the dosage form of the biological agent includes, but is not limited to, a solution, an emulsion, a suspension, an emulsion, a tablet, a pill, a granule, a capsule, a suspension, a syrup, a powder, a sterile aqueous solution, a non-aqueous solution, a lyophilized preparation, or a suppository.

[0048] In some embodiments, the administration method of the pharmaceutical composition or biological agent includes, but is not limited to, subcutaneous, intradermal, intravenous, intramuscular, intrapulmonary, intraperitoneal, oral, topical, intranasal, rectal, etc. When administered orally, it can be formulated with a coating to protect the active ingredients in the pharmaceutical composition or biological agent from degradation in the stomach. In addition, the active ingredients can be administered by any device capable of transferring to the target tissue.

[0049] In some embodiments, the pharmaceutical composition or biological agent provided by the present application can be prepared into various dosage forms according to actual needs, and the dosage beneficial to the patient can be determined by the clinician according to the species, age, weight and general disease condition of the subject, administration method, etc. The administration method can be, for example, injection or any suitable administration method known to those skilled in the art.

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

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

[0052] 2) A method for producing the antibody of the first aspect of the present application or the bispecific antibody of the second aspect of the present application, the method comprising: culturing the recombinant host cell of the fifth aspect of the present application, 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 sample to be tested for non-diagnostic and non-therapeutic purposes, the method comprising: contacting the sample to be tested with the antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, the antibody conjugate of the sixth aspect of the present application, a detection reagent or a detection product, and detecting the formation of an immune complex of B7H3 protein and the antibody.

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

[0055] In some embodiments, the sample or sample to be tested can be selected from blood, serum, plasma, urine, saliva, ascites, brain tissue, cerebrospinal fluid, non-tissue associated cells, tissues, histological preparations, etc. from a subject to be tested, and the present application does not particularly limit the specific type of the sample or sample to be tested, and any sample that can contain B7H3 can be used as the sample or sample to be tested.

[0056] The eighth aspect of the present application provides any one of the following uses:

[0057] 1) Use of the antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the expression vector of the fourth aspect of the present application, or the recombinant host cell of the fifth aspect of the present application in the preparation of an antibody conjugate for detecting B7H3.

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

[0059] 3) The antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the expression vector of the fourth aspect of the present application, the recombinant host cell of the fifth aspect of the present application, the antibody conjugate of the sixth aspect of the present application or the detection reagent are used for preparing a detection product for detecting B7H3.

[0060] 4) The antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the expression vector of the fourth aspect of the present application, the recombinant host cell of the fifth aspect of the present application, the antibody conjugate of the sixth aspect of the present application, the detection reagent or the detection product are used for detecting B7H3 for non-diagnostic and non-therapeutic purposes.

[0061] 5) The antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the expression vector of the fourth aspect of the present application, the recombinant host cell of the fifth aspect of the present application, the antibody conjugate of the sixth aspect of the present application, the detection reagent or the detection product are used for preparing a diagnostic product for diagnosing or assisting in diagnosing a disease with abnormal expression of B7H3.

[0062] 6) The antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the expression vector of the fourth aspect of the present application or the recombinant host cell of the fifth aspect of the present application are used for preparing a drug for treating and / or preventing a disease with abnormal expression of B7H3.

[0063] 7) The antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the expression vector of the fourth aspect of the present application, the recombinant host cell of the fifth aspect of the present application or the pharmaceutical composition of the sixth aspect of the present application are used for preparing a biological agent for treating and / or preventing a disease with abnormal expression of B7H3.

[0064] Further, the disease with abnormal expression of B7H3 includes osteosarcoma, glioma, melanoma, hematological malignancy, prostate cancer, esophageal cancer, cervical cancer, ovarian cancer, colorectal cancer, breast cancer or lung cancer.

[0065] In some embodiments, the disease with abnormal expression of B7H3 is a general term of a class of diseases related to abnormal expression of B7H3, and is not limited to the specific disease types listed above, and any disease related to abnormal expression of B7H3 will fall within the protection scope of the present application.

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

[0067] The present application provides a brand new antibody targeting B7H3 for the field, which can specifically bind to B7H3 and has good affinity and specificity. The present application lays a foundation for the development of B7H3 related detection and / or auxiliary detection products in the field, and at the same time, the present application is expected to provide more diagnostic tools and treatment strategies for the diagnosis and / or treatment of B7H3 related diseases, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 Verification of the binding ability of mouse-derived antibody CM0730 to B7H3; wherein A is immunoprecipitation (IP); B is immunohistochemistry (IHC) of frozen sections; C is immunohistochemistry (IHC) of wax blocks.

[0069] Figure 2 Verification of the binding ability, affinity and specificity of human-derived and mouse-derived antibodies to B7H3; wherein A is flow cytometry verification of the binding ability of three antibodies to B7H3 antigen on the surface of 143B cells; B is ELISA verification of the binding affinity and specificity of three antibodies to B7H3 antigen; C is fluorescence probe verification of the in vivo binding ability of antibody 4KQ3 to B7H3 antigen. DETAILED DESCRIPTION

[0070] The present application will be further described below in conjunction with specific embodiments, which are only used to explain the present application and cannot be understood as limiting the present application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents.

[0071] The actual materials and raw materials used in the present application are easily obtained by those skilled in the art, and if not specially stated, they can be obtained from commercial channels, and the experimental methods of the present application not specified in the present application are generally carried out according to the conventional conditions in the field or according to the conditions suggested by the manufacturer. In particular, the following examples are only used to illustrate the present application and should not limit the scope of the present application in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present application and should not and will not limit the present application described in detail in the claims.

[0072] Example 1 Preparation of antibody targeting B7H3

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

[0074] 1. Preparation and treatment of immunogen

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

[0076] The detection origin is Recombinant Human B7-H3 / CD276 Protein, C-His (Abclonal, cat: #RP01020).

[0077] 2. Animal immunization

[0078] Five healthy Balb / C mice were selected as immunized animals, and all mice were in 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 immunogen. The mice were immunized according to the following time nodes and immunization scheme:

[0079] Table 1 Animal immunization experiment scheme

[0080]

[0081] After the fourth immunization and challenge immunization, the mice were bled, and about 15-30 μl of peripheral blood was collected per mouse each time for the preparation and detection of subsequent polyclonal antibody serum.

[0082] Enzyme-linked immunosorbent assay (ELISA) was used to detect the antibody titer of the serum samples collected after the fourth and fifth (challenge immunization) times. The same immunogen (Human B7-H3 / CD276 Protein, C-His) was used as the coating antigen to evaluate the binding activity of specific polyclonal antibodies in the serum.

[0083] 3. Cell fusion

[0084] Two mice with high specific polyclonal antibody binding activity (one for further development of antibodies and one for standby) were selected. The mouse spleen was fused with mouse sarcoma cell line (SP2 / 0), a total of 20 96-well cell plates, HAT medium for the first screening, and the screening origin for the second screening. ELISA was used to screen fusion supernatant that specifically recognized the screening origin. ELISA positive clones (OD 450 The value is greater than or equal to 5 times the background value), a total of 80 positive clones were selected.

[0085] 4. Subcloning screening

[0086] According to the ELISA binding results, 20 ELISA positive fusion cells were selected for subcloning.

[0087] 5. Sequence fishing

[0088] Subclone cells were collected and lysed to extract RNA. The extracted RNA was used as a template to obtain cDNA in two steps using a reverse transcription kit. The cDNA obtained by reverse transcription was used as a template to amplify by PCR, and the amplified fragments were inserted into an expression vector or a commercial vector to obtain a plasmid containing the correct sequence by sequencing.

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

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

[0091] (1) Identify the CDR region: upload the VH and VL sequences of the parent antibody to IMGT / V-QUEST. Extract the sequences of CDR1, CDR2, and CDR3 from the results (defined according to IMGT numbering).

[0092] (2) Select human framework region (FR): compare the FR region of the parent antibody with the human antibody database using IgBLAST. Select the human FR with the highest homology (IGHV3-23*01). Record the sequence of the human FR.

[0093] (3) Graft the parent CDR region onto the human FR to form the sequence after humanization.

[0094] (4) Construction and expression of humanized antibody: synthesize humanized VH and VL genes, and clone them into an expression vector (such as pcDNA3.4). Express the humanized antibody in CHO cells transiently or stably, and purify it for subsequent experiments.

[0095] 7. Experimental results

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

[0097] Table 2 Sequence information of the antibody targeting B7H3

[0098]

[0099]

[0100] Example 2 Functional study of the antibody targeting B7H3

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

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

[0103] Human osteosarcoma cell line 143B used in the experiment was purchased from the China Academy of Sciences Cell Bank, catalog number TCHu264. Cells were cultured in DMEM (Gibco, C11995500BT) complete medium containing 10% fetal bovine serum (Wisent, 085-150) at 37°C, 5% CO2, and a humidified environment. IP experiments were completed using an immunoprecipitation kit (Protein A+G Agarose Gel method) (Bi Yun Tian, P2197S). Cell lysis, antibody (antibody concentration 50 μg / ml) binding to Protein A+G Agarose Gel, immunoprecipitation, and elution were completed according to the instructions provided with the kit. WB was then used for verification.

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

[0105] Paraffin sections were all derived from the bone tumor department specimen library of Peking University People's Hospital, 4 μm, white sheets stored at -20°C. Immunohistochemical staining was completed on a Bond RX staining machine to remove wax, recover antigen, block endogenous peroxidase, wash, incubate primary antibody (CM0730 concentration 1:100), incubate secondary antibody (DS9800), develop with DAB, restain, dehydrate, clear, and mount.

[0106] Fresh tissues were immediately put into tissue fixative or corresponding special fixative for 24 hours or more, and stored and transported at room temperature. The target tissue was trimmed flat with a scalpel after the tissue was removed from the fixative. The tissue part for frozen section was cut to a thickness of no more than 3 mm, and the length and width of the tissue were no more than 2 cm. The dehydrated tissue was placed on a sample holder with the surface facing up after the surface water was absorbed with filter paper. OCT embedding agent (Yinghua, 4583) was dropped around the tissue, and the sample holder was placed on a quick-freezing table for rapid freezing and embedding. The sample holder was fixed on the sample head of a frozen section machine. The tissue was roughly trimmed to a thickness of 40 μm, and then finely trimmed to a thickness of 8 μm. The tissue was cut to a thickness of 8 μm. The section was taken from the freezer and placed in pre-cooled acetone for 10 minutes in a 4°C refrigerator. After acetone fixation, the glass slide was taken out and air-dried at room temperature for 10-20 minutes. Then the glass slide was soaked in PBS for 5-10 minutes to hydrate the tissue and wash away the fixative. 3% hydrogen peroxide (H2O2) solution was added to cover the tissue. After incubation, the tissue was washed with PBS for 3 times, each for 2 minutes. Blocking solution (normal goat serum for blocking, Servicebio, G1208) was added for blocking for about 30 minutes. CM0730 diluted antibody (concentration 1:100) was added, and the glass slide was incubated at room temperature for 2 hours. MicroStacker™ RX multi-HRP enzyme-labeled secondary antibody polymer (CELNOVTE, CSM1003) was added, and the glass slide was incubated at room temperature for 30 minutes. The glass slide was washed with PBS for 3 times. DAB (diaminobenzidine) developing solution (CELNOVTE, CSK5001) was added, and hematoxylin was added for re-staining. The glass slide was dehydrated with 75%, 85%, 95%, and 100% alcohol, respectively, and transparentized with xylene. The glass slide was sealed with neutral resin.

[0107] (3) Verification of the binding ability of the antibody to B7H3 on the surface of 143B cells by flow cytometry

[0108] The human osteosarcoma cell line 143B used in the experiment was purchased from the Chinese Academy of Sciences Cell Bank, 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 humidified environment with 5% CO2.

[0109] After the supernatant was discarded, the cells were washed with PBS (Gibco, C10010500BT) for 2 times. The cells were digested with trypsin (Gibco, 25200-072) for 2 minutes, and then the digestion was terminated with complete medium. 20 μl of the cell suspension was taken for counting, and 2×10 5Cells / tube, a total of 4 tubes (negative, CM0730, 4KQ3, 3QRG), centrifuge cells at 1200 rpm for 5 min, discard the supernatant.

[0110] Resuspend the cells with 4°C pre-cooled PBS twice, and centrifuge as before. Add 100 μL PBS for negative control, 5 μL (CM0730, 4KQ3, 3QRG) + 95 μL PBS for positive control, 4°C incubate for 1 h, repeat PBS washing and centrifugation for a total of 3 times. Add 100 μl 1:400 prepared secondary antibody (YSFluor™ 488 Goat Anti-Mouse IgG (H+L), 33206ES60) to each tube, 4°C incubate for 1 h, repeat PBS washing and centrifugation for a total of 3 times. Resuspend the cells with 100 μL PBS, and then detect on the BD Accri@ C6 flow cytometer.

[0111] (4) In vivo B7H3 binding ability of antibody 4KQ3 verified by fluorescent probe

[0112] 4KQ3-IRDye800CW fluorescent probe synthesis process is shown in the patent - Optical molecular imaging probe for osteosarcoma detection and preparation method and application (authorized publication number: CN117982685B). BALB / C-nude mice were purchased from Beijing Vito Lihua Experimental Animal Technology Co., Ltd., and 143B cell line was used in this experiment. For subcutaneous OS model, 20 μL of 143B cell suspension (5×10 7 cells per milliliter) was injected subcutaneously into the right rear of the mouse. After the tumor grew to 1 cm, 4KQ3-IRDye800CW group was injected through the tail vein, with a dose of 1 μg per gram of body weight. At 2, 4, 8, 12, 24, 48 and 72 hours after administration of the probe, the 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. Affinity and specificity verification of antibodies targeting B7H3

[0114] ELISA verification of antibody affinity and specificity: add 0.2 μg / ml antigen 100 μl (Recombinant Human B7-H3 / CD276 Protein (Abclonal, cat: #RP01020); Recombinant Human B7-H1 / PD-L1 / CD274 Protein (Abclonal, cat: #RP00068) to a 96-well enzyme-labeled plate (LABSELECT, 31121), respectively, 4°C for 16 h; wash the plate 3 times with PBST (PBS + 0.05% Tween), add blocking solution (SW3015) 300 μl / well, 37°C blocking for 1.5 hours; wash the plate 3 times; 0.5 μg / ml antibody concentration (CM0730, 4KQ3, 3QRG) 4-fold dilution, 37°C 600 rpm for 1 hour, wash the plate 5 times, add enzyme-labeled secondary antibody HRP-IgG (H+L Mouse / human) (A0350 / A0201) 1:250, 37°C 60 rpm for 1 h, wash the plate 5 times, add TMB (Solarbio, PR1200) 100 μl / well, incubate at room temperature for 30 s, add 50 μl / well stop solution (Solarbio, C1058), place the enzyme-labeled plate on the enzyme-labeled instrument to measure the OD value at 450 / 620 nm.

[0115] 3. Experimental results

[0116] Immunoprecipitation (IP) and immunohistochemistry (IHC) results show that the murine antibody CM0730 can effectively bind to B7H3 antigen Figure 1 A, Figure 1 B, Figure 1 C). Subsequently, we proved by flow cytometry that the murine antibody CM0730 and the humanized antibodies 4KQ3 and 3QRG have strong specific binding ability to B7H3 antigen on the surface of 143B cells, and the binding ability of the two humanized antibodies to 143B surface antigen is stronger Figure 2 A). ELISA results show that the three antibodies have high affinity to B7H3 antigen, and the half effective concentration EC 50 values are CM0730: 0.01020 mg / ml; 4KQ3: 0.008758 mg / ml; 3QRG: 0.05768 mg / ml Figure 2 B), we also verified the specificity of the antibody by using B7H1 antigen similar in structure to B7H3 antigen, and found that CM0730 and 4KQ3 had no binding activity to B7H1, proving their binding specificity Figure 2B). Next, we use the humanized antibody 4KQ3 to verify the in vivo binding of B7H3, the results show that the 4KQ3 antibody can effectively bind to the B7H3 antigen on the surface of 143B cells in mice (Fig. 2B). Figure 2 C).

[0117] The above results jointly prove that the mouse-derived antibody and the two corresponding humanized antibodies prepared by the present application all have strong binding capacity to the B7H3 antigen, and can detect the expression of B7H3 in in-vivo and in-vitro samples, wherein the binding of the mouse-derived antibody CM0730 and the humanized antibody 4KQ3 to B7H3 is specific.

[0118] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that, for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. A B7H3 antibody, characterized by, the heavy chain variable region CDR1 of the antibody is as shown in SEQ ID NO: 1; CDR2 is as shown in SEQ ID NO: 2; CDR3 is as shown in SEQ ID NO: 3; the light chain variable region CDR1 of the antibody is as shown in SEQ ID NO: 5; CDR2 is as shown in SEQ ID NO: 6; CDR3 is as shown in SEQ ID NO: 7; or, the heavy chain variable region CDR1 of the antibody is as shown in SEQ ID NO: 9; CDR2 is as shown in SEQ ID NO: 2; CDR3 is as shown in SEQ ID NO: 3; the light chain variable region CDR1 of the antibody is as shown in SEQ ID NO: 5; CDR2 is as shown in SEQ ID NO: 6; CDR3 is as shown in SEQ ID NO: 7; or, the heavy chain variable region CDR1 of the antibody is as shown in SEQ ID NO: 12; CDR2 is as shown in SEQ ID NO: 2; CDR3 is as shown in SEQ ID NO: 3; the light chain variable region CDR1 of the antibody is as shown in SEQ ID NO: 14; CDR2 is as shown in SEQ ID NO: 6; CDR3 is as shown in SEQ ID NO:

7.

2. The antibody of claim 1, wherein the heavy chain variable region of the antibody contains an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence with at least 75% homology to SEQ ID NO: 4; the light chain variable region contains an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence with at least 75% homology to SEQ ID NO: 8; or, the heavy chain variable region of the antibody contains an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence with at least 75% homology to SEQ ID NO: 10; the light chain variable region contains an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence with at least 75% homology to SEQ ID NO: 11; or, the heavy chain variable region of the antibody contains an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence with at least 75% homology to SEQ ID NO: 13; the light chain variable region contains an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence with at least 75% homology to SEQ ID NO:

15.

3. A bispecific antibody, characterized in that, the bispecific antibody comprises the antibody as claimed in any one of claims 1 or 2; Preferably, the bispecific antibody further comprises a second antibody that specifically binds to another antigen.

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

5. An expression vector, characterized by, the expression vector comprises the nucleic acid molecule as claimed in claim 4.

6. A recombinant host cell, characterized in that, the recombinant host cell comprises the expression vector as claimed in claim 5; Preferably, the recombinant host cell comprises a prokaryotic cell, a eukaryotic cell.

7. A product characterized by any one of the product comprises: 1) an antibody conjugate, which is a complex formed by directly or indirectly conjugating the antibody of any one of claims 1 or 2 or the bispecific antibody of claim 3 to a detectable label; 2) a detection reagent comprising the antibody of any one of claims 1 or 2, the bispecific antibody of claim 3 or the antibody conjugate; 3) a detection product comprising the antibody of any one of claims 1 or 2, the bispecific antibody of claim 3, the antibody conjugate or the detection reagent; 4) a pharmaceutical composition comprising the antibody of any one of claims 1 or 2 or the bispecific antibody of claim 3; 5) a biological agent comprising the pharmaceutical composition.

8. The product of claim 7, wherein, The detectable label includes a bioluminescent agent, a chemiluminescent agent, an enzyme, a photosensitive diagnostic agent, a paramagnetic ion or a radionuclide; Preferably, the detection product includes a kit, a chip, a test strip.

9. Any of the following methods, characterized in that, The method comprises: 1) a method for preparing the recombinant host cell of claim 6, which comprises: introducing the expression vector of claim 5 into a host cell to obtain the recombinant host cell of claim 6; 2) A process for the production of an antibody as defined in any of claims 1 or 2 or a bispecific antibody as defined in claim 3, said process comprising: culturing the recombinant host cell of claim 6, and isolating the antibody or bispecific antibody from the culture product of the recombinant host cell; 3) a method for detecting B7H3 in a sample to be tested for non-diagnostic and non-therapeutic purposes, which comprises: contacting the sample to be tested with the antibody of any one of claims 1 or 2, the bispecific antibody of claim 3, the antibody conjugate of claim 7, the detection reagent or the detection product, and detecting the formation of an immune complex of B7H3 protein and the antibody; 4) A method of inhibiting B7H3 activity in a sample in vitro, the method comprising: contacting the sample with the antibody of any one of claims 1 or 2 or the bispecific antibody of claim 3.

10. An application characterized by any one of The application comprises: 1) use of the antibody of any one of claims 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5 or the recombinant host cell of claim 6 in the preparation of an antibody conjugate for detecting B7H3; 2) use of the antibody of any one of claims 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the recombinant host cell of claim 6 or the antibody conjugate of claim 7 in the preparation of a detection reagent for detecting B7H3; 3) use of the antibody of any one of claims 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the recombinant host cell of claim 6, the antibody conjugate of claim 7 or the detection reagent in the preparation of a detection product for detecting B7H3; 4) The use of the antibody of any one of claims 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the recombinant host cell of claim 6, the antibody conjugate of claim 7, a detection reagent or a detection product for detecting B7H3 for non-diagnostic, non-therapeutic purposes; 5) The use of the antibody of any one of claims 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the recombinant host cell of claim 6, the antibody conjugate of claim 7, a detection reagent or a detection product for the preparation of a diagnostic product for diagnosing or aiding in the diagnosis of a disease with abnormal expression of B7H3; 6) The use of the antibody of any one of claims 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5 or the recombinant host cell of claim 6 for the preparation of a medicament for the treatment and / or prevention of a disease with abnormal expression of B7H3; 7) The use of the antibody of any one of claims 1 or 2, the bispecific antibody of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the recombinant host cell of claim 6 or the pharmaceutical composition of claim 7 for the preparation of a biological agent for the treatment and / or prevention of a disease with abnormal expression of B7H3. Preferably, the disease with abnormal expression of B7H3 comprises osteosarcoma, glioma, melanoma, hematological malignancies, prostate cancer, esophageal cancer, cervical cancer, ovarian cancer, colorectal cancer, breast cancer or lung cancer.

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