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WO2023025315A1 - Anti-b7-h3 antibody, and preparation method therefor and use thereof - Google Patents

Anti-b7-h3 antibody, and preparation method therefor and use thereof Download PDF

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Publication number
WO2023025315A1
WO2023025315A1 PCT/CN2022/115318 CN2022115318W WO2023025315A1 WO 2023025315 A1 WO2023025315 A1 WO 2023025315A1 CN 2022115318 W CN2022115318 W CN 2022115318W WO 2023025315 A1 WO2023025315 A1 WO 2023025315A1
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antibody
seq
amino acid
heavy chain
light chain
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French (fr)
Chinese (zh)
Inventor
魏海涛
宁婷婷
李亚男
董国良
李秀兰
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Shanghai Symray Biopharma Co Ltd
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Shanghai Symray Biopharma Co Ltd
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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Definitions

  • the invention relates to the technical field of antibodies, in particular to an anti-B7-H3 antibody, its preparation method and application.
  • Immune checkpoints refer to some inhibitory signaling pathways in the immune system.
  • the body In the case of normal anti-tumor immune response, the body maintains a balance between co-stimulatory signals and co-inhibitory signals, and maintains immunity by regulating the intensity of the autoimmune response. tolerance.
  • the immune checkpoint signaling pathway When the body is invaded by tumors, it usually blocks the immune checkpoint signaling pathway to inhibit autoimmunity and provide opportunities for tumor cell growth and escape.
  • tumor microenvironment and tumor immune escape mechanism it was found that negative B7 family molecules were abnormally expressed in various tumor tissues and tumor infiltrating immune cells, which were an important part of tumor microenvironment and involved in tumor immune escape.
  • B7-H3 also known as CD276, was first cloned from the cDNA library of human dendritic cells by Andrei I.Chapoval et al. in 2001, and has 20–27% homology with the B7 immunoglobulin superfamily at the amino acid level , a member of the immunoglobulin superfamily B7 (Chapoval, Ni et al.2001).
  • B7-H3 protein belongs to type I transmembrane protein, including a signal peptide, a C-terminal immunoglobulin constant region (IgC) and N-terminal variable region (IgV), a transmembrane region and an intracellular region (Vigdorovich, Ramagopal et al. 2013).
  • the B7-H3 protein has two variant splicing variants.
  • the extracellular segment of variant 1 is composed of four immunoglobulin domains of IgV-IgC-IgV-IgC, called 4Ig-B7-H3, and the extracellular segment of variant 2 is composed of IgV -IgC consists of 2 immunoglobulin domains, called 2Ig-B7-H3.
  • B7-H3 is low expressed in healthy tissues, but highly expressed in a large number of malignant tumors. Studies have shown that B7-H3 can play a role in esophageal cancer, melanoma, colorectal cancer, adenocarcinoma, ovarian cancer, non-small cell lung cancer, kidney cancer, gastric cancer, bladder cancer, glioblastoma multiforme and osteosarcoma, etc. Highly expressed in cancer (Tang, Zhao et al.2019, Tang, Liu et al.2020). B7-H3 is not only expressed on tumor cells, but also highly expressed on tumor neovascular endothelial cells, and is a very broad-spectrum tumor marker antigen. High expression of B7-H3 protein can promote cancer progression and is associated with poor prognosis of patients. Therefore, inhibiting the expression of B7-H3 protein has a potential effect on the treatment of cancer.
  • the present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the present invention proposes an anti-B7-H3 antibody capable of binding to B7-H3 with high specificity.
  • the present invention also proposes recombinant proteins, pharmaceutical compositions, polynucleotides, recombinant plasmids and isolated cells related to the above antibodies.
  • the present invention also proposes a preparation method of the above-mentioned antibody.
  • the present invention also proposes the application of the above antibody in the preparation of anticancer drugs.
  • the present invention also proposes the application of the above-mentioned antibody in the preparation of an antibody detection kit.
  • the antibody comprises a heavy chain variable region and/or a light chain variable region:
  • the heavy chain variable region comprises: represented by SEQ ID NO:1
  • the heavy chain complementarity determining region HCDR1 composed of the amino acid sequence
  • the heavy chain complementarity determining region HCDR2 composed of the amino acid sequence shown in SEQ ID NO:2
  • the heavy chain complementarity determining region HCDR3 composed of the amino acid sequence shown in SEQ ID NO:3
  • the light chain variable region which comprises: the light chain complementarity determining region LCDR1 composed of the amino acid sequence shown in SEQ ID NO: 4, the light chain complementarity determining region LCDR2 composed of the WAS amino acid sequence, composed of SEQ ID NO:
  • the light chain complementarity determining region LCDR3 composed of the amino acid sequence shown in 5.
  • the antibody according to the embodiment of the present invention has at least the following beneficial effects: the antibody of the present invention has (1) good binding activity and strong binding ability to B7-H3 protein; (2) cross-reactivity with monkey B7-H3 It has cross-reactivity, which is beneficial to carry out verification experiments and facilitate the subsequent development of products with therapeutic use; (3) has ADCC activity.
  • antibody-dependent cell-mediated cytotoxicity is a cell-mediated immune defense mechanism, which is the elimination of pathogenic target cells bound to specific antibodies by immune cells. mode of action.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the antibody is a murine antibody, a chimeric antibody or a human antibody.
  • the heavy chain variable region is selected from such as SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO The amino acid sequence shown in: 22;
  • the light chain variable region is selected from such as SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID The amino acid sequence shown in NO:27.
  • the antibody comprises a heavy chain constant region and/or a light chain constant region: the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 10; the light chain constant region comprises Amino acid sequence as shown in SEQ ID NO:12.
  • the antibody comprises a heavy chain and/or a light chain: the heavy chain is selected from the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:46 and SEQ ID NO:47; The light chain is selected from the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 48 and SEQ ID NO: 49.
  • the antibody comprises any one of the following properties i to v:
  • the antibody can specifically bind to B7-H3;
  • the antibody has antibody-dependent cell-mediated cytotoxicity
  • the antibody specifically binds to human 4Ig-B7-H3 and cross-reacts with monkey 4Ig-B7-H3;
  • the antibody has reduced or aglycosylated or hypofucosylated.
  • the above antibody sequences have the best binding activity, reaction specificity, species cross-reactivity and ADCC activity. It can be proved that the antibody of the present invention has a strong binding force to the B7-H3 protein through the experiments of the examples; it has the characteristic of species cross-reactivity, which is helpful for the toxicity analysis of the antibody in monkeys, and is conducive to carrying out verification tests in related animals. It is beneficial to the application and development of subsequent therapeutic purposes; it has strong ADCC activity and is more suitable for the subsequent development of antibody drugs.
  • the amino acid positions of the Fc region are modified to enhance Fc ⁇ R binding.
  • the Fc region has a stronger Fc ⁇ RIII binding ability, thereby exerting a stronger antibody-mediated cytotoxicity (ADCC) effect.
  • ADCC antibody-mediated cytotoxicity
  • the recombinant protein, pharmaceutical composition, polynucleotide, vector or isolated cell according to the embodiment of the second aspect of the present invention: the recombinant protein comprises the above-mentioned antibody; the pharmaceutical composition comprises the above-mentioned antibody or the above-mentioned recombinant protein; the multinuclear
  • the nucleotide contains the nucleotide sequence encoding the above-mentioned antibody or recombinant protein; the vector contains the above-mentioned polynucleotide; and the isolated cell produces the above-mentioned antibody.
  • the recombinant protein further comprises a tag sequence to assist expression and/or purification.
  • the recombinant protein is a double antibody, which also includes antibodies capable of binding to other target proteins. Further, the double antibody also includes antibodies that specifically bind to different epitopes of B7-H3.
  • the pharmaceutical composition further includes the above-mentioned double antibody.
  • the pharmaceutical composition further includes an ADC drug containing the above-mentioned antibody.
  • the ADC drug also includes a linker and a toxic molecule.
  • the pharmaceutical composition further includes pharmaceutically available excipients.
  • the preparation method according to the embodiment of the third aspect of the present invention includes the following steps: culturing the above-mentioned isolated cells, and recovering the antibody from the culture.
  • the specific preparation method is as follows: the heavy chain variable region sequence encoding the above-mentioned anti-B7-H3 antibody is cloned into recombinant plasmid 1 containing the IgG1 heavy chain constant region amino acid sequence, and the light chain variable region sequence is cloned into In the recombinant plasmid 2 containing the amino acid sequence of the Kappa light chain constant region, the recombinant plasmid 1 and the recombinant plasmid 2 were simultaneously transfected into cells and cultured, and the anti-B7-H3 antibody was recovered from the culture.
  • the antibody can be applied to the preparation of anticancer drugs and/or antibody detection kits.
  • the anticancer drug is mainly used for preventing or treating cancer, wherein the cancer is breast cancer, endometrial cancer, ovarian cancer, lung cancer, gastric cancer, prostate cancer, kidney cancer, Cancer of the liver, pancreas, colorectum, esophagus, bladder, cervix, blood, lymphoma, or malignant melanoma.
  • anti-cancer drugs also include antibodies targeting other targets, such as: CD3, BCMA, CD38, etc. to construct bispecific antibodies, and develop various methods for regulating tumor cells.
  • the antibody of the present invention can also be coupled to other types of molecules, such as toxins, nucleic acid molecules, etc., and the antibody can specifically bring the coupled molecules into the body of tumor cells, thereby regulating the effect of tumor cells.
  • the B7-H3-specific antibody or antigen-binding fragment can be labeled for use in the method or other methods known to those skilled in the art.
  • the antibodies or antigen-binding fragments thereof of the present invention can be radiolabeled, fluorescently labeled, epitope tags, biotin, chromophore labels, ECL labels, enzymes, ruthenium, 111In-DOTA, 111In-di Ethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, or polyhistidine or similar such labels known in the art.
  • DTPA 111In-DOTA
  • DTPA 111In-di Ethylenetriaminepentaacetic acid
  • horseradish peroxidase alkaline phosphatase and beta-galactosidase
  • polyhistidine or similar such labels known in the art.
  • the antibody can also be developed into a method for detecting the expression level of B7-H3 on the surface of tissue cells by means of immunization.
  • the antibody can also be used for detecting the presence of B7-H3 in a biological sample such as blood or serum, for quantitative analysis of the amount of B7-H3 in a biological sample such as blood or serum, for diagnosing B7-H3 expressing cancer, for To determine a method of treating a subject with cancer, or for monitoring the progression of a B7-H3 expressing cancer in a subject, and the like.
  • the term "antibody” specifically includes antibodies in a narrow sense, and also includes “chimeric” antibodies and antibody fragments, wherein part of the heavy chain and/or light chain corresponds to an antibody derived from a specific species or belonging to a specific antibody type or subclass.
  • the sequence is identical or homologous, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody type or subclass, so long as it specifically binds the target antigen and/or exhibits the desired Biological activity (US Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • antibody in a narrow sense refers to a type of glycosyl-containing globulin that is secreted by antigens entering the body to stimulate B cells to differentiate and proliferate into plasma cells, which can specifically bind to corresponding antigens and produce immune effects.
  • World Health Organization held a meeting and collectively referred to globulins with antibody activity and chemical structure similar to antibodies as immunoglobulins. Modern immunology believes that antibodies and immunoglobulins are equivalent concepts, but antibodies focus on the description of their biological activities, while immunoglobulins focus on their chemical structures.
  • the basic structure of immunoglobulin consists of four peptide chains: two heavy chains (Heavy chain, H chain) and two light chains (Light chain, L chain), the light chain and the heavy chain are connected by disulfide bonds to form a symmetrical tetrapeptide Chain molecules become monomers of immunoglobulin molecules, which are the basic structure of all immunoglobulins.
  • Each heavy and light chain is divided into amino-terminus (N-terminus) and carboxy-terminus (C-terminus).
  • variable region V region
  • C region C-terminal amino acid
  • HVR hypervariable region
  • framework region Framework region
  • HVR1, HVR2 and HVR3 of the heavy chain or light chain respectively.
  • the hypervariable region is the binding site of the antibody and the antigen, and is called the complementarity-determining region (CDR). Therefore, HVR1, HVR2, and HVR3 of the heavy chain or light chain are also called CDR1, CDR2, and CDR3.
  • the term "diabodies”, that is, "bispecific" antibodies refers to an antibody, generally a monoclonal antibody, that has the binding properties of at least two different antigenic epitopes.
  • the epitopes are from the same antigen.
  • the epitopes are from two different antigens.
  • Methods of making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly by co-expressing two immunoglobulin heavy/light chain pairs. See, eg, Milstein et al., Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical linkage. See, eg, Brennan et al., Science 229:81 (1985).
  • Bispecific antibodies include bispecific antibody fragments (eg, Hollinger et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-48 (1993), Gruber et al., J. Immunol. 152:5368 (1994)).
  • humanized antibody refers to a form of antibody that contains sequences derived from non-human (eg, murine) antibodies as well as human antibodies.
  • the antibody is a chimeric antibody containing minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies will comprise substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, all or substantially all of which The FR regions are those of human immunoglobulin sequences.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Cabilly U.S. Patent No. 4,816,567; Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033; Oxford University Press) 1996).
  • Fc immunoglobulin constant region
  • Fig. 1 shows the binding force of chimeric antibody 27B4 and MDA-MB-231 in Example 3 of the present invention
  • Figure 2 shows the ADCC activity of chimeric antibody 27B4 in Example 3 of the present invention
  • Figure 3 shows the binding ability of different 27B4 humanized scFv antibodies to B7-H3 antigen in Example 4 of the present invention
  • Fig. 4 shows the binding force of different Whole IgG forms 27B4 humanized antibody B7-H3 antigen in Example 4 of the present invention
  • Figure 5 shows the ADCC activities of different Whole IgG forms of 27B4 humanized antibodies in Example 4 of the present invention.
  • Fig. 6 shows the binding activity of 27B4 humanized antibody h27B4-H4-L3 (27B4Z02 molecule) in Example 4 of the present invention to B7-H32Ig and 4Ig proteins.
  • Fig. 7 shows the activity of binding human and monkey B7-H3 proteins of the 27B4 humanized antibody h27B4-H4-L3 (27B4Z02 molecule) in Example 4 of the present invention.
  • the human 4Ig-B7-H3 antigen used for screening and detection is a commercial product (Acro, product number B7B-H52E7), and the sequence is as follows:
  • the human 2Ig-B7-H3 antigen used for screening and detection is a commercial product (Acro, product number B73-H52E2), and the sequence is as follows:
  • the macaque 4Ig-B7-H3 antigen used for screening and detection is a commercial product (Acro, product number B73-C52Ha), and the sequence is as follows:
  • mouse 2Ig-B7-H3 antigen used for screening and detection is a commercial product (Acro, product number B73-M52H4), and the sequence is as follows:
  • the horizontal line part is the extracellular region of B7-H3; the italic part is the linker; the black bold part is the His-tag tag.
  • the CDS region encoding human B7-H3 variant 1 (4Ig-B7-H3) was obtained from the NCBI database, and the full-length gene sequence was routinely synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. NotI was constructed into the lentiviral overexpression vector pCDH-EF1-T2A-copGFP, and the sequence was verified to be correct by sequencing, that is, the plasmid pCDH-EF1-h4Ig-B7-H3-T2A-copGFP was successfully constructed.
  • HEK293 cells in the logarithmic growth phase (derived from ATCC) in a good growth state, inoculate 8E6 cells in a 10 cm culture dish, add 10% fetal bovine serum to DMEM, and culture in a 37°C, 5% CO 2 incubator , when the cell fusion rate reaches about 70% to 80%, transfection.
  • the three plasmids psPAX2, pMD2.G and pCDH-EF1-h4Ig-B7-H3-T2A-copGFP were co-transfected using the transfection reagent PEI.
  • the virus liquid was collected 48 hours and 72 hours after transfection, filtered through a 0.45 ⁇ M syringe filter, infected with the target cell CHO-S (derived from ATCC) at an MOI of 10, and cultured in RPMI-1640 plus 10% fetal bovine serum. The medium was changed 48 hours after virus infection. The positive rate of human 4Ig-B7-H3 expression was tested by flow cytometry, and the positive rate was over 95%. The 293-h4Ig-B7-H3-copGFP stably transfected cell line was successfully constructed.
  • mice of SPF grade BALB/C strain purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.
  • the immune antigen was His-tagged human recombinant 4Ig-B7-H3 antigen (Acro, catalog number B7B- H52E7)
  • the antigen was emulsified with an equal volume of adjuvant.
  • the antigen was emulsified with complete Freund's adjuvant (Sigma, product number F5881), and injected subcutaneously at multiple points on the back of the neck of the mouse, and each mouse was immunized with 50 ⁇ g of the antigen.
  • the antigen was emulsified with Freund's incomplete adjuvant (Sigma, product number F5602) for immunization, and each mouse was immunized with 25 ⁇ g of the antigen.
  • Freund's incomplete adjuvant Sigma, product number F5602
  • each mouse was immunized with 25 ⁇ g of the antigen.
  • blood was collected from the orbit of the mouse, and the titer of the mouse immune serum was detected by ELISA according to the method of Examples 2-4.
  • the pre-immune serum was used as a negative control.
  • the immune serum was diluted 1:100, it was then serially diluted to 11 concentrations by 3 times, and the serum dilution corresponding to the OD value of 3 times the negative control was used as its titer.
  • Select the mouse with the highest titer and the serum titer tends to be stable after two booster immunizations.
  • 25 ⁇ g recombinant human 4Ig-B7-H3 antigen is injected intraperitoneally for a booster immunization.
  • the antigen does not need adjuvant emulsification , the buffer is PBS.
  • flow cytometry Flow Cytometry, FACS detection was carried out according to the method in Example 2-5, and the binding force between the supernatant and the overstable cells 293-h4Ig-B7-H3-copGFP was detected.
  • Positive hybridoma cells were monoclonalized by the limiting dilution method, and then detected by ELISA and FACS.
  • the FACS method simultaneously detected the samples and the overstable cells 293-h4Ig-B7-H3-copGFP and endogenous expression cells MDA-MB For the binding status of -231, select positive monoclonal wells and repeat monoclonalization and positive clone screening and identification once.
  • Hybridoma cells were inoculated into a 250mL cell culture flask at 2.5 ⁇ 10 5 /mL, and the culture volume was 50mL. When the cell viability dropped to 30%, the supernatant containing the antibody was harvested by centrifugation, and filtered with a 0.45 ⁇ M filter. Mouse monoclonal antibodies were purified in Protein G medium and replaced by dialysis into PBS pH7.2 buffer. Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.
  • B7-H3 antigen human 4Ig-B7-H3 antigen, Acro, Cat. No. B7B-H52E7 or human 2Ig-B7-H3, Acro, Cat. B73-C52Ha or mouse 2Ig-B7-H3, Acro, Cat. No. B73-M52H4
  • PBS PBS containing 2% skim milk powder
  • the antibody produced by a hybridoma cell 27B4 has the highest binding activity to human 4Ig-B7-H3 antigen and human 2Ig-B7-H3 antigen, and has cross-species reactivity, and can simultaneously bind macaque 4Ig-B7- H3. This cross-reactivity facilitates the application of the antibody in animal experiments.
  • RNAiso Plus RNAiso Plus
  • cDNA was obtained by reverse transcription with 5'RACE method (SMARTer RACE5'/3'Kit, Clontech, product number 634859).
  • Adapter as an upstream primer
  • CL and CH1 as downstream primers for antibody light and heavy chains respectively
  • ExTaq PCR 94°C, 3min; 94°C, 30s, 55°C, 30s, 72°C, 45s, 32cycle; 72°C, 5min
  • Tiangen universal DNA reagent After the cassettes were recovered and purified, they were ligated with the pMD18-T vector (Takara, Cat. No.
  • the ligation products were transformed into TG-1 Escherichia coli competent cells for sequencing.
  • VBASE2 http://www.vbase2.org/vbscAb.php
  • the screened antibody is referred to as 27B4.
  • the amino acid sequence of the 27B4 heavy chain variable region includes heavy chain complementarity determining regions HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2) and HCDR3 (SEQ ID NO: 3); the light chain variable region includes Light chain complementarity determining regions LCDR1 (SEQ ID NO: 4), LCDR2 (WAS) and LCDR3 (SEQ ID NO: 5).
  • Amino acid sequence of 27B4VH of 27B4 heavy chain variable region SEQ ID NO:6.
  • the amino acid sequence hIgG1 (AWK57454.1) of the ⁇ heavy chain constant region of the antibody was obtained from NCBI, and mutations L235V, F243L, R292P, Y300L and P396L were introduced to enhance the ADCC effect (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity cytotoxicity), and at the N-terminal of the sequence, Kozak sequence (5'-GCCACCATGG-3'), signal peptide sequence and multiple cloning site region sequence were sequentially added, and eukaryotic codon optimization was performed to obtain the base sequence, in which the cloning site The dot region contains restriction sites KpnI (5'-GGTACC-3') and MfeI (5'-CAATTG-3'), and the routine synthetic gene sequence of Sangon Bioengineering (Shanghai) Co., Ltd. was constructed into the pTT5 vector (Biovector ), the eukaryotic heavy chain expression vector pTT5-hIgG
  • Optimized hIgG1 base sequence SEQ ID NO: 11.
  • the amino acid sequence HL (CAR58102.1) of the ⁇ light chain constant region of the antibody was obtained from NCBI, and the Kozak sequence (5'-GCCACCATGG-3'), the signal peptide sequence and the sequence of the multiple cloning site region were sequentially added to the N-terminal of the sequence.
  • the base sequence was obtained by codon optimization for eukaryotic expression, in which the cloning site region contains restriction sites KpnI (5'-GGTACC-3') and MfeI (5'-CAATTG-3'), in Sangon Bioengineering (Shanghai ) Co., Ltd. routinely synthesized the gene sequence into the multiple cloning site of the pTT5 vector to obtain the eukaryotic heavy chain expression vector pTT5-HL containing the antibody light chain constant region sequence.
  • Amino acid sequence of constant region of kappa light chain SEQ ID NO:12.
  • the base sequence of the constant region of the ⁇ light chain SEQ ID NO: 13.
  • the Kozak sequence is a nucleic acid sequence located behind the cap structure at the 5' end of eukaryotic mRNA, which can bind to translation initiation factors to mediate translation initiation of mRNA containing the 5' cap structure.
  • the heavy chain and light chain variable region sequences of 27B4 were routinely synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and cloned into pTT5-hIgG1 and pTT5-HL vector plasmids by KpnI and MfeI, respectively, to obtain the heavy chain expression plasmid pTT5- 27B4-VH-hIgG1 and light chain expression plasmid pTT5-27B4-VL-HL.
  • PEI co-transfects light chain expression plasmid pTT5-27B4-VL-HL and heavy chain expression plasmid pTT5-27B4 - 25 ⁇ g each of VH-hIgG1.
  • the cell supernatant on the 7th day after transfection was collected, centrifuged and filtered using a 0.45 ⁇ M filter, the protein A medium was used to purify the antibody, and the antibody was replaced by dialysis into PBS pH7.2 buffer.
  • Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.
  • the binding activity of the chimeric antibody xw.M0883B9.27B4 obtained in Example 3-3 to the B7-H3 antigen was examined by FACS method.
  • the positive reference antibody is MGA-271 (NCT02475213).
  • PBMC peripheral blood mononuclear cell
  • ADCC medium RPMI 1640 containing 1% FBS
  • 40 ⁇ m cell strainer BD Biosciences, Ltd.
  • Collect the MDA-MB-231 cells in the logarithmic growth phase in good growth state wash the cells once with ADCC medium, filter with a 40 ⁇ m cell strainer, count the cells and adjust the cell density to 2x 10 5 /mL, as the target cells.
  • Experimental group experimental well-medium background control well
  • Target cell spontaneous target cell spontaneous LDH release well-medium background control well
  • Effector cell spontaneous effector cell spontaneous LDH release well-medium background control well
  • Maximum LDH release from target cells maximum LDH release pores from target cells - volume correction pores.
  • the mouse anti-human 27B4 monoclonal antibody was humanized according to the so-called CDR grafting method. Briefly, the VH and VK base sequences of the 27B4 antibody were analyzed using the IMGT/V-QUEST tool (http://www.imgt.org/IMGT_vquest/input), and the CDR region sequences of the antibody light chain and heavy chain were determined. Use the IgBlast tool (https://www.ncbi.nlm.nih.gov/igblast/) to analyze the amino acid sequence of the 27B4 antibody to obtain the closest human germline VH and VK sequences of the 27B4 antibody.
  • IMGT/V-QUEST tool http://www.imgt.org/IMGT_vquest/input
  • IgBlast tool https://www.ncbi.nlm.nih.gov/igblast/
  • the CDRs of the 27B4 antibody are grafted into the framework regions of the selected VH and VK human germline sequences, and this sequence is the humanized antibody sequence. Analyze the human germline VH of the 27B4 antibody VH, select 5 different sequences, graft the CDRs of the 27B4 antibody VH into the framework regions of these 5 sequences, and obtain 5 heavy chain variable regions 27B4-H1, 27B4 -H2, 27B4-H3, 27B4-H4 and 27B4-H5. The same method was used to carry out CDR grafting on the light chain, and five light chain variable region sequences 27B4-L1, 27B4-L2, 27B4-L3, 27B4-L4 and 27B4-L5 were obtained.
  • amino acid sequences of 27B4-H1, 27B4-H2, 27B4-H3, 27B4-H4 and 27B4-H5 are respectively denoted as SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 21 and SEQ ID NO: 22.
  • amino acid sequences of 27B4-L1, 27B4-L2, 27B4-L3, 27B4-L4 and 27B4-L5 are respectively denoted as SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 26 and SEQ ID NO: 27.
  • the base sequences of 27B4-H1, 27B4-H2, 27B4-H3, 27B4-H4 and 27B4-H5 are recorded as SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32.
  • the base sequences of 27B4-L1, 27B4-L2, 27B4-L3, 27B4-L4 and 27B4-L5 are shown in the sequence list SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37.
  • the humanized scFv 27B4 antibody obtained through the combination of 27B4-H1-F, linker and 27B4-L1-F is named "scFv-27B4-H1-L1", and the humanized scFv 27B4 antibody obtained through the combination of 27B4-H1-F, linker and 27B4-L2-
  • the humanized scFv 27B4 antibody obtained by combining F is named "scFv-27B4-H1-L2”
  • the humanized scFv 27B4 antibody obtained by combining 27B4-H1-F, linker and 27B4-L3-F is named "scFv -27B4-H1-L3”
  • the humanized scFv 27B4 antibody obtained through the combination of 27B4-H1-F, linker and 27B4-L4-F is named "scFv-27B4-H1-L4"
  • the humanized scFv 27B4 antibody is named "scFv-27B4-H3-L3", and the humanized scFv 27B4 antibody obtained by combining 27B4-H3-F, linker and 27B4-L4-F is named “scFv-27B4-H3 -L4", will be obtained by combining 27B4-H3-F, linker and 27B4-L5-F
  • the humanized scFv 27B4 antibody was named "scFv-27B4-H3-L5", and the humanized scFv 27B4 antibody obtained by combining 27B4-H4-F, linker and 27B4-L1-F was named "scFv-27B4- H4-L1", the humanized scFv 27B4 antibody obtained through the combination of 27B4-H4-F, linker and 27B4-L2-F is named "scFv-27B4-H4-L2", and
  • the humanized scFv 27B4 antibody obtained by combining 27B4-H5-F, linker and 27B4-L1-F is named "scFv-27B4-H5-L1", and will be obtained by combining 27B4-H5-F, linker and 27B4-L2-F
  • the combined humanized scFv 27B4 antibody was named "scFv-27B4-H5-L2”
  • the humanized scFv 27B4 antibody obtained by combining 27B4-H5-F, linker and 27B4-L3-F was named "scFv- 27B4-H5-L3”
  • the humanized scFv 27B4 antibody obtained through the combination of 27B4-H5-F, linker and 27B4-L4-F is named "scFv-27B4-H5-L4"
  • 25 scFv single-chain antibodies constructed in Example 4-2 with humanized sequences of 27B4 were expressed and purified.
  • the centrifuged sample on the column wash the column with 25mL washing buffer containing 10mM and 20mM imidazole in turn, and finally wash the column with 5mL washing buffer containing 250mM imidazole, and collect the eluate.
  • Antibodies were exchanged by dialysis into PBS pH 7.2 buffer. Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining
  • amino acid sequences of scFv-27B4-H2-L2, scFv-27B4-H2-L3, scFv-27B4-H4-L2 and scFv-27B4-H4-L3 are SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41.
  • the base sequences of scFv-27B4-H2-L2, scFv-27B4-H2-L3, scFv-27B4-H4-L2 and scFv-27B4-H4-L3 are SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45.
  • the heavy chain humanized sequence 27B4-H2 and 27B4-H4 were conventionally synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and cloned into pTT5-hIgG1 through KpnI and MfeI respectively, and the light chain humanized sequence 27B4- L2 and 27B4-L3 were routinely synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and cloned into the pTT5-HL vector plasmid by KpnI and MfeI, respectively, to obtain heavy chain expression plasmids pTT5-27B4-H2-hIgG1, pTT5-27B4- H4-hIgG1 and light chain expression plasmids pTT5-27B4-L2-HL, pTT5-27B4-L3-HL.
  • the 293F cells in the logarithmic growth phase in good growth state were inoculated into 250 mL cell culture flasks and cultured in 50 mL medium, and 25 ⁇ g of each light and heavy chain expression plasmids were co-transfected with PEI.
  • the cell supernatant on the 7th day of culture after transfection was collected, centrifuged and filtered using a 0.45 ⁇ M filter, the protein A medium was used to purify the antibody, and the antibody was replaced by dialysis into PBS pH7.2 buffer.
  • Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.
  • the antibody obtained by the combination of pTT5-27B4-H2-hIgG1 and pTT5-27B4-L2-HL was named "h27B4-H2-L2", and the antibody obtained by the combination of pTT5-27B4-H2-hIgG1 and pTT5-27B4-L3-HL
  • the combined antibody was named "h27B4-H2-L3”
  • the antibody obtained by combining pTT5-27B4-H4-hIgG1 and pTT5-27B4-L2-HL was named "h27B4-H4-L2”
  • the antibody obtained from the combination of 27B4-H4-hIgG1 and pTT5-27B4-L3-HL was named "h27B4-H4-L3".
  • the heavy chain amino acid sequences of h27B4-H2 and h27B4-H4 SEQ ID NO: 46, SEQ ID NO: 47.
  • Heavy chain base sequences of h27B4-H2 and h27B4-H4 SEQ ID NO: 50, SEQ ID NO: 51.
  • the light chain base sequences of h27B4-L2 and h27B4-L3 SEQ ID NO: 52, SEQ ID NO: 53.
  • the ADCC activity of the whole IgG form 27B4 human antibody was detected according to the method of Example 3-5.
  • four Whole IgG forms of 27B4 humanized antibodies h27B4-H2-L2, h27B4-H4-L3, h27B4-H2-L3, and h27B4-H4-L2 (identified in the figure as zw.M0883B9.27B4Z01, zw .M0883B9.27B4Z02, zw.M0883B9.27B4Z03, zw.M0883B9.27B4Z04) all have ADCC activity, which is comparable to the ADCC activity of the 27B4 antibody before humanization.
  • the results are shown in Figure 5.
  • the molecule zw.M0883B9.27B4Z02 (h27B4-H4-L3, referred to as molecule Z02) had the highest activity, and the cross-reactivity of molecule Z02 was detected according to the method of Example 2-4. Further binding EC50 and special cross-binding experiments on the Z02 molecule proved that the molecule can simultaneously recognize human 4Ig-B7-H3 antigen and human 2Ig-B7-H3 antigen, and has the ability to bind to monkey 4Ig-B7-H3 protein. Reactivity facilitates the use of antibodies in animal experiments. The results are shown in Figures 6 and 7.
  • the anti-B7-H3 antibodies prepared in the present invention have high B7-H3 antigen binding activity and ADCC activity, and can be applied in the fields of cancer treatment and the like.

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Abstract

Provided in the present application are an anti-B7-H3 antibody, and a preparation method therefor and the use thereof. The anti-B7-H3 antibody has a relatively high binding activity, species cross-reactivity and ADCC activity, and can be used for treating diseases such as cancer.

Description

抗B7-H3抗体、其制备方法及用途Anti-B7-H3 antibody, its preparation method and use 技术领域technical field

本发明涉及抗体技术领域，具体涉及抗B7-H3抗体、其制备方法及用途。The invention relates to the technical field of antibodies, in particular to an anti-B7-H3 antibody, its preparation method and application.

背景技术Background technique

免疫检查点(immune checkpoints)是指免疫系统中存在的一些抑制性信号通路，机体在正常抗肿瘤免疫应答情况下，共刺激信号和共抑制信号保持平衡，通过调节自身免疫反应的强度来维持免疫耐受。机体在受到肿瘤侵袭时，通常会阻断免疫检查点信号通路从而抑制自身免疫，给肿瘤细胞的生长和逃逸提供机会。随着对肿瘤微环境和肿瘤免疫逃逸机制的研究，发现负性B7家族分子异常表达于多种肿瘤组织及肿瘤浸润免疫细胞中，是肿瘤微环境的重要组成部分，并参与肿瘤免疫逃逸。目前已有CTLA-4、PD-1以及PD-L1(Bowyer,Prithviraj et al.2016,Pento 2017,Fujita,Uchida et al.2019)等抗免疫检查点的单克隆抗体应用于肿瘤治疗并取得了显著疗效。这些成果使阻断免疫检查点的治疗策略备受关注。Immune checkpoints refer to some inhibitory signaling pathways in the immune system. In the case of normal anti-tumor immune response, the body maintains a balance between co-stimulatory signals and co-inhibitory signals, and maintains immunity by regulating the intensity of the autoimmune response. tolerance. When the body is invaded by tumors, it usually blocks the immune checkpoint signaling pathway to inhibit autoimmunity and provide opportunities for tumor cell growth and escape. With the study of tumor microenvironment and tumor immune escape mechanism, it was found that negative B7 family molecules were abnormally expressed in various tumor tissues and tumor infiltrating immune cells, which were an important part of tumor microenvironment and involved in tumor immune escape. At present, monoclonal antibodies against immune checkpoints such as CTLA-4, PD-1 and PD-L1 (Bowyer, Prithviraj et al. 2016, Pento 2017, Fujita, Uchida et al. 2019) have been used in tumor treatment and have achieved Significant curative effect. These results have drawn much attention to therapeutic strategies that block immune checkpoints.

B7-H3又名CD276，最早由Andrei I.Chapoval等人于2001年从人树突状细胞的cDNA文库中克隆获得，在氨基酸水平与B7免疫球蛋白超家族存在20–27％的同源性，属于免疫球蛋白超家族B7成员(Chapoval,Ni et al.2001)。B7-H3蛋白属于I型跨膜蛋白，包含一个信号肽，一个C端的免疫球蛋白恒定区(IgC)和N端的可变区(IgV)、一个跨膜区和一个胞内区(Vigdorovich,Ramagopal et al.2013)。B7-H3蛋白有两种变异剪接体，变体1胞外段由IgV-IgC-IgV-IgC 4个免疫球蛋白结构域组成，称为4Ig-B7-H3，变体2胞外段由IgV-IgC 2个免疫球蛋白结构域组成，称为2Ig-B7-H3。在人体中有两种不同的变体形式存在，其主要形式为4Ig-B7-H3，而在小鼠中只含有2Ig-B7-H3形式(Y-H,Y-J et al.2007)。B7-H3, also known as CD276, was first cloned from the cDNA library of human dendritic cells by Andrei I.Chapoval et al. in 2001, and has 20–27% homology with the B7 immunoglobulin superfamily at the amino acid level , a member of the immunoglobulin superfamily B7 (Chapoval, Ni et al.2001). B7-H3 protein belongs to type I transmembrane protein, including a signal peptide, a C-terminal immunoglobulin constant region (IgC) and N-terminal variable region (IgV), a transmembrane region and an intracellular region (Vigdorovich, Ramagopal et al. 2013). The B7-H3 protein has two variant splicing variants. The extracellular segment of variant 1 is composed of four immunoglobulin domains of IgV-IgC-IgV-IgC, called 4Ig-B7-H3, and the extracellular segment of variant 2 is composed of IgV -IgC consists of 2 immunoglobulin domains, called 2Ig-B7-H3. Two different variant forms exist in humans, the predominant form being 4Ig-B7-H3, whereas in mice only the 2Ig-B7-H3 form is present (Y-H, Y-J et al. 2007).

临床上已经报道，B7-H3在健康组织中低表达，但在大量的恶性肿瘤中高表达。研究表明，B7-H3可在食管癌、黑色素瘤、结直肠癌、腺癌、卵巢癌、非小细胞肺癌、肾癌、胃癌、膀胱癌、多形性胶质母细胞瘤以及骨肉瘤等诸多癌症中高表达(Tang,Zhao et al.2019,Tang,Liu et al.2020)。B7-H3不仅表达于肿瘤细胞上，在肿瘤新生血管内皮细胞上同样高表达，是一个非常广谱的肿瘤标志性抗原。B7-H3蛋白高表达可促进癌症进展并与患者的不良预后 有关，因此，抑制B7-H3蛋白的表达对治疗癌症具有潜在作用。It has been clinically reported that B7-H3 is low expressed in healthy tissues, but highly expressed in a large number of malignant tumors. Studies have shown that B7-H3 can play a role in esophageal cancer, melanoma, colorectal cancer, adenocarcinoma, ovarian cancer, non-small cell lung cancer, kidney cancer, gastric cancer, bladder cancer, glioblastoma multiforme and osteosarcoma, etc. Highly expressed in cancer (Tang, Zhao et al.2019, Tang, Liu et al.2020). B7-H3 is not only expressed on tumor cells, but also highly expressed on tumor neovascular endothelial cells, and is a very broad-spectrum tumor marker antigen. High expression of B7-H3 protein can promote cancer progression and is associated with poor prognosis of patients. Therefore, inhibiting the expression of B7-H3 protein has a potential effect on the treatment of cancer.

目前，还没有靶向B7-H3的抗体药物上市，因此，有必要进一步开发具有更高活性、更高亲和力和治疗效果的B7-H3抗体，以进行相关疾病的治疗研究和应用。At present, there is no antibody drug targeting B7-H3 on the market. Therefore, it is necessary to further develop B7-H3 antibodies with higher activity, higher affinity and therapeutic effect for the treatment research and application of related diseases.

发明内容Contents of the invention

本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明提出一种抗B7-H3抗体，能够高特异性结合B7-H3。The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the present invention proposes an anti-B7-H3 antibody capable of binding to B7-H3 with high specificity.

本发明还提出与上述抗体相关的重组蛋白、药物组合物、多核苷酸、重组质粒和分离细胞。The present invention also proposes recombinant proteins, pharmaceutical compositions, polynucleotides, recombinant plasmids and isolated cells related to the above antibodies.

本发明还提出上述抗体的制备方法。The present invention also proposes a preparation method of the above-mentioned antibody.

本发明还提出上述抗体在制备抗癌药物中的应用。The present invention also proposes the application of the above antibody in the preparation of anticancer drugs.

本发明还提出上述抗体在制备抗体检测试剂盒中的应用。The present invention also proposes the application of the above-mentioned antibody in the preparation of an antibody detection kit.

根据本发明的第一方面实施方式的抗体，所述抗体包含重链可变区和/或轻链可变区：所述重链可变区，其包含：由SEQ ID NO:1所示的氨基酸序列组成的重链互补决定区HCDR1，由SEQ ID NO:2所示的氨基酸序列组成的重链互补决定区HCDR2，由SEQ ID NO:3所示的氨基酸序列组成的重链互补决定区HCDR3；所述轻链可变区，其包含：由SEQ ID NO:4所示的氨基酸序列组成的轻链互补决定区LCDR1，由WAS氨基酸序列组成的轻链互补决定区LCDR2，由SEQ ID NO:5所示的氨基酸序列组成的轻链互补决定区LCDR3。According to the antibody of the first aspect embodiment of the present invention, the antibody comprises a heavy chain variable region and/or a light chain variable region: the heavy chain variable region comprises: represented by SEQ ID NO:1 The heavy chain complementarity determining region HCDR1 composed of the amino acid sequence, the heavy chain complementarity determining region HCDR2 composed of the amino acid sequence shown in SEQ ID NO:2, the heavy chain complementarity determining region HCDR3 composed of the amino acid sequence shown in SEQ ID NO:3 The light chain variable region, which comprises: the light chain complementarity determining region LCDR1 composed of the amino acid sequence shown in SEQ ID NO: 4, the light chain complementarity determining region LCDR2 composed of the WAS amino acid sequence, composed of SEQ ID NO: The light chain complementarity determining region LCDR3 composed of the amino acid sequence shown in 5.

根据本发明实施方式的抗体，至少具有如下有益效果：本发明的抗体具有(1)结合活性好，与B7-H3蛋白较强的结合能力；(2)物种交叉反应性，与猴B7-H3具有交叉反应性，利于开展验证实验和便于后续有治疗用途的产品开发；(3)具有ADCC活性。The antibody according to the embodiment of the present invention has at least the following beneficial effects: the antibody of the present invention has (1) good binding activity and strong binding ability to B7-H3 protein; (2) cross-reactivity with monkey B7-H3 It has cross-reactivity, which is beneficial to carry out verification experiments and facilitate the subsequent development of products with therapeutic use; (3) has ADCC activity.

在本发明中，抗体依赖的细胞介导的细胞毒作用(ADCC，antibody-dependent cell-mediated cytotoxicity)是一种细胞介导的免疫防御机制，是免疫细胞消灭已结合特异性抗体的病原靶细胞的作用方式。现如今，ADCC作用机制被用来检测、评定抗体或靶细胞的功效。In the present invention, antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity) is a cell-mediated immune defense mechanism, which is the elimination of pathogenic target cells bound to specific antibodies by immune cells. mode of action. Today, the ADCC mechanism of action is used to detect and evaluate the efficacy of antibodies or target cells.

根据本发明的一些实施方式，所述为鼠源抗体、嵌合抗体或人源抗体。According to some embodiments of the present invention, the antibody is a murine antibody, a chimeric antibody or a human antibody.

根据本发明的一些实施方式，所述重链可变区选自如SEQ ID NO:6、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21或SEQ ID NO:22所示的氨基酸序列；所述轻链可 变区由选自如SEQ ID NO:7、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26或SEQ ID NO:27所示的氨基酸序列。According to some embodiments of the present invention, the heavy chain variable region is selected from such as SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO The amino acid sequence shown in: 22; The light chain variable region is selected from such as SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID The amino acid sequence shown in NO:27.

根据本发明的一些实施方式，所述抗体包含重链恒定区和/或轻链恒定区：所述重链恒定区包含如SEQ ID NO:10所示的氨基酸序列；所述轻链恒定区包含如SEQ ID NO:12所示的氨基酸序列。According to some embodiments of the present invention, the antibody comprises a heavy chain constant region and/or a light chain constant region: the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 10; the light chain constant region comprises Amino acid sequence as shown in SEQ ID NO:12.

根据本发明的一些实施方式，所述抗体包含重链和/或轻链：所述重链选自如SEQ ID NO:14、SEQ ID NO:46和SEQ ID NO:47所示的氨基酸序列；所述轻链选自如SEQ ID NO:15、SEQ ID NO:48和SEQ ID NO:49所示的氨基酸序列。According to some embodiments of the present invention, the antibody comprises a heavy chain and/or a light chain: the heavy chain is selected from the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:46 and SEQ ID NO:47; The light chain is selected from the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 48 and SEQ ID NO: 49.

根据本发明的一些实施方式，所述抗体包含以下i至v中任一种性质：According to some embodiments of the present invention, the antibody comprises any one of the following properties i to v:

i、所述抗体能够特异性结合B7-H3；i, the antibody can specifically bind to B7-H3;

ii、所述抗体具有抗体依赖的细胞介导的细胞毒作用；ii. The antibody has antibody-dependent cell-mediated cytotoxicity;

iii、所述抗体特异性地结合人4Ig-B7-H3并与猴4Ig-B7-H3交叉反应；iii. The antibody specifically binds to human 4Ig-B7-H3 and cross-reacts with monkey 4Ig-B7-H3;

iv、所述抗体具有减少的糖基化或无糖基化或被低岩藻糖基化。iv. The antibody has reduced or aglycosylated or hypofucosylated.

在本发明中，上述抗体序列具有最佳的结合活性、反应特异性、物种交叉反应性和ADCC活性。通过实施例实验可以证明本发明的抗体与B7-H3蛋白结合力强；具有物种交叉反应这一特点，有助于抗体在猴子体内进行毒性分析，有利于在相关动物中开展验证试验，进而有利于后续的治疗用途的应用开发；有较强的ADCC活性，更适合抗体药物的后续开发。In the present invention, the above antibody sequences have the best binding activity, reaction specificity, species cross-reactivity and ADCC activity. It can be proved that the antibody of the present invention has a strong binding force to the B7-H3 protein through the experiments of the examples; it has the characteristic of species cross-reactivity, which is helpful for the toxicity analysis of the antibody in monkeys, and is conducive to carrying out verification tests in related animals. It is beneficial to the application and development of subsequent therapeutic purposes; it has strong ADCC activity and is more suitable for the subsequent development of antibody drugs.

在本发明中，与未经修饰的Fc区相比，所述Fc区的氨基酸位点被修饰以增强FcγR结合。在一些实施方式中，所述Fc区具有更强的FcγRIII结合的能力，从而发挥更强的抗体介导的细胞毒(ADCC)作用。In the present invention, compared with the unmodified Fc region, the amino acid positions of the Fc region are modified to enhance FcγR binding. In some embodiments, the Fc region has a stronger FcγRIII binding ability, thereby exerting a stronger antibody-mediated cytotoxicity (ADCC) effect.

根据本发明第二方面的实施方式的重组蛋白、药物组合物、多核苷酸、载体或分离细胞：所述重组蛋白包含上述抗体；所述药物组合物包含上述抗体或上述重组蛋白；所述多核苷酸包含编码上述抗体或重组蛋白的核苷酸序列；所述载体包含上述多核苷酸；所述分离细胞产生上述抗体。The recombinant protein, pharmaceutical composition, polynucleotide, vector or isolated cell according to the embodiment of the second aspect of the present invention: the recombinant protein comprises the above-mentioned antibody; the pharmaceutical composition comprises the above-mentioned antibody or the above-mentioned recombinant protein; the multinuclear The nucleotide contains the nucleotide sequence encoding the above-mentioned antibody or recombinant protein; the vector contains the above-mentioned polynucleotide; and the isolated cell produces the above-mentioned antibody.

根据本发明的一些实施方式，所述重组蛋白还包含协助表达和/或纯化的标签序列。According to some embodiments of the present invention, the recombinant protein further comprises a tag sequence to assist expression and/or purification.

根据本发明的一些实施方式，所述重组蛋白为双抗，即还包括能够与其他靶蛋白结合的抗体。进一步地，所述双抗还包括特异性结合B7-H3不同表位的抗体。According to some embodiments of the present invention, the recombinant protein is a double antibody, which also includes antibodies capable of binding to other target proteins. Further, the double antibody also includes antibodies that specifically bind to different epitopes of B7-H3.

根据本发明的一些实施方式，所述药物组合物还包括上述双抗。According to some embodiments of the present invention, the pharmaceutical composition further includes the above-mentioned double antibody.

根据本发明的一些实施方式，所述药物组合物还包括含有上述抗体的ADC药物。进一步地，所述ADC药物还包括连接臂和毒性分子。According to some embodiments of the present invention, the pharmaceutical composition further includes an ADC drug containing the above-mentioned antibody. Further, the ADC drug also includes a linker and a toxic molecule.

根据本发明地一些实施方式，所述药物组合物还包括药学上可用的辅料。According to some embodiments of the present invention, the pharmaceutical composition further includes pharmaceutically available excipients.

根据本发明第三方面的实施方式的制备方法，包括以下步骤：培养上述分离细胞，从培养物中回收所述抗体。The preparation method according to the embodiment of the third aspect of the present invention includes the following steps: culturing the above-mentioned isolated cells, and recovering the antibody from the culture.

在本发明中，具体的制备方法为：将编码上述抗B7-H3抗体的重链可变区序列克隆到含有IgG1重链恒定区氨基酸序列的重组质粒1中，轻链可变区序列克隆到含有Kappa轻链恒定区氨基酸序列的重组质粒2中，将重组质粒1和重组质粒2同时转染细胞并进行培养，从培养物中回收所述抗B7-H3抗体。In the present invention, the specific preparation method is as follows: the heavy chain variable region sequence encoding the above-mentioned anti-B7-H3 antibody is cloned into recombinant plasmid 1 containing the IgG1 heavy chain constant region amino acid sequence, and the light chain variable region sequence is cloned into In the recombinant plasmid 2 containing the amino acid sequence of the Kappa light chain constant region, the recombinant plasmid 1 and the recombinant plasmid 2 were simultaneously transfected into cells and cultured, and the anti-B7-H3 antibody was recovered from the culture.

根据本发明第四方面的实施方式的应用，所述抗体可应用于抗癌药物和/或抗体检测试剂盒的制备中。According to the application of the embodiment of the fourth aspect of the present invention, the antibody can be applied to the preparation of anticancer drugs and/or antibody detection kits.

在本发明中，所述抗癌药物主要用于预防或治疗癌症，其中所述癌症是表达B7-H3蛋白的乳腺癌、子宫内膜癌、卵巢癌、肺癌、胃癌、前列腺癌、肾癌、肝癌、胰腺癌、结肠直肠癌、食道癌、膀胱癌、子宫颈癌、血液癌、淋巴瘤或恶性黑色素瘤。In the present invention, the anticancer drug is mainly used for preventing or treating cancer, wherein the cancer is breast cancer, endometrial cancer, ovarian cancer, lung cancer, gastric cancer, prostate cancer, kidney cancer, Cancer of the liver, pancreas, colorectum, esophagus, bladder, cervix, blood, lymphoma, or malignant melanoma.

在本发明中，抗癌药物还包含与靶向其它靶点的抗体，如：CD3、BCMA、CD38等构建成双特异性抗体，开发成各种具有调节肿瘤细胞作用的方法。In the present invention, anti-cancer drugs also include antibodies targeting other targets, such as: CD3, BCMA, CD38, etc. to construct bispecific antibodies, and develop various methods for regulating tumor cells.

在本发明中，本发明的抗体还可以通过偶联其它类型的分子，如：毒素、核酸分子等，通过该抗体特异地将偶联的分子带到肿瘤细胞体内，从而调节肿瘤细胞的作用。In the present invention, the antibody of the present invention can also be coupled to other types of molecules, such as toxins, nucleic acid molecules, etc., and the antibody can specifically bring the coupled molecules into the body of tumor cells, thereby regulating the effect of tumor cells.

在本发明中，可对所述的B7-H3特异性抗体或抗原结合片段进行标记以用于所述方法或本领域技术人员已知的其它方法。例如，本发明所述的抗体或其抗原结合片段可用放射标记物、荧光标记物、表位标签、生物素、发色团标记物、ECL标记物、酶、钌、111In-DOTA、111In-二乙烯三胺五乙酸(DTPA)、辣根过氧化物酶、碱性磷酸酶和β-半乳糖苷酶，或者聚组氨酸或本领域已知的类似此类标记物进行标记。In the present invention, the B7-H3-specific antibody or antigen-binding fragment can be labeled for use in the method or other methods known to those skilled in the art. For example, the antibodies or antigen-binding fragments thereof of the present invention can be radiolabeled, fluorescently labeled, epitope tags, biotin, chromophore labels, ECL labels, enzymes, ruthenium, 111In-DOTA, 111In-di Ethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, or polyhistidine or similar such labels known in the art.

在本发明中，该抗体还可以开发成通过免疫手段，对组织细胞表面B7-H3表达量进行检测的方法。例如，该抗体还可用于检测生物样品诸如血液或血清中B7-H3的存在、用于定量分析生物样品诸如血液或血清中B7-H3的量、用于诊断B7-H3表达型癌症、用于确定治疗患 有癌症的受治疗者的方法、或用于监测受治疗者中B7-H3表达型癌症的进展等。In the present invention, the antibody can also be developed into a method for detecting the expression level of B7-H3 on the surface of tissue cells by means of immunization. For example, the antibody can also be used for detecting the presence of B7-H3 in a biological sample such as blood or serum, for quantitative analysis of the amount of B7-H3 in a biological sample such as blood or serum, for diagnosing B7-H3 expressing cancer, for To determine a method of treating a subject with cancer, or for monitoring the progression of a B7-H3 expressing cancer in a subject, and the like.

在本发明中，术语“抗体”具体包括狭义抗体，还包括“嵌合”抗体以及该抗体片段，其中部分重链和/轻链与衍生自特定物种或属于特定抗体类型或亚类抗体的对应序列相同或同源，而该链的剩余部分与衍生自另一物种或属于另一抗体类型或亚类抗体的对应序列相同或同源，只要其特异性结合靶抗原和/或显示所需的生物活性(美国专利号4,816,567，和Morrison等，Proc.Natl.Acad.Sci.USA 81:6851-6855(1984))。其中，“狭义抗体”是指由抗原进入机体刺激B细胞分化增殖为浆细胞而合并分泌的一类能与相应抗原发生特异性结合并产生免疫效应的含有糖基的球蛋白。1964年世界卫生组织召开会议，将具有抗体活性及化学结构与抗体相似的球蛋白统称为免疫球蛋白。现代免疫学认为，抗体与免疫球蛋白是等同概念，只是抗体侧重于其生物学活性的描述，而免疫球蛋白侧重强调其化学结构。In the present invention, the term "antibody" specifically includes antibodies in a narrow sense, and also includes "chimeric" antibodies and antibody fragments, wherein part of the heavy chain and/or light chain corresponds to an antibody derived from a specific species or belonging to a specific antibody type or subclass. The sequence is identical or homologous, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody type or subclass, so long as it specifically binds the target antigen and/or exhibits the desired Biological activity (US Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Among them, "antibody in a narrow sense" refers to a type of glycosyl-containing globulin that is secreted by antigens entering the body to stimulate B cells to differentiate and proliferate into plasma cells, which can specifically bind to corresponding antigens and produce immune effects. In 1964, the World Health Organization held a meeting and collectively referred to globulins with antibody activity and chemical structure similar to antibodies as immunoglobulins. Modern immunology believes that antibodies and immunoglobulins are equivalent concepts, but antibodies focus on the description of their biological activities, while immunoglobulins focus on their chemical structures.

免疫球蛋白的基本结构包含四条肽链：两条重链(Heavy chain，H链)和两条轻链(Light chain，L链)，轻链与重链由二硫键连接形成一个对称四肽链分子，成为免疫球蛋白分子单体，单体是构成所有免疫球蛋白的基本结构。每条重链和轻链分为氨基端(N端)和羧基端(C端)。通过对H链或L链的氨基酸序列比较分析发现：其N端序列变化很大，称此区为可变区(Variable region，V区)；C端氨基酸相对稳定，变化很小，称此区为恒定区(Constant region，C区)。可变区可分为高可变区(Hypervariable region，HVR)和骨架区(Framework region，FR)。重链可变区和轻链可变区的分别有3个HVR，从N端往C端，分别称为重链或轻链的HVR1、HVR2和HVR3。高可变区为抗体与抗原的结合位置，称为互补决定区(Complementarity-determining region，CDR)，因此，重链或轻链的HVR1、HVR2和HVR3又称CDR1、CDR2和CDR3。The basic structure of immunoglobulin consists of four peptide chains: two heavy chains (Heavy chain, H chain) and two light chains (Light chain, L chain), the light chain and the heavy chain are connected by disulfide bonds to form a symmetrical tetrapeptide Chain molecules become monomers of immunoglobulin molecules, which are the basic structure of all immunoglobulins. Each heavy and light chain is divided into amino-terminus (N-terminus) and carboxy-terminus (C-terminus). Through the comparison and analysis of the amino acid sequences of the H chain or L chain, it is found that the N-terminal sequence changes greatly, and this region is called the variable region (Variable region, V region); the C-terminal amino acid is relatively stable, and the change is small, called this region. It is a constant region (Constant region, C region). The variable region can be divided into hypervariable region (HVR) and framework region (Framework region, FR). There are three HVRs in the heavy chain variable region and the light chain variable region, respectively, from the N-terminal to the C-terminal, called HVR1, HVR2 and HVR3 of the heavy chain or light chain, respectively. The hypervariable region is the binding site of the antibody and the antigen, and is called the complementarity-determining region (CDR). Therefore, HVR1, HVR2, and HVR3 of the heavy chain or light chain are also called CDR1, CDR2, and CDR3.

在本发明中，术语“双抗”，即“双特异性”抗体，指某一抗体，一般是单克隆抗体，具有至少两个不同抗原性表位的结合特性。在一个实施方式中，该表位来自相同抗原。在另一个实施方式中，该表位来自两个不同抗原。制备双特异性抗体的方法为本领域已知。例如，双特异性抗体可通过共同表达两种免疫球蛋白重/轻链对来重组生产。参见例如，Milstein等，Nature305:537-39(1983)。或者，可利用化学连接制备双特异性抗体。参见例如，Brennan等，Science229:81(1985)。双特异性抗体包括双特异性抗体片段(例如，Hollinger等，Proc.Natl.Acad.Sci.U.S.A.90:6444-48(1993)，Gruber等，J.Immunol.152:5368(1994))。In the present invention, the term "diabodies", that is, "bispecific" antibodies, refers to an antibody, generally a monoclonal antibody, that has the binding properties of at least two different antigenic epitopes. In one embodiment, the epitopes are from the same antigen. In another embodiment, the epitopes are from two different antigens. Methods of making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly by co-expressing two immunoglobulin heavy/light chain pairs. See, eg, Milstein et al., Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical linkage. See, eg, Brennan et al., Science 229:81 (1985). Bispecific antibodies include bispecific antibody fragments (eg, Hollinger et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-48 (1993), Gruber et al., J. Immunol. 152:5368 (1994)).

本发明中，术语“人源化抗体”指含有来自非人(例如鼠)抗体以及人抗体序列的抗体形式。该抗体是含有衍生自非人免疫球蛋白最小序列的嵌合抗体。通常，所述人源化抗体包含几乎所有的至少一个且通常两个可变区，其中全部或基本上全部的高变环对应于非人免疫球蛋白的高变环，全部或基本上全部的FR区是人免疫球蛋白序列的FR区。人源化抗体还任选包含至少一部分免疫球蛋白恒定区(Fc)，一般是人免疫球蛋白的Fc。参见例如，Cabilly美国专利号4,816,567；Queen等(1989)Proc.Natl.Acad.Sci.USA86:10029-10033；和Antibody Engineering:A Practical Approach(《抗体工程：实践方法》)(牛津大学出版社(OxfordUniversityPress)1996)。In the present invention, the term "humanized antibody" refers to a form of antibody that contains sequences derived from non-human (eg, murine) antibodies as well as human antibodies. The antibody is a chimeric antibody containing minimal sequence derived from non-human immunoglobulin. Typically, such humanized antibodies will comprise substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, all or substantially all of which The FR regions are those of human immunoglobulin sequences. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Cabilly U.S. Patent No. 4,816,567; Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033; Oxford University Press) 1996).

本发明所用氨基酸三字母代码和单字母代码如J.Biol.Chem,243,P3558(1968)中所述。The three-letter codes and one-letter codes of amino acids used in the present invention are as described in J. Biol. Chem, 243, P3558 (1968).

本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

附图说明Description of drawings

图1示出了本发明实施例3中嵌合抗体27B4与MDA-MB-231的结合力；Fig. 1 shows the binding force of chimeric antibody 27B4 and MDA-MB-231 in Example 3 of the present invention;

图2示出了本发明实施例3中嵌合抗体27B4的ADCC活性；Figure 2 shows the ADCC activity of chimeric antibody 27B4 in Example 3 of the present invention;

图3示出了本发明实施例4中不同27B4人源化scFv抗体与B7-H3抗原的结合力；Figure 3 shows the binding ability of different 27B4 humanized scFv antibodies to B7-H3 antigen in Example 4 of the present invention;

图4示出了本发明实施例4中不同Whole IgG形式27B4人源化抗体B7-H3抗原的结合力；Fig. 4 shows the binding force of different Whole IgG forms 27B4 humanized antibody B7-H3 antigen in Example 4 of the present invention;

图5示出了本发明实施例4中不同Whole IgG形式27B4人源化抗体ADCC活性。Figure 5 shows the ADCC activities of different Whole IgG forms of 27B4 humanized antibodies in Example 4 of the present invention.

图6示出了本发明实施例4中27B4人源化抗体h27B4-H4-L3(27B4Z02分子)结合B7-H32Ig与4Ig蛋白的活性。Fig. 6 shows the binding activity of 27B4 humanized antibody h27B4-H4-L3 (27B4Z02 molecule) in Example 4 of the present invention to B7-H32Ig and 4Ig proteins.

图7示出了本发明实施例4中27B4人源化抗体h27B4-H4-L3(27B4Z02分子)结合人和猴B7-H3蛋白的活性。Fig. 7 shows the activity of binding human and monkey B7-H3 proteins of the 27B4 humanized antibody h27B4-H4-L3 (27B4Z02 molecule) in Example 4 of the present invention.

具体实施方式Detailed ways

为详细说明本发明的技术内容、所实现目的及效果，以下结合实施方式并配合附图予以说明。In order to describe the technical content, achieved goals and effects of the present invention in detail, the following descriptions will be made in conjunction with the embodiments and accompanying drawings.

实施例1.B7-H3抗原、表达载体及稳转细胞株的制备Example 1. Preparation of B7-H3 antigen, expression vector and stable transfection cell line

1-1.B7-H3抗原及检测用蛋白的制备1-1. Preparation of B7-H3 antigen and detection protein

筛选及检测用人4Ig-B7-H3抗原为商品化产品(Acro，货号B7B-H52E7)，序列如下：The human 4Ig-B7-H3 antigen used for screening and detection is a commercial product (Acro, product number B7B-H52E7), and the sequence is as follows:

Figure PCTCN2022115318-appb-000001
Figure PCTCN2022115318-appb-000001

筛选及检测用人2Ig-B7-H3抗原为商品化产品(Acro，货号B73-H52E2)，序列如下：The human 2Ig-B7-H3 antigen used for screening and detection is a commercial product (Acro, product number B73-H52E2), and the sequence is as follows:

Figure PCTCN2022115318-appb-000002
Figure PCTCN2022115318-appb-000002

筛选及检测用食猕猴4Ig-B7-H3抗原为商品化产品(Acro，货号B73-C52Ha)，序列如下：The macaque 4Ig-B7-H3 antigen used for screening and detection is a commercial product (Acro, product number B73-C52Ha), and the sequence is as follows:

Figure PCTCN2022115318-appb-000003
Figure PCTCN2022115318-appb-000003

筛选及检测用鼠2Ig-B7-H3抗原为商品化产品(Acro，货号B73-M52H4)，序列如下：The mouse 2Ig-B7-H3 antigen used for screening and detection is a commercial product (Acro, product number B73-M52H4), and the sequence is as follows:

Figure PCTCN2022115318-appb-000004
Figure PCTCN2022115318-appb-000004

注释：划横线部分为B7-H3胞外区；斜体部分为linker；黑色加粗部分为His-tag标签。Note: The horizontal line part is the extracellular region of B7-H3; the italic part is the linker; the black bold part is the His-tag tag.

1-2.人4Ig-B7-H3表达载体的制备1-2. Preparation of human 4Ig-B7-H3 expression vector

从NCBI数据库中获取编码人B7-H3变体1(4Ig-B7-H3)的CDS区，由生工生物工程(上海)股份有限公司常规合成全长基因序列，并通过酶切位点XbaI与NotI构建到慢病毒过表达载体pCDH-EF1-T2A-copGFP中，经测序验证序列正确，即成功构建质粒pCDH-EF1-h4Ig-B7-H3-T2A-copGFP。在液体LB培养基中培养菌种pCDH-EF1-h4Ig-B7-H3-T2A-copGFP，并使用天根生化科技(北京)有限公司无内毒素质粒大提试剂盒(DP117)，按照说明书常规步骤提取质粒DNA。The CDS region encoding human B7-H3 variant 1 (4Ig-B7-H3) was obtained from the NCBI database, and the full-length gene sequence was routinely synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. NotI was constructed into the lentiviral overexpression vector pCDH-EF1-T2A-copGFP, and the sequence was verified to be correct by sequencing, that is, the plasmid pCDH-EF1-h4Ig-B7-H3-T2A-copGFP was successfully constructed. Cultivate strain pCDH-EF1-h4Ig-B7-H3-T2A-copGFP in liquid LB medium, and use Tiangen Biochemical Technology (Beijing) Co., Ltd. Endotoxin-free Plasmid Large-scale Extraction Kit (DP117), follow the routine steps in the manual Extract plasmid DNA.

人B7-H3变体1(4Ig-B7-H3)的CDS区碱基序列：The base sequence of the CDS region of human B7-H3 variant 1 (4Ig-B7-H3):

Figure PCTCN2022115318-appb-000005
Figure PCTCN2022115318-appb-000005

Figure PCTCN2022115318-appb-000006
Figure PCTCN2022115318-appb-000006

人B7-H3变体1(4Ig-B7-H3)的CDS区氨基酸序列：The amino acid sequence of the CDS region of human B7-H3 variant 1 (4Ig-B7-H3):

Figure PCTCN2022115318-appb-000007
Figure PCTCN2022115318-appb-000007

1-3.表达人4Ig-B7-H3抗原的293稳转细胞株制备1-3. Preparation of 293 stably transfected cell line expressing human 4Ig-B7-H3 antigen

选取生长状态良好的对数增长期的HEK293细胞(来源于ATCC)，在一个10cm培养皿中接种8E6数量的细胞，DMEM中添加10％胎牛血清，37℃、5％CO 2培养箱内培养，当细胞融合率达到70％～80％左右时转染。使用转染试剂PEI共转染三质粒psPAX2、pMD2.G和pCDH-EF1-h4Ig-B7-H3-T2A-copGFP。分别收集转染后48小时与72小时的病毒液，0.45μM针头滤器过滤病毒，按照MOI为10感染目的细胞CHO-S(来源于ATCC)，RPMI-1640加10％胎牛血清培养。病毒感染48小时后换液。构建的细胞株通过流式检测人4Ig-B7-H3表达阳性率，测试阳性率在95％以上，293-h4Ig-B7-H3-copGFP稳转细胞株构建成功。 Select HEK293 cells in the logarithmic growth phase (derived from ATCC) in a good growth state, inoculate 8E6 cells in a 10 cm culture dish, add 10% fetal bovine serum to DMEM, and culture in a 37°C, 5% CO 2 incubator , when the cell fusion rate reaches about 70% to 80%, transfection. The three plasmids psPAX2, pMD2.G and pCDH-EF1-h4Ig-B7-H3-T2A-copGFP were co-transfected using the transfection reagent PEI. The virus liquid was collected 48 hours and 72 hours after transfection, filtered through a 0.45 μM syringe filter, infected with the target cell CHO-S (derived from ATCC) at an MOI of 10, and cultured in RPMI-1640 plus 10% fetal bovine serum. The medium was changed 48 hours after virus infection. The positive rate of human 4Ig-B7-H3 expression was tested by flow cytometry, and the positive rate was over 95%. The 293-h4Ig-B7-H3-copGFP stably transfected cell line was successfully constructed.

实施例2.单克隆抗体的制备和抗体的筛选Example 2. Preparation of Monoclonal Antibodies and Screening of Antibodies

2-1.免疫接种2-1. Immunization

选择4只6周龄SPF级BALB/C品系的雌性健康小鼠(购自上海吉辉实验动物饲养有限公司)，免疫抗原为His标签的人重组4Ig-B7-H3抗原(Acro，货号B7B-H52E7)，抗原与等体积佐剂乳化。首次(第0天)免疫使用弗氏完全佐剂(Sigma，货号F5881)乳化抗原， 于小鼠颈背部皮下进行多点注射，每只小鼠免疫50μg抗原。第14天、28天、42天及56天用弗氏不完全佐剂(Sigma，货号F5602)乳化抗原进行免疫，每只小鼠免疫25μg抗原。于第20天、34天、48天及62天对小鼠进行眼眶采血，按照实施例2-4的方法通过ELISA检测小鼠免疫血清效价。免疫前血清作为阴性对照，将免疫血清按1:100稀释后，再3倍梯度稀释11个浓度，以3倍阴性对照OD值对应的血清稀释度作为其效价。挑选效价最高且两次加强免疫后血清效价趋于平稳的小鼠，在细胞融合前3天采用25μg重组人4Ig-B7-H3抗原腹腔注射进行一次加强免疫，此次抗原无需佐剂乳化，缓冲液为PBS。Four 6-week-old female healthy mice of SPF grade BALB/C strain (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were selected, and the immune antigen was His-tagged human recombinant 4Ig-B7-H3 antigen (Acro, catalog number B7B- H52E7), the antigen was emulsified with an equal volume of adjuvant. For the first (day 0) immunization, the antigen was emulsified with complete Freund's adjuvant (Sigma, product number F5881), and injected subcutaneously at multiple points on the back of the neck of the mouse, and each mouse was immunized with 50 μg of the antigen. On the 14th day, 28th day, 42th day and 56th day, the antigen was emulsified with Freund's incomplete adjuvant (Sigma, product number F5602) for immunization, and each mouse was immunized with 25 μg of the antigen. On the 20th day, 34th day, 48th day and 62nd day, blood was collected from the orbit of the mouse, and the titer of the mouse immune serum was detected by ELISA according to the method of Examples 2-4. The pre-immune serum was used as a negative control. After the immune serum was diluted 1:100, it was then serially diluted to 11 concentrations by 3 times, and the serum dilution corresponding to the OD value of 3 times the negative control was used as its titer. Select the mouse with the highest titer and the serum titer tends to be stable after two booster immunizations. Three days before cell fusion, 25 μg recombinant human 4Ig-B7-H3 antigen is injected intraperitoneally for a booster immunization. The antigen does not need adjuvant emulsification , the buffer is PBS.

2-2.杂交瘤制备及筛选鉴定2-2. Hybridoma preparation and screening identification

收集生长状态良好的对数增长期的SP2/0骨髓瘤细胞(购自中科院细胞库)与脾细胞按照1：1的比例进行电融合，96孔细胞培养板每孔接种15000个细胞，HAT完全培养基37℃5％CO 2培养箱中培养，第4天换成HT完全培养基继续培养。当杂交瘤细胞培养到第7天时，按照实施例2-4的方法通过ELISA法检测细胞上清与人4Ig-B7-H3重组抗原的结合力。对ELISA检测阳性的克隆，按照实施例2-5的方法进行流式细胞仪(Flow Cytometry，FACS)检测，检测上清与过稳转细胞293-h4Ig-B7-H3-copGFP的结合力。采用有限稀释法对阳性杂交瘤细胞进行单克隆化，然后进行ELISA检测及FACS检测，FACS方法同时检测了样品与过稳转细胞293-h4Ig-B7-H3-copGFP及内源表达细胞MDA-MB-231的结合情况，挑选阳性单克隆孔重复进行1次单克隆化及阳性克隆筛选鉴定。 Collect well-growing SP2/0 myeloma cells in the logarithmic growth phase (purchased from the Cell Bank of the Chinese Academy of Sciences) and spleen cells for electrofusion at a ratio of 1:1. 15,000 cells are seeded in each well of a 96-well cell culture plate, and HAT is completely complete. The culture medium was cultured in a 5% CO 2 incubator at 37°C, and replaced with HT complete medium on the 4th day to continue the culture. When the hybridoma cells were cultured to the 7th day, the binding ability of the cell supernatant to the human 4Ig-B7-H3 recombinant antigen was detected by ELISA according to the method of Example 2-4. For the positive clones detected by ELISA, flow cytometry (Flow Cytometry, FACS) detection was carried out according to the method in Example 2-5, and the binding force between the supernatant and the overstable cells 293-h4Ig-B7-H3-copGFP was detected. Positive hybridoma cells were monoclonalized by the limiting dilution method, and then detected by ELISA and FACS. The FACS method simultaneously detected the samples and the overstable cells 293-h4Ig-B7-H3-copGFP and endogenous expression cells MDA-MB For the binding status of -231, select positive monoclonal wells and repeat monoclonalization and positive clone screening and identification once.

2-3.单克隆抗体的制备2-3. Preparation of monoclonal antibody

将杂交瘤细胞以2.5×10 5/mL接种至250mL细胞培养瓶中，培养体积为50mL，待细胞活率降至30％时，离心收获含有抗体的上清液，并使用0.45μM滤器过滤，Protein G介质纯化小鼠单克隆抗体并通过透析方法将抗体置换至PBS pH7.2缓冲液中。通过Nanodrop测定吸光度来确定抗体浓度和纯度，并通过十二烷基硫酸钠凝胶电泳和考马斯染色来检查纯度。 Hybridoma cells were inoculated into a 250mL cell culture flask at 2.5×10 5 /mL, and the culture volume was 50mL. When the cell viability dropped to 30%, the supernatant containing the antibody was harvested by centrifugation, and filtered with a 0.45 μM filter. Mouse monoclonal antibodies were purified in Protein G medium and replaced by dialysis into PBS pH7.2 buffer. Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.

2-4.ELISA方法确定单克隆抗体与不同种属B7-H3及其变体的交叉反应性2-4. ELISA method to determine the cross-reactivity of monoclonal antibodies with different species of B7-H3 and its variants

将商品化的重组B7-H3抗原(人4Ig-B7-H3抗原，Acro，货号B7B-H52E7或人2Ig-B7-H3，Acro，货号B73-H52E2或食猕猴4Ig-B7-H3，Acro，货号B73-C52Ha或鼠2Ig-B7-H3，Acro，货号B73-M52H4)用PBS稀释至1μg/mL，25μL/孔包被384孔酶标板，4℃孵育过夜。弃去包被液并加入80μg/孔封闭液(含2％脱脂奶粉的PBS)室温封闭2小时。弃去封闭液，PBST (1‰Tween-20)洗板3次。系列稀释单克隆抗体，25μL/孔加入酶标板，室温孵育1小时。PBST洗板3次后，加入HRP标记的羊抗鼠IgG(Jackson，货号115-035-071)，室温孵育1小时。弃去二抗，PBST洗板7次后加入底物TMB显色12min。最后每孔加入25μL终止液(2M HCl)终止反应，用酶标仪读取450nm处吸光度值。Commercialized recombinant B7-H3 antigen (human 4Ig-B7-H3 antigen, Acro, Cat. No. B7B-H52E7 or human 2Ig-B7-H3, Acro, Cat. B73-C52Ha or mouse 2Ig-B7-H3, Acro, Cat. No. B73-M52H4) was diluted with PBS to 1 μg/mL, 25 μL/well was coated on a 384-well microtiter plate, and incubated overnight at 4°C. The coating solution was discarded and 80 μg/well of blocking solution (PBS containing 2% skim milk powder) was added to block at room temperature for 2 hours. Discard the blocking solution and wash the plate 3 times with PBST (1‰Tween-20). Serially dilute the monoclonal antibody, add 25 μL/well to the microtiter plate, and incubate at room temperature for 1 hour. After the plate was washed three times with PBST, HRP-labeled goat anti-mouse IgG (Jackson, Cat. No. 115-035-071) was added and incubated at room temperature for 1 hour. The secondary antibody was discarded, the plate was washed 7 times with PBST, and the substrate TMB was added for color development for 12 minutes. Finally, 25 μL of stop solution (2M HCl) was added to each well to terminate the reaction, and the absorbance at 450 nm was read with a microplate reader.

作为结果，发现了1株杂交瘤细胞27B4生产的抗体与人4Ig-B7-H3抗原及人2Ig-B7-H3抗原结合活性最高，且具有物种交叉反应性，能同时结合食猕猴4Ig-B7-H3。该交叉反应活性有利于抗体应用于动物实验中。As a result, it was found that the antibody produced by a hybridoma cell 27B4 has the highest binding activity to human 4Ig-B7-H3 antigen and human 2Ig-B7-H3 antigen, and has cross-species reactivity, and can simultaneously bind macaque 4Ig-B7- H3. This cross-reactivity facilitates the application of the antibody in animal experiments.

2-5.FACS方法确定单克隆抗体与癌细胞系的结合活性2-5. FACS method to determine the binding activity of monoclonal antibodies to cancer cell lines

通过FACS的方法，检验实施例2-4中获得的单克隆抗体是否结合过表达4Ig-B7-H3的稳转细胞293-h4Ig-B7-H3-copGFP及高表达4Ig-B7-H3的癌细胞MDA-MB-231(ATCC)。每孔加入5×10 5个293-h4Ig-B7-H3-copGFP细胞或MDA-MB-231细胞于96孔V型微孔板中，1500r/min离心1min，弃上清。稀释单克隆抗体到20μg/mL，每孔50μL重悬细胞，冰上孵育30min。然后每孔150μL PBS，1500r/min离心1min，弃上清，重复洗板4次。加入APC标记的羊抗鼠IgG(Jackson，货号115-605-164，PBS 1:800稀释)，每孔50μL，冰上孵育30min。用PBS洗板4次后每孔加入150μL PBS重悬细胞，用CytoFLEX进行检测(Beckman)。结果证实，单克隆抗体27B4结合稳转细胞293-h4Ig-B7-H3-copGFP及内源高表达细胞MDA-MB-231。 By FACS method, check whether the monoclonal antibody obtained in Example 2-4 binds to the stable transfection cell 293-h4Ig-B7-H3-copGFP overexpressing 4Ig-B7-H3 and the cancer cell highly expressing 4Ig-B7-H3 MDA-MB-231 (ATCC). Add 5×10 5 293-h4Ig-B7-H3-copGFP cells or MDA-MB-231 cells to each well in a 96-well V-shaped microplate, centrifuge at 1500r/min for 1min, and discard the supernatant. Dilute the monoclonal antibody to 20 μg/mL, resuspend the cells in 50 μL per well, and incubate on ice for 30 min. Then, 150 μL of PBS per well was centrifuged at 1500 r/min for 1 min, the supernatant was discarded, and the plate was repeatedly washed 4 times. Add APC-labeled goat anti-mouse IgG (Jackson, Cat. No. 115-605-164, diluted in PBS 1:800), 50 μL per well, and incubate on ice for 30 min. After washing the plate 4 times with PBS, 150 μL of PBS was added to each well to resuspend the cells, and the CytoFLEX was used for detection (Beckman). The results confirmed that the monoclonal antibody 27B4 binds to the stably transfected cell 293-h4Ig-B7-H3-copGFP and the endogenous highly expressed cell MDA-MB-231.

2-6.杂交瘤测序2-6. Hybridoma sequencing

收集对数增长期的27B4杂交瘤细胞，用RNAiso Plus(TAKARA，货号9109)提取总RNA，5’RACE法(SMARTer RACE5’/3’Kit，Clontech，货号634859)反转录获得cDNA。以cDNA为模板，Adaptor为上游引物，CL和CH1分别为抗体轻重链的下游引物(参考IMGT恒定区CL和CH1设计下游引物，http://www.imgt.org/genedb/)，使用ExTaq PCR(94℃，3min；94℃，30s，55℃，30s，72℃，45s，32cycle；72℃，5min)扩增抗体轻重链的可变区序列，PCR产物经天根通用型DNA纯化回收试剂盒回收纯化后，以摩尔比3:1分别与pMD18-T载体(Takara，货号6011)连接。连接产物转化到TG-1大肠杆菌感受态细胞进行测序。用VBASE2(http://www.vbase2.org/vbscAb.php)分析测序结果，获得抗体轻重链可变区序列。以下将该筛选出来的抗体记为27B4。27B4 hybridoma cells in logarithmic growth phase were collected, total RNA was extracted with RNAiso Plus (TAKARA, product number 9109), and cDNA was obtained by reverse transcription with 5'RACE method (SMARTer RACE5'/3'Kit, Clontech, product number 634859). Using cDNA as a template, Adapter as an upstream primer, CL and CH1 as downstream primers for antibody light and heavy chains respectively (refer to IMGT constant region CL and CH1 to design downstream primers, http://www.imgt.org/genedb/), using ExTaq PCR (94°C, 3min; 94°C, 30s, 55°C, 30s, 72°C, 45s, 32cycle; 72°C, 5min) to amplify the variable region sequence of the light and heavy chain of the antibody, and the PCR product is purified and recovered by Tiangen universal DNA reagent After the cassettes were recovered and purified, they were ligated with the pMD18-T vector (Takara, Cat. No. 6011) at a molar ratio of 3:1. The ligation products were transformed into TG-1 Escherichia coli competent cells for sequencing. VBASE2 (http://www.vbase2.org/vbscAb.php) was used to analyze the sequencing results to obtain the antibody light and heavy chain variable region sequences. Hereinafter, the screened antibody is referred to as 27B4.

其中，27B4重链可变区的氨基酸序列包括重链互补决定区HCDR1(SEQ ID NO：1)、HCDR2(SEQ ID NO：2)和HCDR3(SEQ ID NO：3)；轻链可变区包括轻链互补决定区LCDR1(SEQ ID NO：4)、LCDR2(WAS)和LCDR3(SEQ ID NO：5)。Among them, the amino acid sequence of the 27B4 heavy chain variable region includes heavy chain complementarity determining regions HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2) and HCDR3 (SEQ ID NO: 3); the light chain variable region includes Light chain complementarity determining regions LCDR1 (SEQ ID NO: 4), LCDR2 (WAS) and LCDR3 (SEQ ID NO: 5).

27B4重链可变区27B4VH的氨基酸序列：SEQ ID NO：6。Amino acid sequence of 27B4VH of 27B4 heavy chain variable region: SEQ ID NO:6.

27B4轻链可变区27B4VL的氨基酸序列：SEQ ID NO：7。Amino acid sequence of the 27B4 light chain variable region 27B4VL: SEQ ID NO:7.

表达27B4重链可变区27B4VH的碱基序列：SEQ ID NO：8。Express the base sequence of the 27B4 heavy chain variable region 27B4VH: SEQ ID NO: 8.

表达27B4轻链可变区27B4VL的碱基序列：SEQ ID NO：9。Express the base sequence of the 27B4 light chain variable region 27B4VL: SEQ ID NO: 9.

实施例3.嵌合抗体27B4的制备及活性检测Example 3. Preparation and Activity Detection of Chimeric Antibody 27B4

3-1.重链表达载体pTT5-hIgG1及轻链表达载体pTT5-HL的构建3-1. Construction of heavy chain expression vector pTT5-hIgG1 and light chain expression vector pTT5-HL

从NCBI获取抗体的γ重链恒定区氨基酸序列hIgG1(AWK57454.1)，并引进突变L235V,F243L,R292P,Y300L和P396L以增强ADCC效应(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)，并在序列N端依次加入Kozak序列(5'-GCCACCATGG-3')、信号肽序列和多克隆位点区序列，进行真核密码子优化获得碱基序列，其中克隆位点区包含酶切位点KpnI(5'-GGTACC-3')及MfeI(5'-CAATTG-3’)，在生工生物工程(上海)股份有限公司常规合成基因序列构建到pTT5载体(Biovector)的多克隆位点中，获得含抗体重链恒定区序列的真核重链表达载体pTT5-hIgG1。The amino acid sequence hIgG1 (AWK57454.1) of the γ heavy chain constant region of the antibody was obtained from NCBI, and mutations L235V, F243L, R292P, Y300L and P396L were introduced to enhance the ADCC effect (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity cytotoxicity), and at the N-terminal of the sequence, Kozak sequence (5'-GCCACCATGG-3'), signal peptide sequence and multiple cloning site region sequence were sequentially added, and eukaryotic codon optimization was performed to obtain the base sequence, in which the cloning site The dot region contains restriction sites KpnI (5'-GGTACC-3') and MfeI (5'-CAATTG-3'), and the routine synthetic gene sequence of Sangon Bioengineering (Shanghai) Co., Ltd. was constructed into the pTT5 vector (Biovector ), the eukaryotic heavy chain expression vector pTT5-hIgG1 containing the antibody heavy chain constant region sequence was obtained.

优化的hIgG1氨基端序列：SEQ ID NO：10。Optimized hIgG1 N-terminal sequence: SEQ ID NO:10.

优化的hIgG1碱基序列：SEQ ID NO：11。Optimized hIgG1 base sequence: SEQ ID NO: 11.

从NCBI获取抗体的κ轻链恒定区氨基酸序列HL(CAR58102.1)，并在序列N端依次加入Kozak序列(5'-GCCACCATGG-3')、信号肽序列和多克隆位点区序列，进行真核表达密码子优化获得碱基序列，其中克隆位点区包含酶切位点KpnI(5'-GGTACC-3')及MfeI(5'-CAATTG-3’)，在生工生物工程(上海)股份有限公司常规合成基因序列构建到pTT5载体的多克隆位点中，获得含抗体轻链恒定区序列的真核重链表达载体pTT5-HL。The amino acid sequence HL (CAR58102.1) of the κ light chain constant region of the antibody was obtained from NCBI, and the Kozak sequence (5'-GCCACCATGG-3'), the signal peptide sequence and the sequence of the multiple cloning site region were sequentially added to the N-terminal of the sequence. The base sequence was obtained by codon optimization for eukaryotic expression, in which the cloning site region contains restriction sites KpnI (5'-GGTACC-3') and MfeI (5'-CAATTG-3'), in Sangon Bioengineering (Shanghai ) Co., Ltd. routinely synthesized the gene sequence into the multiple cloning site of the pTT5 vector to obtain the eukaryotic heavy chain expression vector pTT5-HL containing the antibody light chain constant region sequence.

κ轻链恒定区氨基酸序列：SEQ ID NO：12。Amino acid sequence of constant region of kappa light chain: SEQ ID NO:12.

κ轻链恒定区碱基序列：SEQ ID NO：13。The base sequence of the constant region of the κ light chain: SEQ ID NO: 13.

Kozak序列是位于真核生物mRNA 5’端帽子结构后面的一段核酸序列，它可以与翻译起始因子结合而介导含有5’帽子结构的mRNA翻译起始。The Kozak sequence is a nucleic acid sequence located behind the cap structure at the 5' end of eukaryotic mRNA, which can bind to translation initiation factors to mediate translation initiation of mRNA containing the 5' cap structure.

3-2.27B4嵌合表达载体的构建3-2.2 Construction of 7B4 chimeric expression vector

将27B4重链和轻链可变区序列在生工生物工程(上海)股份有限公司常规合成，并通过KpnI及MfeI分别克隆进入pTT5-hIgG1及pTT5-HL载体质粒，得到重链表达质粒pTT5-27B4-VH-hIgG1及轻链表达质粒pTT5-27B4-VL-HL。The heavy chain and light chain variable region sequences of 27B4 were routinely synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and cloned into pTT5-hIgG1 and pTT5-HL vector plasmids by KpnI and MfeI, respectively, to obtain the heavy chain expression plasmid pTT5- 27B4-VH-hIgG1 and light chain expression plasmid pTT5-27B4-VL-HL.

嵌合抗体xw.M0883B9.27B4重链氨基酸序列：SEQ ID NO：14。Chimeric antibody xw.M0883B9.27B4 heavy chain amino acid sequence: SEQ ID NO: 14.

嵌合抗体xw.M0883B9.27B4轻链氨基酸序列：SEQ ID NO：15。Chimeric antibody xw.M0883B9.27B4 light chain amino acid sequence: SEQ ID NO: 15.

嵌合抗体xw.M0883B9.27B4重链碱基序列：SEQ ID NO：16。Chimeric antibody xw.M0883B9.27B4 heavy chain base sequence: SEQ ID NO: 16.

嵌合抗体xw.M0883B9.27B4轻链碱基序列：SEQ ID NO：17。Chimeric antibody xw.M0883B9.27B4 light chain base sequence: SEQ ID NO: 17.

3-3.嵌合抗体27B4(xw.M0883B9.27B4抗体)的表达纯化3-3. Expression and purification of chimeric antibody 27B4 (xw.M0883B9.27B4 antibody)

收集生长状态良好的对数增长期的293F细胞接种至250mL细胞培养瓶中并在50mL培养基中培养，PEI共转染轻链表达质粒pTT5-27B4-VL-HL与重链表达质粒pTT5-27B4-VH-hIgG1各25μg。收集转染后培养第7天的细胞上清，离心并使用0.45μM过滤器过滤，Protein A介质纯化抗体并通过透析方法将抗体置换至PBS pH7.2缓冲液中。通过Nanodrop测定吸光度来确定抗体浓度和纯度，并通过十二烷基硫酸钠凝胶电泳和考马斯染色来检查纯度。Collect well-growing 293F cells in the logarithmic growth phase and inoculate them into 250mL cell culture flasks and culture them in 50mL culture medium. PEI co-transfects light chain expression plasmid pTT5-27B4-VL-HL and heavy chain expression plasmid pTT5-27B4 - 25 µg each of VH-hIgG1. The cell supernatant on the 7th day after transfection was collected, centrifuged and filtered using a 0.45 μM filter, the protein A medium was used to purify the antibody, and the antibody was replaced by dialysis into PBS pH7.2 buffer. Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.

3-4.嵌合抗体结合活性检测3-4. Detection of chimeric antibody binding activity

通过FACS的方法，检验实施例3-3中获得的嵌合抗体xw.M0883B9.27B4与B7-H3抗原的结合活性。阳性参照抗体为MGA-271(NCT02475213)，每孔加入5×10 5个MDA-MB-231于96孔V型微孔板中，1500r/min离心1min，弃上清。稀释xw.M0883B9.27B4抗体，浓度为100、50、25、12.5、3.125、0.78、0.19、0.0488、0.0122、0.003、0.0007和0.00038μg/mL，每孔50μL，冰上孵育30min。然后每孔150μL PBS，1500r/min离心1min，弃上清，重复洗板4次。加入APC标记的羊抗鼠IgG(Jackson，货号115-605-164，PBS 1:800稀释)，每孔50μL，冰上孵育30min。用PBS洗板4次后每孔加入150μL PBS重悬细胞，用CytoFLEX进行检测(Beckman)。经GraphPad 8.0.2分析，xw.M0883B9.27B4的EC50为3.2μg/mL，MGA-271的EC50为23.8μg/mL，xw.M0883B9.27B4有更高的结合活性，结果如图1所示。因此，说明xw.M0883B9.27B4与B7-H3抗原具有极好的结合活性。 The binding activity of the chimeric antibody xw.M0883B9.27B4 obtained in Example 3-3 to the B7-H3 antigen was examined by FACS method. The positive reference antibody is MGA-271 (NCT02475213). Add 5×10 5 MDA-MB-231 to each well in a 96-well V-shaped microplate, centrifuge at 1500 r/min for 1 min, and discard the supernatant. Dilute xw.M0883B9.27B4 antibody at concentrations of 100, 50, 25, 12.5, 3.125, 0.78, 0.19, 0.0488, 0.0122, 0.003, 0.0007 and 0.00038 μg/mL, 50 μL per well, and incubate on ice for 30 min. Then, 150 μL of PBS per well was centrifuged at 1500 r/min for 1 min, the supernatant was discarded, and the plate was repeatedly washed 4 times. Add APC-labeled goat anti-mouse IgG (Jackson, Cat. No. 115-605-164, diluted in PBS 1:800), 50 μL per well, and incubate on ice for 30 min. After washing the plate 4 times with PBS, 150 μL of PBS was added to each well to resuspend the cells, and the CytoFLEX was used for detection (Beckman). According to GraphPad 8.0.2 analysis, the EC50 of xw.M0883B9.27B4 is 3.2 μg/mL, the EC50 of MGA-271 is 23.8 μg/mL, xw.M0883B9.27B4 has higher binding activity, the results are shown in Figure 1. Therefore, it shows that xw.M0883B9.27B4 has excellent binding activity to B7-H3 antigen.

3-5.xw.M0883B9.27B4抗体的ADCC活性ADCC activity of 3-5.xw.M0883B9.27B4 antibody

根据常规方法分离健康捐赠者的人外周血单核细胞(PBMC，peripheral blood mononuclear cell)，并将其悬于含1％FBS的RPMI 1640(称为ADCC培养基)中，使用40μm细胞滤网(BD Biosciences,Ltd.)过滤细胞并计数，调整细胞密度为2x 10 7/mL，作为效应细胞。收集生长状态良好的对数增长期的MDA-MB-231细胞，使用ADCC培养基洗细胞一遍，40μm细胞滤网过滤，细胞计数并调整细胞密度为2x 10 5/mL，作为靶细胞。96孔板中每孔加入靶细胞50μL，即每孔细胞数为10000个。稀释xw.M0883B9.27B4抗体及阳性对照抗体MGA-271，最高浓度为180nM/mL，依次十倍稀释，共8个浓度，即180、18、1.8、0.18、0.018、0.0018、0.00018和0.000018nM/mL，每孔加入50μL，与靶细胞混匀，37℃，5％CO2培养箱培养0.5小时。然后每孔加入效应细胞50μL，即每孔细胞500000个，效靶比为50:1，混匀，37℃，5％CO 2培养箱培养4小时。使用试剂CytoTox

Figure PCTCN2022115318-appb-000008
Non-Radioactive(Promega，货号G1780)按照说明书常规方法检测细胞毒性。根据如下公式进行计算，xw.M0883B9.27B4抗体具有较对照抗体更强的ADCC活性，结果见图2。 Human peripheral blood mononuclear cells (PBMC, peripheral blood mononuclear cell) from healthy donors were isolated according to conventional methods, and suspended in RPMI 1640 (called ADCC medium) containing 1% FBS, using a 40 μm cell strainer ( BD Biosciences, Ltd.) filtered and counted the cells, adjusted the cell density to 2x 10 7 /mL, and used them as effector cells. Collect the MDA-MB-231 cells in the logarithmic growth phase in good growth state, wash the cells once with ADCC medium, filter with a 40 μm cell strainer, count the cells and adjust the cell density to 2x 10 5 /mL, as the target cells. Add 50 μL of target cells to each well of the 96-well plate, that is, the number of cells per well is 10,000. Dilute the xw.M0883B9.27B4 antibody and the positive control antibody MGA-271, the highest concentration is 180nM/mL, ten-fold dilution in sequence, a total of 8 concentrations, namely 180, 18, 1.8, 0.18, 0.018, 0.0018, 0.00018 and 0.000018nM/ mL, add 50 μL to each well, mix with the target cells, and incubate for 0.5 hours at 37°C in a 5% CO2 incubator. Then add 50 μL of effector cells to each well, that is, 500,000 cells per well, and the effector-to-target ratio is 50:1, mix well, and incubate for 4 hours at 37°C in a 5% CO 2 incubator. Using Reagent CytoTox
Figure PCTCN2022115318-appb-000008
Non-Radioactive (Promega, Cat. No. G1780) was used to detect cytotoxicity according to the conventional method of the instruction manual. Calculated according to the following formula, the xw.M0883B9.27B4 antibody has stronger ADCC activity than the control antibody, and the results are shown in Figure 2.

Figure PCTCN2022115318-appb-000009
Figure PCTCN2022115318-appb-000009

备注：实验组＝实验孔-培养基背景对照孔；Remarks: Experimental group = experimental well-medium background control well;

靶细胞自发＝靶细胞自发LDH释放孔-培养基背景对照孔；Target cell spontaneous = target cell spontaneous LDH release well-medium background control well;

效应细胞自发＝效应细胞自发LDH释放孔-培养基背景对照孔；Effector cell spontaneous = effector cell spontaneous LDH release well-medium background control well;

靶细胞最大LDH释放＝靶细胞最大LDH释放孔-体积校正孔。Maximum LDH release from target cells = maximum LDH release pores from target cells - volume correction pores.

实施例4.小鼠抗人抗体27B4的人源化Example 4. Humanization of mouse anti-human antibody 27B4

4-1.27B4抗体的人源化设计方案4-1. Humanization design scheme of 27B4 antibody

根据通常称作的CDR移植方法对鼠抗人27B4单克隆抗体进行人源化。简言之，使用IMGT/V-QUEST工具(http://www.imgt.org/IMGT_vquest/input)分析27B4抗体VH和VK碱基序列，确定抗体轻链和重链的CDR区序列。使用IgBlast tool工具(https://www.ncbi.nlm.nih.gov/igblast/)分析27B4抗体的氨基酸序列，获得27B4抗体最接近的人种系VH和VK序列。将27B4抗体的CDR移植到选定的VH和VK人种系序列的框架区中，该序列即为人源化的抗体序列。对27B4抗体VH的人种系VH进行分析，选择5个不 同的序列，将27B4抗体VH的CDR移植到这5个序列的框架区中，得到5条重链序列可变区27B4-H1、27B4-H2、27B4-H3、27B4-H4和27B4-H5。以相同的方法对轻链进行CDR移植，得到5条轻链可变区序列27B4-L1、27B4-L2、27B4-L3、27B4-L4和27B4-L5。The mouse anti-human 27B4 monoclonal antibody was humanized according to the so-called CDR grafting method. Briefly, the VH and VK base sequences of the 27B4 antibody were analyzed using the IMGT/V-QUEST tool (http://www.imgt.org/IMGT_vquest/input), and the CDR region sequences of the antibody light chain and heavy chain were determined. Use the IgBlast tool (https://www.ncbi.nlm.nih.gov/igblast/) to analyze the amino acid sequence of the 27B4 antibody to obtain the closest human germline VH and VK sequences of the 27B4 antibody. The CDRs of the 27B4 antibody are grafted into the framework regions of the selected VH and VK human germline sequences, and this sequence is the humanized antibody sequence. Analyze the human germline VH of the 27B4 antibody VH, select 5 different sequences, graft the CDRs of the 27B4 antibody VH into the framework regions of these 5 sequences, and obtain 5 heavy chain variable regions 27B4-H1, 27B4 -H2, 27B4-H3, 27B4-H4 and 27B4-H5. The same method was used to carry out CDR grafting on the light chain, and five light chain variable region sequences 27B4-L1, 27B4-L2, 27B4-L3, 27B4-L4 and 27B4-L5 were obtained.

27B4-H1、27B4-H2、27B4-H3、27B4-H4和27B4-H5的氨基酸序列分别记作SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21和SEQ ID NO：22。The amino acid sequences of 27B4-H1, 27B4-H2, 27B4-H3, 27B4-H4 and 27B4-H5 are respectively denoted as SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 21 and SEQ ID NO: 22.

27B4-L1、27B4-L2、27B4-L3、27B4-L4和27B4-L5的氨基酸序列分别记作SEQ ID NO：23、SEQ ID NO：24、SEQ ID NO：25、SEQ ID NO：26和SEQ ID NO：27。The amino acid sequences of 27B4-L1, 27B4-L2, 27B4-L3, 27B4-L4 and 27B4-L5 are respectively denoted as SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 26 and SEQ ID NO: 27.

27B4-H1、27B4-H2、27B4-H3、27B4-H4和27B4-H5的碱基序列分别记作SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31和SEQ ID NO：32。The base sequences of 27B4-H1, 27B4-H2, 27B4-H3, 27B4-H4 and 27B4-H5 are recorded as SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32.

27B4-L1、27B4-L2、27B4-L3、27B4-L4和27B4-L5的碱基序列见序列表SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36和SEQ ID NO：37。The base sequences of 27B4-L1, 27B4-L2, 27B4-L3, 27B4-L4 and 27B4-L5 are shown in the sequence list SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37.

4-2.27B4人源化scFv的构建4-2.27 Construction of humanized scFv of B4

在重链可变区序列5’端添加碱基ttactcgcggcccagccggccatggcc，在重链可变区序列3’端添加碱基acagtctcgagtggtggtgg，得到对应的重链可变区序列27B4-H1-F、27B4-H2-F、27B4-H3-F、27B4-H4-F和27B4-H5-F。设计linker序列。在轻链可变区序列5’端添加碱基gtggcggtggcggtgctagc，在轻链可变区序列3’端添加碱基gcggccgcaggcgcggaacaaaaac，得到对应的轻链可变区序列27B4-L1-F、27B4-L2-F、27B4-L3-F、27B4-L4-F和27B4-L5-F。Add the base ttactcgcggcccagccggccatggcc to the 5' end of the heavy chain variable region sequence, and add the base acagtctcgagtggtggtgg to the 3' end of the heavy chain variable region sequence to obtain the corresponding heavy chain variable region sequences 27B4-H1-F, 27B4-H2-F , 27B4-H3-F, 27B4-H4-F and 27B4-H5-F. Design the linker sequence. Add the base gtggcggtggcggtgctagc to the 5' end of the light chain variable region sequence, and add the base gcggccgcaggcgcggaacaaaaac to the 3' end of the light chain variable region sequence to obtain the corresponding light chain variable region sequences 27B4-L1-F, 27B4-L2-F , 27B4-L3-F, 27B4-L4-F, and 27B4-L5-F.

在生工生物工程(上海)股份有限公司常规合成序列，分别选择一条重链可变区序列、linker(碱基序列信息为：ggtggtggcggttctggtggtggtggtagcggtggcggtggtagtggcggtggcggtgctagc)及一条轻链可变区序列与经过NcoI及NotI酶切处理的噬菌体展示载体pXY，使用重组酶(

Figure PCTCN2022115318-appb-000010
HiFi DNA Assembly Master Mix，NEB，货号E2621S)，按照说明书常规方法进行重组。将通过27B4-H1-F、linker及27B4-L1-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H1-L1”，将通过27B4-H1-F、linker及27B4-L2-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H1-L2”，将通过27B4-H1-F、linker及27B4-L3-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H1-L3”，将通过27B4-H1-F、linker及27B4-L4-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H1-L4”，将通过27B4-H1-F、linker及27B4-L5-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H1-L5”，将通过27B4-H2-F、 linker及27B4-L1-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H2-L1”，将通过27B4-H2-F、linker及27B4-L2-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H2-L2”，将通过27B4-H2-F、linker及27B4-L3-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H2-L3”，将通过27B4-H2-F、linker及27B4-L4-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H2-L4”，将通过27B4-H2-F、linker及27B4-L5-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H2-L5”，将通过27B4-H3-F、linker及27B4-L1-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H3-L1”，将通过27B4-H3-F、linker及27B4-L2-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H3-L2”，将通过27B4-H3-F、linker及27B4-L3-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H3-L3”，将通过27B4-H3-F、linker及27B4-L4-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H3-L4”，将通过27B4-H3-F、linker及27B4-L5-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H3-L5”，将通过27B4-H4-F、linker及27B4-L1-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H4-L1”，将通过27B4-H4-F、linker及27B4-L2-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H4-L2”，将通过27B4-H4-F、linker及27B4-L3-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H4-L3”，将通过27B4-H4-F、linker及27B4-L4-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H4-L4”，将通过27B4-H4-F、linker及27B4-L5-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H4-L5”，将通过27B4-H5-F、linker及27B4-L1-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H5-L1”，将通过27B4-H5-F、linker及27B4-L2-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H5-L2”，将通过27B4-H5-F、linker及27B4-L3-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H5-L3”，将通过27B4-H5-F、linker及27B4-L4-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H5-L4”，将通过27B4-H5-F、linker及27B4-L5-F组合得到的人源化scFv 27B4抗体命名为“scFv-27B4-H5-L5”。 In the routine synthesis sequence of Sangon Bioengineering (Shanghai) Co., Ltd., select a heavy chain variable region sequence, linker (base sequence information: ggtggtggcggttctggtggtggtggtagcggtggcggtggtagtggcggtggcggtgctagc) and a light chain variable region sequence and digested with NcoI and NotI Treated phage display vector pXY, using recombinase (
Figure PCTCN2022115318-appb-000010
HiFi DNA Assembly Master Mix, NEB, Cat. No. E2621S), recombined according to the routine method in the manual. The humanized scFv 27B4 antibody obtained through the combination of 27B4-H1-F, linker and 27B4-L1-F is named "scFv-27B4-H1-L1", and the humanized scFv 27B4 antibody obtained through the combination of 27B4-H1-F, linker and 27B4-L2- The humanized scFv 27B4 antibody obtained by combining F is named "scFv-27B4-H1-L2", and the humanized scFv 27B4 antibody obtained by combining 27B4-H1-F, linker and 27B4-L3-F is named "scFv -27B4-H1-L3", the humanized scFv 27B4 antibody obtained through the combination of 27B4-H1-F, linker and 27B4-L4-F is named "scFv-27B4-H1-L4", and the humanized scFv 27B4 antibody obtained through the combination of 27B4-H1- The humanized scFv 27B4 antibody obtained by combining F, linker and 27B4-L5-F is named "scFv-27B4-H1-L5", and the humanized antibody obtained by combining 27B4-H2-F, linker and 27B4-L1-F The ScFv 27B4 antibody is named "scFv-27B4-H2-L1", and the humanized scFv 27B4 antibody obtained by combining 27B4-H2-F, linker and 27B4-L2-F is named "scFv-27B4-H2-L2 ", the humanized scFv 27B4 antibody obtained by combining 27B4-H2-F, linker and 27B4-L3-F was named "scFv-27B4-H2-L3", and the humanized scFv 27B4 antibody obtained by combining 27B4-H2-F, linker and 27B4- The humanized scFv 27B4 antibody obtained by the combination of L4-F is named "scFv-27B4-H2-L4", and the humanized scFv 27B4 antibody obtained by the combination of 27B4-H2-F, linker and 27B4-L5-F is named "scFv-27B4-H2-L5", the humanized scFv 27B4 antibody obtained through the combination of 27B4-H3-F, linker and 27B4-L1-F is named "scFv-27B4-H3-L1", and the humanized scFv 27B4 antibody obtained through the combination of 27B4- The humanized scFv 27B4 antibody obtained by the combination of H3-F, linker and 27B4-L2-F is named "scFv-27B4-H3-L2". The humanized scFv 27B4 antibody is named "scFv-27B4-H3-L3", and the humanized scFv 27B4 antibody obtained by combining 27B4-H3-F, linker and 27B4-L4-F is named "scFv-27B4-H3 -L4", will be obtained by combining 27B4-H3-F, linker and 27B4-L5-F The humanized scFv 27B4 antibody was named "scFv-27B4-H3-L5", and the humanized scFv 27B4 antibody obtained by combining 27B4-H4-F, linker and 27B4-L1-F was named "scFv-27B4- H4-L1", the humanized scFv 27B4 antibody obtained through the combination of 27B4-H4-F, linker and 27B4-L2-F is named "scFv-27B4-H4-L2", and the humanized scFv 27B4 antibody obtained through the combination of 27B4-H4-F, linker The humanized scFv 27B4 antibody obtained by combining with 27B4-L3-F is named "scFv-27B4-H4-L3", and the humanized scFv 27B4 antibody obtained by combining 27B4-H4-F, linker and 27B4-L4-F The antibody was named "scFv-27B4-H4-L4", and the humanized scFv 27B4 antibody obtained by combining 27B4-H4-F, linker and 27B4-L5-F was named "scFv-27B4-H4-L5". The humanized scFv 27B4 antibody obtained by combining 27B4-H5-F, linker and 27B4-L1-F is named "scFv-27B4-H5-L1", and will be obtained by combining 27B4-H5-F, linker and 27B4-L2-F The combined humanized scFv 27B4 antibody was named "scFv-27B4-H5-L2", and the humanized scFv 27B4 antibody obtained by combining 27B4-H5-F, linker and 27B4-L3-F was named "scFv- 27B4-H5-L3", the humanized scFv 27B4 antibody obtained through the combination of 27B4-H5-F, linker and 27B4-L4-F is named "scFv-27B4-H5-L4", and the humanized scFv 27B4 antibody obtained through the combination of 27B4-H5-F , linker and 27B4-L5-F, the humanized scFv 27B4 antibody was named "scFv-27B4-H5-L5".

4-3.27B4人源化scFv表达4-3.27B4 humanized scFv expression

本实施例对实施例4-2中构建的25个27B4人源化序列的scFv单链抗体进行表达纯化。将TG1表达上清200mL高速离心去除杂质，使用20mL平衡衡缓冲液(50mM Na 2HPO 4，0.3M NaCl，pH＝8.0)预处理镍柱。将离心后的样品上柱，依次用25mL含有10mM、20mM咪唑 的洗涤缓冲液洗涤柱子，最后用5mL含有250mM咪唑的洗涤缓冲液洗涤柱子，收集洗脱液。通过透析方法将抗体置换至PBS pH7.2缓冲液中。通过Nanodrop测定吸光度来确定抗体浓度和纯度，并通过十二烷基硫酸钠凝胶电泳和考马斯染色来检查纯度。 In this example, 25 scFv single-chain antibodies constructed in Example 4-2 with humanized sequences of 27B4 were expressed and purified. 200 mL of TG1 expression supernatant was centrifuged at high speed to remove impurities, and 20 mL of equilibrium buffer (50 mM Na 2 HPO 4 , 0.3 M NaCl, pH=8.0) was used to pretreat the nickel column. Put the centrifuged sample on the column, wash the column with 25mL washing buffer containing 10mM and 20mM imidazole in turn, and finally wash the column with 5mL washing buffer containing 250mM imidazole, and collect the eluate. Antibodies were exchanged by dialysis into PBS pH 7.2 buffer. Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.

4-4.27B4人源化scFv的结合活性检测4-4.27B4 binding activity detection of humanized scFv

按照实施例2-5的方法进行流式细胞仪(FACS)检测，检测27B4人源化scFv与稳转细胞293-h4Ig-B7-H3-copGFP及内源编表达细胞MDA-MB-231的结合力。作为结果，25个27B4人源化scFv与细胞均有不同程度的结合，其中scFv-27B4-H2-L2、scFv-27B4-H2-L3、scFv-27B4-H4-L2及scFv-27B4-H4-L3结合力与细胞表面的B7-H3抗原的结合力与27B4结合力相当或更高，结果见图3。Perform flow cytometry (FACS) detection according to the method of Example 2-5, and detect the binding of 27B4 humanized scFv to stably transfected cells 293-h4Ig-B7-H3-copGFP and endogenously encoded expression cells MDA-MB-231 force. As a result, the 25 27B4 humanized scFvs all bound to cells to varying degrees, among which scFv-27B4-H2-L2, scFv-27B4-H2-L3, scFv-27B4-H4-L2 and scFv-27B4-H4- The binding ability of L3 to the B7-H3 antigen on the cell surface is equal to or higher than that of 27B4. The results are shown in FIG. 3 .

scFv-27B4-H2-L2、scFv-27B4-H2-L3、scFv-27B4-H4-L2及scFv-27B4-H4-L3的氨基酸序列分别为SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO：41。The amino acid sequences of scFv-27B4-H2-L2, scFv-27B4-H2-L3, scFv-27B4-H4-L2 and scFv-27B4-H4-L3 are SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41.

scFv-27B4-H2-L2、scFv-27B4-H2-L3、scFv-27B4-H4-L2及scFv-27B4-H4-L3的碱基序列分别为SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：45。The base sequences of scFv-27B4-H2-L2, scFv-27B4-H2-L3, scFv-27B4-H4-L2 and scFv-27B4-H4-L3 are SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45.

4-5.Whole IgG形式27B4人源化抗体的制备4-5. Preparation of Whole IgG 27B4 Humanized Antibody

对实施例4-4中获得的scFv-27B4-H2-L2、scFv-27B4-H2-L3、scFv-27B4-H4-L2及scFv-27B4-H4-L3进行Whole IgG形式27B4人源化抗体的制备。将重链人源化序列27B4-H2及27B4-H4在生工生物工程(上海)股份有限公司常规合成，并通过KpnI及MfeI分别克隆进入pTT5-hIgG1中，将轻链人源化序列27B4-L2及27B4-L3在生工生物工程(上海)股份有限公司常规合成，并通过KpnI及MfeI分别克隆进入pTT5-HL载体质粒，得到重链表达质粒pTT5-27B4-H2-hIgG1、pTT5-27B4-H4-hIgG1及轻链表达质粒pTT5-27B4-L2-HL、pTT5-27B4-L3-HL。将生长状态良好的对数增长期的293F细胞接种至250mL细胞培养瓶中并在50mL培养基中培养，PEI共转染轻重链表达质粒各25μg。收集转染后培养第7天的细胞上清，离心并使用0.45μM滤器过滤，Protein A介质纯化抗体并通过透析方法将抗体置换至PBS pH7.2缓冲液中。通过Nanodrop测定吸光度来确定抗体浓度和纯度，并通过十二烷基硫酸钠凝胶电泳和考马斯染色来检查纯度。将通过pTT5-27B4-H2-hIgG1和pTT5-27B4-L2-HL的组合得到的抗体命名为“h27B4-H2-L2”，将通过pTT5-27B4-H2-hIgG1和pTT5-27B4-L3-HL的组合得到的抗体命名为“h27B4-H2-L3”，将通过pTT5-27B4-H4-hIgG1和pTT5-27B4-L2-HL 的组合得到的抗体命名为“h27B4-H4-L2”，将通过pTT5-27B4-H4-hIgG1和pTT5-27B4-L3-HL的组合得到的抗体命名为“h27B4-H4-L3”。The scFv-27B4-H2-L2, scFv-27B4-H2-L3, scFv-27B4-H4-L2 and scFv-27B4-H4-L3 obtained in Example 4-4 were subjected to Whole IgG format 27B4 humanized antibody preparation. The heavy chain humanized sequence 27B4-H2 and 27B4-H4 were conventionally synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and cloned into pTT5-hIgG1 through KpnI and MfeI respectively, and the light chain humanized sequence 27B4- L2 and 27B4-L3 were routinely synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and cloned into the pTT5-HL vector plasmid by KpnI and MfeI, respectively, to obtain heavy chain expression plasmids pTT5-27B4-H2-hIgG1, pTT5-27B4- H4-hIgG1 and light chain expression plasmids pTT5-27B4-L2-HL, pTT5-27B4-L3-HL. The 293F cells in the logarithmic growth phase in good growth state were inoculated into 250 mL cell culture flasks and cultured in 50 mL medium, and 25 μg of each light and heavy chain expression plasmids were co-transfected with PEI. The cell supernatant on the 7th day of culture after transfection was collected, centrifuged and filtered using a 0.45 μM filter, the protein A medium was used to purify the antibody, and the antibody was replaced by dialysis into PBS pH7.2 buffer. Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining. The antibody obtained by the combination of pTT5-27B4-H2-hIgG1 and pTT5-27B4-L2-HL was named "h27B4-H2-L2", and the antibody obtained by the combination of pTT5-27B4-H2-hIgG1 and pTT5-27B4-L3-HL The combined antibody was named "h27B4-H2-L3", the antibody obtained by combining pTT5-27B4-H4-hIgG1 and pTT5-27B4-L2-HL was named "h27B4-H4-L2", and the antibody obtained by pTT5- The antibody obtained from the combination of 27B4-H4-hIgG1 and pTT5-27B4-L3-HL was named "h27B4-H4-L3".

h27B4-H2、h27B4-H4的重链氨基酸序列：SEQ ID NO：46、SEQ ID NO：47。The heavy chain amino acid sequences of h27B4-H2 and h27B4-H4: SEQ ID NO: 46, SEQ ID NO: 47.

h27B4-L2、h27B4-L3的轻链氨基酸序列：SEQ ID NO：48、SEQ ID NO：49。Light chain amino acid sequences of h27B4-L2 and h27B4-L3: SEQ ID NO: 48, SEQ ID NO: 49.

h27B4-H2、h27B4-H4的重链碱基序列：SEQ ID NO：50、SEQ ID NO：51。Heavy chain base sequences of h27B4-H2 and h27B4-H4: SEQ ID NO: 50, SEQ ID NO: 51.

h27B4-L2、h27B4-L3的轻链碱基序列：SEQ ID NO：52、SEQ ID NO：53。The light chain base sequences of h27B4-L2 and h27B4-L3: SEQ ID NO: 52, SEQ ID NO: 53.

4-6.Whole IgG形式27B4人源化抗体结合活性检测4-6. Detection of Binding Activity of Whole IgG 27B4 Humanized Antibody

按照实施例2-5的方法进行流式细胞仪(FACS)检测，检测whole IgG形式27B4人源抗体与稳转细胞293-h4Ig-B7-H3-copGFP及内源表达细胞MDA-MB-231的结合力。作为结果，4个Whole IgG形式27B4人源化抗体体h27B4-H2-L2、h27B4-H4-L3、h27B4-H2-L3和h27B4-H4-L2(在图中依次标识为zw.M0883B9.27B4Z01、zw.M0883B9.27B4Z02、zw.M0883B9.27B4Z03、zw.M0883B9.27B4Z04)与细胞表面的B7-H3抗原均有较强结合，与人源化之前的27B4嵌合抗体(图中标识为：xw.M0883B9.27B4)结合力相当，比MGA271结合力强，结果见图4。Carry out flow cytometry (FACS) detection according to the method for embodiment 2-5, detect whole IgG form 27B4 human source antibody and stably transfer cell 293-h4Ig-B7-H3-copGFP and endogenous expression cell MDA-MB-231 Binding force. As a result, four Whole IgG forms of 27B4 humanized antibody bodies h27B4-H2-L2, h27B4-H4-L3, h27B4-H2-L3 and h27B4-H4-L2 (identified in the figure as zw.M0883B9.27B4Z01, zw.M0883B9.27B4Z02, zw.M0883B9.27B4Z03, zw.M0883B9.27B4Z04) have a strong binding to the B7-H3 antigen on the cell surface, and the 27B4 chimeric antibody before humanization (marked as: xw. M0883B9.27B4) has the same binding force and is stronger than MGA271. The results are shown in Figure 4.

4-7.Whole IgG形式27B4人源化抗体ADCC活性检测4-7. Detection of ADCC activity of 27B4 humanized antibody in Whole IgG form

按照实施例3-5的方法检测whole IgG形式27B4人源抗体的ADCC活性。作为结果，4个Whole IgG形式27B4人源化抗体h27B4-H2-L2、h27B4-H4-L3、h27B4-H2-L3和h27B4-H4-L2(在图中依次标识为zw.M0883B9.27B4Z01、zw.M0883B9.27B4Z02、zw.M0883B9.27B4Z03、zw.M0883B9.27B4Z04)均有ADCC活性，与人源化之前的27B4抗体ADCC活性相当，结果见图5。The ADCC activity of the whole IgG form 27B4 human antibody was detected according to the method of Example 3-5. As a result, four Whole IgG forms of 27B4 humanized antibodies h27B4-H2-L2, h27B4-H4-L3, h27B4-H2-L3, and h27B4-H4-L2 (identified in the figure as zw.M0883B9.27B4Z01, zw .M0883B9.27B4Z02, zw.M0883B9.27B4Z03, zw.M0883B9.27B4Z04) all have ADCC activity, which is comparable to the ADCC activity of the 27B4 antibody before humanization. The results are shown in Figure 5.

4-8.Whole IgG形式27B4人源化抗体的交叉结合实验4-8. Cross-binding experiment of 27B4 humanized antibody in Whole IgG form

根据ADCC活性确定zw.M0883B9.27B4Z02号分子(h27B4-H4-L3,简称Z02号分子)的活性最高，按照实施例2-4的方法检测Z02号分子的交叉反应性。通过对Z02分子进一步的结合EC50和特种交叉结合实验证明，该分子能同时识别人4Ig-B7-H3抗原及人2Ig-B7-H3抗原，且具有同猴4Ig-B7-H3蛋白结合，该交叉反应活性有利于抗体应用于动物实验中。结果见图6和图7。According to the ADCC activity, the molecule zw.M0883B9.27B4Z02 (h27B4-H4-L3, referred to as molecule Z02) had the highest activity, and the cross-reactivity of molecule Z02 was detected according to the method of Example 2-4. Further binding EC50 and special cross-binding experiments on the Z02 molecule proved that the molecule can simultaneously recognize human 4Ig-B7-H3 antigen and human 2Ig-B7-H3 antigen, and has the ability to bind to monkey 4Ig-B7-H3 protein. Reactivity facilitates the use of antibodies in animal experiments. The results are shown in Figures 6 and 7.

由上述实施例的实验结果可知，本发明制备得到的抗B7-H3抗体均具有较高的B7-H3抗 原结合活性和ADCC活性，能够应用于癌症治疗等领域。From the experimental results of the above examples, it can be seen that the anti-B7-H3 antibodies prepared in the present invention have high B7-H3 antigen binding activity and ADCC activity, and can be applied in the fields of cancer treatment and the like.

以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等同变换，或直接或间接运用在相关的技术领域，均同理包括在本发明的专利保护范围内。The above description is only an embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent transformations made by using the description of the present invention and the contents of the accompanying drawings, or directly or indirectly used in related technical fields, are all included in the same principle. Within the scope of patent protection of the present invention.

Claims (14)

一种抗体，其特征在于，所述抗体包含重链可变区和/或轻链可变区：An antibody, characterized in that the antibody comprises a heavy chain variable region and/or a light chain variable region: 所述重链可变区，其包含：The heavy chain variable region, which comprises: 由SEQ ID NO:1所示的氨基酸序列组成的重链互补决定区HCDR1，The heavy chain complementarity determining region HCDR1 consisting of the amino acid sequence shown in SEQ ID NO:1, 由SEQ ID NO:2所示的氨基酸序列组成的重链互补决定区HCDR2，The heavy chain complementarity determining region HCDR2 consisting of the amino acid sequence shown in SEQ ID NO:2, 由SEQ ID NO:3所示的氨基酸序列组成的重链互补决定区HCDR3；The heavy chain complementarity determining region HCDR3 consisting of the amino acid sequence shown in SEQ ID NO:3; 所述轻链可变区，其包含：The light chain variable region, which comprises: 由SEQ ID NO:4所示的氨基酸序列组成的轻链互补决定区LCDR1，The light chain complementarity determining region LCDR1 consisting of the amino acid sequence shown in SEQ ID NO:4, 由WAS氨基酸序列组成的轻链互补决定区LCDR2，Light chain complementarity determining region LCDR2 consisting of WAS amino acid sequence, 由SEQ ID NO:5所示的氨基酸序列组成的轻链互补决定区LCDR3。A light chain complementarity determining region LCDR3 consisting of the amino acid sequence shown in SEQ ID NO:5. 根据权利要求1所述的抗体，其特征在于，所述抗体为鼠源抗体、嵌合抗体或人源抗体。The antibody according to claim 1, wherein the antibody is a murine antibody, a chimeric antibody or a human antibody. 根据权利要求1或2所述的抗体，其特征在于，所述重链可变区选自如SEQ ID NO:6、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21或SEQ ID NO:22所示的氨基酸序列；所述轻链可变区由选自如SEQ ID NO:7、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26或SEQ ID NO:27所示的氨基酸序列。The antibody according to claim 1 or 2, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID The amino acid sequence shown in NO: 21 or SEQ ID NO: 22; The light chain variable region is selected from such as SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 25, SEQ ID NO: The amino acid sequence shown in ID NO:26 or SEQ ID NO:27. 根据权利要求1或2所述的抗体，其特征在于，所述抗体包含重链恒定区和/或轻链恒定区：所述重链恒定区包含如SEQ ID NO:10所示的氨基酸序列；所述轻链恒定区包含如SEQ ID NO:12所示的氨基酸序列。The antibody according to claim 1 or 2, wherein the antibody comprises a heavy chain constant region and/or a light chain constant region: the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 10; The light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 12. 根据权利要求1或2所述的抗体，其特征在于，所述抗体包含重链和/或轻链：The antibody according to claim 1 or 2, wherein the antibody comprises a heavy chain and/or a light chain: 所述重链选自如SEQ ID NO:14、SEQ ID NO:46和SEQ ID NO:47所示的氨基酸序列；The heavy chain is selected from the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:46 and SEQ ID NO:47; 所述轻链选自如SEQ ID NO:15、SEQ ID NO:48和SEQ ID NO:49所示的氨基酸序列。The light chain is selected from the amino acid sequences shown in SEQ ID NO:15, SEQ ID NO:48 and SEQ ID NO:49. 根据权利要求1或2所述的抗体，其特征在于，所述包含以下i至iv中任一种性质：The antibody according to claim 1 or 2, characterized in that it comprises any one of the following properties from i to iv: i、所述抗体能够特异性结合B7-H3；i, the antibody can specifically bind to B7-H3; ii、所述抗体具有抗体依赖的细胞介导的细胞毒作用；ii. The antibody has antibody-dependent cell-mediated cytotoxicity; iii、所述抗体特异性地结合人4Ig-B7-H3并与猴4Ig-B7-H3交叉反应；iii. The antibody specifically binds to human 4Ig-B7-H3 and cross-reacts with monkey 4Ig-B7-H3; iv、所述抗体具有减少的糖基化或无糖基化或被低岩藻糖基化。iv. The antibody has reduced or aglycosylated or hypofucosylated. 一种重组蛋白，其特征在于，所述重组蛋白包含如权利要求1或2所述的抗体。A recombinant protein, characterized in that the recombinant protein comprises the antibody according to claim 1 or 2. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1或2所述的抗体或如权利要求7所述的重组蛋白。A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the antibody according to claim 1 or 2 or the recombinant protein according to claim 7. 一种多核苷酸，其特征在于，所述多核苷酸包含编码如权利要求1或2所述抗体或如权利要求7所述的重组蛋白的核苷酸序列。A polynucleotide, characterized in that the polynucleotide comprises a nucleotide sequence encoding the antibody as claimed in claim 1 or 2 or the recombinant protein as claimed in claim 7. 一种载体，其特征在于，所述载体包含如权利要求9所述的多核苷酸。A vector, characterized in that the vector comprises the polynucleotide according to claim 9. 一种分离细胞，其特征在于，所述细胞产生如权利要求1或2所述的抗体或如权利要求7所述的重组蛋白。An isolated cell, characterized in that the cell produces the antibody according to claim 1 or 2 or the recombinant protein according to claim 7. 一种抗体的制备方法，其特征在于，包括以下步骤：培养权利要求11所述的分离细胞，从培养物中回收所述抗体。A method for preparing an antibody, characterized by comprising the following steps: cultivating the isolated cell according to claim 11, and recovering the antibody from the culture. 权利要求1或2的抗体或权利要求7所述的重组蛋白在制备治疗癌症的药物或药物组合物中的应用。The use of the antibody of claim 1 or 2 or the recombinant protein of claim 7 in the preparation of medicines or pharmaceutical compositions for treating cancer. 权利要求1或2的抗体或权利要求7所述的重组蛋白在制备抗体检测试剂盒中的应用。Application of the antibody of claim 1 or 2 or the recombinant protein of claim 7 in the preparation of an antibody detection kit.
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