WO2023001154A1 - 一种b7-h3抗体及其应用 - Google Patents

一种b7-h3抗体及其应用 Download PDF

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WO2023001154A1
WO2023001154A1 PCT/CN2022/106546 CN2022106546W WO2023001154A1 WO 2023001154 A1 WO2023001154 A1 WO 2023001154A1 CN 2022106546 W CN2022106546 W CN 2022106546W WO 2023001154 A1 WO2023001154 A1 WO 2023001154A1
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antibody
heavy chain
cancer
seq
light chain
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French (fr)
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张文军
刘敏
田文武
李峰
华亚南
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广州爱思迈生物医药科技有限公司
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Publication of WO2023001154A1 publication Critical patent/WO2023001154A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the disclosure belongs to the technical fields of tumor treatment and molecular immunology, and relates to a B7-H3 antibody and its application.
  • B7-H3 (B7 homolog 3 protein, also known as CD276) belongs to the B7/CD28 immunoglobulin superfamily and is a protein with a unidirectional transmembrane structure (see: Steinberger P, Majdic O, Derdak S V, et al.
  • the receptor of B7-H3 has not been finally confirmed.
  • TLT-2 myeloid cells
  • TLT-2 myeloid cells
  • TREM family molecules function of TREM family molecules is to regulate cellular responses, and play a role in innate and acquired immunity (see: Klesney-Tait J, Turnbull I R, Colonna M.The TREM receptor family and signal integration.[J].Nature Immunology,2006,7(12):1266), TLT-2 protein is structurally expressed in CD8+T cells and can be induced when CD4+T cells are activated.
  • B7-H3 is a T cell co-inhibitory molecule with partial co-stimulatory function, in the presence of anti-CD3 antibody, B7-H3 can promote the proliferation of CD4+ T cells and CD8+ T cell populations, selectively stimulate ⁇ -interference After TLT-2 is transferred into T cells, it can promote the production of interleukin (IL)-2 and IFN- ⁇ by interacting with B7-H3, and block B7-H3 and TLT- 2 interactions can control hypersensitivity responses mediated by CD8+ T cells.
  • IL interleukin
  • B7-H3 also has a co-inhibitory effect, which can inhibit Treg cells, thereby allowing tumors to escape immune responses, and B7-H3 can also inhibit the activity of NK cells in tissue culture, thereby Reduce the function of NK cells.
  • B7-H3 is low expressed in normal tissues but overexpressed in various cancers, especially non-small cell lung cancer, renal cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme , ovarian and pancreatic cancers. B7-H3 plays an important role in tumor immune escape by inhibiting T cells, and has been proven to promote tumor progression and cancer cell metastasis. In addition, B7-H3 is not only expressed in cancer cells, but also in tumor or It is also expressed in peripheral blood vessels. B7-H3 is also associated with autoimmune diseases.
  • B7-H3 plays an important role in the interaction between fibroblast-like synoviocytes and activated T cells (see: Tran C N, Thacker S G, Louie D M, et al.Interactions of T Cells with Fibroblast-Like Synoviocytes: Role of the B7Family Costimulatory Ligand B7-H3[J].Journal of Immunology,2008,180(5):2989-2998.) , and B7-H3 acts as a co-stimulator when macrophages release cytokines and is thus implicated in the emergence of sepsis.
  • the present disclosure provides a B7-H3 antibody, the heavy chain complementarity determining region of the B7-H3 antibody includes the amino acid sequences shown in SEQ ID No.6-8;
  • the light chain complementarity determining region of the B7-H3 antibody includes the amino acid sequences shown in SEQ ID No. 14-16.
  • the heavy chain variable region of the B7-H3 antibody includes the amino acid sequence shown in SEQ ID No.1;
  • the light chain variable region of the B7-H3 antibody includes the amino acid sequence shown in SEQ ID No.9.
  • the present disclosure also provides an affinity-matured B7-H3 antibody, which is prepared from the B7-H3 antibody through mutation treatment.
  • the heavy chain complementarity determining region CDR1 of the affinity matured B7-H3 antibody includes the amino acid sequence shown in any one of SEQ ID No.6 or SEQ ID No.17-25
  • the heavy chain complementarity determining region CDR2 includes the amino acid sequence shown in any one of SEQ ID No.7 or SEQ ID No.26-37
  • the heavy chain complementarity determining region CDR3 includes any one of SEQ ID No.8 or SEQ ID No.38-48 The amino acid sequence shown in the species;
  • the light chain complementarity determining region of the affinity matured B7-H3 antibody includes the amino acid sequences shown in SEQ ID No.14-16.
  • the light chain variable region of the affinity matured B7-H3 antibody comprises the amino acid sequence shown in SEQ ID No.9;
  • the heavy chain framework region of the affinity matured B7-H3 antibody includes the amino acid sequences shown in SEQ ID No.2-5.
  • the present disclosure also provides a bispecific antibody comprising two heavy chains and two light chains;
  • the heavy chain includes an anti-B7-H3 heavy chain and an anti-CD3 heavy chain;
  • the anti-B7-H3 heavy chain includes the heavy chain of the B7-H3 antibody or the affinity matured B7-H3 antibody described in any of the above the heavy chain;
  • the light chain includes an anti-B7-H3 light chain and an anti-CD3 light chain;
  • the anti-B7-H3 light chain includes the light chain of the B7-H3 antibody or the affinity matured B7-H3 antibody described in any of the above the light chain;
  • the heavy chain is bound to the light chain by a disulfide bond
  • the anti-B7-H3 heavy chain is combined with the anti-CD3 heavy chain through a disulfide bond.
  • variable region of the anti-CD3 heavy chain comprises the amino acid sequence shown in SEQ ID No.49;
  • variable region of the anti-CD3 light chain includes the amino acid sequence shown in SEQ ID No.9.
  • the bispecific antibody comprises a B7-H3-binding immunoglobulin Fab domain, a CD3-binding immunoglobulin Fab domain, and a heterodimeric Fc region;
  • the immunoglobulin Fab domain binding to B7-H3 includes the variable region VH and constant region CH1 of the anti-B7-H3 light chain and anti-B7-H3 heavy chain;
  • the CD3-binding immunoglobulin Fab domain includes the variable region VH and the constant region CH1 of the anti-CD3 light chain and anti-CD3 heavy chain;
  • the heterodimer Fc region includes an anti-B7-H3 heavy chain-linked Fc fragment and an anti-CD3 heavy chain-linked Fc fragment.
  • the anti-B7-H3 heavy chain linked Fc fragment comprises a human Fc fragment or a humanized Fc fragment.
  • the CH3 of the anti-B7-H3 heavy chain-linked Fc fragment contains a T394D mutation, a P395D mutation, and a P396D mutation.
  • the CH3 of the anti-CD3 heavy chain-linked Fc fragment contains a P395K mutation, a P396K mutation, and a V397K mutation.
  • the CH2 of the anti-B7-H3 heavy chain-linked Fc fragment contains a L234A mutation, an L235A mutation, and a P329G mutation.
  • the present disclosure also provides a biological material that is any of the following B1) to B6):
  • B1) A nucleic acid molecule encoding the heavy chain and/or light chain of the antibody of any one of (b1) to (b3) or the antigen-binding portion of the antibody of any one of (b1) to (b3):( b1) the B7-H3 antibody described above, (b2) the affinity matured B7-H3 antibody described in any of the above, or (b3) the bispecific antibody described in any of the above;
  • B2 an expression cassette containing the nucleic acid molecule of B1);
  • B3 a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);
  • B4 A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing a recombinant vector described in B3);
  • B5 the cell line containing the nucleic acid molecule described in B1), or the cell line containing the expression cassette described in B2), or the cell line containing the recombinant vector described in B3);
  • the present disclosure also provides a pharmaceutical composition, which includes any of the B7-H3 antibody, any of the affinity matured B7-H3 antibodies described above, or any of the bispecific antibodies described above. One or a combination of at least two.
  • the present disclosure also provides the B7-H3 antibody, the affinity-matured B7-H3 antibody described in any of the above, the bispecific antibody described in any of the above, or the pharmaceutical composition in the preparation of anti-tumor drugs Applications;
  • the tumor includes any one or at least two of nervous system tumors, colorectal cancer, liver cancer, head and neck cancer, lung cancer, melanoma, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer or prostate cancer The combination.
  • the present disclosure also provides the B7-H3 antibody, the affinity-matured B7-H3 antibody described in any of the above, the bispecific antibody described in any of the above, or the pharmaceutical composition described above, for treating tumors the use of.
  • the tumor comprises any of nervous system tumors, colorectal cancer, liver cancer, head and neck cancer, lung cancer, melanoma, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, or prostate cancer One or a combination of at least two.
  • the present disclosure also provides a method for treating tumor-related diseases, administering the B7-H3 antibody, the affinity-matured B7-H3 antibody described in any one of the above, the above-mentioned Any one of the bispecific antibody or the pharmaceutical composition.
  • the tumor comprises any of nervous system tumors, colorectal cancer, liver cancer, head and neck cancer, lung cancer, melanoma, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, or prostate cancer One or a combination of at least two.
  • Figure 1 is a diagram of the binding activity of 5D9 and B7-H3 antigen detected by ELISA
  • Figure 2A is a graph showing the binding activity of affinity matured variants (44F2, 43G11, 46H7 and 45B11) to B7-H3 antigen detected by ELISA;
  • Figure 2B is a graph showing the binding activity of affinity matured variants (43D4, 42F7 and 45F4) to B7-H3 antigen detected by ELISA;
  • Figure 2C is a graph showing the binding activity of affinity matured variants (46H9, 47H4, 46D4 and 43G5) to B7-H3 antigen detected by ELISA;
  • Figure 2D is a graph showing the binding activity of affinity matured variants (44C5, 42A1, 47C8) to B7-H3 antigen detected by ELISA;
  • Figure 2E is a graph showing the binding activity of affinity matured variants (48E9, 49C3, 49C8, 49E1) to B7-H3 antigen detected by ELISA;
  • Figure 2F is a graph showing the binding activity of affinity matured variants (49F6, 50A1, 50E3, 49H5) to B7-H3 antigen detected by ELISA;
  • Figure 2G is a graph showing the binding activity of affinity matured variants (50B10, 48G2, 49D12, 48D10) to B7-H3 antigen detected by ELISA;
  • Figure 2H is a graph showing the binding activity of affinity matured variants (48A6, 48B12, 49B2, 49C7) to B7-H3 antigen detected by ELISA;
  • Figure 2I is a diagram of the binding activity of affinity matured variants (50A4, 50A6, 50A11) and B7-H3 antigen detected by ELISA;
  • Fig. 3A is a diagram of cell binding activity of affinity matured variants (44F2, 43G11) and overexpressed B7-H3 proximal membrane antigen detected by FACS;
  • Figure 3B is a diagram of the cell binding activity of affinity matured variants (45B11, 43D4, 46H7) and overexpressed B7-H3 proximal membrane antigen detected by FACS;
  • Figure 3C is a diagram of the cell binding activity of affinity matured variants (43G5, 42F7, 45F4) and overexpressed B7-H3 proximal membrane antigen detected by FACS;
  • Figure 3D is a diagram of the cell binding activity of affinity matured variants (46H9, 47H4, 46D4) and overexpressed B7-H3 proximal membrane antigen detected by FACS;
  • Figure 3E is a diagram of the cell binding activity of the affinity matured variants (44C5, 42A1 and 47C8) detected by FACS with the overexpressed B7-H3 proximal membrane antigen;
  • Figure 4A is a graph showing the killing effect of PBMCs on B7-H3 positive tumors mediated by affinity matured variants (44F2, 43G11, 46H7, 45B11, 43D4, 42F7, 45F4 and 46H9);
  • Figure 4B is a graph showing the killing effect of PBMCs on B7-H3 positive tumors mediated by affinity matured variants (46D4, 47H4, 48E9, 49C3, 49E1, 50A1, 50A6 and 5D9);
  • Figure 5 is a schematic diagram of the structure of the B7-H3 ⁇ CD3 bispecific antibody
  • Figure 6 is a diagram of the binding activity of the B7-H3 ⁇ CD3 bispecific antibody
  • Figure 7 is a graph showing the activation effect of B7-H3 ⁇ CD3 bispecific antibody on T cells
  • Figure 8A is a graph showing the effect of B7-H3 ⁇ CD3 bispecific antibodies (49E1 ⁇ CD3, 42F7 ⁇ CD3, 43G11 ⁇ CD3, 8H9 ⁇ CD3 and 46H7 ⁇ CD3) mediated killing of NCI-N87 cells by PBMC;
  • Figure 8B is a graph showing the effect of B7-H3 ⁇ CD3 bispecific antibodies (42F7 ⁇ CD3, 43G11 ⁇ CD3, 49E1 ⁇ CD3, 8H9 ⁇ CD3 and 46H7 ⁇ CD3) mediated killing of A498 cells by PBMC;
  • Figure 8C is a graph showing the effect of B7-H3 ⁇ CD3 bispecific antibodies (42F7 ⁇ CD3, 43G11 ⁇ CD3, 49E1 ⁇ CD3, 8H9 ⁇ CD3 and 46H7 ⁇ CD3) mediated killing of HepG2 cells by PBMC;
  • Figure 9A is a graph showing the stability test results of the 46H7 ⁇ CD3 bispecific antibody.
  • Figure 9B is a graph showing the stability test results of the 43G11 ⁇ CD3 bispecific antibody.
  • Figure 9C is a graph showing the stability test results of the 49E1 ⁇ CD3 bispecific antibody.
  • Figure 9D is a graph showing the stability test results of the 42F7 ⁇ CD3 bispecific antibody.
  • Fig. 10 is a graph showing the anti-tumor effect of B7-H3 ⁇ CD3 bispecific antibody in mice.
  • Antibody antibody, Ab.
  • Immunoglobulin immunoglobulin, Ig.
  • Heavy chain heavy chain, HC.
  • Light chain light chain, LC.
  • Heavy chain variable domain heavy chain variable domain, VH.
  • Heavy chain constant domain heavy chain constant domain, CH.
  • Light chain variable domain light chain variable domain, VL.
  • Light chain constant domain light chain constant domain, CL.
  • Antigen binding region antigen binding fragment, Fab.
  • Hinge area hinge region.
  • Fc fragment fragment crystallizable region, Fc region.
  • Monoclonal antibodies monoclonal antibodies, mAbs.
  • Antibody-dependent cytotoxicity antibody-dependent cell-mediated cytotoxicity, ADCC.
  • Complement-dependent cytotoxicity complement dependent cytotoxicity, CDC.
  • Natural killer cells natural killing cells, NK cells.
  • Bispecific antibody bispecific antibody, BsAb.
  • T cell receptor T cell receptor, TCR.
  • Major histocompatibility complex major histocompatibility complex, MHC.
  • Complementarity determining region refers to the antigen complementary binding region of an antibody.
  • Immunoreceptor tyrosine-based activation motif immunoreceptor tyrosine-based activation motif, ITAM.
  • Single-chain variable region antibody fragment also known as single-chain antibody: single-chain variable fragment, scFv;
  • Adoptive immunotherapy adoptive cellular immunotherapy, ACI.
  • Lymphokine-activated killer cells lymphokine-activated killer cells, LAK cells.
  • Tumor infiltrating lymphocytes Tumor Infiltrating Lymphocyte, TIL cells.
  • Cytokine-induced killer cells cytokine-induced killer cell, CIK cells.
  • nucleotide sequences described in this disclosure are arranged and written left to right in a 5' to 3' orientation.
  • amino acid sequences described in this disclosure are arranged and written left to right in a direction from amino terminus (N-terminus) to carboxy-terminus (C-terminus).
  • amino acid refers to one of the 20 naturally occurring amino acids or any unnatural analogue that may be present at a specific defined position.
  • amino acid mutation in the present disclosure refers to amino acid substitution, insertion, deletion and modification in the polypeptide sequence, and any combination of amino acid substitution, insertion, deletion and modification. Preferred amino acid modifications herein are substitutions.
  • amino acid substitution or “substitution” in the present disclosure refers to the replacement of an amino acid at a specific position in a parent polypeptide sequence with another amino acid.
  • a substitution C220S refers to a variant polypeptide in which the aminocysteine at position 220 of the polypeptide has been replaced by the amino acid serine.
  • Amino acid mutations can be achieved by molecular cloning or chemical methods. Molecular cloning methods include PCR, site-directed mutagenesis, and total gene synthesis.
  • protein refers to molecules with two or more amino acids linked by peptide bonds, including natural proteins, artificial proteins, protein fragments, mutant proteins and fusion proteins.
  • structural domain refers to a specific structural region with independent functions in a biomacromolecule.
  • the domain has an independent tertiary structure, and its function does not depend on the rest of the biomacromolecule.
  • the domain in this disclosure refers specifically to a protein In such regions, such as the VH domain of the heavy chain variable region and the VL domain of the light chain variable region, the combination of domains can form a large domain.
  • antibody refers to an immunoglobulin molecule that contains at least one antigen recognition site and is capable of specifically binding an antigen.
  • antigen is a substance that can induce an immune response in the body and specifically binds to an antibody, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, hapten, or a combination of the above substances .
  • the binding of antibody and antigen is mediated by the interaction formed between them, including hydrogen bond, van der Waals force, ionic bond and hydrophobic bond.
  • the region on the surface of an antigen that binds to an antibody is called an "antigenic determinant" or "epitope".
  • each antigen has multiple determinants.
  • antibody referred to in this disclosure includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, multispecific antibodies comprising at least two different epitope binding domains (eg, bispecific antibodies), human antibodies, humanized antibodies, post-translationally modified antibodies, camelid antibodies, chimeric antibodies, fusion proteins comprising antibody epitopes, and any other modified immunoglobulin molecule comprising an antigen recognition site, provided These antibodies exhibit the desired biological activity.
  • Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain at least one antigen binding site.
  • bispecific antibody refers to an antibody that contains two different antigen-binding sites and can bind to two different antigen-binding sites simultaneously.
  • Fab fragment
  • Fab region fragment
  • VH variable domain
  • VL variable domain
  • Fc is the effector region of an antibody, capable of eliciting, for example, CDC, ADCC, ADCP, cytokine release, and the like.
  • Natural antibody Fc is usually composed of two identical protein fragments, which contain two or three immunoglobulin constant region domains.
  • the Fc described in the present disclosure includes native Fc as well as mutated Fc. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined to comprise residues starting at C226 or P230 to its carboxyl terminus. Under the experimental conditions, the fragments generated by papain digestion of immunoglobulin monomers are Fab and Fc, respectively.
  • the "hinge” or “hinge region” of an antibody refers to a flexible polypeptide comprising the amino acids between the first and second constant domains (CH1 and CH2) of the antibody.
  • the amino acid numbering of antibody variable regions described in the present disclosure uses the coding scheme described by Kabat et al. in 1991, namely "Kabat index” or “Kabat numbering” (Kabat, E.A. et al. Immunological Interest, 5th ed., NIH Publication No. 91-3242, Bethesda, MD.: 1991).
  • the amino acid numbering of the constant regions of antibodies described in this disclosure uses the EU index (Edelman GM, et. al. Proc Natl Acad Sci US A 1969, 63:78-85.).
  • antigen-binding site refers to one or more amino acid residues that directly interact with an antigen in an antigen-binding molecule.
  • the antigen-binding site of an antibody is composed of complementarity-determining regions (CDRs).
  • CDRs complementarity-determining regions
  • Natural immunoglobulin molecules usually contain two Fab molecules usually contain one antigen-binding site.
  • T cell activation refers to one or more immune responses of T lymphocytes, especially killer T lymphocytes, including: proliferation, differentiation, release of cytokines, secretion of killer effector molecules, cell killing, etc.
  • EC 50 namely the concentration for 50% of maximal effect, refers to the concentration of antibody corresponding to causing 50% of the maximal effect.
  • Specific binding refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.
  • an antibody that specifically binds to an antigen refers to an antibody that binds to an antigen at less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Binds the antigen with an affinity (KD) of 10 ⁇ 8 M, 10 ⁇ 9 M, or 10 ⁇ 10 M or less.
  • affinity affinity
  • targeting refers to specific binding.
  • KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • the antibody dissociates at an equilibrium constant (KD) of less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less Binding antigen.
  • single-chain variable region antibody fragment refers to a fusion protein of an immunoglobulin heavy chain variable region VH and a light chain variable region VL, including different combinations of N-terminal VH and N-terminal VL, It can be prepared by conventional molecular cloning methods for constructing recombinant proteins (Sambrook JF, E.F. et al. Molecular cloning: a laboratory manual. 4th ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York: 2012).
  • humanized antibody refers to an antibody or antibody fragment obtained by replacing part or all of the CDR regions of a human immunoglobulin (recipient antibody) with a CDR region of a non-human antibody (donor antibody), wherein
  • the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody of desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, so as to further improve or optimize one of the antibodies. or multiple properties.
  • the term "host cell” refers to a cell into which an exogenous nucleic acid has been introduced, and its progeny can be transformed or transfected with a nucleotide encoding the polypeptide, thereby expressing the exogenous polypeptide.
  • the host cells described in the present disclosure include but are not limited to CHO cells (Chinese hamster ovary cells, Chinese hamster ovary cells), HEK293 cells (Human embryonic kidney cells 293, human embryonic kidney cells 293), BHK cells (Baby Hamster Kidney, young hamster cells) kidney cells), myeloma cells, yeast, insect cells or prokaryotic cells such as Escherichia coli, etc.
  • the "host cell” mentioned in the present disclosure not only refers to the cell into which the exogenous nucleic acid has been introduced, but also includes the offspring of the cell. Since the offspring cells will undergo mutations during cell division, they still belong to the host cell described in the present disclosure. Term scope.
  • an "effective amount” may refer to an amount of a composition described herein or a pharmaceutical formulation described herein that will cause the desired effect in a tissue, system, animal, plant, plant, or animal, as the researcher, veterinarian, physician, or other clinician is seeking. Desired biological or medical response of protozoa, bacteria, yeast or humans.
  • the term "subject” refers to a vertebrate, optionally a mammal, optionally a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.
  • Some embodiments of the present disclosure provide a B7-H3 antibody
  • the heavy chain complementarity determining region (VH-CDR1, VH-CDR2 and VH-CDR3) of the B7-H3 antibody includes SEQ ID No.6-8 Amino acid sequence
  • the light chain complementarity determining region (VL-CDR1, VL-CDR2 and VL-CDR3) of the B7-H3 antibody includes the amino acid sequence shown in SEQ ID No.14-16.
  • the B7-H3 antibody of the present disclosure can bind to the B7-H3 antigen with high affinity, can activate T cells, and has important application value in the fields of immunotherapy and cancer treatment.
  • the heavy chain variable region of the B7-H3 antibody includes the amino acid sequence shown in SEQ ID No.1.
  • the light chain variable region of the B7-H3 antibody includes the amino acid sequence shown in SEQ ID No.9.
  • SEQ ID No.8 (VH-CDR3): ARDQVVATSGVNFGMDV.
  • SEQ ID No.15 VL-CDR2: GTN.
  • Some embodiments of the present disclosure also provide DNA fragments encoding the B7-H3 antibody described above.
  • the light chain of the B7-H3 antibody has the nucleotide sequence shown in SEQ ID NO.50, and the heavy chain of the B7-H3 antibody has the nucleotide sequence of SEQ ID NO.51.
  • Some embodiments of the present disclosure also provide an expression vector comprising at least one copy of the above-mentioned DNA fragment.
  • Some embodiments of the present disclosure also provide a host cell comprising the expression vector as described above.
  • Some embodiments of the present disclosure also provide an affinity-matured B7-H3 antibody, which is prepared by mutating the above-mentioned B7-H3 antibody.
  • the mutagenesis process comprises random mutations of the light chain variable region and/or the heavy chain variable region of the B7-H3 antibody. It is believed, without being bound by theory, that the mutational treatments of the present disclosure can further increase the affinity of the B7-H3 antibody.
  • the heavy chain complementarity determining region CDR1 of the affinity matured B7-H3 antibody includes the amino acid sequence shown in any one of SEQ ID No.6 or SEQ ID No.17-25
  • the heavy chain complementarity determining region CDR2 includes the amino acid sequence shown in any one of SEQ ID No.7 or SEQ ID No.26-37
  • the heavy chain complementarity determining region CDR3 includes any one of SEQ ID No.8 or SEQ ID No.38-48 The amino acid sequence shown.
  • the light chain complementarity determining region of the affinity matured B7-H3 antibody includes the amino acid sequences shown in SEQ ID No. 14-16.
  • SEQ ID No. 17 GGTLRPVL.
  • SEQ ID No. 18 GGTSGVAL.
  • SEQ ID No. 19 GGTRPAAL.
  • SEQ ID No. 20 GGTVRPAL.
  • SEQ ID No. 21 GGTGGIYL.
  • SEQ ID No. 22 GGTGGAYL.
  • SEQ ID No. 23 GGTSRPVL.
  • SEQ ID No. 24 GGTRPPAL.
  • SEQ ID No. 25 GGTTGRYL.
  • SEQ ID No. 26 IIPGFYSL.
  • SEQ ID No. 27 IIPMWQSV.
  • SEQ ID No. 32 IIPVFRSV.
  • SEQ ID No. 34 IIPDF NSA.
  • SEQ ID No. 36 IIPNFHSS.
  • SEQ ID No. 37 IIPNFYSV.
  • SEQ ID No. 38 ARDRFTPRSGVNFGMDV.
  • SEQ ID No. 39 ARDREARESGVNFGMDV.
  • SEQ ID No. 40 ARDFDAPGSGVNFGMDV.
  • SEQ ID No. 41 ARDVVVPRSGVNFGMDV.
  • SEQ ID No. 42 ARDIEVMASGVNFGMDV.
  • SEQ ID No. 43 ARDQVVPLSGVNFGMDV.
  • SEQ ID No. 44 ARDVNNPGSGVNFGMDV.
  • SEQ ID No. 45 ARDVEVFNSGVNFGMDV.
  • SEQ ID No. 46 ARDEVVVGSGVNFGMDV.
  • SEQ ID No. 47 ARDAVVPNSGVNFGMDV.
  • SEQ ID No. 48 ARDQVVPRSGVNFGMDV.
  • the light chain variable region of the affinity matured B7-H3 antibody comprises the amino acid sequence shown in SEQ ID No.9.
  • the heavy chain framework region of the affinity matured B7-H3 antibody includes the amino acid sequences shown in SEQ ID No. 2-5.
  • SEQ ID No. 2 QVQLVQSGAEVKKPGSSVKVSCKAS.
  • SEQ ID No. 3 INWVRQAPGQGLEWMGG.
  • SEQ ID No. 4 NYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYC.
  • the light chain framework region of the affinity matured B7-H3 antibody comprises the amino acid sequences shown in SEQ ID Nos. 10-13.
  • SEQ ID No. 10 DIQMTQEPSLTTSPGGTVTLTCRSS.
  • SEQ ID No. 11 ANWVQEKPGQAPRGLIG.
  • SEQ ID No. 12 KRAPGTPARFSGSLIGGKAALTITGVQPEDEAIYFC.
  • SEQ ID No. 13 FGGGTKLEIK.
  • B7-H3 antibodies described above in this disclosure and the affinity matured B7-H3 antibodies described above have the following properties:
  • Some embodiments of the present disclosure also provide a bispecific antibody, the bispecific antibody includes two heavy chains and two light chains, the heavy chains include an anti-B7-H3 heavy chain and an anti-CD3 heavy chain, and the anti-B7-H3 heavy chain
  • the chain includes the heavy chain of the B7-H3 antibody described above or the heavy chain of the affinity matured B7-H3 antibody described above, and the light chain includes an anti-B7-H3 light chain and an anti-CD3 light chain;
  • an anti-B7-H3 light chain Chains include the light chain of the B7-H3 antibody described above or the light chain of the affinity matured B7-H3 antibody described above.
  • the heavy chain is associated with the light chain by a disulfide bond.
  • the anti-B7-H3 heavy chain is disulfide bonded to the anti-CD3 heavy chain.
  • variable region of the anti-CD3 heavy chain comprises the amino acid sequence shown in SEQ ID No.49.
  • variable region of the anti-CD3 light chain includes the amino acid sequence shown in SEQ ID No.9.
  • the structure of the bispecific antibody is shown in Figure 5, and the bispecific antibody includes an immunoglobulin Fab domain binding to B7-H3, an immunoglobulin Fab domain binding to CD3, and a heterodimerization Body Fc region, the immunoglobulin Fab domain that binds B7-H3 includes the variable region VH and the constant region CH1 of the anti-B7-H3 light chain and anti-B7-H3 heavy chain, and has the ability to specifically bind B7-H3 The ability of antigen; the immunoglobulin Fab domain that binds to CD3 includes the variable region VH and constant region CH1 of the anti-CD3 light chain and anti-CD3 heavy chain, and has the ability to specifically bind to the immune cell surface antigen CD3;
  • the heterodimer Fc region includes an anti-B7-H3 heavy chain-linked Fc fragment and an anti-CD3 heavy chain-linked Fc fragment, and the Fc fragment consists of constant regions CH2 and CH3.
  • the anti-B7-H3 heavy chain linked Fc fragment comprises a human Fc fragment or a humanized Fc fragment.
  • the anti-B7-H3 heavy chain-linked Fc fragment comprises a human IgG1 Fc fragment.
  • the anti-CD3 heavy chain-linked Fc fragment comprises a human Fc fragment or a humanized Fc fragment.
  • the anti-CD3 heavy chain-linked Fc fragment comprises a human IgG1 Fc fragment.
  • the CH3 of the anti-B7-H3 heavy chain-linked Fc fragment contains a T394D mutation, a P395D mutation, and a P396D mutation.
  • the CH3 of the anti-CD3 heavy chain-linked Fc fragment contains a P395K mutation, a P396K mutation, and a V397K mutation.
  • the CH2 of the anti-B7-H3 heavy chain-linked Fc fragment contains a L234A mutation or an L235A mutation and a P329G mutation.
  • the present disclosure also provides a biological material that is any of the following B1) to B6):
  • B1 Nucleic acid molecule encoding the heavy chain and/or light chain of the antibody described in any one of (b1) to (b3), or in the antigen-binding portion of the antibody described in any one of the encoding (b1) to (b3)
  • B2 an expression cassette containing the nucleic acid molecule of B1);
  • B3 a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);
  • B4 A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing a recombinant vector described in B3);
  • B5 the cell line containing the nucleic acid molecule described in B1), or the cell line containing the expression cassette described in B2), or the cell line containing the recombinant vector described in B3);
  • Some embodiments of the present disclosure provide a pharmaceutical composition, which includes the above-mentioned B7-H3 antibody, the above-mentioned affinity matured B7-H3 antibody or the above-mentioned bispecific antibody Any one or a combination of at least two.
  • the pharmaceutical composition further includes any one or a combination of at least two of pharmaceutically acceptable carriers, diluents or excipients.
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody or antibody fragment, bispecific antibody or antibody conjugate of the present disclosure, and optionally a pharmaceutically acceptable carrier, surfactant and/or thinner.
  • the pharmaceutical composition comprises, in addition to an antibody, bispecific antibody, or antibody conjugate of the disclosure, one or more additional therapeutic agents.
  • additional therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents for radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other Agents for the treatment of cancer.
  • Some embodiments of the present disclosure also provide the above-mentioned B7-H3 antibody, the above-mentioned affinity matured B7-H3 antibody, the above-mentioned bispecific antibody or the above-mentioned pharmaceutical composition in the preparation of anti- Application in tumor medicine.
  • Some embodiments of the present disclosure also provide the B7-H3 antibody, the affinity-matured B7-H3 antibody described in any of the above, the bispecific antibody described in any of the above, or the pharmaceutical composition described above, for use in for the treatment of tumors.
  • the present disclosure also provide a method for treating tumor-related diseases, administering the B7-H3 antibody or the affinity-matured B7-H3 antibody described above to the subject in need , the bispecific antibody or the pharmaceutical composition described in any one of the above.
  • the tumor comprises any of nervous system tumors, colorectal cancer, liver cancer, head and neck cancer, lung cancer, melanoma, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, or prostate cancer One or a combination of at least two.
  • the disclosure provides a B7-H3 antibody and its application.
  • the B7-H3 antibody binds to the B7-H3 antigen with high affinity, has important application value in the fields of immunotherapy and cancer treatment, solves the deficiencies of the prior art and satisfies the actual needs.
  • the B7-H3 antibody of the present disclosure can bind to B7-H3 antigen and cells expressing B7-H3 antigen with high affinity, and can efficiently mediate PBMC to kill B7-H3 positive tumors;
  • the present disclosure further improves the affinity of the B7-H3 antibody by mutating the complementarity determining region of the heavy chain of the B7-H3 antibody;
  • the disclosure utilizes the B7-H3 antibody to construct a B7-H3 ⁇ CD3 bispecific antibody.
  • the bispecific antibody has high affinity and stability, can effectively mediate PBMC to kill tumor cells, and has a significant effect of inhibiting tumor growth.
  • the present disclosure further encompasses nucleic acid sequences encoding these polypeptide chains.
  • the nucleic acid sequence is inserted into a suitable vector, including but not limited to: plasmid, phage expression vector, cosmid, artificial chromosome, phage and animal virus.
  • the expression vector contains elements for regulating expression, including but not limited to promoter, transcription initiation sequence, enhancer, signal peptide sequence and the like. Promoters include, but are not limited to, T7 promoter, T3 promoter, SP6 promoter, ⁇ -actin promoter, EF-1 ⁇ promoter, CMV promoter, and SV40 promoter.
  • Appropriate methods known in the art can be used to transfer the expression vector into host cells, including but not limited to: calcium phosphate precipitation method, polyethylenimine transfection method, liposome transfection method, electroporation method, PEI ( Polyethyleneimine) transfection method.
  • This embodiment provides a B7-H3 antibody, and the preparation method of the B7-H3 antibody includes the following steps:
  • the human VH gene and CD3VL gene were assembled into a scFv library by G4S-linker through overlapping extension PCR technology, and a human-VH:CD3-VL antibody library was constructed.
  • the library was cloned into a phage vector, the host was transformed by electric shock Cells (TG1 Electrocompetent Cells: Lucigen.) obtained human-VH:CD3-VL antibody library phage library.
  • the phage library was packaged with auxiliary phage and precipitated with PEG for later use. Take a 96-well plate and add appropriate amount of recombinant human B7-H3 antigen to incubate overnight.
  • Example 1 the antibody 5D9 described in Example 1 was expressed, purified and verified.
  • All the antibodies in this disclosure were transiently expressed in 293F cells, and the DNA encoding the heavy chain and light chain of the 5D9 antibody were synthesized, cloned into the expression vector pcDNA3.1, and the heavy chain of the antibody 5D9 was transfected by PEI transfection. and light chain expression plasmids were transfected into 293F cells, and after 6 days of culture, the cell culture supernatant was collected by centrifugation, and then the antibody in the supernatant was purified by protein A affinity chromatography column (GE healthcare).
  • GE healthcare protein A affinity chromatography column
  • the sequence of the positive control antibody Enoblituzumab (Enoblituzumab) mimic in this disclosure is derived from the patent: WO2011US26689, which is expressed and purified by the above method; the sequence of the positive control antibody 8H9 monoclonal antibody mimic in the disclosure is derived from the patent WO2003US07004 is also expressed and purified by the above method.
  • 5D9 binds better to the B7-H3 antigen, which is slightly weaker than that of Enoblituzumab mimics to the B7-H3 antigen.
  • This embodiment provides an affinity matured B7-H3 antibody.
  • the B7-H3 antibody 5D9 obtained in the present disclosure has a slightly weaker binding activity to the B7-H3 antigen than the enoblituzumab mimic.
  • the affinity matured B7-H3 antibody was obtained by in vitro affinity maturation of the variable region of the heavy chain of the antibody 5D9, and the preparation method included the following steps:
  • the phage library of the affinity maturation library was enriched for positive clones and sequenced to obtain 33 affinity maturation variants, wherein, the light chain variable region (VL) of 33 affinity maturation variants has the amino acid sequence shown in SEQ ID No.9, heavy chain framework region VH-FR1, VH-FR2, VH -FR3 and VH-FR4 have the amino acid sequences shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5 respectively.
  • VL light chain variable region
  • amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2, and VH-CDR3 of each variant are shown in Table 2.
  • This example expresses and purifies the affinity-matured B7-H3 antibody described in Example 3.
  • the heavy chain and light chain DNAs encoding the variants of affinity maturation prepared in Example 4 were synthesized respectively, and cloned into the expression vector pcDNA3.1, and the affinity maturation was performed by PEI transfection.
  • the heavy chain and light chain expression plasmids of each variant were transfected into 293F cells, and after 5-6 days of culture, the cell culture supernatant was collected by centrifugation, and then the protein A affinity chromatography column (GE healthcare) was used to purify the supernatant in the supernatant.
  • Antibodies, a total of 33 anti-affinity matured B7-H3 antibodies were obtained.
  • the variants of affinity maturation have higher binding activity to human B7-H3 antigen, most of which are similar to enoblituzumab mimics, and the binding activity (EC 50 )as shown in Table 3.
  • the present disclosure uses FACS to detect its binding activity to cells overexpressing the B7-H3 proximal membrane antigen.
  • Collect positive (Positive) cells and negative (Negative) cells wash once with 4°C pre-cooled 2% FBS/PBS (diluent), and then resuspend the cells in the diluent to make the cell density 5.0 ⁇ 106 cells/ mL, inoculate the cells on a 96-well plate, 100 ⁇ L/well (well), that is, 5.0 ⁇ 10 5 /well, prepare each variant of the affinity maturation with the dilution solution, the concentration is 5 ⁇ g/mL, take 100 ⁇ L antibody solution and mix with Mix an equal volume of negative (Negative) or positive (Positive) cells, that is, the final concentration is 2.5 ⁇ g/mL, incubate the 96-well plate at 4°C for 1 hour, and centrifuge the 96-well plate at 500 ⁇ g, 4°C for 5 minutes , remove the supernatant, wash twice with 200 ⁇ L of diluent, resuspend the cells
  • affinity matured variants 44F2, 43G11, 46H7, 45B11, 43D4, 42F7, 45F4, 46H9, 46D4, 47H4, 48E9, 49C3, 46D4, 47H4, 48E9, 49C3, 49E1, 50A1 and 50A6
  • human gastric cancer cells NCI-N87 with B7-H3 positive expression were used as target cells
  • human peripheral blood mononuclear cells PBMC were used as effector cells
  • the affinity maturation variants mediated by PBMCs to kill NCI-N87 tumors were detected cell activity.
  • NCI-N87 cells were digested with trypsin to prepare a single cell suspension, and the cell density was adjusted to 0.40 ⁇ 106 cells/mL with 5 % FBS-RPMI1640 medium without phenol red, and 50 ⁇ L/well was added to a 96-well plate; Adjust the cell density of effector cell PBMC to 4.00 ⁇ 106 cells/mL, add 100 ⁇ L/well into a 96-well plate; dilute the antibody to be tested to 40 ⁇ g/mL with 5% FBS-RPMI1640 medium without phenol red, and then press the volume Doubling dilution at a ratio of 1:4 to obtain 10 concentrations, which are 40 ⁇ g/mL, 10 ⁇ g/mL, 2.5 ⁇ g/mL, 0.625 ⁇ g/mL, 0.1563 ⁇ g/mL, 0.0391 ⁇ g/mL, 0.0098 ⁇ g/mL , 0.0024 ⁇ g/mL, 0.0006 ⁇ g/mL and 0.00015 ⁇ g/mL,
  • each of the 15 variants of affinity maturation can mediate PBMC killing of B7-H3 positive human gastric cancer cell NCI-N87.
  • This example provides a bispecific antibody targeting CD3 and B7-H3.
  • the aforementioned affinity-matured B7-H3 antibodies (42F7, 43G11, 49E1, 46H7) were selected to prepare bispecific antibodies targeting CD3 and B7-H3, the bispecific antibodies in the practice of the present disclosure, based on a common light chain and two The construction of different heavy chains, the structure diagram is shown in Figure 5, including 4 polypeptide chains, respectively named anti-B7-H3 heavy chain (from N-terminal to C-terminal is VH-CH1-hinge (Hinge)-CH2-CH3 -), anti-CD3 heavy chain (VH-CH1-hinge-CH2-CH3- from N-terminal to C-terminal), anti-B7-H3 light chain (VL-CL from N-terminal to C-terminal), anti-CD3
  • the light chain (VL-CL from N-terminus to C-terminus) forms an immunoglobulin Fab domain that specifically binds B7-H3, an immunoglobulin Fab domain that specifically binds CD3, and
  • the Fc part of the antibody is partially sequenced according to the method disclosed by the inventor in the PCT (WO2017034770A1) patent, wherein the CH3 domain of the Fc part of the anti-B7-H3 heavy chain is mutated as follows: T394D, P395D, P396D (EU) , the mutation is marked as OB, negatively charged; the CH3 domain of the Fc part of the anti-CD3 heavy chain is mutated as follows: P395K, P396K, V397K (EU), the mutation is marked as OA, positively charged; in order to reduce the interaction between Fc and its receptor Fc ⁇ R
  • the CH2 domains of the anti-B7-H3 heavy chain and the Fc portion of the anti-CD3 heavy chain both contain L234A, L235A and P329G (LALA-PG) mutations.
  • DNAs encoding heavy chains and light chains of B7-H3 antibodies 42F7, 43G11, 49E1, and 46H7 were synthesized respectively, and their Fab gene fragments were constructed into the eukaryotic expression vector pFUSEss-CHIg-hG1-Fc1 (including OB, L234A, L235A mutation and P329G mutation), the obtained expression plasmid was named plasmid 1; the anti-CD3 heavy chain was cloned into the plasmid pFUSE-hIgG1-Fc2 (including OA, N106T mutation, L234A mutation, L235A mutation and P329G mutation), and the obtained expression The plasmid was named plasmid 2, and the anti-CD3 light chain was cloned into pCDNA3.1, and the obtained plasmid was named plasmid 3.
  • the transfected cell suspension is placed in a culture shaker at 37°C, 5% CO 2 , 120rpm and cultured in the dark for 6 days , after high-speed centrifugation, the culture supernatant was applied to a protein A affinity chromatography column for purification, and SDS-PAGE analysis was performed to detect and confirm the size and purity of each BsAb, and a total of B7-H3 named below was obtained ⁇ CD3 bispecific antibodies: 42F7 ⁇ CD3, 43G11 ⁇ CD3, 49E1 ⁇ CD3 and 46H7 ⁇ CD3, positive control antibody 8H9 monoclonal antibody mimic, and 8H9 ⁇ CD3 bispecific antibody was also constructed by the same technical means.
  • the detection system composed of Jurkat/NFAT-Luc reporter gene cell line and B7-H3 positive human renal cancer cell A498 was used to evaluate the activation effect of B7-H3 ⁇ CD3 on T cells.
  • Jurkat/NFAT-Luc cell line fluorescence
  • the luciferase gene is regulated by NFAT (Nuclear factor of activated T-cell) transcription factor.
  • NFAT Nuclear factor of activated T-cell
  • B7-H3 positive A498 cells are used as target cells.
  • B7-H3 on A498 cells can activate the CD3 signaling pathway in Jurkat/NFAT-Luc cells through B7-H3 ⁇ CD3, and promote the expression of luciferase, which can pass the detection
  • the expression of luciferase in the cells was used to judge the degree of activation of Jurkat T cells.
  • B7-H3 ⁇ CD3 was diluted to a concentration of 10 ⁇ g/mL, 2.5 ⁇ g/mL, 0.625 ⁇ g/mL, 0.15625 ⁇ g/mL mL, 0.0390 ⁇ g/mL, 0.0097 ⁇ g/mL, 0.0024 ⁇ g/mL and 0.0006 ⁇ g/mL, add different concentrations of B7-H3 ⁇ CD3 to the above cell culture plate, culture at 37°C for 6h, add ONE-Glo Luciferase detection reagent, use a multi-functional microplate reader to measure the chemiluminescence value, take the logarithmic value of the antibody concentration as the abscissa, and the average chemiluminescence value as the ordinate, perform four-parameter fitting, draw the dose-effect curve, and obtain the EC 50 of each curve The value, EC 50
  • the CD3 binding arm of the bispecific antibody B7-H3 ⁇ CD3 specifically binds to the CD3 complex on the surface of T cells, and the other end of the B7-H3 binding arm specifically binds to B7-H3 molecules on the surface of tumor cells, allowing T cells to interact with tumor cells. Form an immune bridge between them, and then activate T cells, release cell-killing proteins such as perforin (Perforin) and granzyme B (Granzyme B), thereby killing tumor cells.
  • Perforin perforin
  • Gnzyme B granzyme B
  • This disclosure uses human gastric cancer cells NCI-N87, A498, and HepG2 with different expressions of B7-H3 as target cells, and human peripheral blood mononuclear cells (PBMC) as effector cells to detect 4 strains of B7-H3 ⁇ CD3 bispecific antibody-mediated PBMC Killing activity against tumor cells.
  • PBMC peripheral blood mononuclear cells
  • the bispecific antibody B7-H3 ⁇ CD3 constructed in the present disclosure has a good killing effect on NCI-N87, A498, and HepG2 cells with different expressions of B7-H3.
  • Size exclusion column Superdex200 10/300 GL increase
  • UV detection wavelength 280nm
  • the instrument used is an AKTA pure 25L1 chromatograph, and UNICORN software is used to record the spectrum and calculate the proportion of remaining monomers.
  • Figure 9A represents the results of SEC-HPLC testing the purity of 46H7 ⁇ CD3 after being stored at 40°C for 14 days.
  • the peak area ratio was 100%, and the purity of the 46H7 ⁇ CD3 bispecific antibody was >98%
  • Figure 9B represents the results of SEC-HPLC detection of the purity of 43G11 ⁇ CD3 after being stored at 40°C for 14 days, wherein the liquid concentration of the 43G11 ⁇ CD3 bispecific antibody The retention time of the phase elution peak was 28.5min, the peak area ratio was 100%, and the purity of the 43G11 ⁇ CD3 bispecific antibody was >98%
  • Figure 9C represents the results of SEC-HPLC detection of the purity of 49E1 ⁇ CD3 after being placed at 40°C for 14 days, where , the retention time of the liquid-phase elution peak of 49E1 ⁇ CD3 bispecific antibody was 28.5 min, the peak area ratio was 100%, and the purity of 49E1 ⁇ CD3 bispecific antibody was >98%
  • Figure 9D represents
  • one of the B7-H3 ⁇ CD3 bispecific antibodies (ie, 42F7 ⁇ CD3 bispecific antibody) was selected to detect the anti-tumor killing effect in mice.
  • mice 8-week-old female NOD-SCID mice (purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.) were selected to collect MC38/hB7-H3 cells in the logarithmic growth phase and freshly isolated PBMCs, and inoculate 100 ⁇ L/mouse in small The right forelimb of the mice was divided into groups on the next day after inoculation. The day of grouping was defined as D0 day, and the administration began on D0 day.
  • the experiment was divided into 2 groups, with 6 mice in each group, which were the isotype control hIgG group (5mg/kg, iv) , B7-H3 ⁇ CD3 group (5mg/kg, iv), on D1, D5, D8, D11, D15 tail vein injection administration (iv), after starting administration, observe the tumor size and weigh the mouse body weight, tumor
  • B7-H3 ⁇ CD3 has a significant inhibitory effect on tumor growth in the MC38/hB7-H3 xenograft model established subcutaneously in NOD/SCID mice with PBMC-reconstituted human immune system.
  • the B7-H3 antibody of the present disclosure can bind to the B7-H3 antigen with high affinity, and the affinity of the B7-H3 antibody is further improved by mutating the complementarity-determining region of the heavy chain, which can efficiently mediate the killing of B7-H3 by PBMC. H3-positive tumors.
  • the disclosure utilizes the B7-H3 antibody to construct a B7-H3 ⁇ CD3 bispecific antibody.
  • the bispecific antibody has high affinity and stability, can effectively mediate PBMC to kill tumor cells, and can significantly inhibit tumor growth. role.
  • the present disclosure illustrates the detailed methods of the present disclosure through the above-mentioned embodiments, but the present disclosure is not limited to the above-mentioned detailed methods, that is, it does not mean that the present disclosure must rely on the above-mentioned detailed methods to be implemented.
  • Those skilled in the art should understand that any improvement to the present disclosure, the equivalent replacement of each raw material of the disclosed product, the addition of auxiliary components, the selection of the method, etc., all fall within the scope of protection and disclosure of the present disclosure.
  • the disclosure provides a B7-H3 antibody and its application.
  • the disclosure provides that the B7-H3 antibody can bind to B7-H3 antigen and cells expressing B7-H3 antigen with high affinity, and can efficiently mediate PBMC to kill B7-H3 positive tumors , using the B7-H3 antibody to construct a B7-H3 ⁇ CD3 bispecific antibody.
  • the bispecific antibody has high affinity and stability, can effectively mediate PBMC to kill tumor cells, and has a significant effect of inhibiting tumor growth. Therefore, this
  • the publicly available B7-H3 antibodies have excellent utility.

Abstract

本申请提供了一种B7-H3抗体及其应用。所述B7-H3抗体的重链互补决定区包括SEQ ID No.6-8所示的氨基酸序列，所述B7-H3抗体的轻链互补决定区包括SEQ ID No.14-16所示的氨基酸序列。所述B7-H3抗体能够以高亲和力结合B7-H3抗原以及表达B7-H3抗原的细胞，能够高效介导PBMC杀伤B7-H3阳性肿瘤，利用所述B7-H3抗体构建B7-H3×CD3双特异性抗体，所述双特性抗体具备高亲和力和稳定性，能够有效介导PBMC杀伤肿瘤细胞。

Description

一种B7-H3抗体及其应用
相关申请的交叉引用
本公开要求于2021年07月20日提交中国专利局的申请号为CN 202110817833.7、名称为“一种B7-H3抗体及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开属于肿瘤治疗和分子免疫学技术领域，涉及一种B7-H3抗体及其应用。
背景技术
B7-H3(B7 homolog 3 protein，又名CD276)属于B7/CD28免疫球蛋白超家族，是一种拥有单向跨膜结构的蛋白质(参见：Steinberger P,Majdic O,Derdak S V,et al.Molecular Characterization of Human 4Ig-B7-H3,a Member of the B7 Family with Four Ig-Like Domains[J].Journal of Immunology,2004,172(4):2352-2359.)，由316个氨基酸组成，在氨基端有一信号肽，包括细胞外的免疫球蛋白样可变区(IgV)、恒定区(IgC)、跨膜区和45个氨基酸的胞浆区，在单核细胞、树突细胞以及激活的T细胞中表达。
目前，B7-H3的受体尚未得到最终确认，有研究认为，B7-H3的受体是I类跨膜蛋白TLT-2，它属于髓细胞表达的触发受体(TREM)家族(参见：King R G,Herrin B R,Justement L B.Trem-like transcript 2 is expressed on cells of the myeloid/granuloid and B lymphoid lineage and is up-regulated in response to inflammation.[J].Journal of Immunology,2006,176(10):6012-6021.)，TREM家族分子的功能是调节细胞应答，而且在先天性和获得性免疫中发挥作用(参见：Klesney-Tait J,Turnbull I R,Colonna M.The TREM receptor family and signal integration.[J].Nature Immunology,2006,7(12):1266)，TLT-2蛋白结构性地表达于CD8+T细胞，在CD4+T细胞活化时可被诱导表达。
B7-H3是一种T细胞共抑制分子，具有部分共刺激功能，当存在抗CD3抗体时，B7-H3可促进CD4+T细胞和CD8+T细胞群的增殖，选择性地刺激γ-干扰素(IFN-γ)的产生，TLT-2转入T细胞后，通过与B7-H3相互作用可以促进白细胞介素(IL)-2和IFN-γ的产生，阻断B7-H3和TLT-2的相互作用可以控制由CD8+T细胞介导的超敏反应。另一方面，在人类和小鼠中的实验证明，B7-H3也具有共抑制作用，可以抑制Treg细胞，从而使肿瘤逃逸免疫反应，B7-H3还可抑制组织培养中的NK细胞活性，从而降低NK细胞的功能。
B7-H3在正常组织中表达量较低，但在多种癌症中过表达，尤其是非小细胞肺癌、肾癌、尿路上皮癌、结肠直肠癌、前列腺癌、多形性成胶质细胞瘤、卵巢癌和胰腺癌。B7-H3通过对T细胞的抑制作用，在肿瘤免疫逃逸中发挥重要作用，并已被证明能促进肿瘤的进展和癌细胞的转移，此外，B7-H3不仅在癌细胞中表达，在肿瘤或周围血管中亦表达。B7-H3还与自体免疫疾病相关，在风湿及其他自体免疫疾病中，B7-H3在成纤维细胞样的滑膜细胞和激活的T细胞相互作用中起重要的作用(参见：Tran C N,Thacker S G,Louie D M,et al.Interactions of T Cells with Fibroblast-Like Synoviocytes:Role of the B7Family Costimulatory Ligand B7-H3[J].Journal of Immunology,2008,180(5):2989-2998.)，并且B7-H3在巨噬细胞释放细胞因子时作为共刺激因子，因此与败血症的出现有关。
目前，免疫疗法和癌症治疗领域存在着开发新B7-H3抗体的需求。
发明内容
本公开提供一种B7-H3抗体，所述B7-H3抗体的重链互补决定区包括SEQ ID No.6-8所示的氨基酸序列；
所述B7-H3抗体的轻链互补决定区包括SEQ ID No.14-16所示的氨基酸序列。
在一些实施方式中，所述B7-H3抗体的重链可变区包括SEQ ID No.1所示的氨基酸序列；
所述B7-H3抗体的轻链可变区包括SEQ ID No.9所示的氨基酸序列。
本公开还提供一种亲和力成熟B7-H3抗体，所述亲和力成熟B7-H3抗体由所述的B7-H3抗体经过突变处理制备得到。
在一些实施方式中，所述亲和力成熟B7-H3抗体的重链互补决定区CDR1包括SEQ ID No.6或SEQ ID No.17～25中任意一种所示的氨基酸序列，重链互补决定区CDR2包括如SEQ ID No.7或SEQ ID No.26～37中任意一种所示的氨基酸序列，重链互补决定区CDR3包括如SEQ ID No.8或SEQ ID No.38～48中任意一种所示的氨基酸序列；
所述亲和力成熟B7-H3抗体的轻链互补决定区包括SEQ ID No.14-16所示的氨基酸序列。
在一些实施方式中，所述亲和力成熟B7-H3抗体的轻链可变区包括如SEQ ID No.9所示的氨基酸序列；
所述亲和力成熟B7-H3抗体的重链框架区包括SEQ ID No.2-5所示的氨基酸序列。
本公开还提供一种双特异性抗体所述双特异性抗体包括两条重链和两条轻链；
所述重链包括抗B7-H3重链和抗CD3重链；所述抗B7-H3重链包括所述B7-H3抗体的重链或上文任一项所述的亲和力成熟B7-H3抗体的重链；
所述轻链包括抗B7-H3轻链和抗CD3轻链；所述抗B7-H3轻链包括所述B7-H3抗体的轻链或上文任一项所述的亲和力成熟B7-H3抗体的轻链；
所述重链通过二硫键与所述轻链结合；
所述抗B7-H3重链通过二硫键与所述抗CD3重链结合。
在一些实施方式中，所述抗CD3重链的可变区包括SEQ ID No.49所示的氨基酸序列；
所述抗CD3轻链的可变区包括SEQ ID No.9所示的氨基酸序列。
在一些实施方式中，所述双特异性抗体包括结合B7-H3的免疫球蛋白Fab结构域、结合CD3的免疫球蛋白Fab结构域和异源二聚体Fc区域；
所述结合B7-H3的免疫球蛋白Fab结构域包括所述抗B7-H3轻链和抗B7-H3重链的可变区VH和恒定区CH1；
所述结合CD3的免疫球蛋白Fab结构域包括所述抗CD3轻链和抗CD3重链的可变区VH和恒定区CH1；
所述异源二聚体Fc区域包括抗B7-H3重链连接的Fc片段和抗CD3重链连接的Fc片段。
在一些实施方式中，所述抗B7-H3重链连接的Fc片段包括人源Fc片段或人源化的Fc片段。
在一些实施方式中，所述抗B7-H3重链连接的Fc片段的CH3含有T394D突变、P395D突变和P396D突变。
在一些实施方式中，所述抗CD3重链连接的Fc片段的CH3含有P395K突变、P396K突变和V397K突变。
在一些实施方式中，所述抗B7-H3重链连接的Fc片段的CH2含有L234A突变、L235A突变和P329G突变。
本公开还提供生物材料，所述生物材料为以下B1)至B6)中的任一种：
B1)编码(b1)至(b3)中任一项所述抗体或者(b1)至(b3)中任一项所述抗体的抗原结合部分中的重链和/或轻链的核酸分子：(b1)上文所述的B7-H3抗体，(b2)上文任一项所述的亲和力成熟B7-H3抗体，或者(b3)上文任一项所 述的双特异性抗体；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；
B5)含有B1)所述核酸分子的细胞系、或含有B2)所述表达盒的细胞系、或含有B3)所述重组载体的细胞系；
B6)编码(b1)至(b3)中任一项所述抗体或者(b1)至(b3)中任一项所述抗体的抗原结合部分中的重链可变区和/或轻链可变区的核酸分子。
本公开还提供一种药物组合物，所述药物组合物包括所述B7-H3抗体、上文任一项所述的亲和力成熟B7-H3抗体或上文所述的双特异性抗体中的任意一种或至少两种的组合。
本公开还提供所述B7-H3抗体、上文任一项所述的亲和力成熟B7-H3抗体、上文任一项所述的双特异性抗体或所述药物组合物在制备抗肿瘤药物中的应用；
所述肿瘤包括神经系统肿瘤、结直肠癌、肝癌、头颈癌、肺癌、黑色素瘤、胰腺癌、胃癌、肾癌、膀胱癌、乳腺癌、卵巢癌或前列腺癌中的任意一种或至少两种的组合。
本公开还提供所述B7-H3抗体、上文任一项所述的亲和力成熟B7-H3抗体、上文任一项所述的双特异性抗体或所述的药物组合物，用于治疗肿瘤的用途。
在一些实施方式中，所述肿瘤包括神经系统肿瘤、结直肠癌、肝癌、头颈癌、肺癌、黑色素瘤、胰腺癌、胃癌、肾癌、膀胱癌、乳腺癌、卵巢癌或前列腺癌中的任意一种或至少两种的组合。
本公开还提供一种治疗与肿瘤有关疾病的方法，向所述有此需要的受试者给药所述B7-H3抗体、上文任一项所述的亲和力成熟B7-H3抗体、上文任一项所述的双特异性抗体或所述药物组合物。
在一些实施方式中，所述肿瘤包括神经系统肿瘤、结直肠癌、肝癌、头颈癌、肺癌、黑色素瘤、胰腺癌、胃癌、肾癌、膀胱癌、乳腺癌、卵巢癌或前列腺癌中的任意一种或至少两种的组合。
附图说明
图1为ELISA检测5D9与B7-H3抗原的结合活性图；
图2A为ELISA法检测亲和力成熟变体(44F2、43G11、46H7和45B11)与B7-H3抗原的结合活性图；
图2B为ELISA法检测亲和力成熟变体(43D4、42F7和45F4)与B7-H3抗原的结合活性图；
图2C为ELISA法检测亲和力成熟变体(46H9、47H4、46D4和43G5)与B7-H3抗原的结合活性图；
图2D为ELISA法检测亲和力成熟变体(44C5、42A1、47C8)与B7-H3抗原的结合活性图；
图2E为ELISA法检测亲和力成熟变体(48E9、49C3、49C8、49E1)与B7-H3抗原的结合活性图；
图2F为ELISA法检测亲和力成熟变体(49F6、50A1、50E3、49H5)与B7-H3抗原的结合活性图；
图2G为ELISA法检测亲和力成熟变体(50B10、48G2、49D12、48D10)与B7-H3抗原的结合活性图；
图2H为ELISA法检测亲和力成熟变体(48A6、48B12、49B2、49C7)与B7-H3抗原的结合活性图；
图2I为ELISA法检测亲和力成熟变体(50A4、50A6、50A11)与B7-H3抗原的结合活性图；
图3A为FACS检测亲和力成熟变体(44F2、43G11)与过表达B7-H3近膜端抗原的细胞结合活性图；
图3B为FACS检测亲和力成熟变体(45B11、43D4、46H7)与过表达B7-H3近膜端抗原的细胞结合活性图；
图3C为FACS检测亲和力成熟变体(43G5、42F7、45F4)与过表达B7-H3近膜端抗原的细胞结合活性图；
图3D为FACS检测亲和力成熟变体(46H9、47H4、46D4)与过表达B7-H3近膜端抗原的细胞结合活性图；
图3E为FACS检测亲和力成熟变体(44C5、42A1和47C8)与过表达B7-H3近膜端抗原的细胞结合活性图；
图4A为亲和力成熟变体(44F2、43G11、46H7、45B11、43D4、42F7、45F4和46H9)介导PBMC对B7-H3阳性肿瘤的杀伤效果图；
图4B为亲和力成熟变体(46D4、47H4、48E9、49C3、49E1、50A1、50A6和5D9)介导PBMC对B7-H3阳性肿瘤的杀伤效果图；
图5为B7-H3×CD3双特异性抗体结构示意图；
图6为B7-H3×CD3双特异性抗体的结合活性图；
图7为B7-H3×CD3双特异性抗体对T细胞的激活效果图；
图8A为B7-H3×CD3双特异性抗体(49E1×CD3、42F7×CD3、43G11×CD3、8H9×CD3和46H7×CD3)介导PBMC杀伤NCI-N87细胞效果图；
图8B为B7-H3×CD3双特异性抗体(42F7×CD3、43G11×CD3、49E1×CD3、8H9×CD3和46H7×CD3)介导PBMC杀伤A498细胞效果图；
图8C为B7-H3×CD3双特异性抗体(42F7×CD3、43G11×CD3、49E1×CD3、8H9×CD3和46H7×CD3)介导PBMC杀伤HepG2细胞效果图；
图9A为46H7×CD3双特异性抗体稳定性测试结果图；
图9B为43G11×CD3双特异性抗体稳定性测试结果图；
图9C为49E1×CD3双特异性抗体稳定性测试结果图；
图9D为42F7×CD3双特异性抗体稳定性测试结果图；
图10为B7-H3×CD3双特异性抗体小鼠体内抗肿瘤作用图。
具体实施方式
为进一步阐述本公开所采取的技术手段及其效果，以下结合实施方式和实施例和附图对本公开作进一步地说明。可以理解的是，此处所描述的实施方式仅仅用于解释本公开，而非对本公开的限定。
实施方式和实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
除非另有定义，与本公开结合使用的科学和技术术语及其缩略语应具有本公开所属领域的普通技术人员通常理解的含义，以下列举了本公开中使用的部分术语和缩略语。
抗体：antibody，Ab。
免疫球蛋白：immunoglobulin，Ig。
重链：heavy chain，HC。
轻链：light chain，LC。
重链可变区：heavy chain variable domain，VH。
重链恒定区：heavy chain constant domain，CH。
轻链可变区：light chain variable domain，VL。
轻链恒定区：light chain constant domain，CL。
抗原结合区：antigen binding fragment，Fab。
铰链区：hinge region。
Fc片段：fragment crystallizable region，Fc region。
单克隆抗体：monoclonal antibodies，mAbs。
抗体依赖性细胞毒作用：antibody-dependent cell-mediated cytotoxicity，ADCC。
补体依赖性细胞毒性作用：complement dependent cytotoxicity，CDC。
自然杀伤细胞：natural killing cell，NK细胞。
双特异性抗体：bispecific antibody，BsAb。
T细胞受体：T cell receptor，TCR。
主要组织相容性复合体：major histocompatibility complex，MHC。
互补决定区：complementarity determining region，CDR，是指抗体的抗原互补结合区。
免疫受体酪氨酸活化基序：immunoreceptor tyrosine-based activation motif，ITAM。
单链可变区抗体片段(又称单链抗体)：single-chain variable fragment，scFv；
过继免疫治疗：adoptive cellular immunotherapy，ACI。
淋巴因子激活的杀伤细胞：lymphokine-activated killer cell，LAK细胞。
肿瘤浸润淋巴细胞：Tumor Infiltrating Lymphocyte，TIL细胞。
细胞因子诱导的杀伤细胞：cytokine-induced killer cell，CIK细胞。
本公开所述分子克隆、细胞培养、蛋白纯化、免疫学实验、微生物学、动物模型等试验的操作步骤为该领域内被广泛应用的常规步骤。除非上下文另有说明，否则本公开单数术语包含复数且复数含义包括单数含义。除非另外指明，否则本公开所述核苷酸序列按照5’端至3’端的方向从左至右排列并书写。除非另外指明，否则本公开所述氨基酸序列按照从氨基端(N末端)到羧基端(C末端)的方向从左至右排列并书写。本公开所提及的氨基酸三字母缩写及核苷酸单字母缩写为该技术领域普遍接受的形式，氨基酸单字母缩写为IUPAC-IUB生物化学命名委员会(IUPAC-IUB Biochemical Nomenclature Commission)推荐的形式。
术语“氨基酸”是指20种天然存在的氨基酸之一或可能存在于特定的限定位置的任何非天然类似物。本公开所述“氨基酸突变”是指多肽序列中的氨基酸取代、插入、缺失和修饰，以及氨基酸取代、插入、缺失和修饰的任意组合。本文中优选的氨基酸修饰是取代。本公开中“氨基酸取代”或“取代”是指将亲本多肽序列中特定位置的氨基酸替代为另一种氨基酸。例如，取代C220S是指变体多肽，其中多肽的位置220处的氨基半胱氨酸已被氨基酸丝氨酸替代。氨基酸突变可以通过分子克隆或化学的方法实现，分子克隆的方法包括PCR、定点突变、全基因合成等。
术语“蛋白”、“肽链”、“多肽链”是指两个以上的氨基酸通过肽键连接的分子，包含天然蛋白、人工蛋白、蛋白片段、突变蛋白和融合蛋白等。
术语“结构域”是指生物大分子中具有独立功能的特异结构区域，结构域具有独立的三级结构，其功能不依赖于生物大分子中的其余部分，本公开中的结构域特指蛋白中这样的区域，如重链可变区VH结构域、轻链可变区VL结构域，结构域之间相互结合可以构成一个大的结构域。
术语“抗体”指包含至少一个抗原识别位点并能特异性结合抗原的免疫球蛋白分子。在此，术语“抗原”是在机体内能诱发免疫应答且与抗体特异性结合的物质，如蛋白质、多肽、肽、碳水化合物、多聚核苷酸、脂质、半抗原或上述物质的组合。抗体与抗原的结合依靠二者间形成的相互作用来介导，包括氢键、范德华力、离子键以及疏水键。抗原表面与抗体结合的区域为“抗原决定簇”或“表位”，一般来说，每个抗原有多个决定簇。本公开所提及的术语“抗体”包含单克隆抗体(包括全长单克隆抗体)、多克隆抗体、抗体片段、包含至少两个不同表位结合结构域的多特异性抗体(例如，双特异性抗体)、人抗体、人源化抗体、翻译后修饰抗体、骆驼抗体、嵌合抗体、包含抗体抗原决定簇的融合蛋白、以及包含抗原识别位点的任何其他修饰的免疫球蛋白分子，只要这些抗体展现出所期望的生物活性。抗体包括免疫球蛋白分子和免疫球蛋白分子的免疫活性片段，即至少含有一个抗原结合位点的分子。
术语“双特异性抗体”指含有两个不同抗原结合位点的抗体，可同时与两种不同的抗原结合位点结合。
术语“Fab”、“Fab区域”、“Fab片段”或“Fab分子”是抗原结合片段，包含免疫球蛋白重链的VH结构域、CH1结构域以及轻链的VL结构域、CL结构域，重链第一个恒定区结构域CH1与轻链的恒定区结构域CL结合，重链的可变区结构域VH和轻链的可变区结构域VL结合。
术语“Fc”、“Fc区域”、“Fc片段”或“Fc分子”是抗体的效应区，能够引起如CDC、ADCC、ADCP、细胞因子释放等。天然的抗体Fc通常是由两个相同的蛋白片段结合构成，该蛋白片段包含两个或三个免疫球蛋白恒定区结构域。本公开中所述Fc包含天然Fc以及突变的Fc。虽然Fc区的边界可以变化，但人IgG重链Fc区通常定义为包含从C226或P230开始到其羧基末端的残基。在实验条件下，免疫球蛋白单体经木瓜蛋白酶酶切生成的片段分别为Fab和Fc。抗体的“铰链”或“铰链区”是指包含抗体的第一和第二恒定结构域(CH1和CH2)之间的氨基酸的柔性多肽。
除非另有说明，否则本公开所述抗体可变区氨基酸编号使用Kabat等人在1991年所阐述的编码方案，即“Kabat索引”或“Kabat编号”(Kabat,E.A.et al.Sequences of Proteins of Immunological Interest,5th ed.,NIH Publication No.91–3242,Bethesda,MD.:1991)。除非另有说明，否则本公开所述抗体恒定区氨基酸编号使用EU索引(Edelman GM,et.al.Proc Natl Acad Sci U S A 1969,63:78-85.)。
术语“抗原结合位点”指抗原结合分子与抗原产生直接相互作用的一个或多个氨基酸残基，抗体的抗原结合位点由抗原互补决定区(CDR)构成，天然免疫球蛋白分子通常包含两个抗原结合位点，Fab分子通常包含一个抗原结合位点。
术语“T细胞激活”指T淋巴细胞尤其是杀伤性T淋巴细胞的一种或多种免疫反应，包括：增值、分化、细胞因子的释放、杀伤性效应分子的分泌、细胞杀伤等。
术语“EC 50”即半最大效应浓度(concentration for 50％of maximal effect)，是指引起50％最大效应所对应的抗体浓度。
本公开所用“特异性结合”是指，两种分子间的非随机的结合反应，如抗体及其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、 10 -7M、10 -8M、10 -9M、或10 -10M或更小的亲和力(KD)结合该抗原。在本公开的一些实施方式中，术语“靶向”指特异性结合。
本公开所用“KD”是指，特定抗体-抗原相互作用的解离平衡常数，用于描述抗体与抗原间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M、或10 -10M或更小的平衡解离常(KD)结合抗原。
术语“单链可变区抗体片段”或“scFv”指免疫球蛋白重链可变区VH和轻链可变区VL的融合蛋白，包括N端为VH以及N端为VL的不同组合形式，利用构建重组蛋白的常规分子克隆方法即可制备(Sambrook JF,E.F.et al.Molecular cloning:a laboratory manual.4th ed.Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New York:2012)。
术语“人源化抗体”是指人源免疫球蛋白(受体抗体)的部分或者全部CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或者抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或者反应性的非人源(如小鼠、大鼠或兔)抗体。此外，受体抗体的架构区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或者被其他抗体的氨基酸残基替换，以进一步改善或者优化抗体的一种或多种特性。
术语“宿主细胞”指导入外源核酸的细胞及其后代，可通过编码多肽的核苷酸转化或转染，从而表达外源多肽。本公开所述的宿主细胞包括但不限于CHO细胞(Chinese hamster ovary cells，中国仓鼠卵巢细胞)、HEK293细胞(Human embryonic kidney cells 293，人胚肾细胞293)、BHK细胞(Baby Hamster Kidney，幼仓鼠肾细胞)、骨髓瘤细胞、酵母、昆虫细胞或原核细胞如大肠杆菌(Escherichia coli)等。应当指出，本公开所述“宿主细胞”不仅指导入了外源核酸的细胞，同时还包括了该细胞的后代，由于在细胞分裂过程中后代细胞会发生突变，但仍属于本公开所述的术语范围。
如本文所用，“有效量”可以指本文所述的组合物或本文所述的药物制剂的量，其将引起研究者、兽医、医师或其他临床医生正在寻求的组织、系统、动物、植物、原生动物、细菌、酵母或人类的期望的生物学或医学响应。
如本文所用，术语“受试者”是指脊椎动物，可选地为哺乳动物，可选地为人类。哺乳动物包括但不限于鼠类、猿类、人类、家畜、竞技动物和宠物。在体内获得的或在体外培养的生物实体的组织、细胞及其子代也包括在内。
应当理解本文所用术语仅用于描述本公开上下文诸如实施方式和实施例的目的，并不旨在限制本发明的范围。
本公开的一些实施方式提供一种B7-H3抗体，所述B7-H3抗体的重链互补决定区(VH-CDR1、VH-CDR2和VH-CDR3)包括SEQ ID No.6-8所示的氨基酸序列，所述B7-H3抗体的轻链互补决定区(VL-CDR1、VL-CDR2和VL-CDR3)包括SEQ ID No.14-16所示的氨基酸序列。
本公开的B7-H3抗体能够以高亲和力与B7-H3抗原结合，可激活T细胞，在免疫疗法和癌症治疗领域具有重要应用价值。
在一些实施方式中，所述B7-H3抗体的重链可变区包括SEQ ID No.1所示的氨基酸序列。
在一些实施方式中，所述B7-H3抗体的轻链可变区包括SEQ ID No.9所示的氨基酸序列。
SEQ ID No.1：
Figure PCTCN2022106546-appb-000001
SEQ ID No.6(VH-CDR1)：GGTFSNYA。
SEQ ID No.7(VH-CDR2)：IIPLFGTA。
SEQ ID No.8(VH-CDR3)：ARDQVVATSGVNFGMDV。
SEQ ID No.9：
Figure PCTCN2022106546-appb-000002
SEQ ID No.14(VL-CDR1)：TGAVTTSNY。
SEQ ID No.15(VL-CDR2)：GTN。
SEQ ID No.16(VL-CDR3)：ALWYSNLWV。
本公开一些实施方式还提供编码上文所述B7-H3抗体的DNA片段。
在一些实施方式中，B7-H3抗体的轻链具有SEQ ID NO.50所示的核苷酸序列，B7-H3抗体的重链具有SEQ ID NO.51的核苷酸序列。
SEQ ID NO.50：
Figure PCTCN2022106546-appb-000003
SEQ ID NO.51：
Figure PCTCN2022106546-appb-000004
本公开一些实施方式还提供一种表达载体，所述表达载体包含至少一个拷贝的如上文所述的DNA片段。
本公开一些实施方式还提供一种宿主细胞，所述宿主细胞包含如上文所述的表达载体。
本公开一些实施方式还提供一种亲和力成熟B7-H3抗体，所述亲和力成熟B7-H3抗体由上文所述的B7-H3抗体经过突变处理制备得到。
在一些实施方式中，改突变处理包括对B7-H3抗体的轻链可变区和/或重链可变区进行随机突变。据信，不受理论的约束，本公开的突变处理可以进一步提高B7-H3抗体的亲和力。
在一些实施方式中，所述亲和力成熟B7-H3抗体的重链互补决定区CDR1包括SEQ ID No.6或SEQ ID No.17～25中任意一种所示的氨基酸序列，重链互补决定区CDR2包括如SEQ ID No.7或SEQ ID No.26～37中任意一种所示的氨基酸序列，重链互补决定区CDR3包括如SEQ ID No.8或SEQ ID No.38～48中任意一种所示的氨基酸序列。
在一些实施方式中，所述亲和力成熟B7-H3抗体的轻链互补决定区包括SEQ ID No.14-16所示的氨基酸序列。
SEQ ID No.17：GGTLRPVL。
SEQ ID No.18：GGTSGVAL。
SEQ ID No.19：GGTRPAAL。
SEQ ID No.20：GGTVRPAL。
SEQ ID No.21：GGTGGIYL。
SEQ ID No.22：GGTGGAYL。
SEQ ID No.23：GGTSRPVL。
SEQ ID No.24：GGTRPPAL。
SEQ ID No.25：GGTTGRYL。
SEQ ID No.26：IIPGFYSL。
SEQ ID No.27：IIPMWQSV。
SEQ ID No.28：IIPLFRSS。
SEQ ID No.29：IIPRFGGV。
SEQ ID No.30：IIPNFESV。
SEQ ID No.31：IIPKFRSQ。
SEQ ID No.32：IIPVFRSV。
SEQ ID No.33：IIPGFDAV。
SEQ ID No.34：IIPDFNSA。
SEQ ID No.35：IIPRFASR。
SEQ ID No.36：IIPNFHSS。
SEQ ID No.37：IIPNFYSV。
SEQ ID No.38：ARDRFTPRSGVNFGMDV。
SEQ ID No.39：ARDREARESGVNFGMDV。
SEQ ID No.40：ARDFDAPGSGVNFGMDV。
SEQ ID No.41：ARDVVVPRSGVNFGMDV。
SEQ ID No.42：ARDIEVMASGVNFGMDV。
SEQ ID No.43：ARDQVVPLSGVNFGMDV。
SEQ ID No.44：ARDVNNPGSGVNFGMDV。
SEQ ID No.45：ARDVEVFNSGVNFGMDV。
SEQ ID No.46：ARDEVVVGSGVNFGMDV。
SEQ ID No.47：ARDAVVPNSGVNFGMDV。
SEQ ID No.48：ARDQVVPRSGVNFGMDV。
在一些实施方式中，所述亲和力成熟B7-H3抗体的轻链可变区包括如SEQ ID No.9所示的氨基酸序列。
在一些实施方式中，所述亲和力成熟B7-H3抗体的重链框架区包括SEQ ID No.2-5所示的氨基酸序列。
SEQ ID No.2：QVQLVQSGAEVKKPGSSVKVSCKAS。
SEQ ID No.3：INWVRQAPGQGLEWMGG。
SEQ ID No.4：NYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYC。
SEQ ID No.5：WGQGTTVTVSS。
在一些实施方式中，亲和力成熟B7-H3抗体的轻链框架区包括SEQ ID No.10-13所示的氨基酸序列。
SEQ ID No.10：DIQMTQEPSLTTSPGGTVTLTCRSS。
SEQ ID No.11：ANWVQEKPGQAPRGLIG。
SEQ ID No.12：KRAPGTPARFSGSLIGGKAALTITGVQPEDEAIYFC。
SEQ ID No.13：FGGGTKLEIK。
据信，不受理论的约束，本公开上文所述B7-H3抗体和上文所述亲和力成熟B7-H3抗体具有以下性质：
a.特异性结合人B7-H3抗原；
b.特异性结合过表达人B7-H3近膜端抗原的细胞；
c.介导PBMC杀伤B7-H3阳性肿瘤。
本公开一些实施方式还提供一种双特异性抗体，该双特异性抗体包括两条重链和两条轻链，重链包括抗B7-H3重链和抗CD3重链，抗B7-H3重链包括上文所述的B7-H3抗体的重链或上文所述的亲和力成熟B7-H3抗体的重链，轻链包括抗B7-H3轻链和抗CD3轻链；抗B7-H3轻链包括上文所述的B7-H3抗体的轻链或上文所述的亲和力成熟B7-H3抗体的轻链。
在一些实施方式中，所述重链通过二硫键与所述轻链结合。
在一些实施方式中，所述抗B7-H3重链通过二硫键与所述抗CD3重链结合。
在一些实施方式中，所述抗CD3重链的可变区包括SEQ ID No.49所示的氨基酸序列。
SEQ ID No.49：
Figure PCTCN2022106546-appb-000005
所述抗CD3轻链的可变区包括SEQ ID No.9所示的氨基酸序列。
在一些实施方式中，双特异性抗体的结构如图5所示，该双特异性抗体包括结合B7-H3的免疫球蛋白Fab结构域、结合CD3的免疫球蛋白Fab结构域和异源二聚体Fc区域，所述结合B7-H3的免疫球蛋白Fab结构域包括所述抗B7-H3轻链和抗B7-H3重链的可变区VH和恒定区CH1，具有特异性结合B7-H3抗原的能力；所述结合CD3的免疫球蛋白Fab结构域包括所述抗CD3轻链和抗CD3重链的可变区VH和恒定区CH1，具有特异性结合免疫细胞表面抗原CD3的能力；所述异源二聚体Fc区域包括抗B7-H3重链连接的Fc片段和抗CD3重链连接的Fc片段，所述Fc片段由恒定区CH2和CH3组成。
在一些实施方式中，所述抗B7-H3重链连接的Fc片段包括人Fc片段或人源化的Fc片段。
在一些实施方式中，所述抗B7-H3重链连接的Fc片段包含人IgG1Fc片段。
在一些实施方式中，所述抗CD3重链连接的Fc片段包含人Fc片段或者人源化的Fc片段。
在一些实施方式中，所述抗CD3重链连接的Fc片段包含人IgG1Fc片段。
在一些实施方式中，所述抗B7-H3重链连接的Fc片段的CH3含有T394D突变、P395D突变和P396D突变。
在一些实施方式中，所述抗CD3重链连接的Fc片段的CH3含有P395K突变、P396K突变和V397K突变。
在一些实施方式中，所述抗B7-H3重链连接的Fc片段的CH2含有L234A突变或L235A突变和P329G突变。
本公开一些实施方式提供的双特异性抗体具有以下性质：
a.结合B7-H3和CD3；
b.对T细胞有激活作用；
c.介导T细胞杀伤B7-H3阳性的肿瘤细胞；
d.体内抑制B7-H3阳性肿瘤的生长。
本公开还提供生物材料，所述生物材料为以下B1)至B6)中的任一种：
B1)编码(b1)至(b3)中任一项所述抗体的重链和/或轻链的核酸分子，或者编码(b1)至(b3)中任一项所述抗体的抗原结合部分中的重链和/或轻链的核酸分子：(b1)上文所述的B7-H3抗体，(b2)上文任一项所述的亲和力成熟B7-H3抗体，或者(b3)上文任一项所述的双特异性抗体；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；
B5)含有B1)所述核酸分子的细胞系、或含有B2)所述表达盒的细胞系、或含有B3)所述重组载体的细胞系；
B6)编码上述(b1)至(b3)中任一项所述抗体的重链可变区和/或轻链可变区的核酸分子，或者编码上述(b1)至(b3)中任一项所述抗体的抗原结合部分中的重链可变区和/或轻链可变区的核酸分子。
本公开一些实施方式提供一种药物组合物，该药物组合物包括上文所述的B7-H3抗体、上文所述的亲和力成熟B7-H3抗体或上文所述的双特异性抗体中的任意一种或至少两种的组合。
在一些实施方式中，所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂中的任意一种或至少两种的组合。
在一些实施方式中，本公开涉及药物组合物，其包含本公开的抗体或抗体片段、双特异性抗体或抗体偶联物，和任选地药学上可接受的载体，表面活性剂和/或稀释剂。在一些实施方式中，除本公开的抗体、双特异性抗体或抗体偶联物外，药物组合物还包含一种或多种另外的治疗剂。在一些实施方式中，所述另外的治疗剂包括但不限于化疗剂、生长抑制剂、细胞毒性剂、用于放射疗法的试剂、抗血管生成剂、凋亡剂、抗微管蛋白剂和其它治疗癌症的试剂。
本公开一些实施方式还提供上文所述的B7-H3抗体、上文所述的亲和力成熟B7-H3抗体、上文所述的双特异性抗体或上文所述的药物组合物在制备抗肿瘤药物中的应用。
本公开一些实施方式还提供所述B7-H3抗体、上文任一项所述的亲和力成熟B7-H3抗体、上文任一项所述的双特异性抗体或所述的药物组合物，用于治疗肿瘤的用途。
本公开一些实施方式还提供一种治疗与肿瘤有关疾病的方法，向所述有此需要的受试者给药所述B7-H3抗体、上文任一项所述的亲和力成熟B7-H3抗体、上文任一项所述的双特异性抗体或所述药物组合物。在一些实施方式中，所述肿瘤包括神经系统肿瘤、结直肠癌、肝癌、头颈癌、肺癌、黑色素瘤、胰腺癌、胃癌、肾癌、膀胱癌、乳腺癌、卵巢癌或前列腺癌中的任意一种或至少两种的组合。
本公开提供一种B7-H3抗体及其应用，所述B7-H3抗体以高亲和力与B7-H3抗原结合，在免疫疗法和癌症治疗领域具有重要应用价值，解决了现有技术的不足并且满足了实际需求。
(1)本公开的B7-H3抗体能够以高亲和力结合B7-H3抗原以及表达B7-H3抗原的细胞，能够高效介导PBMC杀伤B7-H3阳性肿瘤；
(2)本公开通过B7-H3抗体重链互补决定区进行突变，进一步提高了B7-H3抗体的亲和力；
(3)本公开利用B7-H3抗体构建B7-H3×CD3双特异性抗体，所述双特性抗体具备高亲和力和稳定性，能够有效介导PBMC杀伤肿瘤细胞，具有显著抑制肿瘤生长的作用。
本公开还进一步包含编码这些多肽链的核酸序列。在表达抗体的过程中，将核酸序列插入合适的载体中，载体包括但不限于：质粒、噬菌表达载体、柯斯质粒、人工染色体、噬菌体以及动物病毒。表达载体中包含用于调控表达的元件，包括但不限于启动子、转录起始序列、增强子、信号肽序等。启动子包括但不限于T7启动子、T3启动子、SP6启动子、β-actin启动子、EF-1α启动子、CMV启动子以及SV40启动子。将表达载体转入宿主细胞中可使用本领域内已知的合适方法，包括但不限于：磷酸钙沉淀法、聚乙烯亚胺转染法、脂质体转染法、电穿孔法、PEI(聚乙烯亚胺)转染法。
实施例
实施例1
本实施例提供一种B7-H3抗体，所述B7-H3抗体的制备方法包括以下步骤：
(1)制备B7-H3胞外区(29-461)抗原，序列来源于UniProtKB:Q5ZPR3(CD276_HUMAN)将抗原序列克隆至真核表达载体pcDNA3.1-TEV-cFc-His，建立hB7-H3-TEV-cFc-His重组真核表达质粒，以PEI转染法转染至293F宿主细胞，经5天培养后收集细胞上清液，上清经蛋白A(protein A)亲和纯化得到B7-H3-TEV-cFc-His融合蛋白，再利用TEV蛋白酶酶切，镍柱纯化后得到人B7-H3抗原；
(2)取人外周血，分离外周单核细胞，提取总RNA，用逆转录试剂盒提取得到cDNA，以该cDNA为模板，参考Lim,T.S.,et al.(Lim T S,Mollova S,Rubelt F,et al.An optimised procedure for amplification of rearranged human antibody genes of different isotypes[J].New Biotechnology,2010,27(2):108-117.)设计特异引物进行PCR，PCR扩增其VH片段，获得人源VH基因；
(3)取本发明人已公布的专利(CN110551221A)中结合CD3的轻链序列(SEQ ID No：9)，设计特异引物进行PCR并扩增，获得CD3VL基因；
(4)将所述人源VH基因及CD3VL基因以G4S-linker经过重叠延伸PCR技术组装成scFv文库，构建人-VH:CD3-VL抗体文库，将文库克隆至噬菌体载体后，经电击转化宿主细胞(TG1Electrocompetent Cells：Lucigen.)获得人-VH:CD3-VL抗体文库噬菌体文库，噬菌体文库经辅助噬菌体包装、PEG沉淀后备用，取96孔板，加入适量重组人B7-H3抗原孵育过夜，PBST(含吐温-20的磷酸盐缓冲溶液)洗涤数次，加入噬菌体文库进行筛淘，经多轮筛淘后对富集的阳性克隆测序，最终获得B7-H3抗体，命名为5D9，抗体5D9的重链可变区(VH)、轻链可变区(VL)的氨基酸序列如表1所示。
表1
Figure PCTCN2022106546-appb-000006
实施例2
本实施例对实施例1所述的抗体5D9进行表达、纯化及验证。
本公开中的所有抗体均采用293F细胞瞬转表达，分别合成编码5D9抗体重链和轻链DNA，且将其克隆至表达载体pcDNA3.1中，用PEI转染法，将抗体5D9的重链和轻链表达质粒转染至293F细胞中，培养6天后，离心收集细胞培养上清，随后用蛋白A亲和层析柱(GE healthcare)纯化上清液中的抗体。
本公开中的阳性对照抗体依诺妥珠单抗(Enoblituzumab)模拟物，序列来源于专利：WO2011US26689，采用上述方法表达、纯化；本公开中的阳性对照抗体8H9单抗模拟物，序列来源于专利WO2003US07004，亦采用上述方法表达、纯化。
采用ELISA法检测5D9与B7-H3抗原的结合，将人B7-H3抗原包被于96孔板上，置于4℃过夜，将抗体5D9以及对照样品稀释至10μg/mL，再进行4倍梯度稀释，共8个浓度，并加入到抗原包被孔进行孵育，洗涤后加入HRP(辣根过氧化物酶)标记的山羊抗人IgG(H+L)二抗，TMB(3,3',5,5'-四甲基联苯胺)显色，酶标仪检测波长为450nm，读取吸光度OD值，以样品浓度的对数值为X轴，吸光度OD 450值为Y轴，绘制剂量反应曲线，计算EC 50，以EC 50代表抗体与B7-H3抗原结合的能力，实验同时设置阴性对照IgG组、阳性抗体依诺妥珠单抗(Enoblituzumab)模拟物对照组，结果见图1。
如图1所示，5D9与B7-H3抗原结合较好，稍弱于依诺妥珠单抗(Enoblituzumab)模拟物与B7-H3抗原的结合。
实施例3
本实施例提供一种亲和力成熟B7-H3抗体。
由前述可知，本公开获得的B7-H3抗体5D9，与B7-H3抗原的结合活性稍弱于依诺妥珠单抗(Enoblituzumab)模拟物，为进一步获得与B7-H3抗原有高亲和力的抗体，本实施例通过对抗体5D9重链的可变区进行体外亲和力成熟，获得亲和力成熟B7-H3抗体，制备方法包括以下步骤：
(1)亲和力成熟文库的生成，分别合成5D9重链互补决定区HC-CDR1、HC-CDR2、HC-CDR3的三联密码子上下游随机引物，经SOE-PCR获得5D9重链互补决定区随机突变文库，然后，将所得的随机突变文库克隆至噬菌粒载体并电击转化宿主细胞(TG1Electrocompetent Cells：Lucigen.)，获得5D9重链互补决定区随机突变噬菌体文库；
(2)亲和力成熟文库的筛淘，利用噬菌体展示技术实施亲和力成熟的抗体的生成，如前述实施例方法，亲和力成熟文库噬菌体文库经包装、三轮淘洗后，富集阳性克隆并测序，得到33个亲和力成熟变体，其中，33个亲和力成熟变体的轻链可变区(VL)具有如SEQ ID No.9所示的氨基酸序列，重链框架区VH-FR1、VH-FR2、VH-FR3、VH-FR4分别具有如SEQ ID No.2、SEQ ID No.3、SEQ ID No.4和SEQ ID No.5所示的氨基酸序列。
各个变体的重链互补决定区VH-CDR1、VH-CDR2、VH-CDR3具有的氨基酸序列如表2所示。
表2
Figure PCTCN2022106546-appb-000007
Figure PCTCN2022106546-appb-000008
Figure PCTCN2022106546-appb-000009
实施例4
本实施例对实施例3所述的亲和力成熟B7-H3抗体进行表达和纯化。
根据实施例2所述方法，分别合成实施例4制备的编码亲和力成熟各变体的重链和轻链DNA，且将其克隆至表达载体pcDNA3.1中，用PEI转染法，将亲和力成熟各变体的重链和轻链表达质粒转染至293F细胞中，培养5～6天后，离心收集细胞培养上清，随后用蛋白A亲和层析柱(GE healthcare)纯化上清液中的抗体，共获得33株抗亲和力成熟B7-H3抗体。
实施例5
本实施例对实施例4制备的亲和力成熟各变体进行功能验证。
(1)ELISA法测定亲和力成熟各变体与人B7-H3抗原的结合活性
采用ELISA方法检测亲和力成熟变体与人B7-H3抗原的结合活性，以样品浓度的对数值为X轴，吸光度OD 450值为Y轴，绘制剂量反应曲线，计算EC 50，以EC 50代表样品与人B7-H3抗原结合的能力，实验设置阳性对照组依诺妥珠单抗(Enoblituzumab)模拟物以及阴性对照组hIgG，结果见图2A-图2I。
如图2A-图2I所示，亲和力成熟各变体与人B7-H3抗原有较高的结合活性，其结合活性大多与依诺妥珠单抗(Enoblituzumab)模拟物相似，结合活性(EC 50)如表3所示。
表3
Figure PCTCN2022106546-appb-000010
(2)流式细胞术(FACS)测定亲和力成熟各变体与过表达B7-H3近膜端抗原细胞的结合活性
为了进一步研究上述的亲和力成熟各变体对人B7-H3抗原功能区的结合活性，本公开使用FACS检测其与过表达B7-H3近膜端抗原的细胞的结合活性。首先，构建稳定表达B7-H3近膜端(243-534bp)的细胞株，命名为阳性(Positive)细胞；不表达B7-H3近膜端的同种细胞命名为阴性(Negative)细胞。
收集阳性(Positive)细胞和阴性(Negative)细胞，以4℃预冷的2％FBS/PBS(稀释液)洗涤一次，再以稀释液重悬细胞，使细胞密度为5.0×10 6个细胞/mL，将细胞接种于96孔板上，100μL/孔(well)，即5.0×10 5/孔，以稀释液配制所述亲和力成熟各变体，浓度为5μg/mL，取100μL抗体溶液分别与等体积的阴性(Negative)细胞或阳性(Positive)细胞混匀，即终浓度为2.5μg/mL，把96孔板置于4℃孵育1h，将96孔板在500×g、4℃离心5min，去除上清，再以200μL稀释液洗涤两次，用PE anti-human IgG FC(Invitrogen)稀释液重悬细胞，4℃避光孵育1h，将96孔板在500×g、4℃下离心5min，去除上清，再用200μL稀释液洗涤两次，用200μL稀释液重悬细胞后立刻上机检测，结果见图3A-图3E。
部分结果如3A-图3E所示，亲和力成熟各变体均可与表达B7-H3近膜端抗原的阳性(Positive)细胞结合(图中右侧实线)，而不与阴性(Negative)细胞结合(图中左侧实线)。
(3)检测亲和力成熟变体介导PBMC对B7-H3阳性肿瘤细胞杀伤活性
根据前述ELISA和FACS结果，选择与人B7-H3抗原有较高亲和力的15株亲和力成熟变体(44F2、43G11、46H7、45B11、43D4、42F7、45F4、46H9、46D4、47H4、48E9、49C3、49E1、50A1和50A6)，采用B7-H3阳性表达的细胞人胃癌细胞NCI-N87作为靶细胞，人外周血单核细胞PBMC作为效应细胞，检测亲和力成熟各变体介导PBMC杀伤NCI-N87肿瘤细胞的活性。
用胰酶消化NCI-N87细胞，制备单细胞悬液，用无酚红的5％FBS-RPMI1640培养基调整细胞密度为0.40×10 6个细胞/mL，50μL/孔加入96孔板中；将效应细胞PBMC调整细胞密度为4.00×10 6个细胞/mL，100μL/孔加入96孔板中；用无酚红的5％FBS-RPMI1640培养基将待测抗体稀释至40μg/mL，然后按体积比1:4的比例进行倍比稀释，得到10个浓度，分别为40μg/mL、10μg/mL、2.5μg/mL、0.625μg/mL、0.1563μg/mL、0.0391μg/mL、0.0098μg/mL、0.0024μg/mL、0.0006μg/mL和0.00015μg/mL，50μL/孔加入96孔板中。96孔板混匀后置于37℃、5％CO 2培养箱中培养过夜，第二天(约20小时后)用乳酸脱氢酶细胞毒性试剂盒(Beyotime)来检测细胞的毒性，进而检测抗体的杀伤活性，杀伤率的计算公式为：细胞毒性(Cytotoxicity)％＝(OD样品-SR)/(MR-SR)×100％，其中，SR＝OD自发释放孔(靶细胞+效应细胞)，MR＝OD最大释放孔(靶细胞)，结果见图4A和图4B。
如图4A和图4B所示，15株亲和力成熟各变体均可介导PBMC杀伤B7-H3阳性的人胃癌细胞NCI-N87。
实施例6
本实施例提供一种靶向CD3和B7-H3的双特异性抗体。
(1)B7-H3×CD3双特异性抗体的序列设计
选择前述制备的亲和力成熟B7-H3抗体(42F7、43G11、49E1、46H7)以制备靶向CD3和B7-H3的双特异性抗体，本公开实施中的双特异性抗体，基于共同轻链和两条不同重链构建，结构示意图如图5所示，包括4条多肽链，分别命名为抗B7-H3重链(从N端至C端依次为VH-CH1-铰链(Hinge)-CH2-CH3-)、抗CD3重链(从N端至C端依次为VH-CH1-铰链-CH2-CH3-)、抗B7-H3轻链(从N端至C端依次为VL-CL)，抗CD3轻链((从N端至C端依次为VL-CL)，形成一个特异性结合B7-H3的免疫球蛋白Fab结构域，一个特异性结合CD3的免疫球蛋白Fab结构域以及一个异源二聚体Fc区域，其中抗B7-H3轻链和抗CD3轻链为共同轻链，两条重链的CH3结构域包含带相反电荷的不对称氨基酸修饰，形成上述所述的异源二聚体Fc区域。
特异性结合B7-H3的免疫球蛋白Fab结构域，包括抗B7-H3的重链和轻链，序列来源于前述实施例中制备亲和力成熟B7-H3抗体(42F7、43G11、49E1、46H7)的重链和轻链。
特异性结合CD3的免疫球蛋白Fab结构域，包括抗CD3的重链(SEQ ID No.49)和轻链(SEQ ID No.9)。
SEQ ID No.49：
Figure PCTCN2022106546-appb-000011
SEQ ID No.9：
Figure PCTCN2022106546-appb-000012
抗体Fc部分按照本发明人已经公开的PCT(WO2017034770A1)专利中的方法进行部分序列的改造，其中，抗B7-H3重链的Fc部分CH3结构域做如下突变：T394D、P395D、P396D(EU)，突变标记为OB，带负电荷；抗CD3重链的Fc部分CH3结构域做如下突变：P395K、P396K、V397K(EU)，突变标记为OA，带正电荷；为减少Fc与其受体FcγR的结合，抗B7-H3重链及抗CD3重链的Fc部分其CH2结构域都包含L234A、L235A和P329G(LALA-PG)突变。
(2)表达质粒的分子克隆
分别合成编码B7-H3抗体42F7、43G11、49E1、46H7重链和轻链的DNA，将其Fab形式基因片段基因片段构建至真核表达载体pFUSEss-CHIg-hG1-Fc1(包含OB、L234A、L235A突变及P329G突变)上，得到表达质粒命名为质粒1；将抗CD3的重链克隆到质粒pFUSE-hIgG1-Fc2(包含OA、N106T突变、L234A突变、L235A突变和P329G突变)上，得到的表达质粒命名为质粒2，将抗CD3的轻链克隆至pCDNA3.1上，得到的质粒命名为质粒3。
(3)表达、纯化
提取重组质粒1、质粒2和质粒3，使用PEI试剂与HEK293F细胞进行共转染，转染后的细胞悬浮液放置在37℃、5％CO 2、120rpm的培养摇床中避光培养6天，将培养上清液通过高速离心后，上样至蛋白A亲和层析柱纯化，并进行SDS-PAGE分析，以检测并确认每种BsAb的大小和纯度，共获得以下命名的B7-H3×CD3双特异性抗体：42F7×CD3、43G11×CD3、49E1×CD3和46H7×CD3，阳性对照抗体8H9单抗模拟物，亦通过同样的技术手段，构建8H9×CD3双特异性抗体。
实施例7
本实施例对实施例6制备的B7-H3×CD3双特异性抗体进行功能验证。
(1)B7-H3×CD3双特异性抗体的结合活性
采用间接ELISA法检测前述制备的B7-H3×CD3双特异性抗体结合B7-H3抗原活性，方法包括：将稀释为1μg/mL的B7-H3抗原，100μL/孔加入酶标板中，4℃包被过夜，次日，0.05％PBST洗涤后，室温封闭2h，再次用0.05％PBST洗涤后，加入梯度稀释的B7-H3×CD3双特异性抗体(PBS稀释)，室温孵育1h，使用0.05％PBST洗涤未结合物，随后加入HRP标记的羊抗人IgG(H+L)二抗(Proteintech，Cat.SA0001-17)，室温孵育1h，以0.05％PBST洗涤，加入TMB显色液，室温避光孵育5min，使用H 2SO 4终止反应，在酶标仪450nm处检测吸光度，以log(抗体浓度)为横坐标，吸光度OD 450为纵坐标，绘制剂量效应曲线，结果如图6所示，由图6可知，所有测试的B7-H3×CD3双特异性抗体均能以高活性结合人B7-H3抗原。
(2)B7-H3×CD3双特异性抗体对T细胞的激活作用检测
采用Jurkat/NFAT-Luc报告基因细胞系和B7-H3阳性的人肾癌细胞A498组成的检测系统，评价B7-H3×CD3对T细胞的激活作用，在Jurkat/NFAT-Luc细胞系中，荧光素酶基因受NFAT(Nuclear factor of activated T-cell)转录因子调控，作为效应细胞，B7-H3阳性的A498细胞作为靶细胞，当Jurkat/NFAT-Luc细胞与A498细胞共培养时，荧光素酶不表达，而当加入B7-H3×CD3时，A498细胞上的B7-H3可以通过B7-H3×CD3激活Jurkat/NFAT-Luc细胞内的CD3信号通路，促使荧光素酶表达，从而可以通过检测细胞中的荧光素酶表达量来判断Jurkat T细胞激活程度的强弱。
实验中，在96孔细胞培养板中依次加入A498细胞、Jurkat/NFAT-Luc细胞，再将B7-H3×CD3稀释到浓度为10μg/mL、2.5μg/mL、0.625μg/mL、0.15625μg/mL、0.0390μg/mL、0.0097μg/mL、0.0024μg/mL和0.0006μg/mL，将不同浓度的B7-H3×CD3加入到上述细胞培养板中，37℃静置培养6h，加入ONE-Glo Luciferase检测试剂，使用多功能酶标仪测定化学发光值，以抗体浓度的对数值为横坐标，平均化学发光值为纵坐标，进行四参数拟合，绘制剂量效应曲线，获得各曲线的EC 50值，以EC 50代表B7-H3×CD3激活T细胞的活性，实验设置阳性对照抗体8H9×CD3组。
结果如图7所示，当与B7-H3阳性肿瘤细胞A498、Jurkat/NFAT-Luc细胞共同孵育时，本公开构建的4株B7-H3×CD3双特异性抗体对Jurkat T细胞均有较好的激活作用。
(3)检测B7-H3×CD3双特异性抗体对B7-H3阳性细胞的杀伤作用
双特异性抗体B7-H3×CD3的CD3结合臂特异性地结合T细胞表面的CD3复合物，另一端B7-H3结合臂特异性地结合肿瘤细胞表面B7-H3分子，使T细胞与肿瘤细胞之间形成免疫桥联，继而激活T细胞，释放穿孔素(Perforin)和颗粒酶B(Granzyme B)等细胞杀伤蛋白，从而杀伤肿瘤细胞，当肿瘤细胞膜受损后，细胞膜通透性增大，胞浆中的乳酸脱氢酶(LDH)被释放到培养上清中。取一定量的上清，加入乳酸脱氢酶的反应底物乳酸后，乳酸脱氢酶催化脱氢反应，产生红色产物甲臜(Formazan)，可在490nm波长下产生吸收峰。细胞上清的乳酸脱氢酶含量越多，其颜色越深，吸光值越大；故可通过测定吸光值来定量肿瘤细胞释放乳酸脱氢酶的量，并计算B7-H3×CD3介导PBMC杀伤肿瘤细胞的杀伤活性，杀伤率的计算公式为：Cytotoxicity％＝(OD样品-SR)/(MR-SR)×100％，其中，SR＝OD自发释放孔(靶细胞+效应细胞)，MR＝OD最大释放孔(靶细胞)。
本公开采用B7-H3不同表达的细胞人胃癌细胞NCI-N87、A498、HepG2作为靶细胞，人外周血单核细胞PBMC作为效应细胞，检测4株B7-H3×CD3双特异性抗体介导PBMC杀伤肿瘤细胞的杀伤活性。
取新鲜分离的人外周血单核细胞PBMC，调整密度为2.00×10 6个细胞/mL，取生长状态良好的NCI-N87、A498、HepG2细胞，调整细胞浓度为2.00×10 5个细胞/mL，用1×PBS缓冲液(pH 7.4)将样品稀释为30μg/mL，再进行4倍梯度稀释， 共10个梯度，其浓度依次为30μg/mL、7.5μg/mL、1.875μg/mL、0.4688μg/mL、0.1172μg/mL、0.0293μg/mL、0.0073μg/mL、0.0018μg/mL、0.0005μg/mL和0.0001μg/mL，取96孔细胞培养板，依次加入50μL/孔靶细胞、100μL/孔PBMC和50μL/孔梯度稀释的样品，混匀，使得PBMC与靶细胞的体积细胞数目比例为20:1，样品的起始浓度为10μg/mL，对照孔用无酚红RPMI 1640培养基补足200μL，把培养板置于37℃、5％CO 2培养箱内孵育，21±1h后，用乳酸脱氢酶细胞毒性试剂盒(碧云天)检测细胞的杀伤毒性，以抗体浓度的对数值为横坐标，杀伤活性为纵坐标，进行四参数拟合，绘制剂量效应曲线，获得各曲线的EC 50值，以EC 50代表B7-H3×CD3对靶细胞的杀伤活性，结果见图8A-图8C。
如8A-图8C所示，本公开构建的双特异性抗体B7-H3×CD3对B7-H3不同表达的NCI-N87、A498、HepG2细胞都有较好的杀伤效果。
(4)B7-H3×CD3双特异性抗体的稳定性研究
评估B7-H3×CD3双特异性抗体在40℃条件下放置不同时间的稳定性，将充分密封的样品(1mg/mL)放置于40℃恒温箱(BINDER KBF240)，在相应时间点(基线(第0天)、第14天)取20μg样品进行SEC-HPLC检测纯度。所述SEC-HPLC条件如下：
排阻色谱柱：Superdex200 10/300 GL increase；
流动相：50mM NaAc,51mM NaCl,0.05mM EDTA pH 5.5；
流速：0.5mL/min；
紫外检测波长：280nm；
采集时间：35min。
所用仪器是AKTA pure 25L1色谱仪，利用UNICORN软件记录图谱并计算剩余单体的比例。
如图9A-图9D所示，图9A代表46H7×CD3在40℃放置14天后SEC-HPLC检测纯度的结果，其中，46H7×CD3双特异性抗体的液相出峰的保留时间为28.5min、峰面积比为100％，46H7×CD3双特异性抗体纯度＞98％；图9B代表43G11×CD3在40℃放置14天后SEC-HPLC检测纯度的结果，其中，43G11×CD3双特异性抗体的液相出峰的保留时间为28.5min、峰面积比为100％，43G11×CD3双特异性抗体纯度＞98％；图9C代表49E1×CD3在40℃放置14天后SEC-HPLC检测纯度的结果，其中，49E1×CD3双特异性抗体的液相出峰的保留时间为28.5min、峰面积比为100％，49E1×CD3双特异性抗体纯度＞98％；图9D代表42F7×CD3在40℃放置14天后SEC-HPLC检测纯度的结果，其中，42F7×CD3双特异性抗体的液相出峰的保留时间为28.5min、峰面积比为100％，42F7×CD3双特异性抗体纯度＞98％，以上结果说明，在40℃实验条件下，所述抗体的多聚体比例未随时间延长而增加，故认为所述双特异四价抗体具备较好的热稳定性，具有可开发性。
(5)检测B7-H3×CD3双特异性抗体小鼠体内抗肿瘤作用
根据前述实施例结果，选择其中一种B7-H3×CD3双特异性抗体(即42F7×CD3双特异性抗体)，进行小鼠体内抗肿瘤杀伤作用的检测。
选用8周龄的雌性NOD-SCID小鼠(购自北京维通利华实验动物有限公司)，收集对数生长期的MC38/hB7-H3细胞和新鲜分离的PBMC，以100μL/只接种于小鼠右前肢，接种次日分组，分组当天定义为D0天，并于D0天开始给药，实验共分2组，每组6只小鼠，分别是同型对照hIgG组(5mg/kg，i.v.)、B7-H3×CD3组(5mg/kg，i.v.)，于D1、D5、D8、D11、D15尾静脉注射给药(i.v.)，开始给药后，观测肿瘤大小并称量小鼠体重，瘤体积计算方式为：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)，利用肿瘤体积计算肿瘤增殖率(T/C％)和肿瘤抑制率(TGI)，根据肿瘤抑制率(TGI)进行疗效评价，结果见图10。
如图10所示，B7-H3×CD3对PBMC重建人免疫系统的NOD/SCID小鼠皮下建立的MC38/hB7-H3异种移植模型中小鼠肿瘤模型具有显著抑制肿瘤生长的作用。
综上所述，本公开的B7-H3抗体能够以高亲和力结合B7-H3抗原，通过对重链互补决定区进行突变，进一步提高了B7-H3抗体的亲和力，能够高效介导PBMC杀伤B7-H3阳性肿瘤，同时，本公开利用B7-H3抗体构建B7-H3×CD3双特异性抗体，所述双特性抗体具备高亲和力和稳定性，能够有效介导PBMC杀伤肿瘤细胞，具有显著抑制肿瘤生长的作用。
申请人声明，本公开通过上述实施例来说明本公开的详细方法，但本公开并不局限于上述详细方法，即不意味着本公开必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本公开的任何改进，对本公开产品各原料的等效替换及辅助成分的添加、方式的选择等，均落在本公开的保护范围和公开范围之内。
工业实用性
本公开提供了一种B7-H3抗体及其应用，本公开提供B7-H3抗体能够以高亲和力结合B7-H3抗原以及表达B7-H3抗原的细胞，能够高效介导PBMC杀伤B7-H3阳性肿瘤，利用所述B7-H3抗体构建B7-H3×CD3双特异性抗体，所述双特性抗体具备高亲和力和稳定性，能够有效介导PBMC杀伤肿瘤细胞，具有显著抑制肿瘤生长的作用，因此本公开提供的B7-H3抗体具有优异的实用性。

Claims (17)

  1. 一种B7-H3抗体，其特征在于，所述B7-H3抗体的重链互补决定区包括SEQ ID No.6-8所示的氨基酸序列；
    所述B7-H3抗体的轻链互补决定区包括SEQ ID No.14-16所示的氨基酸序列。
  2. 根据权利要求1所述的B7-H3抗体，其特征在于，所述B7-H3抗体的重链可变区包括SEQ ID No.1所示的氨基酸序列；
    所述B7-H3抗体的轻链可变区包括SEQ ID No.9所示的氨基酸序列。
  3. 一种亲和力成熟B7-H3抗体，其特征在于，所述亲和力成熟B7-H3抗体由权利要求1或2所述的B7-H3抗体经过突变处理制备得到。
  4. 根据权利要求3所述的亲和力成熟B7-H3抗体，其特征在于，所述亲和力成熟B7-H3抗体的重链互补决定区CDR1包括SEQ ID No.6或SEQ ID No.17～25中任意一种所示的氨基酸序列，重链互补决定区CDR2包括如SEQ ID No.7或SEQ ID No.26～37中任意一种所示的氨基酸序列，重链互补决定区CDR3包括如SEQ ID No.8或SEQ ID No.38～48中任意一种所示的氨基酸序列；
    所述亲和力成熟B7-H3抗体的轻链互补决定区包括SEQ ID No.14-16所示的氨基酸序列。
  5. 根据权利要求3或4所述的亲和力成熟B7-H3抗体，其特征在于，所述亲和力成熟B7-H3抗体的轻链可变区包括如SEQ ID No.9所示的氨基酸序列；
    所述亲和力成熟B7-H3抗体的重链框架区包括SEQ ID No.2-5所示的氨基酸序列。
  6. 一种双特异性抗体，其特征在于，所述双特异性抗体包括两条重链和两条轻链；
    所述重链包括抗B7-H3重链和抗CD3重链；所述抗B7-H3重链包括权利要求1或2所述的B7-H3抗体的重链或权利要求3-5任一项所述的亲和力成熟B7-H3抗体的重链；
    所述轻链包括抗B7-H3轻链和抗CD3轻链；所述抗B7-H3轻链包括权利要求1或2所述的B7-H3抗体的轻链或权利要求3-5任一项所述的亲和力成熟B7-H3抗体的轻链；
    所述重链通过二硫键与所述轻链结合；
    所述抗B7-H3重链通过二硫键与所述抗CD3重链结合。
  7. 根据权利要求6所述的双特异性抗体，其特征在于，所述抗CD3重链的可变区包括SEQ ID No.49所示的氨基酸序列；
    所述抗CD3轻链的可变区包括SEQ ID No.9所示的氨基酸序列。
  8. 根据权利要求6或7所述的双特异性抗体，其特征在于，所述双特异性抗体包括结合B7-H3的免疫球蛋白Fab结构域、结合CD3的免疫球蛋白Fab结构域和异源二聚体Fc区域；
    所述结合B7-H3的免疫球蛋白Fab结构域包括所述抗B7-H3轻链和抗B7-H3重链的可变区VH和恒定区CH1；
    所述结合CD3的免疫球蛋白Fab结构域包括所述抗CD3轻链和抗CD3重链的可变区VH和恒定区CH1；
    所述异源二聚体Fc区域包括抗B7-H3重链连接的Fc片段和抗CD3重链连接的Fc片段。
  9. 根据权利要求8所述的双特异性抗体，其特征在于，所述抗B7-H3重链连接的Fc片段包括人源Fc片段或人源化的Fc片段。
  10. 根据权利要求8或9所述的双特异性抗体，其特征在于，所述抗B7-H3重链连接的Fc片段的CH3含有T394D突变、P395D突变和P396D突变。
  11. 根据权利要求8-10中任一项所述的双特异性抗体，其特征在于，所述抗CD3重链连接的Fc片段的CH3含有P395K突变、P396K突变和V397K突变。
  12. 根据权利要求8-11中任一项所述的双特异性抗体，其特征在于，所述抗B7-H3重链连接的Fc片段的CH2含有L234A突变、L235A突变和P329G突变。
  13. 生物材料，其特征在于，所述生物材料为以下B1)至B6)中的任一种：
    B1)编码(b1)至(b3)中任一项所述抗体或者(b1)至(b3)中任一项所述抗体的抗原结合部分中的重链和/或轻链的核酸分子：(b1)权利要求1或2所述的B7-H3抗体，(b2)权利要求3-5任一项所述的亲和力成熟B7-H3抗体，或者(b3)权利要求6-12任一项所述的双特异性抗体；
    B2)含有B1)所述核酸分子的表达盒；
    B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；
    B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；
    B5)含有B1)所述核酸分子的细胞系、或含有B2)所述表达盒的细胞系、或含有B3)所述重组载体的细胞系；
    B6)编码(b1)至(b3)中任一项所述抗体或者(b1)至(b3)中任一项所述抗体的抗原结合部分中的重链可变区和/或轻链可变区的核酸分子。
  14. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1或2所述的B7-H3抗体、权利要求3-5任一项所述的亲和力成熟B7-H3抗体或权利要求6-12所述的双特异性抗体中的任意一种或至少两种的组合。
  15. 权利要求1或2所述的B7-H3抗体、权利要求3-5任一项所述的亲和力成熟B7-H3抗体、权利要求6-12任一项所述的双特异性抗体或权利要求14所述的药物组合物在制备抗肿瘤药物中的应用；
    所述肿瘤包括神经系统肿瘤、结直肠癌、肝癌、头颈癌、肺癌、黑色素瘤、胰腺癌、胃癌、肾癌、膀胱癌、乳腺癌、卵巢癌或前列腺癌中的任意一种或至少两种的组合。
  16. 权利要求1或2所述的B7-H3抗体、权利要求3-5任一项所述的亲和力成熟B7-H3抗体、权利要求6-12任一项所述的双特异性抗体或权利要求14所述的药物组合物，用于治疗肿瘤的用途；
    优选地，所述肿瘤包括神经系统肿瘤、结直肠癌、肝癌、头颈癌、肺癌、黑色素瘤、胰腺癌、胃癌、肾癌、膀胱癌、乳腺癌、卵巢癌或前列腺癌中的任意一种或至少两种的组合。
  17. 一种治疗与肿瘤有关疾病的方法，其特征在于，向所述有此需要的受试者给药根据权利要求1或2所述的B7-H3抗体、权利要求3-5任一项所述的亲和力成熟B7-H3抗体、权利要求6-12任一项所述的双特异性抗体或权利要求14所述的药物组合物；
    优选地，所述肿瘤包括神经系统肿瘤、结直肠癌、肝癌、头颈癌、肺癌、黑色素瘤、胰腺癌、胃癌、肾癌、膀胱癌、乳腺癌、卵巢癌或前列腺癌中的任意一种或至少两种的组合。
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CN114539420A (zh) * 2022-01-20 2022-05-27 同济大学苏州研究院 抗b7-h3单克隆抗体、抗b7-h3×cd3双特异性抗体、制备方法及其应用

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