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WO2024149346A1 - Binding protein of immunoregulation protein molecule b7-h3 and use thereof - Google Patents

Binding protein of immunoregulation protein molecule b7-h3 and use thereof Download PDF

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Publication number
WO2024149346A1
WO2024149346A1 PCT/CN2024/071909 CN2024071909W WO2024149346A1 WO 2024149346 A1 WO2024149346 A1 WO 2024149346A1 CN 2024071909 W CN2024071909 W CN 2024071909W WO 2024149346 A1 WO2024149346 A1 WO 2024149346A1
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antibody
seq
amino acid
acid sequence
tumor
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PCT/CN2024/071909
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French (fr)
Chinese (zh)
Inventor
王少雄
吕品
黄璐
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迈博斯（香港）科技有限公司
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Publication of WO2024149346A1 publication Critical patent/WO2024149346A1/en

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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C07K2317/50—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
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention belongs to the field of immunology; specifically, it relates to a binding protein of immunomodulatory protein molecule B7-H3 and application thereof.
  • B7-H3 (also known as CD276) is a type I transmembrane protein that belongs to the B7 immunomodulatory molecule family.
  • B7-H3 has 20%-27% similarity with other family members and has a similar molecular structure to B7-H1 (PD-L1).
  • the B7-H3 protein is encoded by the chromosome 15q24 gene and consists of 316 amino acids (2IgB7-H3 subtype) including an extracellular domain, a transmembrane domain, and a short intracellular domain, with a molecular weight of 45kDa.
  • human B7-H3 protein contains one or two pairs of identical extracellular domains, resulting in two subtypes: 2IgB7-H3 consists of a pair of immunoglobulin variable region (IgV)-like and immunoglobulin constant region (IgC)-like extracellular domains; 4IgB7-H3 contains two pairs of identical IgV-like and IgC-like extracellular domains, which is also the main subtype in human cells.
  • B7-H3 is a member of the B7 family of immunoregulatory molecules and is overexpressed in a variety of solid tumors, including head and neck tumors, bladder cancer, colorectal cancer, pancreatic cancer, and ovarian cancer.
  • B7-H3 plays a major role in the anti-tumor activity of immune cells, which helps tumor cells escape immune surveillance.
  • Tumor expression of B7-H3 can inhibit T cell-mediated anti-tumor immune responses, and high expression of B7-H3 is associated with the lack of response to PD-1 antibodies in NSCLC patients.
  • high expression of this protein is also associated with reduced CD8T cell infiltration and exhaustion in ovarian cancer.
  • B7-H3 protein can indirectly activate NF-kB, PI3K/Akt, and JAK/STAT3 pathways to induce cell survival and proliferation; inhibit transcription factor NRF2, leading to increased levels of reactive oxygen species (ROS) and HIF1a, thereby inducing aerobic glycolysis and leading to tumor growth. Therefore, B7-H3 also has the effect of promoting tumor proliferation, metabolism, and metastasis.
  • ROS reactive oxygen species
  • B7-H3 is widely and highly expressed in many solid tumors, and its co-inhibitory effect on the anti-tumor activity of immune cells means that antibody drugs targeting B7-H3, and their derived bispecific antibodies and ADC drugs, and CAR-T cell therapy methods based on the B7-H3 antibody-antigen binding sequence, are feasible for use in the treatment of a wide range of solid tumors.
  • B7-H3 is highly expressed in a variety of refractory and recurrent solid tumors, including central nervous system tumors, head and neck tumors, triple-negative breast cancer, non-small cell lung cancer and kidney tumors, and high expression of B7-H3 is correlated with poor tumor immunotherapy. Therefore, B7-H3 antibodies can not only target and treat refractory and recurrent solid tumors such as central nervous system tumors, head and neck tumors, triple-negative breast cancer, non-small cell lung cancer and kidney tumors, but can also be combined with immunotherapy to treat solid tumors that are not sensitive to immunotherapy.
  • MGA271 MacroGenics' B7-H3 antibody MGA271 (Enoblituzumab) combined with PD-1 monoclonal antibody (Pembrolizumab)
  • MGA271 performed better than the single-drug PD-1 monoclonal antibody Pembrolizumab and Nivolumab. Despite this, MGA271 still has room for improvement in terms of affinity and other aspects.
  • the purpose of the present invention is to provide a binding protein of the immunomodulatory protein molecule B7-H3 and its application.
  • an antibody (including an antigen-binding fragment thereof) binding to the immunomodulatory molecule B7-H3 which has a significantly improved effect of inhibiting the binding of the immunomodulatory molecule B7-H3 to its receptor molecule (cell surface receptor molecule).
  • the antibody has a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 13, the amino acid sequence of CDR2 is shown in SEQ ID NO: 14, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 15; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 16, the amino acid sequence of CDR2 is shown in SEQ ID NO: 17, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 18.
  • the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO: 19
  • the amino acid sequence of CDR2 is shown as SEQ ID NO: 20
  • the amino acid sequence of CDR3 is shown as SEQ ID NO: 21
  • the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO: 22
  • the amino acid sequence of CDR2 is shown as SEQ ID NO: 23
  • the amino acid sequence of CDR3 is shown as SEQ ID NO: 24.
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 3.
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 5
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 6.
  • the antibody comprises a heavy chain variable region and a light chain variable region: its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 8; or, its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 10; or, its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 11, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 12.
  • the antibody also includes an antibody whose heavy chain variable region amino acid sequence is more than 80% (such as 85%, 90%, 93%, 95%, 97% or 99% or more) identical to the sequence shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11, and a light chain variable region amino acid sequence is more than 80% (such as 85%, 90%, 93%, 95%, 97% or 99% or more) identical to the sequence shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12 and retains binding activity.
  • the antibody comprises: a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody.
  • the antibody comprises: Fab, Fab'-SH, Fv, Fd, scFv or (Fab')2 fragment.
  • the antibody is an IgG antibody.
  • an isolated polynucleotide or a construct containing the polynucleotide wherein the polynucleotide encodes the antibody that binds to the immunomodulatory molecule B7-H3; preferably, the construct is an expression vector.
  • an antibody expression system wherein the expression system contains the construct or the exogenous polynucleotide integrated into the genome; preferably, the expression system is a cell (expression system).
  • the cell is a mammalian cell.
  • the mammalian cells include (but are not limited to): Chinese hamster ovary (CHO) cells, Vero cells, HEK-293 cells, NS0 cells, SP2/0 cells, BHK cells, PER-C6 cells; preferably, CHO cells.
  • CHO Chinese hamster ovary
  • a method for preparing the antibody comprising: expressing the antibody using the antibody expression system under conditions suitable for expressing the antibody, thereby expressing the antibody; preferably, it also comprises purifying and isolating the antibody.
  • the use of the antibody is provided for: preparing an anti-tumor drug that specifically targets cells expressing the immunomodulatory molecule B7-H3, wherein the drug inhibits tumors by stimulating antigen-specific immune cells (such as T cells) to respond; preparing antibody-drug conjugates or immunoconjugates; preparing bifunctional (bispecific) or multifunctional (multispecific) antibodies; preparing immune cells modified with chimeric antigen receptors.
  • antigen-specific immune cells such as T cells
  • a fusion protein or immunoconjugate which comprises the antibody and a functional molecule operably linked thereto.
  • the functional molecules include: tumor-suppressing molecules, molecules targeting tumor surface markers, molecules or detectable markers targeting surface markers of immune cells, cytotoxins, radioactive isotopes, biologically active proteins, fusion partners, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or a combination thereof.
  • the molecule targeting a surface marker of an immune cell is an antibody or a ligand that binds to a surface marker of an immune cell.
  • the tumor-suppressing molecule is an anti-tumor toxin or an anti-tumor cytokine.
  • the molecule targeting a tumor surface marker is an antibody or a ligand that binds to a tumor surface marker.
  • the fusion partner includes (but is not limited to): a protein or active domain that has the effect of extending the half-life in vivo.
  • the antigens bound by the antibody that binds to the immune cell surface marker include (but are not limited to): CD3, CD20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1 or TenB2.
  • the antibody that binds to the tumor surface marker is an antibody that recognizes antigens other than the immune regulatory molecule B7-H3, and the other antigens include (but are not limited to): EGFR, EGFRvIII, mesothelin, HER2, EphA2, cMet, EpCAM, MUC1, MUC16, CEA, Claudin 18.2, Claudin 6, WT1, NY-ESO-1, MAGE 3, CD47, ASGPR1 or CDH16.
  • the anti-tumor cytokines include (but are not limited to): IL-2, IL-12, IL-15, IFN-beta, TNF-alpha.
  • the anti-tumor toxin includes (but is not limited to): maytansine, auristatin, monomethyl auristatin, maytansinoid alkaloid, dolastatin, calicheamicin, methotrexate, vindesine, taxanes such as docetaxel, paclitaxel, lalopaclitaxel, tecitaxel or octataxel, trichothecenes, CC1065, DXd, Duocarmycin, Calicheamicin, Pyrrolobenzodiazepines or SN-38.
  • the protein or active domain having the effect of extending the in vivo half-life includes (but is not limited to): immunoglobulin Fc region, preferably human immunoglobulin Fc region, serum albumin (such as human HSA) or a fragment thereof.
  • the immunoglobulin is a combination of one or more selected from IgG, IgA1, IgA2, IgD, IgE, and IgM, and the IgG is a combination of one or more selected from IgG1, IgG2, IgG3, or IgG4 subtypes.
  • a connecting peptide is provided between the antibody and the functional molecule operably connected thereto; the connecting peptide is preferably selected from a flexible polypeptide chain composed of alanine and/or serine and/or glycine, and the length of the connecting peptide is preferably 3 to 30 amino acids.
  • a pharmaceutical composition comprising the antibody, the fusion protein or the immunoconjugate; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the tumor includes a solid tumor; preferably, the tumor includes a tumor expressing the immunomodulatory molecule B7-H3; preferably, the tumor includes (but is not limited to): ovarian cancer, breast cancer (triple-negative breast cancer), central nervous system tumors, head and neck tumors, bladder cancer, rectal cancer, pancreatic cancer, non-small cell lung cancer, and kidney tumors.
  • kits or a medicine box comprising the antibody, the fusion protein or the immunoconjugate, or a pharmaceutical composition containing the same.
  • test kit or pharmaceutical kit further comprises: an immunostimulatory antibody, a chemotherapeutic agent, and an antiviral drug.
  • the immunostimulatory antibody is selected from one or more of the following: an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, and an anti-CTLA-4 antibody.
  • test kit or medicine box further contains one or more containers for containing the antibody, fusion protein, immunoconjugate or pharmaceutical composition.
  • the kit or pharmaceutical kit further contains instructions for use (package insert) describing the method for use/administration of the antibody, fusion protein, immunoconjugate or pharmaceutical composition.
  • a method for stimulating an immune response in a subject comprising administering to the subject any of the antibodies described above, thereby stimulating the immune response of the subject.
  • the subject is a subject bearing a tumor, and administering the antibody to the subject stimulates an immune response against the tumor.
  • the subject is a subject carrying a virus, and the subject is given the Antibodies stimulate an immune response against the virus.
  • a method of inhibiting tumor cell growth in a subject comprising administering the above-described antibody to the subject.
  • a method of treating a viral infection in a subject comprising administering to the subject the antibody described above.
  • FIG. 1 ELISA test results of 5 IgG4 monoclonal antibodies binding to human B7-H3 tag fusion protein.
  • FIG. 1 SEC-HPLC purity test of B7-H3 monoclonal antibody produced by transiently transfected ExpiCHO-S cells. From top to bottom in the figure are: BM (PC), 13725, 14479.
  • FIG. 7 ELISA test results of the binding of B7-H3 monoclonal antibody to mouse B7-H4-huFc.
  • FIG8 The results of the cell binding activity test of B7-H3 monoclonal antibody and breast cancer cell line MCF-7.
  • FIG. 9 The results of the cell binding activity test of B7-H3 monoclonal antibody to the ovarian cancer cell line OVCAR-3.
  • Figure 10 Detection results of the binding activity of B7-H3 monoclonal antibody to human insulin.
  • FIG. 12 ADCC detection results of B7-H3 humanized monoclonal antibody.
  • FIG. 13 In vivo antitumor efficacy in mouse tumor-bearing models. The figure shows the mean tumor volume ⁇ SEM.
  • FIG. 14A-B the efficacy of ADC prepared with B7-H3 monoclonal antibody in inhibiting tumor cell growth in vivo.
  • B7-H3-specific antibody that can specifically bind to B7-H3 and stimulate antigen-specific immune cells (such as T cells) to respond, and can be used to inhibit diseases related to B7-H3 expression (including overexpression) or affected by B7-H3 function, such as tumors or viral infections.
  • the present invention relates to anti-B7-H3 antibodies.
  • the present invention provides an anti-B7-H3 antibody comprising a binding domain comprising at least 1, 2, 3, 4, 5 or 6 hypervariable regions (HVRs) or complementary determining regions (CDRs) selected from the following: (a) HVR-H1 comprising an amino acid sequence of SEQ ID NO: 13 or 19 or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% homologous to the sequence; (b) HVR-H2 comprising an amino acid sequence of SEQ ID NO: 14 or 20 or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% homologous to the sequence; (c) HVR-H3 comprising an amino acid sequence of SEQ ID NO: 15 or 21, or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% homologous thereto; (d) HVR-L1 comprising an amino acid sequence of SEQ ID NO: 16 or
  • the anti-B7-H3 antibody may have a heavy chain variable (VH) domain (region) that includes an amino acid sequence having at least 90% sequence homology (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology) to SEQ ID NO: 1 or 5, or the amino acid sequence of SEQ ID NO: 1 or 5, and/or a light chain variable (VL) domain (region) that includes an amino acid sequence having at least 90% sequence homology (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology) to SEQ ID NO: 3 or 6, or the amino acid sequence of SEQ ID NO: 3 or 6.
  • VH heavy chain variable
  • the anti-B7-H3 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11.
  • the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12.
  • the anti-B7-H3 antibody comprises the following combination of heavy chain variable region and light chain variable region:
  • the anti-B7-H3 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 5, and the light chain variable region comprises the amino acid sequence SEQ ID NO: 3 or SEQ ID NO: 6.
  • the antibodies provided herein are antibody fragments.
  • Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, (Fab')2, Fv and scFv fragments and other fragments described below.
  • Fab fragment antigen binding protein
  • Fab' fragment antigen binding protein
  • Fv fragment antigen binding protein
  • scFv fragments see, for example, Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds. (Springer-Verlag, Berlin), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458.
  • Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. See, e.g., EP0404097A2; WO 1993/011161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).
  • a single-domain antibody is an antibody fragment that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody.
  • the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).
  • Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage) as described herein.
  • recombinant host cells eg, E. coli or phage
  • the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984).
  • chimeric antibodies are humanized antibodies.
  • non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibodies.
  • humanized antibodies include one or more variable domains, wherein HVR, such as CDR, (or a portion thereof) is derived from non-human antibodies, and FR (or a portion thereof) is derived from human antibody sequences.
  • HVR such as CDR
  • FR or a portion thereof
  • Humanized antibodies will optionally also include at least a portion of human constant regions.
  • some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (e.g., antibodies that obtain HVR residues), for example, to repair or improve antibody specificity or affinity.
  • the antibodies provided herein are human antibodies.
  • Human antibodies can be produced using various techniques known in the art.
  • Human antibodies can be prepared by administering an immunogen to a modified transgenic animal and then attacking it with an antigen to prepare a complete human antibody or a complete antibody with a human variable region.
  • Such animals typically contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci or are present outside the chromosomes or randomly integrated into the animal chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated.
  • U.S. Patents 6,075,181 and 6,150,584 describing XENOMOUSE TM technology
  • U.S. Patent 5,770,429 U.S. Patent 7,041,870 (describing KM technology)
  • U.S. Application Publication No. US 2007/0061900 The human variable regions derived from the complete antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.
  • Human antibodies can also be made by hybridoma-based methods.
  • Human myeloma and mouse-human hybrid myeloma cell lines for producing human monoclonal antibodies have been described, see, e.g., Kozbor et al., J. Immunol., 133:3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63; Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86-95 (1991).
  • Human antibodies produced by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).
  • Human antibodies can also be prepared by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains.
  • the present invention describes in part the process of screening human antibodies through phage display libraries.
  • antibodies of the present invention with high affinity can be isolated by screening combinatorial libraries for antibodies with binding activity to B7-H3.
  • various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics. Such methods can be found, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are further described in, for example, the following literature: McCafferty et al., Nature 348: 552-554 (1990); Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol.
  • VH and VL gene repertoires are individually cloned by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol. 12: 433-455 (1994).
  • Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments.
  • scFv single-chain Fv
  • Libraries from immune sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas.
  • Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373 and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936.
  • Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.
  • the anti-B7-H3 antibodies provided herein are multispecific antibodies, such as bispecific antibodies.
  • Multispecific antibodies are monoclonal antibodies that have binding specificity for at least two different sites.
  • one binding specificity is for B7-H3
  • the other binding specificity is for any other antigen (e.g., a second biological molecule, such as an immune cell surface antigen, such as a tumor antigen).
  • bispecific anti-B7-H3 antibodies may have binding specificity for B7-H3 and other antigens, such as CD3, CD20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1 or TenB2.
  • Bispecific antibodies can also be prepared as full-length antibodies or antibody fragments.
  • Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537-540 (1983)); WO 93/08829; and Traunecker et al., EMBO J. 10: 3655-3659 (1991)) WO 2009/080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108: 11187-11192 (2011)), WO 2009/089004, etc.
  • the antibodies of the present invention encompass amino acid sequence variants of the anti-B7-H3 antibodies of the present invention. For example, it may be necessary to further improve the binding affinity and/or other biological properties of the antibody and prepare the antibody variant.
  • the amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, residue deletions and/or insertions and/or substitutions within the antibody amino acid sequence. Any combination of deletions, insertions and substitutions can be performed to obtain the final construct, which can give the final construct the desired characteristics, such as antigen binding.
  • antibody variants with one or more amino acid substitutions are provided.
  • the relevant sites induced by substitution mutations include HVR and FR.
  • Conservative substitutions are shown below under the heading "Preferred Substitutions”. More substantial changes are provided below under the heading "Exemplary Substitutions” and are further described below with reference to amino acid side chain categories.
  • Amino acid substitutions can be introduced into related antibodies and the product can be screened for the desired activity (e.g., retaining/improving antigen binding or improving ADCC or CDC).
  • Amino acids can be grouped according to common side chain properties:
  • Non-conservative substitutions will necessarily entail exchanging a member of one of these classes for another class.
  • substitutional variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody).
  • a parent antibody e.g., a humanized antibody or a human antibody.
  • the resulting variants selected for further study will be modified (e.g., improved) relative to the parent antibody in terms of certain biological properties (e.g., increased affinity) and/or will substantially retain certain biological properties of the parent antibody.
  • Exemplary substitutional variants are affinity matured antibodies, which can be conveniently produced using, for example, affinity maturation techniques based on phage display (such as those described herein).
  • affinity maturation techniques based on phage display (such as those described herein).
  • one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for specific biological activities (e.g., binding affinity).
  • substitutions, insertions, or deletions may occur within one or more HVRs (CDRs), as long as such changes do not substantially impair the ability of the antibody to bind B7H3.
  • CDRs HVRs
  • conservative changes e.g., conservative substitutions as provided herein
  • HVRs that do not substantially reduce binding affinity may be made in HVRs that contact the antigen in the
  • each HVR is unchanged or contains no more than 1, 2 or 3 amino acid substitutions.
  • a useful method for identifying antibody residues or regions that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • the anti-B7-H3 antibodies of the present invention can be prepared using recombinant methods, for example, as described in U.S. Pat. No. 4,816,567.
  • an isolated nucleic acid encoding an anti-B7-H3 antibody described herein is provided.
  • Such nucleic acid may encode an amino acid sequence comprising the VL of the antibody (e.g., the light chain and/or heavy chain of the antibody) and/or an amino acid sequence comprising its VH.
  • one or more vectors e.g., expression vectors
  • a host cell comprising such nucleic acids is provided.
  • the host cell comprises (e.g., transformed to have): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody.
  • the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, SP2/0 cell).
  • a method of making an anti-B7-H3 antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and comprises recovering the antibody from the host cell (or host cell culture medium).
  • nucleic acids encoding the antibodies are isolated (e.g., as described above) and inserted into one or more vectors for further cloning and/or expression in host cells.
  • Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibodies).
  • Suitable host cells for cloning or expressing antibody encoding vectors include prokaryotes or eukaryotic cells described herein.
  • antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required.
  • For expressing antibody fragments and polypeptides in bacteria see, for example, U.S. Patents Nos. 5,648,237, 5,789,199, and 5,840,523. After expression, antibodies in the soluble portion can be separated from the bacterial cell slurry and can be further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized” to produce antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004); and Li et al., Nat. Biotech. 24: 210-215 (2006).
  • Suitable host cells for expressing glycosylated antibodies also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used to bind to insect cells, especially for transfecting Spodoptera frugiperda cells.
  • Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES TM technology for producing antibodies in transgenic plants).
  • Vertebrate cells can also be used as hosts.
  • mammalian cell lines adapted to growth in suspension may be suitable.
  • suitable mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7); human embryonic kidney cell line (e.g., 293 or 293 cells as described in Graham et al., J. Gen. Virol. 36:59-74 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod.
  • Suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci.
  • myeloma cell lines such as Y0, NS0 and SP2/0.
  • myeloma cell lines such as Y0, NS0 and SP2/0.
  • the affinity KD of the B7-H3 antibody of the present invention to human B7-H3 is lower than 1.0 pM, which is superior to B7-H3 antibodies such as MGA271 of Macrogenics, and has excellent application prospects in the treatment of solid tumors including central nervous system tumors, head and neck tumors, triple-negative breast cancer, non-small cell lung cancer and kidney tumors.
  • the present invention also provides immunoconjugates comprising an anti-B7-H3 antibody herein combined with one or more cytotoxic agents, such as chemotherapeutic agents or chemotherapeutic drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin or fragments thereof), or radioactive isotopes, etc.
  • cytotoxic agents such as chemotherapeutic agents or chemotherapeutic drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin or fragments thereof), or radioactive isotopes, etc.
  • the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is bound to one or more drugs, including but not limited to maytansine (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP0425235B1); auristatin, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos.
  • ADC antibody-drug conjugate
  • the immunoconjugate comprises a conjugate of an anti-B7-H3 antibody as described herein and an enzymatically active toxin or a fragment thereof, including but not limited to diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, and trichothecenes, etc.
  • the immunoconjugate comprises a radioconjugate formed by combining an anti-B7-H3 antibody as described herein with a radioactive atom.
  • a radioconjugate formed by combining an anti-B7-H3 antibody as described herein with a radioactive atom.
  • radioisotopes can be used to produce radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioisotopes of Lu.
  • Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, esters (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (such as dimethyl adipate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5
  • the pharmaceutical preparation of the anti-B7-H3 antibody of the present invention is prepared by mixing an antibody having a desired purity with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) into a lyophilized preparation or an aqueous solution.
  • Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid
  • Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958.
  • Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,86 and WO 2006/044908, the latter formulations including histidine-acetate buffer.
  • the preparations herein may also contain more than one active ingredient that must be present for the specific indication being treated, preferably with active ingredients that have complementary activities that do not adversely affect each other.
  • active ingredients that have complementary activities that do not adversely affect each other.
  • additional therapeutic agents e.g., chemotherapeutic agents, cytotoxic agents, growth inhibitors and/or anti-hormonal agents.
  • Such active ingredients are suitable for being present in a combined form in an amount effective for a predetermined purpose.
  • the present invention also provides an article containing an antibody or pharmaceutical composition of the present invention.
  • the article comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. Such containers can be made of various materials, such as glass or plastic.
  • the container holds the composition of the present invention itself or a combination of the composition and another composition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle).
  • At least one active agent in the composition is an antibody of the present invention.
  • the label or package insert indicates that the composition is used to treat a selected tumor or viral infection.
  • the article may include (a) a first container containing a composition, wherein the composition comprises an antibody of the present invention; and (b) a second container containing a composition, wherein the composition comprises another cytotoxic agent or other therapeutic agent.
  • the article in this embodiment of the present invention may further include a package insert indicating that such a composition can be used to treat a tumor or viral infection.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further comprise any other pharmaceutically acceptable buffer as deemed desirable from a commercial and user standpoint. Other materials required, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • single-chain antibodies with high affinity binding to human B7-H3 recombinant fusion protein are selected from a fully human single-chain scFv phage display library.
  • the method involves utilizing the fully human single-chain scFv phage display library as described below, and using human B7-H3-6*His recombinant fusion protein (Nearshore protein, cat#CK62), Escherichia coli TG1 (Lucigen, cat#60502-1) and M13KO7 helper phage (Thermo Fisher, cat#18311019).
  • the fully human single-chain scFv phage display library was constructed from the phagemid vector pCLS1.0 containing the pBR3.22 promoter and the M13 phage pIII protein display system, and its C-terminus contained a Myc-His tag. Escherichia coli TG1 was used for library construction.
  • the first strand of cDNA was used as a template to amplify the human antibody heavy chain variable region and light chain variable region genes by PCR.
  • the human antibody heavy chain variable region and light chain variable region gene fragments were cloned into the phagemid vector pCLS1.0 using molecular cloning technology. Specifically, the human antibody light chain variable region was cloned into the vector pCLS1.0 by double digestion with Nhe I/Not I, and electrotransformed into Escherichia coli TG1 to obtain a phage light chain sublibrary and a vector pCLS1.0-VL.
  • the human antibody heavy chain variable region was cloned into the vector pCLS1.0-VL by double digestion with Sfi I/Xho I). The same method was used to obtain fully human scFv phage display sub-libraries with heavy chain and light chain variable region combinations. The total library capacity of each sub-library was 9 ⁇ 10 9 .
  • the human B7-H3-6*His tag fusion proteins in the panning experiment were all labeled with biotin using the Biotin Labeling Kit-NH2 (Dojindo, cat#LK03).
  • the panning experiment was briefly described as follows: M13KO7 helper phage was prepared using E. coli TG1, and the titer was determined to be about 1.0 ⁇ 10 13 , and it was used to infect the above-mentioned fully human scFv single-chain phage display library with an OD600 value of 0.5-0.6 to obtain the first round of input phage.
  • the first round of output phages were used to infect TG1 Escherichia coli with an OD600 value of 0.5-0.6 to obtain the second round of input phages, and the above-mentioned bio-panning method was continued to obtain the second round of output phages binding to the human B7-H3-6*His tag recombinant fusion protein.
  • the second round of bio-panning process was repeated for the third or fourth round of panning, and the output phages enriched in the third or fourth round of panning were used to infect E. coli TG1 to obtain single clones, and the single clones of E. coli with high affinity for human B7-H3-6*His tag recombinant fusion protein were identified and selected by phage ELISA, and sequenced and analyzed to obtain 20 clones, as shown in Table 1.
  • the E. coli monoclonal clone carrying the scFv single-chain antibody was induced to express soluble scFv single-chain antibody protein with IPTG, and the supernatant after filtration was taken to detect the binding activity with CHO cells overexpressing B7-H3.
  • the 20 soluble scFv single-chain antibody proteins induced by IPTG, 2YT medium (Shanghai Biotechnology Co., Ltd., cat#SD7019) and blank control PBS (Hyclone, cat#SH30256.01) were incubated with CHO cells overexpressing B7-H3 at 4°C for 1 hour.
  • the cells were then washed twice with cold 1 ⁇ PBS buffer.
  • Anti-His-PE labeled antibody (Miltenyi Biotec, cat#130-092-691) diluted 1:50 was added, and the mixture was incubated at 4°C for 30 minutes, then washed twice with cold 1 ⁇ PBS buffer, and the binding activity of the induced expressed scFv single-chain antibody to CHO cells overexpressing B7-H3 was analyzed using Guava easyCyte HT flow cytometer (MERCK MILLIPORE). During flow analysis, gates were set for CHO cells overexpressing B7-H3, and the binding activity was examined by PE signal intensity.
  • the binding activity of 20 IPTG-induced expressed soluble scFv single-chain antibody proteins to human CHO cells overexpressing B7-H3 is shown in Table 2.
  • Five antibody proteins (13725, 14479, 13683, 14463, 14471) have the highest binding activity to CHO cells overexpressing B7-H3.
  • amino acid sequence SEQ ID NO: 3
  • nucleotide sequence SEQ ID NO: 4
  • Amino acid sequence of the VH chain of the 14479 cloned single-chain antibody (SEQ ID NO: 5):
  • Amino acid sequence of the VL chain of the 14479 cloned single-chain antibody (SEQ ID NO: 6):
  • Amino acid sequence of the VH chain of the 13683 cloned single-chain antibody (SEQ ID NO: 7):
  • Amino acid sequence of the VL chain of the 13683 cloned single-chain antibody (SEQ ID NO: 8):
  • Amino acid sequence of the VH chain of the 14463 cloned single-chain antibody (SEQ ID NO: 9):
  • Amino acid sequence of the VL chain of the 14463 cloned single-chain antibody (SEQ ID NO: 10):
  • Amino acid sequence of the VL chain of the 14471 cloned single-chain antibody (SEQ ID NO: 12):
  • the above was molecularly cloned and converted into IgG4.
  • the sequence containing the leader peptide, Nhe I/Not I restriction site, heavy chain gene constant region and human IgG4-Fc was cloned into pCDNA3.3+ to obtain the vector pCDNA3.3-IgG4.
  • the sequence containing the leader peptide, Nhe I/BsiW I restriction site, and light chain Kappa gene constant region was cloned into pCDNA3.3+ to obtain the vector pCDNA3.3-VKappa, or the sequence containing the leader peptide, BamH I/Hind III restriction site, and light chain lambda gene constant region was cloned into pCDNA3.3+ to obtain the vector pCDNA3.3-VLambda.
  • the recombinant plasmids containing the heavy chain variable region and the light chain variable region listed in Table 3 above and the recombinant plasmids containing the heavy chain variable region and the light chain variable region of the control antibody BM (PC) were transiently transfected into suspension cultured Expi CHO-S TM cells (Thermo Fisher, cat#A29127) using the ExpiFectamine TM CHO Transfection Kit (Thermo Fisher, cat#A29129) liposome method.
  • the transfected ExpiCHO-S cells obtained above were cultured in 30 ml of ExpiCHO Expression medium (Thermo Fisher, cat#A2910001) at 37°C, 8% CO 2 , and 120 rpm.
  • first centrifugation condition 20min, 400g; second centrifugation condition 20min, 10000g The clarified supernatant was loaded into a protein A affinity chromatography column (GE Healthcare, cat#GE-17-5438-04), and impurities were removed by three steps of elution (elution buffer was phosphate buffer containing 150mM NaCl, pH 5.0; 20mM sodium citrate-1M sodium chloride pH 5.0; 20mM sodium citrate pH 5.0 elution buffer), and then the target antibody was separated and captured by 20mM sodium citrate solution at pH 3.0. Finally, the target antibody was replaced into 1 ⁇ PBS buffer at pH 7.4 by ultrafiltration concentration step.
  • elution buffer was phosphate buffer containing 150mM NaCl, pH 5.0; 20mM sodium citrate-1M sodium chloride pH 5.0; 20mM sodium citrate pH 5.0 elution buffer
  • the purified 5 IgG antibodies (13683, 13725, 14463, 14471, 14479) were tested for their in vitro binding activity with human B7-H3*His tag recombinant fusion protein antigen by ELISA.
  • This experiment used a commercially available human B7-H3-6*His tag fusion protein (Near Shore Protein, cat#CK62).
  • the rinse solution carbonate buffer
  • the anti-human B7-H3 fully human monoclonal antibody (13683, 13725, 14463, 14471, 14479) was diluted 3 times from 10 ⁇ g/ml with 1xPBS buffer at pH 7.4 and incubated with the coated antibody at 25°C for 2h. The binding of the antibody to the human B7-H3-6*His tag fusion protein antigen was then detected with an HRP-labeled anti-hIGFc detection antibody.
  • the fully human monoclonal antibodies 13725 and 14479 have particularly ideal affinities for the human B7-H3-6*His tag fusion protein antigen, among which 13725 has the best affinity.
  • the CDR region sequences of the fully human monoclonal antibodies 13725 and 14479 are shown in Table 4.
  • the recombinant plasmid containing the heavy and light chain genes of the preferred clone B7-H3 was transiently transfected into a larger scale (200-600 ml) suspension culture of ExpiCHO-S cells (Thermo Fisher, cat#A29127) using the ExpiFectamine TM CHO Transfection Kit (Thermo Fisher, cat#A29129) liposome method.
  • the transfection, culture and purification methods were as described in Example 1.3 to prepare the target antibody.
  • the target antibody was replaced into 1 ⁇ PBS buffer at pH 7.4 by ultrafiltration concentration step for subsequent isomerization.
  • Example 4 Binding of B7-H3 monoclonal antibody to human B7-H3-His protein, human B7-H4-His protein, human PD-L1-ECD-mFc protein, mouse B7-H3-His protein, and mouse B7-H4-huFc protein
  • the experiment used commercially available human B7-H3-His protein (nearshore protein, cat#CK62), human B7-H4-His protein (Acro, B74-H5222), human PD-L1-ECD-mFc protein (nearshore protein, cat#CM06), mouse B7-H3-His protein (nearshore protein, cat#CM83), and mouse B7-H4-huFc protein (nearshore protein, cat#CS04).
  • the protein antigens to be tested were diluted to a concentration of 1 ⁇ g/ml with 1xPBS coating buffer at pH 7.4 and coated in 96-well plates (CORNING, cat#9018) at 4°C overnight.
  • the anti-human B7-H3 fully human monoclonal antibodies 13725 and 14479 to be tested were diluted 3 times from 10 ⁇ g/ml in 1xPBS buffer at pH 7.4 and incubated with the coated protein antigen at 25°C for 2 hours. The binding of the antibodies to the respective protein antigens was then detected using an HRP-labeled anti-human IgG-Fab detection antibody diluted 1:4000.
  • the test results are shown in Figures 3 to 7.
  • the anti-B7-H3 fully human monoclonal antibodies 13725 and 14479 have high binding activity with human B7-H3-His protein, and the EC50 values of binding can be seen in Tables 6 and 7. It was also found that 13725 and 14479 also bind to mouse B7-H3-His protein, but almost do not bind to human B7-H4-His, mouse B7-H4-huFc, and human PD-L1-ECD-mFc protein. This shows that 13725 and 14479 have good specificity.
  • B7-H3 mAb The ability of B7-H3 mAb to bind to human B7-H3-His protein was tested using the Octet Red96 Biomolecular Interaction Instrument System (Octet Red96, ForteBio) with BM (PC) as the positive control antibody.
  • Anti-human IgG Fc (AHC) kinetic-grade biosensors (Fortebio, #18-5063) were pretreated with glycine at pH 1.7 and then immersed in the assay PBS buffer.
  • Anti-B7-H3 mAb was immobilized to the AHC biosensor at a concentration of 10 ⁇ g/ml.
  • the AHC biosensor loaded with B7-H3 mAb was then immersed in different concentrations of human B7-H3-His antigen and buffer.
  • the last dilution point of the analyte column contained only the assay buffer to test for nonspecific binding between the buffer and the loaded biosensor. Antigen-antibody binding was detected from 0 to 300 seconds, followed by dissociation from 300 to 900 seconds. A baseline of 60 seconds was established with the assay buffer. The affinity curve of the monoclonal antibody against B7-H3 was fitted with a kinetic sensing monovalent binding model of 1:1 binding. The binding kinetic analysis is shown in Table 8.
  • the antibodies cloned 13725 and 14479 obtained in the present invention have good affinity to human B7-H3-His protein, among which the affinity of 13725 is particularly ideal, reaching a surprisingly low KD value of less than 1.0E-12.
  • This example conducts an in vitro binding activity test (Concentration for 50% of maximal Effect, EC50), which can illustrate that the anti-human B7-H3 fully human monoclonal antibodies 13725, 14479 have strong binding to human tumor cell lines that highly express B7-H3.
  • MCF-7 is a human breast cancer cell line
  • OVCAR-3 is a human ovarian cancer cell line. Both cell surfaces highly express B7-H3.
  • MCF-7 and OVCAR-3 were cultured in 96-well plates at a cell plating density of 10 6 /well.
  • 13725 and 14479 were diluted 3 times from 30 ⁇ g/ml with 1xPBS buffer, and the IgG4 negative control (IgG4iso) was diluted to 10 ⁇ g/ml, and incubated with MCF-7 and OVCAR-3 cells at 37°C for 2h. Then, the cells were incubated with goat anti-human IgG-Fc-PE labeled antibody (Abcam, cat# ab98596) diluted 1:200 for 1 hour, and the binding activity of the antibody to the cells was analyzed using Guava easyCyte HT flow cytometer (MERCK MILLIPORE). The results are shown in Figures 8, 9 and Table 9. B7-H3 monoclonal antibodies 13725 and 14479 can bind to tumor cell lines MCF-7 and OVCAR-3 that highly express B7-H3. High affinity binding, good targeting and low non-specificity.
  • an in vitro binding activity test was conducted to test the binding activity of anti-human B7-H3 fully human monoclonal antibodies 13725 and 14479 with human insulin, and compared with the marketed products Hercepin and Rituxan to analyze the potential non-specific binding of B7-H3 fully human monoclonal antibodies in clinical applications.
  • human insulin (Sigma, cat.I2643) was first diluted to a concentration of 10 ⁇ g/ml with 1xPBS coating buffer at pH 7.4, and coated in a 96-well plate (CORNING, cat#9018) at 4°C overnight. After washing three times with 1xPBS buffer at pH 7.4, the plate was blocked with 5% skim milk for 2 hours.
  • the anti-human B7-H3 fully human monoclonal antibodies 13725, 14479, Herceptin and Rituxan to be tested were diluted 3 times from 10 ⁇ g/ml with 1xPBS buffer at pH 7.4, and incubated with the coated insulin at 25°C for 2 hours.
  • an in vitro binding activity test was conducted to test the binding activity of anti-human B7-H3 fully human monoclonal antibodies 13725 and 14479 to salmon DNA, and compared with the marketed products Hercepin and Rituxan to analyze the potential non-specific binding of B7-H3 fully human monoclonal antibodies in clinical applications.
  • the experiment first diluted salmon DNA (Sigma, cat.D1626) with 1xPBS coating buffer at pH 7.4 to a concentration of 10 ⁇ g/ml and coated in a 96-well plate (CORNING, cat#9018) at 4°C overnight. After washing three times with 1xPBS buffer at pH 7.4, the plate was blocked with 5% skim milk for 2 hours.
  • the anti-human B7-H3 fully human monoclonal antibodies 13725, 14479, Herceptin and Rituxan to be tested were diluted 3 times from 10 ⁇ g/ml with 1xPBS buffer at pH 7.4 and coated with DNA at 4°C. Incubate at 25°C for 2 hours.
  • sheep anti-human IgG-HRP detection antibody (1:5000) was used to detect the binding of the antibody to DNA, and the multiple of the OD450 detection signal of each concentration point of the sample relative to the OD450 detection signal of PBS was calculated as the score value, and the 4-parameter fitting dose-effect curve was used.
  • the test results are shown in Figure 11.
  • the binding ability of anti-B7-H3 fully human monoclonal antibodies 13725 and 14479 to salmon DNA is comparable to that of Herceptin and Rituxan to salmon DNA, and the EC50 values can be seen in Table 11.
  • humanized anti-B7-H3 monoclonal antibodies 13725 and 14479 were subjected to DSF detection to investigate thermal stability.
  • Anti-B7-H3 monoclonal antibody and fluorescent group SYPRO Orange 5000 ⁇ (Thermo, cat. S6650) were diluted and mixed in PCR eight-tubes using PBS, with a mixed system of 20 ⁇ l/tube, where the final concentration of the antibody was 0.2 mg/ml and the final concentration of SYPRO Orange was 5 ⁇ . Then the eight-tube PCR was placed in the fluorescent PCR instrument ABI 7500, and the melting curve experiment was performed in continuous mode. The scanning temperature was 25-99°C, equilibrated at 25°C for 5 minutes, the heating rate was 1%, and data was collected during the heating process. The reporting group was ROX, and the quenching group was selected as None.
  • ADCC activity was detected for humanized anti-B7-H3 monoclonal antibodies 13725, 14479 and positive control antibody BM (PC).
  • the target cells were human breast cancer cell line MCF-7, and the effector cells were human PBMC.
  • the experimental process is described as follows: human PBMC (Auscells, cat#PB-003F-S) suspension was diluted to 1 ⁇ 10 7 cells/mL with RPMI 1640 analysis medium and added to a 96-well plate at 50 ⁇ l/well.
  • Anti-B7-H3 monoclonal antibody was diluted to 5 ⁇ g/mL with RPMI 1640 analysis medium as the first concentration point S01.
  • a total of 10 concentration points S01 to S10 were obtained by 3-fold gradient dilution from S01 and added to the wells containing PBMC at 50 ⁇ l/well.
  • MCF-7 cell suspension was diluted to 2 ⁇ 10 5 cells/mL with RPMI 1640 medium and added to the wells containing monoclonal antibodies and PBMC at 50 ⁇ l/well.
  • the 96-well plate was placed in a constant temperature incubator at 37°C and 5% CO 2 for 5 to 6 hours.
  • the color development was performed using an LDH color development kit (Promega, Cytotoxicity Assay (Non-Radioactive), cat. G1780), and the target cell killing rate of each well was calculated.
  • the ADCC activity of the B7-H3 monoclonal antibody was analyzed by performing a 4-parameter fitting nonlinear regression based on the dose-effect relationship between the added monoclonal antibody concentration and the corresponding target cell killing rate using GraphPad Prism.
  • Humanized anti-B7-H3 monoclonal antibodies 13725 and 14479 have strong ADCC activity, among which 13725 is particularly ideal.
  • Example 10 Efficacy of B7-H3 monoclonal antibody in inhibiting tumor cell growth in vivo
  • SCID mice were subcutaneously implanted with approximately 1 ⁇ 10 6 human breast cancer MDA-MB-231 cells on day 0.
  • group administration began, and the administration was continued twice a week for three consecutive weeks.
  • the mean tumor volume was observed and measured for three to four consecutive weeks from the start of administration.
  • B7-H3 humanized monoclonal antibodies 13725, 14479 and BM (PC) daily control can effectively inhibit tumor growth.
  • the average tumor growth inhibition rate is calculated as Ti or Vi represents the average tumor volume of the treatment group or control group at a specific time point; T0 or V0 represents the average tumor volume of the treatment group or control group before drug administration after grouping).
  • the tumor growth inhibition effects of monoclonal antibodies 13725 and 14479 are both relatively ideal; among them, 13725 is the strongest, with an average tumor growth inhibition rate of 41.48%.
  • Example 11 In vivo efficacy of ADC prepared with B7-H3 monoclonal antibody in inhibiting tumor cell growth
  • an antibody-drug conjugate ADC was prepared.
  • the structure of the ADC is similar to that of the B7-H3 ADC drug DS7300a developed by Daiichi Sankyo.
  • the B7-H3 fully human monoclonal antibody 13725 of the present invention is used as the antibody part, coupled by cysteine; DAR is 4; Linker is a tetrapeptide cleavable linker; positive control DS7300a.
  • the in vivo drug-drug interaction of the ADC molecule was performed on the mouse CDX model of human ovarian teratoma cells (PA-1) and human lung adenocarcinoma cells (NCI-H1975) that highly express human B7-H3.
  • PA-1 human ovarian teratoma cells
  • NCI-H1975 human lung adenocarcinoma cells
  • the efficacy was investigated and a positive control DS7300a was set up.
  • the experimental process of investigating the in vivo efficacy of the CDX model is briefly described as follows: using highly immunodeficient mice, approximately 1 to 5 ⁇ 10 6 human PA-1 or NCI-H1975 cells were subcutaneously injected on day 0. When the tumor grew to about 100 to 200 (mm 3 ), the drugs were divided into groups and administered in a single dose. The injection dose for PA-1 was 4.5 mg/kg, and the injection dose for NCI-H1975 was 5 mg/kg. The mean tumor volume was measured and observed for 2-3 consecutive weeks from the start of administration.
  • the ADC molecule based on 13725 had a significantly better inhibitory effect on tumor growth than DS7300a; 11 days after a single administration in the CDX model of NCI-H1975, the ADC molecule based on 13725 had a significantly better inhibitory effect on tumor growth than DS7300a.

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Abstract

The present invention relates to a binding protein of immunoregulation protein molecules B7-H3 and the use thereof. Disclosed is a B7-H3-specific antibody, which can specifically bind to B7-H3 to stimulate response of antigen-specific immune cells (e.g., T cells), and can be applied for the inhibition of diseases associated with B7-H3 expression (including overexpression) or affected by B7-H3 functions, such as tumors or viral infections.

Description

免疫调节蛋白分子B7-H3的结合蛋白及其应用B7-H3 binding protein and its application
本申请要求2023年1月12日提交、发明名称为“免疫调节蛋白分子B7-H3的结合蛋白及其应用”、申请号为CN 202310041822.3的优先权，其全部内容并入本文。This application claims priority to the invention entitled “Binding protein of immunomodulatory protein molecule B7-H3 and its application” filed on January 12, 2023 and application number CN 202310041822.3, all contents of which are incorporated herein.
技术领域Technical Field
本发明属于免疫学领域；具体地，涉及一种免疫调节蛋白分子B7-H3的结合蛋白及其应用。The present invention belongs to the field of immunology; specifically, it relates to a binding protein of immunomodulatory protein molecule B7-H3 and application thereof.
背景技术Background technique
B7-H3(又称CD276)是一种I型跨膜蛋白，属于B7免疫调节分子家族。B7-H3与其他家族成员存在20％-27％相似性，与B7-H1(PD-L1)有相似的分子结构。B7-H3蛋白由染色体15q24基因编码，结构上包括由涵盖胞外域、跨膜域和短胞内域在内的316个氨基酸(2IgB7-H3亚型)组成，分子量为45kDa。它包含一个28AA信号肽，一个由免疫球蛋白恒定(IgC)和可变(IgV)结构组成的217AA细胞外区域，一个跨膜区域和45个氨基酸的胞质结构域。由于外显子重复，人B7-H3蛋白包含一对或两对相同的胞外结构域造成了两种亚型：2IgB7-H3由一对免疫球蛋白可变区(IgV)样和免疫球蛋白恒定区(IgC)样胞外结构域组成；4IgB7-H3包含两对相同的IgV样和IgC样胞外结构域，也是人类细胞中的主要亚型。B7-H3 (also known as CD276) is a type I transmembrane protein that belongs to the B7 immunomodulatory molecule family. B7-H3 has 20%-27% similarity with other family members and has a similar molecular structure to B7-H1 (PD-L1). The B7-H3 protein is encoded by the chromosome 15q24 gene and consists of 316 amino acids (2IgB7-H3 subtype) including an extracellular domain, a transmembrane domain, and a short intracellular domain, with a molecular weight of 45kDa. It contains a 28AA signal peptide, a 217AA extracellular region composed of immunoglobulin constant (IgC) and variable (IgV) structures, a transmembrane region, and a 45-amino acid cytoplasmic domain. Due to exon duplication, human B7-H3 protein contains one or two pairs of identical extracellular domains, resulting in two subtypes: 2IgB7-H3 consists of a pair of immunoglobulin variable region (IgV)-like and immunoglobulin constant region (IgC)-like extracellular domains; 4IgB7-H3 contains two pairs of identical IgV-like and IgC-like extracellular domains, which is also the main subtype in human cells.
B7-H3是免疫调节分子B7家族成员，在头颈部肿瘤，膀胱癌，直肠癌，胰腺癌，卵巢癌等多种实体瘤中过表达。B7-H3在免疫细胞抗肿瘤活性中主要发挥共抑制作用，有利于肿瘤细胞逃避免疫监视。肿瘤表达B7-H3可抑制T细胞介导的抗肿瘤免疫反应，B7-H3高表达与NSCLC患者对PD-1抗体无应答有关。此外，该蛋白高表达还与卵巢癌中CD8T细胞浸润降低以及耗竭有关。因此，B7-H3的过度表达与肿瘤患者的免疫治疗的不良预后有关。B7-H3蛋白能够间接激活NF-kB、PI3K/Akt和JAK/STAT3通路以诱导细胞存活和增殖；抑制转录因子NRF2，导致活性氧(ROS)和HIF1a水平增加，从而诱导有氧糖酵解并导致肿瘤生长。因此，B7-H3还有促进肿瘤增殖代谢和转移的作用。B7-H3 is a member of the B7 family of immunoregulatory molecules and is overexpressed in a variety of solid tumors, including head and neck tumors, bladder cancer, colorectal cancer, pancreatic cancer, and ovarian cancer. B7-H3 plays a major role in the anti-tumor activity of immune cells, which helps tumor cells escape immune surveillance. Tumor expression of B7-H3 can inhibit T cell-mediated anti-tumor immune responses, and high expression of B7-H3 is associated with the lack of response to PD-1 antibodies in NSCLC patients. In addition, high expression of this protein is also associated with reduced CD8T cell infiltration and exhaustion in ovarian cancer. Therefore, overexpression of B7-H3 is associated with poor prognosis of tumor patients with immunotherapy. B7-H3 protein can indirectly activate NF-kB, PI3K/Akt, and JAK/STAT3 pathways to induce cell survival and proliferation; inhibit transcription factor NRF2, leading to increased levels of reactive oxygen species (ROS) and HIF1a, thereby inducing aerobic glycolysis and leading to tumor growth. Therefore, B7-H3 also has the effect of promoting tumor proliferation, metabolism, and metastasis.
B7-H3在多种实体瘤种中广泛高表达，及其对免疫细胞抗肿瘤活性中的共抑制效应，意味着靶向B7-H3的抗体药物，及其衍生的双抗和ADC药物和基于B7-H3抗体抗原结合序列的CAR-T细胞治疗方法，具备应用于广阔的实体瘤治疗的可行性。B7-H3 is widely and highly expressed in many solid tumors, and its co-inhibitory effect on the anti-tumor activity of immune cells means that antibody drugs targeting B7-H3, and their derived bispecific antibodies and ADC drugs, and CAR-T cell therapy methods based on the B7-H3 antibody-antigen binding sequence, are feasible for use in the treatment of a wide range of solid tumors.
作为泛癌细胞靶标，B7-H3在多种难治、易复发实体瘤中高表达，其中包括中枢神经肿瘤，头颈部肿瘤，三阴乳腺癌，非小细胞肺癌和肾脏肿瘤，且B7-H3的高表达与肿瘤免疫治疗效果不佳有相关性。因此，B7-H3抗体既可以靶向治疗中枢神经肿瘤，头颈部肿瘤，三阴乳腺癌，非小细胞肺癌和肾脏肿瘤等难治、易复发实体瘤，也可以联合免疫治疗针对性治疗免疫治疗不敏感的实体瘤病例。As a pan-cancer cell target, B7-H3 is highly expressed in a variety of refractory and recurrent solid tumors, including central nervous system tumors, head and neck tumors, triple-negative breast cancer, non-small cell lung cancer and kidney tumors, and high expression of B7-H3 is correlated with poor tumor immunotherapy. Therefore, B7-H3 antibodies can not only target and treat refractory and recurrent solid tumors such as central nervous system tumors, head and neck tumors, triple-negative breast cancer, non-small cell lung cancer and kidney tumors, but can also be combined with immunotherapy to treat solid tumors that are not sensitive to immunotherapy.
MacroGenics的B7-H3抗体MGA271(Enoblituzumab)联合PD-1单抗(Pembrolizumab)在 治疗头颈鳞状细胞癌和非小细胞肺癌的临床研究中表现优于PD-1单抗Pembrolizumab和Nivolumab的单药。尽管如此，MGA271在亲和力等方面的表现还有需要提高的空间。MacroGenics' B7-H3 antibody MGA271 (Enoblituzumab) combined with PD-1 monoclonal antibody (Pembrolizumab) In clinical studies of treating head and neck squamous cell carcinoma and non-small cell lung cancer, MGA271 performed better than the single-drug PD-1 monoclonal antibody Pembrolizumab and Nivolumab. Despite this, MGA271 still has room for improvement in terms of affinity and other aspects.
发明内容Summary of the invention
本发明的目的在于提供免疫调节蛋白分子B7-H3的结合蛋白及其应用。The purpose of the present invention is to provide a binding protein of the immunomodulatory protein molecule B7-H3 and its application.
在本发明的第一方面，提供结合免疫调节分子B7-H3的抗体(包括其抗原结合片段)，其具有显著改善的抑制免疫调节分子B7-H3与其受体分子(细胞表面受体分子)的结合的作用。In a first aspect of the present invention, an antibody (including an antigen-binding fragment thereof) binding to the immunomodulatory molecule B7-H3 is provided, which has a significantly improved effect of inhibiting the binding of the immunomodulatory molecule B7-H3 to its receptor molecule (cell surface receptor molecule).
在一种或多种实施方式中，该抗体具有轻链可变区和重链可变区，其中，重链CDR1的氨基酸序列如SEQ ID NO：13所示，CDR2的氨基酸序列如SEQ ID NO：14所示，CDR3的氨基酸序列如SEQ ID NO：15所示；轻链CDR1的氨基酸序列如SEQ ID NO：16所示，CDR2的氨基酸序列如SEQ ID NO：17所示，CDR3的氨基酸序列如SEQ ID NO：18所示。In one or more embodiments, the antibody has a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 13, the amino acid sequence of CDR2 is shown in SEQ ID NO: 14, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 15; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 16, the amino acid sequence of CDR2 is shown in SEQ ID NO: 17, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 18.
在一种或多种实施方式中，重链CDR1的氨基酸序列如SEQ ID NO：19所示，CDR2的氨基酸序列如SEQ ID NO：20所示，CDR3的氨基酸序列如SEQ ID NO：21所示；轻链CDR1的氨基酸序列如SEQ ID NO：22所示，CDR2的氨基酸序列如SEQ ID NO：23所示，CDR3的氨基酸序列如SEQ ID NO：24所示。In one or more embodiments, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO: 19, the amino acid sequence of CDR2 is shown as SEQ ID NO: 20, and the amino acid sequence of CDR3 is shown as SEQ ID NO: 21; the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO: 22, the amino acid sequence of CDR2 is shown as SEQ ID NO: 23, and the amino acid sequence of CDR3 is shown as SEQ ID NO: 24.
在一种或多种实施方式中，所述抗体的重链可变区具有SEQ ID NO：1所示氨基酸序列，轻链可变区具有SEQ ID NO：3所示氨基酸序列。In one or more embodiments, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 3.
在一种或多种实施方式中，所述抗体的重链可变区具有SEQ ID NO：5所示氨基酸序列，轻链可变区具有SEQ ID NO：6所示氨基酸序列。In one or more embodiments, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 5, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 6.
在一种或多种实施方式中，所述抗体包含重链可变区和轻链可变区：其重链可变区具有SEQ ID NO：7所示的氨基酸序列，轻链可变区具有SEQ ID NO：8所示的氨基酸序列；或，其重链可变区具有SEQ ID NO：9所示的氨基酸序列，轻链可变区具有SEQ ID NO：10所示的氨基酸序列；或，其重链可变区具有SEQ ID NO：11所示的氨基酸序列，轻链可变区具有SEQ ID NO：12所示的氨基酸序列。In one or more embodiments, the antibody comprises a heavy chain variable region and a light chain variable region: its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 8; or, its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 10; or, its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 11, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 12.
在一种或多种实施方式中，所述抗体还包括重链可变区氨基酸序列与SEQ ID NO：1、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：9或SEQ ID NO：11所示序列具有80％以上(如85％、90％、93％、95％、97％或99％以上)相同性、轻链可变区氨基酸序列与SEQ ID NO：3、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：10或SEQ ID NO：12所示具有80％以上(如85％、90％、93％、95％、97％或99％以上)相同性且保留结合活性的抗体。In one or more embodiments, the antibody also includes an antibody whose heavy chain variable region amino acid sequence is more than 80% (such as 85%, 90%, 93%, 95%, 97% or 99% or more) identical to the sequence shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11, and a light chain variable region amino acid sequence is more than 80% (such as 85%, 90%, 93%, 95%, 97% or 99% or more) identical to the sequence shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12 and retains binding activity.
在一种或多种实施方式中，所述抗体包括：单克隆抗体、人类抗体、人源化抗体或嵌合抗体。In one or more embodiments, the antibody comprises: a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody.
在一种或多种实施方式中，所述抗体包括：Fab、Fab’-SH、Fv、Fd、scFv或(Fab’)2片段。In one or more embodiments, the antibody comprises: Fab, Fab'-SH, Fv, Fd, scFv or (Fab')2 fragment.
在一种或多种实施方式中，所述抗体为IgG抗体。In one or more embodiments, the antibody is an IgG antibody.
在本发明的另一方面，提供一种分离的多核苷酸或含有该多核苷酸的构建体，所述多核苷酸编码所述的结合免疫调节分子B7-H3的抗体；较佳地，所述的构建体为表达载体。 In another aspect of the present invention, an isolated polynucleotide or a construct containing the polynucleotide is provided, wherein the polynucleotide encodes the antibody that binds to the immunomodulatory molecule B7-H3; preferably, the construct is an expression vector.
在本发明的另一方面，提供一种抗体表达系统，所述表达系统含有所述的构建体或基因组中整合有外源的所述的多核苷酸；较佳地所述表达系统为细胞(表达系统)。In another aspect of the present invention, an antibody expression system is provided, wherein the expression system contains the construct or the exogenous polynucleotide integrated into the genome; preferably, the expression system is a cell (expression system).
在一种或多种实施方式中，所述细胞(宿主细胞)为哺乳动物细胞。In one or more embodiments, the cell (host cell) is a mammalian cell.
在一种或多种实施方式中，所述哺乳动物细胞包括(但不限于)：中国仓鼠卵巢(CHO)细胞，Vero细胞，HEK-293细胞，NS0细胞，SP2/0细胞，BHK细胞，PER-C6细胞；较佳的，为CHO细胞。In one or more embodiments, the mammalian cells include (but are not limited to): Chinese hamster ovary (CHO) cells, Vero cells, HEK-293 cells, NS0 cells, SP2/0 cells, BHK cells, PER-C6 cells; preferably, CHO cells.
在本发明的另一方面，提供一种制备所述的抗体的方法，包括：在适合表达所述抗体的条件下，利用所述的抗体表达系统进行表达，从而表达出所述的抗体；较佳地，还包括纯化分离出所述抗体。In another aspect of the present invention, a method for preparing the antibody is provided, comprising: expressing the antibody using the antibody expression system under conditions suitable for expressing the antibody, thereby expressing the antibody; preferably, it also comprises purifying and isolating the antibody.
在本发明的另一方面，提供所述的抗体的用途，用于：制备特异性靶向表达免疫调节分子B7-H3的细胞的抗肿瘤药物，所述药物通过刺激抗原特异性免疫细胞(如T细胞)应答抑制肿瘤；制备抗体药物偶联物或免疫缀合物；制备双功能(双特异性)或多功能(多特异性)抗体；制备嵌合抗原受体修饰的免疫细胞。In another aspect of the present invention, the use of the antibody is provided for: preparing an anti-tumor drug that specifically targets cells expressing the immunomodulatory molecule B7-H3, wherein the drug inhibits tumors by stimulating antigen-specific immune cells (such as T cells) to respond; preparing antibody-drug conjugates or immunoconjugates; preparing bifunctional (bispecific) or multifunctional (multispecific) antibodies; preparing immune cells modified with chimeric antigen receptors.
在本发明的另一方面，提供一种融合蛋白或免疫缀合物，其包括所述的抗体，以及与之操作性连接的功能性分子。In another aspect of the present invention, a fusion protein or immunoconjugate is provided, which comprises the antibody and a functional molecule operably linked thereto.
在一种或多种实施方式中，所述功能性分子包括：抑制肿瘤的分子，靶向肿瘤表面标志物的分子，靶向免疫细胞的表面标志物的分子或可检测标记物，细胞毒素，放射性同位素，生物活性蛋白质，融合伴侣，基于嵌合抗原受体技术的胞外铰链区、跨膜区及胞内信号区，或其组合。In one or more embodiments, the functional molecules include: tumor-suppressing molecules, molecules targeting tumor surface markers, molecules or detectable markers targeting surface markers of immune cells, cytotoxins, radioactive isotopes, biologically active proteins, fusion partners, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or a combination thereof.
在一种或多种实施方式中，所述靶向免疫细胞的表面标志物的分子是结合免疫细胞表面标志物的抗体或配体。In one or more embodiments, the molecule targeting a surface marker of an immune cell is an antibody or a ligand that binds to a surface marker of an immune cell.
在一种或多种实施方式中，所述的抑制肿瘤的分子是抗肿瘤毒素或抗肿瘤细胞因子。In one or more embodiments, the tumor-suppressing molecule is an anti-tumor toxin or an anti-tumor cytokine.
在一种或多种实施方式中，所述靶向肿瘤表面标志物的分子是结合肿瘤表面标志物的抗体或配体。In one or more embodiments, the molecule targeting a tumor surface marker is an antibody or a ligand that binds to a tumor surface marker.
在一种或多种实施方式中，所述的融合伴侣包括(但不限于)：具有延长体内半衰期作用的蛋白或活性结构域。In one or more embodiments, the fusion partner includes (but is not limited to): a protein or active domain that has the effect of extending the half-life in vivo.
在一种或多种实施方式中，所述结合免疫细胞表面标志物的抗体结合的抗原包括(但不限于)：CD3、CD20、FcRH5、HER2、LYPD1、LY6G6D、PMEL17、LY6E、CD19、CD33、CD22、CD79A、CD79B、EDAR、GFRA1、MRP4、RET、Steap1或TenB2。In one or more embodiments, the antigens bound by the antibody that binds to the immune cell surface marker include (but are not limited to): CD3, CD20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1 or TenB2.
在一种或多种实施方式中，所述结合肿瘤表面标志物的抗体是识别免疫调节分子B7-H3以外的其它抗原的抗体，所述的其它抗原包括(但不限于)：EGFR，EGFRvIII，mesothelin，HER2，EphA2，cMet，EpCAM，MUC1，MUC16，CEA，Claudin 18.2，Claudin 6，WT1，NY-ESO-1，MAGE 3，CD47，ASGPR1或CDH16。In one or more embodiments, the antibody that binds to the tumor surface marker is an antibody that recognizes antigens other than the immune regulatory molecule B7-H3, and the other antigens include (but are not limited to): EGFR, EGFRvIII, mesothelin, HER2, EphA2, cMet, EpCAM, MUC1, MUC16, CEA, Claudin 18.2, Claudin 6, WT1, NY-ESO-1, MAGE 3, CD47, ASGPR1 or CDH16.
在一种或多种实施方式中，所述的抗肿瘤细胞因子包括(但不限于)：IL-2、IL-12、IL-15、IFN-beta、TNF-alpha。 In one or more embodiments, the anti-tumor cytokines include (but are not limited to): IL-2, IL-12, IL-15, IFN-beta, TNF-alpha.
在一种或多种实施方式中，所述抗肿瘤毒素包括(但不限于)：美登素，奥利司他汀，单甲基澳瑞他汀，美登木素生物碱，多拉司他汀，卡奇霉素，氨甲蝶呤，长春地辛，紫杉烷如多烯紫杉醇、紫杉醇、拉洛紫杉醇、特西紫杉醇或欧塔紫杉醇，单端孢霉毒素，CC1065，DXd，Duocarmycin，Calicheamicin，Pyrrolobenzodiazepines或SN-38。In one or more embodiments, the anti-tumor toxin includes (but is not limited to): maytansine, auristatin, monomethyl auristatin, maytansinoid alkaloid, dolastatin, calicheamicin, methotrexate, vindesine, taxanes such as docetaxel, paclitaxel, lalopaclitaxel, tecitaxel or octataxel, trichothecenes, CC1065, DXd, Duocarmycin, Calicheamicin, Pyrrolobenzodiazepines or SN-38.
在一种或多种实施方式中，所述具有延长体内半衰期作用的蛋白或活性结构域包括(但不限于)：免疫球蛋白Fc区、优选人免疫球蛋白Fc区，血清白蛋白(如人源的HSA)或其片段。In one or more embodiments, the protein or active domain having the effect of extending the in vivo half-life includes (but is not limited to): immunoglobulin Fc region, preferably human immunoglobulin Fc region, serum albumin (such as human HSA) or a fragment thereof.
在一种或多种实施方式中，所述免疫球蛋白为选自IgG、IgA1、IgA2、IgD、IgE、IgM中的一种或多种的组合，所述IgG选自IgG1、IgG2、IgG3或IgG4亚型中的一种或多种的组合。In one or more embodiments, the immunoglobulin is a combination of one or more selected from IgG, IgA1, IgA2, IgD, IgE, and IgM, and the IgG is a combination of one or more selected from IgG1, IgG2, IgG3, or IgG4 subtypes.
在一种或多种实施方式中，所述抗体以及与之操作性连接的功能性分子之间设有连接肽；所述连接肽优选选自由丙氨酸和/或丝氨酸和/或甘氨酸组成的柔性多肽链，连接肽的长度优选为3～30个氨基酸。In one or more embodiments, a connecting peptide is provided between the antibody and the functional molecule operably connected thereto; the connecting peptide is preferably selected from a flexible polypeptide chain composed of alanine and/or serine and/or glycine, and the length of the connecting peptide is preferably 3 to 30 amino acids.
在本发明的另一方面，提供一种药物组合物，包括所述的抗体、所述的融合蛋白或免疫缀合物；较佳地，所述的药物组合物中还包括药学上可接受的载体。In another aspect of the present invention, a pharmaceutical composition is provided, comprising the antibody, the fusion protein or the immunoconjugate; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
在本发明的另一方面，提供前面任一所述的抗体、所述的融合蛋白或免疫缀合物、或含有它们的药物组合物在制备用于抑制肿瘤或刺激抗原特异性免疫细胞(如T细胞)应答的制剂、试剂盒或药盒中的用途。In another aspect of the present invention, there is provided the use of any of the above-mentioned antibodies, fusion proteins or immunoconjugates, or pharmaceutical compositions containing them in the preparation of preparations, kits or medicine boxes for inhibiting tumors or stimulating antigen-specific immune cell (such as T cell) responses.
在一种或多种实施方式中，所述肿瘤包括实体瘤；较佳地，所述肿瘤包括表达免疫调节分子B7-H3的肿瘤；较佳地，所述肿瘤包括(但不限于)：卵巢癌，乳腺癌(三阴乳腺癌)，中枢神经肿瘤，头颈部肿瘤，膀胱癌，直肠癌，胰腺癌，非小细胞肺癌，肾脏肿瘤。In one or more embodiments, the tumor includes a solid tumor; preferably, the tumor includes a tumor expressing the immunomodulatory molecule B7-H3; preferably, the tumor includes (but is not limited to): ovarian cancer, breast cancer (triple-negative breast cancer), central nervous system tumors, head and neck tumors, bladder cancer, rectal cancer, pancreatic cancer, non-small cell lung cancer, and kidney tumors.
在本发明的另一方面，提供一种试剂盒或药盒，包括所述的抗体、所述的融合蛋白或免疫缀合物、或含有它们的药物组合物。In another aspect of the present invention, a kit or a medicine box is provided, comprising the antibody, the fusion protein or the immunoconjugate, or a pharmaceutical composition containing the same.
在一种或多种实施方式中，所述试剂盒或药盒中还含有：免疫刺激性抗体、化疗剂和抗病毒药物。In one or more embodiments, the test kit or pharmaceutical kit further comprises: an immunostimulatory antibody, a chemotherapeutic agent, and an antiviral drug.
在一种或多种实施方式中，所述免疫刺激性抗体选自如下的一种或多种：抗PD-1抗体、抗PD-L1抗体、抗PD-L2抗体和抗CTLA-4抗体。In one or more embodiments, the immunostimulatory antibody is selected from one or more of the following: an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, and an anti-CTLA-4 antibody.
在一种或多种实施方式中，所述试剂盒或药盒中还含有用于装入所述抗体、融合蛋白、免疫缀合物或药物组合物的容器，所述容器为一个或多个。In one or more embodiments, the test kit or medicine box further contains one or more containers for containing the antibody, fusion protein, immunoconjugate or pharmaceutical composition.
在一种或多种实施方式中，所述试剂盒或药盒中还含有使用说明书(包装插页)，说明所述抗体、融合蛋白、免疫缀合物或药物组合物的使用/服用方法。In one or more embodiments, the kit or pharmaceutical kit further contains instructions for use (package insert) describing the method for use/administration of the antibody, fusion protein, immunoconjugate or pharmaceutical composition.
在本发明的另一方面，提供一种在受试者中刺激免疫反应的方法，该方法包括给予该受试者上面任一所述的抗体，从而刺激该受试者的免疫反应。In another aspect of the present invention, a method for stimulating an immune response in a subject is provided, the method comprising administering to the subject any of the antibodies described above, thereby stimulating the immune response of the subject.
在一种或多种实施方式中，所述受试者为携带肿瘤的受试者，并且给予该受试者所述的抗体刺激了针对该肿瘤的免疫反应。In one or more embodiments, the subject is a subject bearing a tumor, and administering the antibody to the subject stimulates an immune response against the tumor.
在一种或多种实施方式中，所述受试者为携带病毒的受试者，并且给予该受试者所述的 抗体刺激了针对该病毒的免疫反应。In one or more embodiments, the subject is a subject carrying a virus, and the subject is given the Antibodies stimulate an immune response against the virus.
在本发明的另一方面，提供一种抑制受试者中肿瘤细胞生长的方法，该方法包括给予该受试者前面所述的抗体。In another aspect of the present invention, a method of inhibiting tumor cell growth in a subject is provided, the method comprising administering the above-described antibody to the subject.
在本发明的另一方面，提供一种治疗受试者中病毒感染的方法，该方法包括给予该受试者前面所述的抗体。In another aspect of the present invention, a method of treating a viral infection in a subject is provided, the method comprising administering to the subject the antibody described above.
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1、5个IgG4单克隆抗体与人B7-H3标签融合蛋白结合的ELISA检测结果。Figure 1. ELISA test results of 5 IgG4 monoclonal antibodies binding to human B7-H3 tag fusion protein.
图2、瞬时转染的ExpiCHO-S细胞制备的B7-H3单克隆抗体的SEC-HPLC检测纯度。图中从上至下依次是：BM(PC)、13725、14479。Figure 2. SEC-HPLC purity test of B7-H3 monoclonal antibody produced by transiently transfected ExpiCHO-S cells. From top to bottom in the figure are: BM (PC), 13725, 14479.
图3、B7-H3单克隆抗体同人B7-H3-his结合的ELISA检测结果。Figure 3. ELISA test results of B7-H3 monoclonal antibody binding to human B7-H3-his.
图4、B7-H3单克隆抗体同人B7-H4-his结合的ELISA检测结果。Figure 4. ELISA test results of B7-H3 monoclonal antibody binding to human B7-H4-his.
图5、B7-H3单克隆抗体同人PD-L1-ECD-mFc结合的ELISA检测结果。Figure 5. ELISA test results of B7-H3 monoclonal antibody binding to human PD-L1-ECD-mFc.
图6、B7-H3单克隆抗体同小鼠B7-H3-his结合的ELISA检测结果。Figure 6. ELISA test results of the binding of B7-H3 monoclonal antibody to mouse B7-H3-his.
图7、B7-H3单克隆抗体同小鼠B7-H4-huFc结合的ELISA检测结果。FIG. 7 ELISA test results of the binding of B7-H3 monoclonal antibody to mouse B7-H4-huFc.
图8、B7-H3单克隆抗体同乳腺癌细胞系MCF-7的细胞结合活性检测结果。FIG8 . The results of the cell binding activity test of B7-H3 monoclonal antibody and breast cancer cell line MCF-7.
图9、B7-H3单克隆抗体同卵巢癌细胞系OVCAR-3的细胞结合活性检测结果。FIG. 9 . The results of the cell binding activity test of B7-H3 monoclonal antibody to the ovarian cancer cell line OVCAR-3.
图10、B7-H3单克隆抗体同人Insulin结合活性检测结果。Figure 10. Detection results of the binding activity of B7-H3 monoclonal antibody to human insulin.
图11、B7-H3单克隆抗体同DNA结合活性检测结果。Figure 11. Results of DNA binding activity test of B7-H3 monoclonal antibody.
图12、B7-H3人源化单克隆抗体ADCC检测结果。FIG. 12 . ADCC detection results of B7-H3 humanized monoclonal antibody.
图13、小鼠荷瘤模型体内抗肿瘤药效。图中显示为肿瘤的平均体积±SEM。Figure 13. In vivo antitumor efficacy in mouse tumor-bearing models. The figure shows the mean tumor volume ± SEM.
图14A-B、B7-H3单克隆抗体制备ADC的体内抑制肿瘤细胞生长的功效。FIG. 14A-B , the efficacy of ADC prepared with B7-H3 monoclonal antibody in inhibiting tumor cell growth in vivo.
具体实施方式Detailed ways
本发明人经过深入的研究，揭示了一种B7-H3特异性的抗体，其能与B7-H3特异性结合，刺激抗原特异性免疫细胞(如T细胞)应答，可以应用于抑制与B7-H3表达(包括过表达)相关或者是受到B7-H3功能影响的疾病，例如肿瘤或病毒感染。After in-depth research, the inventors have revealed a B7-H3-specific antibody that can specifically bind to B7-H3 and stimulate antigen-specific immune cells (such as T cells) to respond, and can be used to inhibit diseases related to B7-H3 expression (including overexpression) or affected by B7-H3 function, such as tumors or viral infections.
1)抗体1) Antibodies
本发明涉及抗B7-H3抗体。在优选的实施方案中，本发明提供一种抗B7-H3抗体，其包含含有至少1、2、3、4、5或6个选自以下的高变区(HVR)或称为互补决定区(CDR)的结合域：(a)HVR-H1，其包含SEQ ID NO：13或19的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(b)HVR-H2，其包含SEQ ID NO：14或20的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列； (c)HVR-H3，其包含SEQ ID NO：15或21的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(d)HVR-L1，其包含SEQ ID NO：16或22的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(e)HVR-L2，其包含SEQ ID NO：17或23的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；及(f)HVR-L3，其包含SEQ ID NO：18或24的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列。在一些情况下，抗B7-H3抗体所具有的重链可变(VH)域(区)可包括与SEQ ID NO：1或5具有至少90％序列同源性(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同源性)的氨基酸序列或SEQ ID NO：1或5的氨基酸序列，和/或其轻链可变(VL)域(区)包含与SEQ ID NO：3或6具有至少90％序列同源性(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同源性)的氨基酸序列或SEQ ID NO：3或6的氨基酸序列。The present invention relates to anti-B7-H3 antibodies. In a preferred embodiment, the present invention provides an anti-B7-H3 antibody comprising a binding domain comprising at least 1, 2, 3, 4, 5 or 6 hypervariable regions (HVRs) or complementary determining regions (CDRs) selected from the following: (a) HVR-H1 comprising an amino acid sequence of SEQ ID NO: 13 or 19 or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% homologous to the sequence; (b) HVR-H2 comprising an amino acid sequence of SEQ ID NO: 14 or 20 or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% homologous to the sequence; (c) HVR-H3 comprising an amino acid sequence of SEQ ID NO: 15 or 21, or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% homologous thereto; (d) HVR-L1 comprising an amino acid sequence of SEQ ID NO: 16 or 22, or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% homologous thereto; (e) HVR-L2 comprising an amino acid sequence of SEQ ID NO: 17 or 23, or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% homologous thereto; and (f) HVR-L3 comprising an amino acid sequence of SEQ ID NO: 18 or 24, or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% homologous thereto. In some cases, the anti-B7-H3 antibody may have a heavy chain variable (VH) domain (region) that includes an amino acid sequence having at least 90% sequence homology (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology) to SEQ ID NO: 1 or 5, or the amino acid sequence of SEQ ID NO: 1 or 5, and/or a light chain variable (VL) domain (region) that includes an amino acid sequence having at least 90% sequence homology (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology) to SEQ ID NO: 3 or 6, or the amino acid sequence of SEQ ID NO: 3 or 6.
在一些实施方案中，抗B7-H3抗体包含重链可变区和轻链可变区，其中重链可变区包含选自下组的氨基酸序列：SEQ ID NO：1、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11。轻链可变区包含选自下组的氨基酸序列：SEQ ID NO：3、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12。In some embodiments, the anti-B7-H3 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11. The light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12.
在一些实施方案中，抗B7-H3抗体包含如下的重链可变区和轻链可变区的组合：In some embodiments, the anti-B7-H3 antibody comprises the following combination of heavy chain variable region and light chain variable region:
(1)氨基酸序列SEQ ID NO：1和SEQ ID NO：3；(No.13725)；(1) Amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 3; (No. 13725);
(2)氨基酸序列SEQ ID NO：5和SEQ ID NO：6；(No.14479)；(2) Amino acid sequences SEQ ID NO: 5 and SEQ ID NO: 6; (No. 14479);
(3)氨基酸序列SEQ ID NO：7和SEQ ID NO：8；(No.13683)；(3) Amino acid sequences SEQ ID NO: 7 and SEQ ID NO: 8; (No. 13683);
(4)氨基酸序列SEQ ID NO：9和SEQ ID NO：10；(No.14463)；(4) Amino acid sequences SEQ ID NO: 9 and SEQ ID NO: 10; (No. 14463);
(5)氨基酸序列SEQ ID NO：11和SEQ ID NO：12；(No.14471)。(5) Amino acid sequences SEQ ID NO: 11 and SEQ ID NO: 12; (No.14471).
在优选的实施方案中，抗B7-H3抗体包含重链可变区和轻链可变区，其中重链可变区包含氨基酸序列SEQ ID NO：1或SEQ ID NO：5，轻链可变区包含氨基酸序列SEQ ID NO：3或SEQ ID NO：6。In a preferred embodiment, the anti-B7-H3 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 5, and the light chain variable region comprises the amino acid sequence SEQ ID NO: 3 or SEQ ID NO: 6.
2)抗体片段2) Antibody fragments
在某些实施方案中，本文中所提供的抗体为抗体片段。抗体片段包括但不限于Fab、Fab’、Fab’-SH、(Fab’)2、Fv及scFv片段及下文所描述的其他片段。关于某些抗体片段的综述，参见Hudson等人，Nat.Med.9：129-134(2003)。关于scFv片段的综述，参见例如Plückthun，The Pharmacology of Monoclonal Antibodies，第113卷，Rosenburg和Moore编(Springer-Verlag，Berlin)，第269-315页(1994)；也可参见WO 93/16185；及美国专利第5，571，894号及第5,587,458号。In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, (Fab')2, Fv and scFv fragments and other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, for example, Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds. (Springer-Verlag, Berlin), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458.
双功能抗体为具有两个抗原结合位点的抗体片段，其可为二价或双特异性的。参见例如EP0404097A2；WO 1993/011161；Hudson等人，Nat.Med.9：129-134(2003)；及Hollinger等人，Proc.Natl.Acad.Sci.USA 90：6444-6448(1993)。三功能抗体及四功能抗体也描述于Hudson等人，Nat.Med.9：129-134(2003)中。 Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. See, e.g., EP0404097A2; WO 1993/011161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).
单域抗体为包含抗体的所有或部分重链可变域或所有或部分轻链可变域的抗体片段。在某些实施方案中，单域抗体为人类单域抗体(Domantis，Inc.，Waltham，MA；参见例如美国专利第6,248,516 B1号)。A single-domain antibody is an antibody fragment that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).
抗体片段可通过各种技术产生，包括但不限于完整抗体的蛋白水解消化以及由如本文中所描述的重组宿主细胞(例如大肠杆菌或噬菌体)产生。Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage) as described herein.
3)嵌合抗体及人源化抗体3) Chimeric antibodies and humanized antibodies
在某些实施方案中，本文中所提供的抗体为嵌合抗体。某些嵌合抗体描述于例如美国专利第4,816,567号；及Morrison等人，Proc.Natl.Acad.Sci.USA，81：6851-6855(1984))中。In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984).
在某些实施方案中，嵌合抗体为人源化抗体。典型地，非人类抗体经人源化以降低对人类的免疫原性，同时保留亲本非人类抗体的特异性及亲和力。一般而言，人源化抗体包含一个或多个可变域，其中HVR，例如CDR，(或其部分)源自于非人类抗体，且FR(或其部分)源自于人类抗体序列。人源化抗体任选地也将包含人类恒定区的至少一部分。在一些实施方案中，人源化抗体中的一些FR残基经来自于非人类抗体(例如获得HVR残基的抗体)的相应残基取代，例如以修复或改善抗体特异性或亲和力。In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibodies. Generally speaking, humanized antibodies include one or more variable domains, wherein HVR, such as CDR, (or a portion thereof) is derived from non-human antibodies, and FR (or a portion thereof) is derived from human antibody sequences. Humanized antibodies will optionally also include at least a portion of human constant regions. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (e.g., antibodies that obtain HVR residues), for example, to repair or improve antibody specificity or affinity.
人源化抗体及其制造方法可查阅例如Almagro及Fransson，Front.Biosci.13：1619-1633(2008)，且进一步描述于例如以下文献中：Riechmann等人，Nature 332：323-327(1988)；美国专利第5，821，337号、第7,527,791号、第6,982,321号及第7,087,409号；Kashmiri等人，Methods 36：25-34(2005)(描述特异性决定区(SDR)移植)；Padlan，Mol.Immunol.28：489-498(1991)(描述“表面重整”)；Dall’Acqua等人，Methods36：43-60(2005)(描述“FR改组”)；及Osbourn等人，Methods 36：61-68(2005)及Klimka等人，Br.J.Cancer，83：252-260(2000)(描述针对FR改组的“指导选择”法)。Humanized antibodies and methods for making them can be found, for example, in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described in, for example, Riechmann et al., Nature 332: 323-327 (1988); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36: 25-34 (200 5) (describing specificity determining region (SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface remodeling"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing "guided selection" methods for FR shuffling).
4)人类抗体4) Human Antibodies
在某些实施方案中，本文中所提供的抗体为人类抗体。人类抗体可使用本领域中已知的各种技术产生。In certain embodiments, the antibodies provided herein are human antibodies.Human antibodies can be produced using various techniques known in the art.
人类抗体可通过将免疫原施用给经修饰的转基因动物，然后用抗原攻击制备完整人类抗体或具有人类可变区的完整抗体。此类动物典型地含有所有或部分人类免疫球蛋白基因座，其置换内源免疫球蛋白基因座或存在于染色体外或随机整合于动物染色体中。在此类转基因小鼠中，内源免疫球蛋白基因座一般已灭活。关于由转基因动物获得人类抗体的方法，参见例如美国专利第6,075，181号及第6,150,584号(描述XENOMOUSETM技术)；美国专利第5,770,429号；美国专利第7,041,870号(描述K-M技术)；及美国申请公布第US 2007/0061900号。得自于由此类动物产生的完整抗体的人类可变区可经进一步修饰，例如通过与不同的人类恒定区组合。Human antibodies can be prepared by administering an immunogen to a modified transgenic animal and then attacking it with an antigen to prepare a complete human antibody or a complete antibody with a human variable region. Such animals typically contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci or are present outside the chromosomes or randomly integrated into the animal chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For methods of obtaining human antibodies from transgenic animals, see, for example, U.S. Patents 6,075,181 and 6,150,584 (describing XENOMOUSE TM technology); U.S. Patent 5,770,429; U.S. Patent 7,041,870 (describing KM technology); and U.S. Application Publication No. US 2007/0061900. The human variable regions derived from the complete antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.
人类抗体也可通过基于杂交瘤的方法制造。已描述用于产生人类单克隆抗体的人类骨髓瘤及小鼠-人类杂交骨髓瘤细胞株，参见例如Kozbor等人，J.Immunol.，133：3001-3005(1984)；Brodeur等人，Monoclonal Antibody Production Techniques and Applications，第51-63页； Marcel Dekker，Inc.，New York，1987)；及Boerner等人，J.Immunol.，147：86-95(1991)。经由人类B细胞杂交瘤技术产生的人类抗体也描述于Li等人，Proc.Natl.Acad.Sci.USA，103：3557-3562(2006)中。其他方法包括例如美国专利第7，189，826号(描述由杂交瘤细胞株产生单克隆人类IgM抗体)及倪，现代免疫学，26(4)：265-268(2006)(描述人类-人类杂交瘤技术)中所描述的方法。人类杂交瘤技术(Trioma技术)也描述于Vollmers及Histol.Histopathol.，20(3)：927-937(2005)以及Vollmers和Methods and Findings in Experimental and Clinical Pharmacology，27(3)：185-191(2005)中。Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for producing human monoclonal antibodies have been described, see, e.g., Kozbor et al., J. Immunol., 133:3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63; Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86-95 (1991). Human antibodies produced by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include, for example, those described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies by hybridoma cell lines) and Ni, Modern Immunology, 26(4):265-268 (2006) (describing human-human hybridoma technology). Human hybridoma technology (Trioma technology) is also described in Vollmers and Histol. Histopathol., 20(3):927-937 (2005) and Vollmers and Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-191 (2005).
人类抗体也可通过分离选自人类来源噬菌体展示库的Fv克隆可变域序列来制备。接着可将此类可变域序列与所要的人类恒定域组合。本发明实施例部分描述了通过噬菌体展示库筛选人类抗体的过程。Human antibodies can also be prepared by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. The present invention describes in part the process of screening human antibodies through phage display libraries.
具体地，可通过针对具有结合B7-H3活性的抗体筛选组合库来分离具有高亲和力的本发明抗体。例如，本领域中已知用于产生噬菌体展示库及针对具有所要结合特征的抗体筛选此类库的多种方法。此类方法可查阅例如Hoogenboom等人，Methods in Molecular Biology 178：1-37(O’Brien等人编，Human Press，Totowa，NJ，2001)，且进一步描述于例如以下文献中：McCafferty等人，Nature 348：552-554(1990)；Clackson等人，Nature 352：624-628(1991)；Marks等人，J.Mol.Biol.222：581-597(1991)；Marks及Bradbury，Methods in Molecular Biology 248：161-175(Lo编，Human Press，Totowa，NJ，2003)；Sidhu等人，J.Mol.Biol.338(2)：299-310(2004)；Lee等人，J.Mol.Biol.340(5)：1073-1093(2004)；Fellouse，Proc.Natl.Acad.Sci.USA 101(34)：12467-12472(2004)；及Lee等人，J.Immunol.Methods 284(1-2)：119-132(2004)。Specifically, antibodies of the present invention with high affinity can be isolated by screening combinatorial libraries for antibodies with binding activity to B7-H3. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics. Such methods can be found, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are further described in, for example, the following literature: McCafferty et al., Nature 348: 552-554 (1990); Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1991); Marks and Bradbury, Methods i n Molecular Biology 248:161-175 (Lo ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).
在某些噬菌体展示方法中，通过聚合酶链反应(PCR)单独克隆VH及VL基因谱系，且随机重组于噬菌体库中，接着可如Winter等人，Ann.Rev.Immunol.12：433-455(1994)中所描述针对抗原-结合噬菌体进行筛选。噬菌体典型地将抗体片段呈现为单链Fv(scFv)片段或Fab片段。来自于免疫源的库提供针对免疫原的高亲和力抗体，而无需构建杂交瘤。描述人类抗体噬菌体库的专利公布包括例如：美国专利第5,750,373号及美国专利公布第2005/0079574号、第2005/0119455号、第2005/0266000号、第2007/0117126号、第2007/0160598号、第2007/0237764号、第2007/0292936号。In certain phage display methods, VH and VL gene repertoires are individually cloned by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol. 12: 433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373 and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936.
自人类抗体库分离的抗体或抗体片段在本文中被视为人类抗体或人类抗体片段。Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.
5)多特异性抗体5) Multispecific Antibodies
在任何上述方面中，本文中所提供的抗B7-H3抗体为多特异性抗体，例如双特异性抗体。多特异性抗体为对至少两个不同的位点具有结合特异性的单克隆抗体。在某些实施方案中，一个结合特异性针对B7-H3，而另一结合特异性针对任何其他抗原(例如第二生物分子，例如免疫细胞表面抗原，例如肿瘤抗原)。相应地，双特异性抗B7-H3抗体可对B7-H3及其他抗原，例如CD3、CD20、FcRH5、HER2、LYPD1、LY6G6D、PMEL17、LY6E、CD19、CD33、CD22、CD79A、CD79B、EDAR、GFRA1、MRP4、RET、Steap1或TenB2有结合特 异性。双特异性抗体也可制备为全长抗体或抗体片段。In any of the above aspects, the anti-B7-H3 antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity for at least two different sites. In certain embodiments, one binding specificity is for B7-H3, and the other binding specificity is for any other antigen (e.g., a second biological molecule, such as an immune cell surface antigen, such as a tumor antigen). Accordingly, bispecific anti-B7-H3 antibodies may have binding specificity for B7-H3 and other antigens, such as CD3, CD20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1 or TenB2. Bispecific antibodies can also be prepared as full-length antibodies or antibody fragments.
用于制造多特异性抗体的技术包括但不限于具有不同特异性的两个免疫球蛋白重链-轻链配对的重组共表达(参见Milstein及Cuello，Nature 305：537-540(1983))；WO 93/08829；及Traunecker等人，EMBO J.10：3655-3659(1991))WO 2009/080253；Schaefer等人，Proc.Natl.Acad.Sci.USA，108：11187-11192(2011))，WO 2009/089004等。Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537-540 (1983)); WO 93/08829; and Traunecker et al., EMBO J. 10: 3655-3659 (1991)) WO 2009/080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108: 11187-11192 (2011)), WO 2009/089004, etc.
6)抗体变体6) Antibody variants
本发明的抗体涵盖本发明抗B7-H3抗体的氨基酸序列变体。例如，可能需要进一步改善抗体的结合亲和力和/或其他生物学性质而制备的抗体变体。抗体的氨基酸序列变体可通过向编码该抗体的核苷酸序列中引入适当修饰或通过肽合成来制备。此类修饰包括例如抗体氨基酸序列内的残基缺失和/或插入和/或取代。可对缺失、插入及取代进行任何组合以获得最终构建体，可使最终构建体具有所要特征，例如抗原结合。The antibodies of the present invention encompass amino acid sequence variants of the anti-B7-H3 antibodies of the present invention. For example, it may be necessary to further improve the binding affinity and/or other biological properties of the antibody and prepare the antibody variant. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, residue deletions and/or insertions and/or substitutions within the antibody amino acid sequence. Any combination of deletions, insertions and substitutions can be performed to obtain the final construct, which can give the final construct the desired characteristics, such as antigen binding.
a.取代变体、插入变体及缺失变体a. Substitution variants, insertion variants and deletion variants
在某些实施方案中，提供具有一个或多个氨基酸取代的抗体变体。取代型突变诱发的相关位点包括HVR及FR。保守取代显示于以下“优选取代”标题下。更多实质性变化提供于如下“例示性取代”标题下，且如下文参考氨基酸侧链类别进一步描述。可将氨基酸取代引入相关抗体中且针对所希望获得的活性(例如保留/改善抗原结合或改善ADCC或CDC)来筛选产物。In certain embodiments, antibody variants with one or more amino acid substitutions are provided. The relevant sites induced by substitution mutations include HVR and FR. Conservative substitutions are shown below under the heading "Preferred Substitutions". More substantial changes are provided below under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain categories. Amino acid substitutions can be introduced into related antibodies and the product can be screened for the desired activity (e.g., retaining/improving antigen binding or improving ADCC or CDC).
例示性氨基酸取代及优选氨基酸取代Exemplary amino acid substitutions and preferred amino acid substitutions
可根据共同侧链性质对氨基酸进行分组：Amino acids can be grouped according to common side chain properties:
(1)疏水性：正亮氨酸、Met、Ala、Val、Leu、Ile；(1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;
(2)中性亲水性：Cys、Ser、Thr、Asn、Gln；(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;
(3)酸性：Asp、Glu；(3) Acidic: Asp, Glu;
(4)碱性：His、Lys、Arg；(4) Basic: His, Lys, Arg;
(5)影响链取向的残基：Gly、Pro；(5) Residues that affect chain orientation: Gly, Pro;
(6)芳族：Trp、Tyr、Phe。(6) Aromatic: Trp, Tyr, Phe.
非保守取代将必然伴有将这些类别之一的成员交换成另一类别。Non-conservative substitutions will necessarily entail exchanging a member of one of these classes for another class.
一种类型取代型变体涉及取代亲本抗体(例如人源化抗体或人类抗体)的一个或多个高变区残基。一般而言，选择用于进一步研究的所得变体将在某些生物学性质(例如增加亲和力)方面相对于亲本抗体得以修饰(例如改善)和/或将实质上保留亲本抗体的某些生物学性质。例示性取代型变体为亲和力成熟抗体，其可使用例如基于噬菌体展示的亲和力成熟技术(诸如本文中所描述的那些)便利地产生。简言之，使一个或多个HVR残基突变且使变异抗体展现于噬菌体上，且针对特定生物活性(例如结合亲和力)进行筛选。One type of substitutional variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). In general, the resulting variants selected for further study will be modified (e.g., improved) relative to the parent antibody in terms of certain biological properties (e.g., increased affinity) and/or will substantially retain certain biological properties of the parent antibody. Exemplary substitutional variants are affinity matured antibodies, which can be conveniently produced using, for example, affinity maturation techniques based on phage display (such as those described herein). In short, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for specific biological activities (e.g., binding affinity).
在某些实施方案中，一个或多个HVR(CDR)内可发生取代、插入或缺失，只要此类变化实质上不削弱抗体结合B7H3的能力即可。例如，可在HVR中进行实质上不降低结合亲和力的保守变化(例如，如本文中所提供的保守取代)。例如，此类变化可在HVR中的抗原接触 残基以外。在以上所提供的变异VH及VL序列的某些实施方案中，各HVR未改变或含有不超过1、2或3个氨基酸取代。In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs (CDRs), as long as such changes do not substantially impair the ability of the antibody to bind B7H3. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in HVRs that do not substantially reduce binding affinity. For example, such changes may be made in HVRs that contact the antigen in the In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than 1, 2 or 3 amino acid substitutions.
用于鉴别可靶向以进行突变诱发的抗体残基或区域的适用方法称为“丙氨酸扫描突变诱发”，如Cunningham及Wells(1989)Science，244：1081-1085所描述。A useful method for identifying antibody residues or regions that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
7)重组方法7) Recombination method
本发明的抗B7-H3抗体可使用重组方法来制备，例如，如美国专利第4,816,567号中所描述。在一个实施方案中，提供编码本文中所描述的抗B7-H3抗体的经分离核酸。此类核酸可编码包含该抗体(例如该抗体的轻链和/或重链)的VL的氨基酸序列和/或包含其VH的氨基酸序列。在另一实施方案中，提供包含此类核酸的一种或多种载体(例如表达载体)。在另一实施方案中，提供包含此类核酸的宿主细胞。在一个此类实施方案中，宿主细胞包含(例如，经转化而具有)：(1)包含编码包含该抗体的VL的氨基酸序列及包含该抗体的VH的氨基酸序列的核酸的载体；或(2)包含编码包含该抗体的VL的氨基酸序列的核酸的第一载体及包含编码包含该抗体的VH的氨基酸序列的核酸的第二载体。在一个实施方案中，该宿主细胞为真核细胞，例如中国仓鼠卵巢(CHO)细胞或淋巴样细胞(例如Y0、NS0、SP2/0细胞)。在一个实施方案中，提供制造抗B7-H3抗体的方法，其中该方法包括在适合表达该抗体的条件下培养如上文所提供的包含编码该抗体的核酸的宿主细胞，及包括自该宿主细胞(或宿主细胞培养基)回收该抗体。The anti-B7-H3 antibodies of the present invention can be prepared using recombinant methods, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-B7-H3 antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL of the antibody (e.g., the light chain and/or heavy chain of the antibody) and/or an amino acid sequence comprising its VH. In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., transformed to have): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, SP2/0 cell). In one embodiment, a method of making an anti-B7-H3 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and comprises recovering the antibody from the host cell (or host cell culture medium).
为了重组生产抗B7-H3抗体，分离编码抗体的核酸(例如，如上所描述)插入一个或多个载体中以便在宿主细胞中进行进一步克隆和/或表达。此类核酸可使用常规程序(例如，通过使用能够特异性结合编码该抗体的重链及轻链的基因的寡核苷酸探针)容易地分离并测序。For recombinant production of anti-B7-H3 antibodies, nucleic acids encoding the antibodies are isolated (e.g., as described above) and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibodies).
适用于克隆或表达抗体编码载体的宿主细胞包括本文中所描述的原核细胞或真核细胞。例如，可在细菌中产生抗体，尤其在不需要糖基化及Fc效应功能时。对于在细菌中表达抗体片段及多肽，参见例如美国专利第5，648,237号、第5，789，199号及第5，840,523号。在表达之后，可自细菌细胞浆中分离处于可溶性部分中的抗体且可进一步纯化。Suitable host cells for cloning or expressing antibody encoding vectors include prokaryotes or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expressing antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents Nos. 5,648,237, 5,789,199, and 5,840,523. After expression, antibodies in the soluble portion can be separated from the bacterial cell slurry and can be further purified.
除原核生物以外，诸如丝状真菌或酵母的真核微生物也为抗体编码载体的合适克隆或表达宿主，包括糖基化途径已“人源化”从而产生具有部分或完全人类糖基化模式的抗体的真菌及酵母菌株。参见Gerngross，Nat.Biotech.22：1409-1414(2004)；及Li等人，Nat.Biotech.24：210-215(2006)。In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004); and Li et al., Nat. Biotech. 24: 210-215 (2006).
适用于表达糖基化抗体的宿主细胞也来源于多细胞生物体(无脊椎动物及脊椎动物)。无脊椎动物细胞的实例包括植物及昆虫细胞。已鉴别许多可用于结合昆虫细胞，尤其用于转染草地贪夜蛾(Spodoptera frugiperda)细胞的杆状病毒菌株。Suitable host cells for expressing glycosylated antibodies also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used to bind to insect cells, especially for transfecting Spodoptera frugiperda cells.
植物细胞培养物也可用作宿主。参见例如美国专利第5，959，177号、第6,040,498号、第6,420,548号、第7，125,978号及第6,417,429号(描述用于在转基因植物中产生抗体的PLANTIBODIESTM技术)。Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES ™ technology for producing antibodies in transgenic plants).
脊椎动物细胞也可用作宿主。例如，适于在悬浮液中生长的哺乳动物细胞株可能适用。 适用哺乳动物宿主细胞株的其他实例为经SV40转化的猴类肾CV1细胞株(COS-7)；人类胚肾细胞株(例如，如Graham等人，J.Gen.Virol.36：59-74(1977)中所描述的293或293细胞)；幼仓鼠肾细胞(BHK)；小鼠塞尔托利细胞(例如，如Mather，Biol.Reprod.23：243-252(1980)中所描述的TM4细胞)；猴肾细胞(CV1)；非洲绿猴肾细胞(VERO-76)；人类子宫颈癌细胞(HELA)；犬类肾细胞(MDCK)；布法罗大鼠肝细胞(BRL3A)；人类肺细胞(W138)；人类肝细胞(Hep G2)；小鼠乳房肿瘤(MMT 060562)；例如，如Mather等人，Annals N.Y.Acad.Sci.383：44-68(1982)中所描述的TRI细胞；MRC 5细胞；及FS4细胞。其他适用哺乳动物宿主细胞株包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞(Urlaub等人，Proc.Natl.Acad.Sci.USA 77：4216-4220(1980))；及骨髓瘤细胞株，诸如Y0、NS0及SP2/0。关于适用于产生抗体的某些哺乳动物宿主细胞株的综述，参见例如Yazaki及Wu，Methods in Molecular Biology，第248卷(B.K.C.Lo编，Humana Press，Totowa，NJ)，第255-268页(2003)。Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension may be suitable. Other examples of suitable mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7); human embryonic kidney cell line (e.g., 293 or 293 cells as described in Graham et al., J. Gen. Virol. 36:59-74 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-252 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); for example, as described in Mather et al., Annals of Genomics, 1996). TRI cells described in NY Acad. Sci. 383: 44-68 (1982); MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216-4220 (1980)); and myeloma cell lines, such as Y0, NS0 and SP2/0. For a review of certain mammalian host cell lines suitable for producing antibodies, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).
本发明B7-H3抗体与人B7-H3的亲和力KD低于1.0pM，优于Macrogenics的MGA271等B7-H3抗体，在治疗实体瘤包括中枢神经肿瘤，头颈部肿瘤，三阴乳腺癌，非小细胞肺癌和肾脏肿瘤等方面具有优异的应用前景。The affinity KD of the B7-H3 antibody of the present invention to human B7-H3 is lower than 1.0 pM, which is superior to B7-H3 antibodies such as MGA271 of Macrogenics, and has excellent application prospects in the treatment of solid tumors including central nervous system tumors, head and neck tumors, triple-negative breast cancer, non-small cell lung cancer and kidney tumors.
8)免疫缀合物8) Immunoconjugates
本发明也提供包含本文中的抗B7-H3抗体与一种或多种细胞毒性剂结合的免疫缀合物，此类细胞毒性剂为诸如化学治疗剂或化学治疗药物、生长抑制剂、毒素(例如蛋白毒素、细菌、真菌、植物或动物来源的酶活性毒素或其片段)或放射性同位素等。The present invention also provides immunoconjugates comprising an anti-B7-H3 antibody herein combined with one or more cytotoxic agents, such as chemotherapeutic agents or chemotherapeutic drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin or fragments thereof), or radioactive isotopes, etc.
在一个实施方案中，免疫缀合物为抗体-药物缀合物(ADC)，其中抗体与一种或多种药物结合，包括但不限于美登素(参见美国专利第5,208，020号、第5,416,064号及欧洲专利EP0425235B1)；奥利司他汀，诸如单甲基奥利司他汀药物部分DE及DF(MMAE及MMAF)(参见美国专利第5,635,483号及第5,780,588号及第7,498,298号)；多拉司他汀；卡奇霉素或其衍生物(参见美国专利第5,712,374号、第5,714,586号、第5,739，116号、第5,767,285号、第5,770,701号、第5,770,710号、第5,773,001号及第5,877,296号；Hinman等人，Cancer Res.53：3336-3342(1993)；及Lode等人，Cancer Res.58：2925-2928(1998))，氨甲蝶呤；长春地辛；紫杉烷，诸如多烯紫杉醇、紫杉醇、拉洛紫杉醇(larotaxel)、特西紫杉醇(tesetaxel)及欧塔紫杉醇(ortataxel)；单端孢霉毒素(trichothecene)；及CC1065。In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is bound to one or more drugs, including but not limited to maytansine (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP0425235B1); auristatin, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,743,297 and 5,761,298); 767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)), methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.
在另一实施方案中，免疫缀合物包含如本文中所描述的抗B7-H3抗体与酶活性毒素或其片段的缀合物，该酶活性毒素包括但不限于白喉A链、白喉毒素的非结合活性片段、外毒素A链及单端孢霉毒素等。In another embodiment, the immunoconjugate comprises a conjugate of an anti-B7-H3 antibody as described herein and an enzymatically active toxin or a fragment thereof, including but not limited to diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, and trichothecenes, etc.
在另一实施方案中，免疫缀合物包含如本文中所描述的抗B7-H3抗体与放射性原子结合形成的放射性缀合物。多种放射性同位素可用于产生放射性缀合物。实例包括At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32、Pb212及Lu的放射性同位素。In another embodiment, the immunoconjugate comprises a radioconjugate formed by combining an anti-B7-H3 antibody as described herein with a radioactive atom. A variety of radioisotopes can be used to produce radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioisotopes of Lu.
抗体与细胞毒性剂的缀合物可使用多种双官能蛋白偶合剂制造，诸如N-丁二酰亚胺基-3-(2-吡啶基二硫)丙酸酯(SPDP)、丁二酰亚胺基-4-(N-顺丁烯二酰亚胺基甲基)环己烷-1-甲酸 酯(SMCC)、亚胺基硫杂环戊烷(IT)、酰亚胺酯的双官能衍生物(诸如己二酸二甲酯盐酸盐)、活性酯(诸如辛二酸二丁二酰亚胺酯)、醛(诸如戊二醛)、双叠氮基化合物(诸如双(对叠氮基苯甲酰)己二胺)、双重氮衍生物(诸如双(对重氮苯甲酰)乙二胺)、二异氰酸酯(诸如甲苯2，6-二异氰酸酯)及双活性氟化合物(诸如1，5-二氟-2，4-二硝基苯)。Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, esters (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (such as dimethyl adipate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
9)药物制剂9) Pharmaceutical preparations
本发明的抗B7-H3抗体的药物制剂通过将具有所要纯度的抗体与一种或多种任选的药学上可接受的载体(Remington’s Pharmaceutical Sciences第16版，Osol，A.编(1980))混合制成冻干制剂或水溶液形式来制备。药学上可接受的载体在所采用的剂量及浓度下一般对受体无毒，且包括但不限于：缓冲剂，诸如磷酸盐、柠檬酸盐及其他有机酸；抗氧化剂，包括抗坏血酸及甲硫氨酸；防腐剂(诸如十八烷基二甲基苯甲基氯化铵；氯化六烃季铵；苯扎氯铵；苄索氯铵；苯酚、丁醇或苯甲醇；对羟基苯甲酸烷基酯，诸如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；儿茶酚；间苯二酚；环己醇；3-戊醇；及间甲酚)；低分子量(少于约10个残基)多肽；蛋白质，诸如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，诸如聚乙烯吡咯啶酮；氨基酸，诸如甘氨酸、谷酰氨酸、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖及其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，诸如EDTA；糖，诸如蔗糖、甘露醇、岩藻糖或山梨糖醇；成盐相对离子，诸如钠；金属配合物(例如锌-蛋白配合物)；和/或非离子表面活性剂，诸如聚乙二醇(PEG)。The pharmaceutical preparation of the anti-B7-H3 antibody of the present invention is prepared by mixing an antibody having a desired purity with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) into a lyophilized preparation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, fucose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).
例示性冻干抗体制剂描述于美国专利第6,267,958号中。水性抗体制剂包括美国专利第6,171，,86号及WO2006/044908中所描述的那些，后者的制剂包括组氨酸-乙酸盐缓冲液。Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,86 and WO 2006/044908, the latter formulations including histidine-acetate buffer.
本文中的制剂也可含有超过一种对于所治疗的特定适应症而言必需存在的活性成分，优选具有不会对彼此造成不利影响的互补活性的活性成分。例如，可能需要进一步提供额外治疗剂(例如化学治疗剂、细胞毒性剂、生长抑制剂和/或抗激素剂)。此类活性成分适合以组合形式以对预定目的有效的量存在。The preparations herein may also contain more than one active ingredient that must be present for the specific indication being treated, preferably with active ingredients that have complementary activities that do not adversely affect each other. For example, it may be necessary to further provide additional therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, growth inhibitors and/or anti-hormonal agents). Such active ingredients are suitable for being present in a combined form in an amount effective for a predetermined purpose.
10)制品10) Products
本发明还提供了含有本发明抗体或药物组合物的制品。该制品包含容器及处于该容器上或与该容器相关联的标签或包装插页。合适的容器包括例如瓶、小瓶、注射器、IV溶液袋等。此类容器可由各种材料制成，诸如玻璃或塑料。该容器容纳本发明组合物自身或该组合物与另一组合物的组合，且可具有无菌进入口(例如，该容器可为静脉内溶液袋或具有可由皮下注射针刺穿的瓶塞的小瓶)。该组合物中的至少一种活性剂为本发明的抗体。该标签或包装插页指示该组合物用于治疗选定肿瘤或病毒感染。此外，该制品可包含(a)其中含有组合物的第一容器，其中该组合物包含本发明的抗体；及(b)其中含有组合物的第二容器，其中该组合物包含另一细胞毒性剂或其他治疗剂。本发明的此实施方案中的制品可进一步包含指示此类组合物可用于治疗肿瘤或病毒感染的包装插页。可选地，或另外，该制品可进一步包括第二(或第三)容器，该容器包含药学上可接受的缓冲剂，诸如抑菌注射用水(BWFI)、磷酸盐缓冲盐水、林格氏溶液及右旋糖溶液。其可进一步包括自商业及使用者的立场来看合乎需 要的其他材料，包括其他缓冲剂、稀释剂、过滤器、针及注射器。The present invention also provides an article containing an antibody or pharmaceutical composition of the present invention. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. Such containers can be made of various materials, such as glass or plastic. The container holds the composition of the present invention itself or a combination of the composition and another composition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat a selected tumor or viral infection. In addition, the article may include (a) a first container containing a composition, wherein the composition comprises an antibody of the present invention; and (b) a second container containing a composition, wherein the composition comprises another cytotoxic agent or other therapeutic agent. The article in this embodiment of the present invention may further include a package insert indicating that such a composition can be used to treat a tumor or viral infection. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further comprise any other pharmaceutically acceptable buffer as deemed desirable from a commercial and user standpoint. Other materials required, including other buffers, diluents, filters, needles, and syringes.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社(2016)中所述的条件，或按照制造厂商所建议的条件。The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press (2016), or according to the conditions recommended by the manufacturer.
实施例1、抗B7-H3单克隆抗体的制备Example 1. Preparation of anti-B7-H3 monoclonal antibody
1.1高亲和力结合人B7-H3重组融合蛋白的单链抗体的淘选1.1 Selection of single-chain antibodies with high affinity binding to human B7-H3 recombinant fusion protein
本实施例中，从全人源单链scFv噬菌体展示文库中淘选高亲和力结合人B7-H3重组融合蛋白的单链抗体，该方法涉及利用如下所述的全人源单链scFv噬菌体展示文库，以及使用人B7-H3-6*His重组融合蛋白(近岸蛋白，cat#CK62)，大肠杆菌TG1(Lucigen，cat#60502-1)以及M13KO7辅助噬菌体(Thermo Fisher，cat#18311019)。In this embodiment, single-chain antibodies with high affinity binding to human B7-H3 recombinant fusion protein are selected from a fully human single-chain scFv phage display library. The method involves utilizing the fully human single-chain scFv phage display library as described below, and using human B7-H3-6*His recombinant fusion protein (Nearshore protein, cat#CK62), Escherichia coli TG1 (Lucigen, cat#60502-1) and M13KO7 helper phage (Thermo Fisher, cat#18311019).
全人源单链scFv噬菌体展示文库构建自含有pBR3.22启动子和M13噬菌体pIII蛋白展示系统的噬菌粒载体pCLS1.0，其C端含有Myc-His标签。文库构建时使用大肠杆菌TG1。该全人源单链scFv噬菌体展示文库采取常规方法建立，简述如下：利用RNA提取试剂盒(TaKaRa，cat#9767)从商购健康人群PBMCs(澳赛尔斯，cat#PB-003F-S，50个样本)中获得RNA，每个全人源scFv单链噬菌体展示子文库由五或十个样本的RNA混合建立。用逆转录试剂盒(Thermo Fisher，cat#4368814)将RNA逆转录合成cDNA第一链，按文献(CaiX.，GarenA.PNAS，1995.92(14)：6537-6541)以及V-base数据库设计合成扩增人类抗体可变区基因的特异性引物。以cDNA第一链为模板，通过PCR分别扩增人类抗体重链可变区和轻链可变区基因。利用分子克隆技术分别将人类抗体重链可变区及轻链可变区基因片段克隆至噬菌粒载体pCLS1.0。具体的，人类抗体轻链可变区由Nhe I/Not I双酶切克隆至载体pCLS1.0，并电转化至大肠杆菌TG1，获得噬菌体轻链子文库，获得载体pCLS1.0-VL。继续用Sfi I/Xho I双酶切将人类抗体重链可变区克隆至载体pCLS1.0-VL)。利用相同方法获得具有重链和轻链可变区组合的全人源scFv噬菌体展示子文库。各个子文库总计库容为9×109。The fully human single-chain scFv phage display library was constructed from the phagemid vector pCLS1.0 containing the pBR3.22 promoter and the M13 phage pIII protein display system, and its C-terminus contained a Myc-His tag. Escherichia coli TG1 was used for library construction. The fully human single-chain scFv phage display library was established using conventional methods, which are briefly described as follows: RNA was obtained from commercially available PBMCs of healthy people (Ausells, cat#PB-003F-S, 50 samples) using an RNA extraction kit (TaKaRa, cat#9767), and each fully human scFv single-chain phage display sublibrary was established by mixing RNA from five or ten samples. The RNA was reverse transcribed into the first strand of cDNA using a reverse transcription kit (Thermo Fisher, cat#4368814), and specific primers for synthesizing and amplifying human antibody variable region genes were designed and synthesized according to the literature (Cai X., Garen A. PNAS, 1995.92 (14): 6537-6541) and the V-base database. The first strand of cDNA was used as a template to amplify the human antibody heavy chain variable region and light chain variable region genes by PCR. The human antibody heavy chain variable region and light chain variable region gene fragments were cloned into the phagemid vector pCLS1.0 using molecular cloning technology. Specifically, the human antibody light chain variable region was cloned into the vector pCLS1.0 by double digestion with Nhe I/Not I, and electrotransformed into Escherichia coli TG1 to obtain a phage light chain sublibrary and a vector pCLS1.0-VL. The human antibody heavy chain variable region was cloned into the vector pCLS1.0-VL by double digestion with Sfi I/Xho I). The same method was used to obtain fully human scFv phage display sub-libraries with heavy chain and light chain variable region combinations. The total library capacity of each sub-library was 9×10 9 .
淘选实验中的人B7-H3-6*His标签融合蛋白均利用生物素标记试剂盒(Biotin Labeling Kit-NH2(Dojindo，cat#LK03))标记生物素。淘选实验简述如下：利用大肠杆菌TG1制备M13KO7辅助噬菌体，测定滴度约为1.0×1013，并将其侵染OD600值为0.5-0.6的上述全人源scFv单链噬菌体展示文库，以获得第一轮输入(input)噬菌体。利用液相淘选策略，通过事先和M-280Streptavidin(Thermo，cat#11206D)预孵育的经生物素标记的人B7-H3-6*His标签重组融合蛋白富集结合人B7-H3的scFv单链抗体的噬菌体。用pH 2.2，0.1M的甘氨酸-盐酸溶液洗脱富集的所述单链抗体噬菌体，得到第一轮输出(output)噬菌体。用第一轮输出噬菌体侵染OD600值为0.5-0.6的TG1大肠杆菌，以获得第二轮输入噬菌体，继续使用上述生物淘选方法得到与人B7-H3-6*His标签重组融合蛋白结合的第二轮输出噬菌 体。重复第二轮生物淘选过程进行第三轮或第四轮淘选，将经过第三轮或者第四轮淘选富集的输出噬菌体侵染大肠杆菌TG1，获得单克隆，通过噬菌体ELISA鉴定、挑选和人B7-H3-6*His标签重组融合蛋白具有高亲和力的单克隆大肠杆菌进行测序分析，获得20个克隆，见表1。The human B7-H3-6*His tag fusion proteins in the panning experiment were all labeled with biotin using the Biotin Labeling Kit-NH2 (Dojindo, cat#LK03). The panning experiment was briefly described as follows: M13KO7 helper phage was prepared using E. coli TG1, and the titer was determined to be about 1.0×10 13 , and it was used to infect the above-mentioned fully human scFv single-chain phage display library with an OD600 value of 0.5-0.6 to obtain the first round of input phage. Using the liquid phase panning strategy, by pre- and The phages of the scFv single-chain antibody binding to human B7-H3 were enriched with the biotin-labeled human B7-H3-6*His tag recombinant fusion protein pre-incubated with M-280 Streptavidin (Thermo, cat#11206D). The enriched single-chain antibody phages were eluted with a pH 2.2, 0.1M glycine-hydrochloric acid solution to obtain the first round of output phages. The first round of output phages were used to infect TG1 Escherichia coli with an OD600 value of 0.5-0.6 to obtain the second round of input phages, and the above-mentioned bio-panning method was continued to obtain the second round of output phages binding to the human B7-H3-6*His tag recombinant fusion protein. The second round of bio-panning process was repeated for the third or fourth round of panning, and the output phages enriched in the third or fourth round of panning were used to infect E. coli TG1 to obtain single clones, and the single clones of E. coli with high affinity for human B7-H3-6*His tag recombinant fusion protein were identified and selected by phage ELISA, and sequenced and analyzed to obtain 20 clones, as shown in Table 1.
表1、20个克隆
Table 1. 20 clones
1.2通过流式分析选择结合B7-H3蛋白的单链抗体进行IgG转换1.2 Selection of single-chain antibodies binding to B7-H3 protein for IgG conversion by flow cytometry
为了测试20个单克隆scFv单链抗体与细胞表面B7-H3蛋白结合的能力，将带有该scFv单链抗体的大肠杆菌单克隆用IPTG诱导表达可溶scFv单链抗体蛋白，取过滤后的上清与过表达B7-H3的CHO细胞检测结合活性。将所述的20个IPTG诱导表达的可溶scFv单链抗体蛋白、2YT培养基(生工生物工程(上海)股份有限公司，cat#SD7019)及空白对照PBS(Hyclone，cat#SH30256.01)分别与过表达B7-H3的CHO细胞4℃共孵育1h。然后用冷的1×PBS缓冲液将细胞洗涤两次。加入1：50稀释的anti-His-PE标记的抗体(Miltenyi Biotec，cat#130-092-691)，并将混合物在4℃下孵育30分钟，之后用冷的1×PBS缓冲液洗涤两次，用Guava easyCyte HT流式细胞仪(MERCK MILLIPORE)分析所述诱导表达的scFv单链抗体与过表达B7-H3的CHO细胞的结合活性。流式分析时针对过表达B7-H3的CHO细胞框选设门，通过PE信号强度考察结合活性。20个IPTG诱导表达的可溶scFv单链抗体蛋白与过表达B7-H3的人CHO细胞的结合活性如表2所示，有5个抗体蛋白(13725，14479，13683，14463，14471)具有与过表达B7-H3的CHO细胞的最高的结合活性。In order to test the ability of 20 monoclonal scFv single-chain antibodies to bind to cell surface B7-H3 protein, the E. coli monoclonal clone carrying the scFv single-chain antibody was induced to express soluble scFv single-chain antibody protein with IPTG, and the supernatant after filtration was taken to detect the binding activity with CHO cells overexpressing B7-H3. The 20 soluble scFv single-chain antibody proteins induced by IPTG, 2YT medium (Shanghai Biotechnology Co., Ltd., cat#SD7019) and blank control PBS (Hyclone, cat#SH30256.01) were incubated with CHO cells overexpressing B7-H3 at 4°C for 1 hour. The cells were then washed twice with cold 1×PBS buffer. Anti-His-PE labeled antibody (Miltenyi Biotec, cat#130-092-691) diluted 1:50 was added, and the mixture was incubated at 4°C for 30 minutes, then washed twice with cold 1×PBS buffer, and the binding activity of the induced expressed scFv single-chain antibody to CHO cells overexpressing B7-H3 was analyzed using Guava easyCyte HT flow cytometer (MERCK MILLIPORE). During flow analysis, gates were set for CHO cells overexpressing B7-H3, and the binding activity was examined by PE signal intensity. The binding activity of 20 IPTG-induced expressed soluble scFv single-chain antibody proteins to human CHO cells overexpressing B7-H3 is shown in Table 2. Five antibody proteins (13725, 14479, 13683, 14463, 14471) have the highest binding activity to CHO cells overexpressing B7-H3.
表2

Table 2

选取13725，14479，13683，14463，14471这5个scFv单链抗体，序列如下(氨基酸序列中以下划线表示相应抗体的CDR区)：Five scFv single-chain antibodies, 13725, 14479, 13683, 14463, and 14471, were selected, and their sequences are as follows (the underline in the amino acid sequence indicates the CDR region of the corresponding antibody):
13725克隆的单链抗体的VH链的氨基酸(SEQ ID NO：1)及核苷酸序列(SEQ ID NO：2)：
The amino acid sequence (SEQ ID NO: 1) and nucleotide sequence (SEQ ID NO: 2) of the VH chain of the single-chain antibody of clone 13725:
13725克隆单链抗体的VL链的氨基酸(SEQ ID NO：3)及核苷酸序列(SEQ ID NO：4)：
The amino acid sequence (SEQ ID NO: 3) and nucleotide sequence (SEQ ID NO: 4) of the VL chain of the 13725 cloned single-chain antibody:
14479克隆单链抗体的VH链的氨基酸序列(SEQ ID NO：5)：
Amino acid sequence of the VH chain of the 14479 cloned single-chain antibody (SEQ ID NO: 5):
14479克隆单链抗体的VL链的氨基酸序列(SEQ ID NO：6)：
Amino acid sequence of the VL chain of the 14479 cloned single-chain antibody (SEQ ID NO: 6):
13683克隆单链抗体的VH链的氨基酸序列(SEQ ID NO：7)：
Amino acid sequence of the VH chain of the 13683 cloned single-chain antibody (SEQ ID NO: 7):
13683克隆单链抗体的VL链的氨基酸序列(SEQ ID NO：8)：
Amino acid sequence of the VL chain of the 13683 cloned single-chain antibody (SEQ ID NO: 8):
14463克隆单链抗体的VH链的氨基酸序列(SEQ ID NO：9)：
Amino acid sequence of the VH chain of the 14463 cloned single-chain antibody (SEQ ID NO: 9):
14463克隆单链抗体的VL链的氨基酸序列(SEQ ID NO：10)：
Amino acid sequence of the VL chain of the 14463 cloned single-chain antibody (SEQ ID NO: 10):
14471克隆单链抗体的VH链的氨基酸序列(SEQ ID NO：11)：
Amino acid sequence of the VH chain of the 14471 cloned single-chain antibody (SEQ ID NO: 11):
14471克隆单链抗体的VL链的氨基酸序列(SEQ ID NO：12)：
Amino acid sequence of the VL chain of the 14471 cloned single-chain antibody (SEQ ID NO: 12):
对上述进行分子克隆转换成IgG4的形式。将包含前导肽、Nhe I/Not I酶切位点、重链基因恒定区及人IgG4-Fc的序列克隆构建至pCDNA3.3+，得到载体pCDNA3.3-IgG4。同样的，将包含前导肽、Nhe I/BsiW I酶切位点、轻链Kappa基因恒定区序列克隆构建至pCDNA3.3+，得到载体pCDNA3.3-VKappa，或者将包含前导肽、BamH I/Hind III酶切位点、轻链lambda基因恒定区序列克隆构建至pCDNA3.3+，得到载体pCDNA3.3-VLambda。将前述scFv单链抗体的重链可变区基因核苷酸序列两端加上Nhe I/Not I位点，插入到pCDNA3.3-IgG4质粒的相应位点中；将前述scFv单链抗体的轻链可变区核苷酸序列两端加上Nhe I/BsiW I位点，插入到pCDNA3.3-VKappa质粒的相应位点中或是利用BamH I/Hind III酶切位点将scFv单链抗体的轻链可变区核苷酸序列插入到pCDNA3.3-VLambda载体中。获得表达所述单克隆抗体的重组质粒，参见表3，其中所有重链都为IgG4(Padlan EA，Mol.Immunol.，1994，31(3)：169-217.Anatomy of the antibody molecule)；以BM(PC)作为阳性对照抗体(Patent EP2542256，BRCA69D)。The above was molecularly cloned and converted into IgG4. The sequence containing the leader peptide, Nhe I/Not I restriction site, heavy chain gene constant region and human IgG4-Fc was cloned into pCDNA3.3+ to obtain the vector pCDNA3.3-IgG4. Similarly, the sequence containing the leader peptide, Nhe I/BsiW I restriction site, and light chain Kappa gene constant region was cloned into pCDNA3.3+ to obtain the vector pCDNA3.3-VKappa, or the sequence containing the leader peptide, BamH I/Hind III restriction site, and light chain lambda gene constant region was cloned into pCDNA3.3+ to obtain the vector pCDNA3.3-VLambda. Add Nhe I/Not I sites at both ends of the heavy chain variable region gene nucleotide sequence of the aforementioned scFv single-chain antibody and insert it into the corresponding site of the pCDNA3.3-IgG4 plasmid; add Nhe I/BsiW I sites at both ends of the light chain variable region nucleotide sequence of the aforementioned scFv single-chain antibody and insert it into the corresponding site of the pCDNA3.3-VKappa plasmid or use BamH I/Hind III restriction sites to insert the light chain variable region nucleotide sequence of the scFv single-chain antibody into the pCDNA3.3-VLambda vector. Obtain a recombinant plasmid expressing the monoclonal antibody, see Table 3, wherein all heavy chains are IgG4 (Padlan EA, Mol. Immunol., 1994, 31(3): 169-217. Anatomy of the antibody molecule); BM (PC) is used as a positive control antibody (Patent EP2542256, BRCA69D).
1.3 IgG4单克隆抗体的表达、纯化、鉴定1.3 Expression, purification and identification of IgG4 monoclonal antibody
将上述表3所列的含重链可变区和轻链可变区的重组质粒以及含对照抗体BM(PC)的重链可变区和轻链可变区的重组质粒用ExpiFectamineTMCHO Transfection Kit(Thermo Fisher，cat#A29129)脂质体法瞬时转染悬浮培养的Expi CHO-STM细胞(Thermo Fisher，cat#A29127)。The recombinant plasmids containing the heavy chain variable region and the light chain variable region listed in Table 3 above and the recombinant plasmids containing the heavy chain variable region and the light chain variable region of the control antibody BM (PC) were transiently transfected into suspension cultured Expi CHO-S TM cells (Thermo Fisher, cat#A29127) using the ExpiFectamine TM CHO Transfection Kit (Thermo Fisher, cat#A29129) liposome method.
在30ml ExpiCHO Expression培养基(Thermo Fisher，cat#A2910001)中，以37℃，8％CO2，120rpm的转速条件下培养前述获得的转染ExpiCHO-S细胞。The transfected ExpiCHO-S cells obtained above were cultured in 30 ml of ExpiCHO Expression medium (Thermo Fisher, cat#A2910001) at 37°C, 8% CO 2 , and 120 rpm.
经过10-14天培养的转染细胞，通过二级离心处理(第一级离心条件20min，400g；第二级离心条件20min，10000g)，去除细胞和细胞碎片，得到上清液。将澄清的上清液装载入蛋白A亲和层析柱(GE Healthcare，cat#GE-17-5438-04)，通过三步淋洗(淋洗缓冲液依次为含150mM NaCl，pH 5.0的磷酸盐缓冲液；20mM柠檬酸钠-1M氯化钠pH 5.0；20mM柠檬酸钠pH 5.0的淋洗缓冲液)来去除杂质，然后通过pH 3.0的20mM柠檬酸钠溶液分离捕获目标抗体。最后通过超滤浓缩步骤将目标抗体置换至pH 7.4的1×PBS缓冲液中。After 10-14 days of culture, the transfected cells were treated with secondary centrifugation (first centrifugation condition 20min, 400g; second centrifugation condition 20min, 10000g) to remove cells and cell debris and obtain supernatant. The clarified supernatant was loaded into a protein A affinity chromatography column (GE Healthcare, cat#GE-17-5438-04), and impurities were removed by three steps of elution (elution buffer was phosphate buffer containing 150mM NaCl, pH 5.0; 20mM sodium citrate-1M sodium chloride pH 5.0; 20mM sodium citrate pH 5.0 elution buffer), and then the target antibody was separated and captured by 20mM sodium citrate solution at pH 3.0. Finally, the target antibody was replaced into 1× PBS buffer at pH 7.4 by ultrafiltration concentration step.
1.4抗体结合活性分析1.4 Antibody Binding Activity Analysis
将纯化出来的5个IgG抗体(13683，13725，14463，14471，14479)利用ELISA的方法检测其与人B7-H3*His标签重组融合蛋白抗原的体外结合活性。该实验使用了一种商购人B7-H3-6*His标签融合蛋白(近岸蛋白，cat#CK62)。将人B7-H3*His标签融合蛋白用包被缓 冲液(碳酸盐缓冲液)稀释至浓度1μg/ml，4℃过夜包被至96孔板(CORNING，cat#9018)中。经pH 7.4的1xPBS缓冲液洗涤三次后，用3％的BSA封闭2h。将抗人B7-H3全人源单克隆抗体(13683，13725，14463，14471，14479)用pH 7.4的1xPBS缓冲液从10μg/ml开始进行3倍梯度稀释，与包被抗体在25℃共孵育2h。然后用带HRP标签的抗-hIGFc检测抗体检测所述抗体与人B7-H3-6*His标签融合蛋白抗原的结合。The purified 5 IgG antibodies (13683, 13725, 14463, 14471, 14479) were tested for their in vitro binding activity with human B7-H3*His tag recombinant fusion protein antigen by ELISA. This experiment used a commercially available human B7-H3-6*His tag fusion protein (Near Shore Protein, cat#CK62). The rinse solution (carbonate buffer) was diluted to a concentration of 1 μg/ml and coated in a 96-well plate (CORNING, cat#9018) at 4°C overnight. After washing three times with 1xPBS buffer at pH 7.4, it was blocked with 3% BSA for 2h. The anti-human B7-H3 fully human monoclonal antibody (13683, 13725, 14463, 14471, 14479) was diluted 3 times from 10 μg/ml with 1xPBS buffer at pH 7.4 and incubated with the coated antibody at 25°C for 2h. The binding of the antibody to the human B7-H3-6*His tag fusion protein antigen was then detected with an HRP-labeled anti-hIGFc detection antibody.
如图1和表3所示，全人源单克隆抗体13725、14479与人B7-H3-6*His标签融合蛋白抗原亲和力尤为理想，其中13725具有最佳亲和力。As shown in FIG1 and Table 3, the fully human monoclonal antibodies 13725 and 14479 have particularly ideal affinities for the human B7-H3-6*His tag fusion protein antigen, among which 13725 has the best affinity.
表3、IgG4单克隆抗体与人B7-H3-6*His标签融合蛋白体外结合活性
Table 3. In vitro binding activity of IgG4 monoclonal antibody and human B7-H3-6*His tag fusion protein
全人源单克隆抗体13725、14479抗体的CDR区序列如表4。The CDR region sequences of the fully human monoclonal antibodies 13725 and 14479 are shown in Table 4.
表4
Table 4
实施例2、由转染的ExpiCHO-S细胞制备B7-H3单克隆抗体Example 2: Preparation of B7-H3 monoclonal antibody from transfected ExpiCHO-S cells
将含有优选克隆B7-H3的重链和轻链基因的重组质粒用ExpiFectamineTM CHO Transfection Kit(Thermo Fisher，cat#A29129)脂质体法瞬时转染较大规模(200-600ml)悬浮培养的ExpiCHO-S细胞(Thermo Fisher，cat#A29127)，转染，培养，纯化方法如实施例的1.3所述，制备目标抗体。The recombinant plasmid containing the heavy and light chain genes of the preferred clone B7-H3 was transiently transfected into a larger scale (200-600 ml) suspension culture of ExpiCHO-S cells (Thermo Fisher, cat#A29127) using the ExpiFectamine ™ CHO Transfection Kit (Thermo Fisher, cat#A29129) liposome method. The transfection, culture and purification methods were as described in Example 1.3 to prepare the target antibody.
通过超滤浓缩步骤将目标抗体置换至pH7.4的1×PBS缓冲液中，用以进行后续各项体 外和体内活性检测和质量分析实验。The target antibody was replaced into 1× PBS buffer at pH 7.4 by ultrafiltration concentration step for subsequent isomerization. In vitro and in vivo activity assays and quality analysis experiments.
实施例3、瞬时表达、纯化的B7-H3单克隆抗体的纯度分析Example 3. Purity Analysis of Transiently Expressed and Purified B7-H3 Monoclonal Antibody
本实施例中，通过分子筛液相色谱(SEC-HPLC)分析B7-H3单克隆抗体13725、14479的蛋白纯度。In this example, the protein purity of B7-H3 monoclonal antibodies 13725 and 14479 was analyzed by molecular sieve liquid chromatography (SEC-HPLC).
20μg抗人B7-H3全人源单克隆抗体(浓度调整至1mg/ml)加载到HPLC色谱仪(Agilent Technologies)的色谱柱(TOSOH，型号：TSKgel G3000SWXL)，流动相为50mM磷酸盐缓冲液，300mM氯化钠，pH 7.0，流速为0.8ml/min，检测波长为280nm。结果如图2和表5。峰值计算使用ChemStation软件进行分析，本发明的抗人B7-H3全人源单克隆抗体13725、14479的主峰百分比＞99.2％、多聚体峰百分比＜1％，纯度高。20 μg of anti-human B7-H3 fully human monoclonal antibody (concentration adjusted to 1 mg/ml) was loaded into a chromatographic column (TOSOH, model: TSKgel G3000SWXL) of an HPLC chromatograph (Agilent Technologies), the mobile phase was 50 mM phosphate buffer, 300 mM sodium chloride, pH 7.0, the flow rate was 0.8 ml/min, and the detection wavelength was 280 nm. The results are shown in Figure 2 and Table 5. The peak calculation was analyzed using ChemStation software. The main peak percentage of the anti-human B7-H3 fully human monoclonal antibodies 13725 and 14479 of the present invention was greater than 99.2%, and the multimer peak percentage was less than 1%, with high purity.
表5、B7-H3单克隆抗体的SEC主峰纯度百分比
Table 5. SEC main peak purity percentage of B7-H3 monoclonal antibody
实施例4、B7-H3单克隆抗体与人B7-H3-His蛋白、人B7-H4-His蛋白、人PD-L1-ECD-mFc蛋白、小鼠B7-H3-His蛋白、小鼠B7-H4-huFc蛋白的结合Example 4. Binding of B7-H3 monoclonal antibody to human B7-H3-His protein, human B7-H4-His protein, human PD-L1-ECD-mFc protein, mouse B7-H3-His protein, and mouse B7-H4-huFc protein
4.1、ELISA检测B7-H3单克隆抗体13725，14479与多种蛋白的结合4.1. ELISA detection of the binding of B7-H3 monoclonal antibodies 13725 and 14479 to various proteins
该实验使用了商购人B7-H3-His蛋白(近岸蛋白，cat#CK62)、人B7-H4-His蛋白(Acro，B74-H5222)、人PD-L1-ECD-mFc蛋白(近岸蛋白，cat#CM06)、小鼠B7-H3-His蛋白(近岸蛋白，cat#CM83)、小鼠B7-H4-huFc蛋白(近岸蛋白，cat#CS04)。将待测蛋白抗原分别用pH 7.4的1xPBS包被缓冲液稀释至浓度1μg/ml，4℃过夜包被至96孔板(CORNING，cat#9018)中。经pH 7.4的1xPBS缓冲液洗涤三次后，用5％的脱脂牛奶封闭2h。将待测抗人B7-H3全人源单克隆抗体13725，14479用pH 7.4的1xPBS缓冲液从10μg/ml开始进行3倍梯度稀释，与包被蛋白抗原在25℃共孵育2h。然后用稀释1∶4000的带HRP标签的抗-人IgG-Fab检测抗体检测所述抗体与各个蛋白抗原的结合。The experiment used commercially available human B7-H3-His protein (nearshore protein, cat#CK62), human B7-H4-His protein (Acro, B74-H5222), human PD-L1-ECD-mFc protein (nearshore protein, cat#CM06), mouse B7-H3-His protein (nearshore protein, cat#CM83), and mouse B7-H4-huFc protein (nearshore protein, cat#CS04). The protein antigens to be tested were diluted to a concentration of 1 μg/ml with 1xPBS coating buffer at pH 7.4 and coated in 96-well plates (CORNING, cat#9018) at 4°C overnight. After washing three times with 1xPBS buffer at pH 7.4, the plates were blocked with 5% skim milk for 2 hours. The anti-human B7-H3 fully human monoclonal antibodies 13725 and 14479 to be tested were diluted 3 times from 10 μg/ml in 1xPBS buffer at pH 7.4 and incubated with the coated protein antigen at 25°C for 2 hours. The binding of the antibodies to the respective protein antigens was then detected using an HRP-labeled anti-human IgG-Fab detection antibody diluted 1:4000.
检测结果见图3至图7，抗B7-H3全人源单克隆抗体13725、14479与人B7-H3-His蛋白具有高的结合活性，结合的EC50值可见表6，表7。同时发现13725、14479也与小鼠B7-H3-His蛋白结合；而与人B7-H4-His，小鼠B7-H4-huFc，以及人PD-L1-ECD-mFc蛋白几乎不结合。说明13725、14479特异性好。The test results are shown in Figures 3 to 7. The anti-B7-H3 fully human monoclonal antibodies 13725 and 14479 have high binding activity with human B7-H3-His protein, and the EC50 values of binding can be seen in Tables 6 and 7. It was also found that 13725 and 14479 also bind to mouse B7-H3-His protein, but almost do not bind to human B7-H4-His, mouse B7-H4-huFc, and human PD-L1-ECD-mFc protein. This shows that 13725 and 14479 have good specificity.
表6、B7-H3单克隆抗体与人B7-H3-His蛋白的结合活性

Table 6. Binding activity of B7-H3 monoclonal antibody to human B7-H3-His protein

表7、B7-H3单克隆抗体与小鼠B7-H3-His蛋白的结合活性
Table 7. Binding activity of B7-H3 monoclonal antibody to mouse B7-H3-His protein
4.2、B7-H3单克隆抗体的亲和平衡解离常数KD的检测4.2. Detection of affinity equilibrium dissociation constant KD of B7-H3 monoclonal antibody
B7-H3单克隆抗体与人B7-H3-His蛋白相结合的能力采用Octet Red96生物分子相互作用仪系统(Octet Red96，ForteBio)进行检测，以BM(PC)为阳性对照抗体。抗人IgG Fc(AHC)的动力学级生物传感器(Fortebio，#18-5063)经pH 1.7的甘氨酸预处理后浸泡在测定PBS缓冲液中。抗B7-H3单克隆抗体以10μg/ml的浓度固定到AHC生物传感器。然后将加载了B7-H3单克隆抗体的AHC生物传感器浸入到不同浓度的人B7-H3-His抗原和缓冲液中。分析物柱的最后稀释点只包含检测缓冲液，以测试缓冲液与负载生物传感器之间的非特异性结合。从0到300秒检测到了抗原与抗体结合，随后300到900秒发生解离。用检测缓冲液确定了60秒的基线。抗B7-H3的单克隆抗体的亲和力曲线是以1∶1结合的动力学传感单价结合模型拟合的。结合动力学分析如表8所示。The ability of B7-H3 mAb to bind to human B7-H3-His protein was tested using the Octet Red96 Biomolecular Interaction Instrument System (Octet Red96, ForteBio) with BM (PC) as the positive control antibody. Anti-human IgG Fc (AHC) kinetic-grade biosensors (Fortebio, #18-5063) were pretreated with glycine at pH 1.7 and then immersed in the assay PBS buffer. Anti-B7-H3 mAb was immobilized to the AHC biosensor at a concentration of 10 μg/ml. The AHC biosensor loaded with B7-H3 mAb was then immersed in different concentrations of human B7-H3-His antigen and buffer. The last dilution point of the analyte column contained only the assay buffer to test for nonspecific binding between the buffer and the loaded biosensor. Antigen-antibody binding was detected from 0 to 300 seconds, followed by dissociation from 300 to 900 seconds. A baseline of 60 seconds was established with the assay buffer. The affinity curve of the monoclonal antibody against B7-H3 was fitted with a kinetic sensing monovalent binding model of 1:1 binding. The binding kinetic analysis is shown in Table 8.
表8、人B7-H3-His蛋白与B7-H3单克隆抗体的亲和力
Table 8. Affinity of human B7-H3-His protein and B7-H3 monoclonal antibody
根据表8可见，本发明获得的13725与14479克隆的抗体与人B7-H3-His蛋白均具有良好的亲和力，其中13725的亲和力尤为理想，达到了令人意外的低于1.0E-12的KD值。According to Table 8, the antibodies cloned 13725 and 14479 obtained in the present invention have good affinity to human B7-H3-His protein, among which the affinity of 13725 is particularly ideal, reaching a surprisingly low KD value of less than 1.0E-12.
实施例5、B7-H3单克隆抗体与高表达B7-H3的肿瘤细胞系的结合活性Example 5. Binding activity of B7-H3 monoclonal antibody to tumor cell lines that highly express B7-H3
本实施例进行一项体外结合活性测试(Concentration for 50％of maximal Effect，EC50)，该测试能够说明抗人B7-H3全人源单克隆抗体13725，14479与人高表达B7-H3的肿瘤细胞系有较强的结合。MCF-7是人乳腺癌细胞系，OVCAR-3是人卵巢癌细胞系。两种细胞表面均高表达B7-H3。培养MCF-7，OVCAR-3于96孔板中，细胞铺板密度为106/孔。用1xPBS缓冲液将13725，14479从30μg/ml进行3倍梯度稀释，并将IgG4阴性对照(IgG4iso)稀释到10μg/ml，与MCF-7，OVCAR-3细胞在37℃共孵育2h。接着用稀释1∶200的山羊抗人IgG-Fc-PE标记的抗体(Abcam，cat#ab98596)孵育1h，并利用Guava easyCyte HT流式细胞仪(MERCK MILLIPORE)进行所述抗体与细胞的结合活性分析。结果如图8，图9和表9所示，B7-H3单克隆抗体13725、14479与高表达B7-H3的肿瘤细胞系MCF-7和OVCAR-3均能够 高亲和结合，靶向性好、非特异性低。This example conducts an in vitro binding activity test (Concentration for 50% of maximal Effect, EC50), which can illustrate that the anti-human B7-H3 fully human monoclonal antibodies 13725, 14479 have strong binding to human tumor cell lines that highly express B7-H3. MCF-7 is a human breast cancer cell line, and OVCAR-3 is a human ovarian cancer cell line. Both cell surfaces highly express B7-H3. MCF-7 and OVCAR-3 were cultured in 96-well plates at a cell plating density of 10 6 /well. 13725 and 14479 were diluted 3 times from 30 μg/ml with 1xPBS buffer, and the IgG4 negative control (IgG4iso) was diluted to 10 μg/ml, and incubated with MCF-7 and OVCAR-3 cells at 37°C for 2h. Then, the cells were incubated with goat anti-human IgG-Fc-PE labeled antibody (Abcam, cat# ab98596) diluted 1:200 for 1 hour, and the binding activity of the antibody to the cells was analyzed using Guava easyCyte HT flow cytometer (MERCK MILLIPORE). The results are shown in Figures 8, 9 and Table 9. B7-H3 monoclonal antibodies 13725 and 14479 can bind to tumor cell lines MCF-7 and OVCAR-3 that highly express B7-H3. High affinity binding, good targeting and low non-specificity.
表9、B7-H3单克隆抗体与MCF-7，OVCAR-3细胞的结合活性
Table 9. Binding activity of B7-H3 monoclonal antibody to MCF-7 and OVCAR-3 cells
实施例6、B7-H3单克隆抗体与胰岛素的结合活性Example 6. Binding activity of B7-H3 monoclonal antibody to insulin
本实施例进行一项体外结合活性测试，测试抗人B7-H3全人源单克隆抗体13725，14479与人胰岛素(Insulin)的结合活性，并与已上市产品Hercepin，Rituxan比较，分析B7-H3全人源单克隆抗体在临床应用时的潜在的非特异性结合。In this example, an in vitro binding activity test was conducted to test the binding activity of anti-human B7-H3 fully human monoclonal antibodies 13725 and 14479 with human insulin, and compared with the marketed products Hercepin and Rituxan to analyze the potential non-specific binding of B7-H3 fully human monoclonal antibodies in clinical applications.
该实验首先将人胰岛素(Sigma，cat.I2643)用pH 7.4的1xPBS包被缓冲液稀释至浓度10μg/ml，4℃过夜包被至96孔板(CORNING，cat#9018)中。经pH 7.4的1xPBS缓冲液洗涤三次后，用5％的脱脂牛奶封闭2h。将待测抗人B7-H3全人源单克隆抗体13725，14479，Herceptin以及Rituxan用pH 7.4的1xPBS缓冲液从10μg/ml开始进行3倍梯度稀释，与包被Insulin在25℃共孵育2h。然后用羊抗-人IgG-HRP检测抗体(1∶5000)检测所述抗体与Insulin的结合，计算样品各个浓度点的OD450检测信号相对于PBS的OD450检测信号的倍数作为分数(Score)值，4参数拟合量效关系曲线。检测结果见图10，抗B7-H3全人源单克隆抗体13725，14479对人胰岛素蛋白的结合能力与Herceptin，Rituxan对人胰岛素蛋白的结合能力相当，EC50值可见表10。In this experiment, human insulin (Sigma, cat.I2643) was first diluted to a concentration of 10 μg/ml with 1xPBS coating buffer at pH 7.4, and coated in a 96-well plate (CORNING, cat#9018) at 4°C overnight. After washing three times with 1xPBS buffer at pH 7.4, the plate was blocked with 5% skim milk for 2 hours. The anti-human B7-H3 fully human monoclonal antibodies 13725, 14479, Herceptin and Rituxan to be tested were diluted 3 times from 10 μg/ml with 1xPBS buffer at pH 7.4, and incubated with the coated insulin at 25°C for 2 hours. Then, the binding of the antibody to insulin was detected with goat anti-human IgG-HRP detection antibody (1:5000), and the multiple of the OD450 detection signal of each concentration point of the sample relative to the OD450 detection signal of PBS was calculated as the score value, and the 4-parameter fitting dose-effect relationship curve was used. The test results are shown in Figure 10. The binding abilities of anti-B7-H3 fully human monoclonal antibodies 13725 and 14479 to human insulin protein are comparable to those of Herceptin and Rituxan. The EC50 values can be seen in Table 10.
表10、B7-H3单克隆抗体与人胰岛素的结合活性
Table 10. Binding activity of B7-H3 monoclonal antibody to human insulin
实施例7、B7-H3单克隆抗体与DNA的结合活性Example 7. Binding activity of B7-H3 monoclonal antibody to DNA
本实施例进行一项体外结合活性测试，测试抗人B7-H3全人源单克隆抗体13725，14479与鲑鱼DNA的结合活性，并与已上市产品Hercepin，Rituxan比较，分析B7-H3全人源单克隆抗体在临床应用时的潜在的非特异性结合。In this example, an in vitro binding activity test was conducted to test the binding activity of anti-human B7-H3 fully human monoclonal antibodies 13725 and 14479 to salmon DNA, and compared with the marketed products Hercepin and Rituxan to analyze the potential non-specific binding of B7-H3 fully human monoclonal antibodies in clinical applications.
该实验首先将鲑鱼DNA(Sigma，cat.D1626)用pH 7.4的1xPBS包被缓冲液稀释至浓度10μg/ml，4℃过夜包被至96孔板(CORNING，cat#9018)中。经pH 7.4的1xPBS缓冲液洗涤三次后，用5％的脱脂牛奶封闭2h。将待测抗人B7-H3全人源单克隆抗体13725，14479，Herceptin以及Rituxan用pH 7.4的1xPBS缓冲液从10μg/ml开始进行3倍梯度稀释，与包被DNA在 25℃共孵育2h。然后用羊抗-人IgG-HRP检测抗体(1∶5000)检测所述抗体与DNA的结合，计算样品各个浓度点的OD450检测信号相对于PBS的OD450检测信号的倍数作为分数(Score)值，4参数拟合量效关系曲线。检测结果见图11，抗B7-H3全人源单克隆抗体13725，14479对鲑鱼DNA的结合能力与Herceptin，Rituxan对鲑鱼DNA的结合能力相当，EC50值可见表11。The experiment first diluted salmon DNA (Sigma, cat.D1626) with 1xPBS coating buffer at pH 7.4 to a concentration of 10 μg/ml and coated in a 96-well plate (CORNING, cat#9018) at 4°C overnight. After washing three times with 1xPBS buffer at pH 7.4, the plate was blocked with 5% skim milk for 2 hours. The anti-human B7-H3 fully human monoclonal antibodies 13725, 14479, Herceptin and Rituxan to be tested were diluted 3 times from 10 μg/ml with 1xPBS buffer at pH 7.4 and coated with DNA at 4°C. Incubate at 25°C for 2 hours. Then, sheep anti-human IgG-HRP detection antibody (1:5000) was used to detect the binding of the antibody to DNA, and the multiple of the OD450 detection signal of each concentration point of the sample relative to the OD450 detection signal of PBS was calculated as the score value, and the 4-parameter fitting dose-effect curve was used. The test results are shown in Figure 11. The binding ability of anti-B7-H3 fully human monoclonal antibodies 13725 and 14479 to salmon DNA is comparable to that of Herceptin and Rituxan to salmon DNA, and the EC50 values can be seen in Table 11.
表11、B7-H3单克隆抗体与DNA的结合活性
Table 11. Binding activity of B7-H3 monoclonal antibody to DNA
实施例8、B7-H3抗体DSF热稳定性分析Example 8. Analysis of thermal stability of B7-H3 antibody DSF
本实施例对人源化抗B7-H3单克隆抗体13725，14479进行DSF检测，考察热稳定性。将抗B7-H3单克隆抗体与荧光基团SYPRO Orange 5000×(Thermo，cat.S6650)利用PBS稀释混合在PCR八联管中，混合体系20μl/管，其中抗体终浓度0.2mg/ml，SYPRO Orange的终浓度是5×。接着将PCR八联管至于荧光PCR仪ABI 7500，采取连续模式进行溶解曲线实验。扫描温度25-99℃，25℃平衡5min，升温速率为1％，在升温过程中采集数据，报告基团ROX，淬灭基团选择None。In this example, humanized anti-B7-H3 monoclonal antibodies 13725 and 14479 were subjected to DSF detection to investigate thermal stability. Anti-B7-H3 monoclonal antibody and fluorescent group SYPRO Orange 5000× (Thermo, cat. S6650) were diluted and mixed in PCR eight-tubes using PBS, with a mixed system of 20 μl/tube, where the final concentration of the antibody was 0.2 mg/ml and the final concentration of SYPRO Orange was 5×. Then the eight-tube PCR was placed in the fluorescent PCR instrument ABI 7500, and the melting curve experiment was performed in continuous mode. The scanning temperature was 25-99°C, equilibrated at 25°C for 5 minutes, the heating rate was 1%, and data was collected during the heating process. The reporting group was ROX, and the quenching group was selected as None.
检测结果见表12，人源化抗B7-H3单克隆抗体13725，14479具有高的Tm值，热稳定性较好。The test results are shown in Table 12. Humanized anti-B7-H3 monoclonal antibodies 13725 and 14479 have high Tm values and good thermal stability.
表12、B7-H3单克隆抗体热稳定性(DSF)分析结果
Table 12. Results of thermal stability (DSF) analysis of B7-H3 monoclonal antibody
实施例9、B7-H3单克隆抗体的ADCC体外杀伤活性实验Example 9: ADCC in vitro killing activity experiment of B7-H3 monoclonal antibody
本实施例对人源化抗B7-H3单克隆抗体13725，14479以及阳性对照抗体BM(PC)进行ADCC活性检测。靶细胞是人乳腺癌细胞系MCF-7，效应细胞是人PBMC。实验过程描述如下，将人PBMC(澳赛尔斯，cat#PB-003F-S)悬液用RPMI 1640分析培养基稀释至1×107cells/mL，并以50μl/孔加入96孔板中，将抗B7-H3单克隆抗体用RPMI 1640分析培养基稀释至5μg/mL，作为第1个浓度点S01，从S01开始进行3倍梯度稀释一共得到10个浓度点S01～S10，并以50μl/孔加入含有PBMC的孔中，同时将MCF-7细胞悬液用RPMI 1640培养基稀释至2×105cells/mL，50μl/孔加入含有单克隆抗体以及PBMC的孔中。单克隆抗体、效应细胞以及靶细胞添加混合完成后将96孔板置于37℃，5％CO2的恒温培养箱中培养5～6h。 利用LDH显色试剂盒(Promega，Cytotoxicity Assay(Non-Radioactive)，cat.G1780)进行显色，并计算各个加样孔的靶细胞杀伤率。根据加样单克隆抗体浓度和对应靶细胞杀伤率的量效关系用GraphPad Prism进行4-参数拟合非线性回归，分析B7-H3单克隆抗体的ADCC活性。In this example, ADCC activity was detected for humanized anti-B7-H3 monoclonal antibodies 13725, 14479 and positive control antibody BM (PC). The target cells were human breast cancer cell line MCF-7, and the effector cells were human PBMC. The experimental process is described as follows: human PBMC (Auscells, cat#PB-003F-S) suspension was diluted to 1×10 7 cells/mL with RPMI 1640 analysis medium and added to a 96-well plate at 50 μl/well. Anti-B7-H3 monoclonal antibody was diluted to 5 μg/mL with RPMI 1640 analysis medium as the first concentration point S01. A total of 10 concentration points S01 to S10 were obtained by 3-fold gradient dilution from S01 and added to the wells containing PBMC at 50 μl/well. At the same time, MCF-7 cell suspension was diluted to 2×10 5 cells/mL with RPMI 1640 medium and added to the wells containing monoclonal antibodies and PBMC at 50 μl/well. After the monoclonal antibody, effector cells and target cells were added and mixed, the 96-well plate was placed in a constant temperature incubator at 37°C and 5% CO 2 for 5 to 6 hours. The color development was performed using an LDH color development kit (Promega, Cytotoxicity Assay (Non-Radioactive), cat. G1780), and the target cell killing rate of each well was calculated. The ADCC activity of the B7-H3 monoclonal antibody was analyzed by performing a 4-parameter fitting nonlinear regression based on the dose-effect relationship between the added monoclonal antibody concentration and the corresponding target cell killing rate using GraphPad Prism.
检测结果见图12以及表13，人源化抗B7-H3单克隆抗体13725，14479具有强的ADCC活性，其中13725尤为理想。The test results are shown in FIG. 12 and Table 13 . Humanized anti-B7-H3 monoclonal antibodies 13725 and 14479 have strong ADCC activity, among which 13725 is particularly ideal.
表13、B7-H3单克隆抗体ADCC分析结果
Table 13. B7-H3 monoclonal antibody ADCC analysis results
实施例10、B7-H3单克隆抗体体内抑制肿瘤细胞生长的功效Example 10: Efficacy of B7-H3 monoclonal antibody in inhibiting tumor cell growth in vivo
为了测试B7-H3全人源单克隆抗体13725、14479在体内抑制肿瘤细胞生长的功效，使用SCID小鼠在第0天各自皮下注射植入约1×106个人乳腺癌MDA-MB-231细胞，在肿瘤长至均值为50～100(mm3)左右时开始分组给药，连续三周每周给药两次，从给药开始连续三至四周观察测量肿瘤体积均值。动物分为3组(n＝6)：(1)IgG4iso阴性对照，10mg/kg，BIWx3，ip；(2)13725单克隆抗体，10mg/kg，BIWx3，ip；(3)14479单克隆抗体，10mg/kg，BIWx3，ip。In order to test the efficacy of B7-H3 fully human monoclonal antibodies 13725 and 14479 in inhibiting tumor cell growth in vivo, SCID mice were subcutaneously implanted with approximately 1×10 6 human breast cancer MDA-MB-231 cells on day 0. When the tumor grew to an average of about 50 to 100 (mm3), group administration began, and the administration was continued twice a week for three consecutive weeks. The mean tumor volume was observed and measured for three to four consecutive weeks from the start of administration. The animals were divided into 3 groups (n=6): (1) IgG4iso negative control, 10 mg/kg, BIWx3, ip; (2) 13725 monoclonal antibody, 10 mg/kg, BIWx3, ip; (3) 14479 monoclonal antibody, 10 mg/kg, BIWx3, ip.
结果如表14和图13所示，B7-H3人源化单克隆抗体13725，14479以及BM(PC)日性对照均能够有效抑制肿瘤生长，平均肿瘤生长抑制率的计算是Ti或者Vi表示在某一特定时间点处理组或者对照组平均肿瘤体积；T0或V0表示分组后给药前处理组或者对照组平均肿瘤体积)，单克隆抗体13725、14479的肿瘤生长抑制效果均较为理想；其中13725最强，平均肿瘤生长抑制率为41.48％。The results are shown in Table 14 and Figure 13. B7-H3 humanized monoclonal antibodies 13725, 14479 and BM (PC) daily control can effectively inhibit tumor growth. The average tumor growth inhibition rate is calculated as Ti or Vi represents the average tumor volume of the treatment group or control group at a specific time point; T0 or V0 represents the average tumor volume of the treatment group or control group before drug administration after grouping). The tumor growth inhibition effects of monoclonal antibodies 13725 and 14479 are both relatively ideal; among them, 13725 is the strongest, with an average tumor growth inhibition rate of 41.48%.
表14、荷瘤模型体内B7-H3抗体的肿瘤抑制效果
Table 14. Tumor inhibitory effect of B7-H3 antibody in tumor-bearing models
实施例11、B7-H3单克隆抗体制备ADC的体内抑制肿瘤细胞生长的功效Example 11: In vivo efficacy of ADC prepared with B7-H3 monoclonal antibody in inhibiting tumor cell growth
基于B7-H3全人源单克隆抗体13725制备抗体偶联药物ADC，该ADC的结构与第一三共开发的B7-H3 ADC药物DS7300a相似、以本发明的B7-H3全人源单克隆抗体13725作为抗体部分，通过半胱氨酸偶联；DAR是4；Linker是四肽可裂解连接子；阳性对照DS7300a。为了测试该ADC分子在体内抑制肿瘤细胞生长的功效，分别在高表达人B7-H3的人卵巢畸胎瘤细胞(PA-1)和人肺腺癌细胞(NCI-H1975)的小鼠CDX模型上对ADC分子的体内药 效进行考察，同时设置阳性对照DS7300a。Based on B7-H3 fully human monoclonal antibody 13725, an antibody-drug conjugate ADC was prepared. The structure of the ADC is similar to that of the B7-H3 ADC drug DS7300a developed by Daiichi Sankyo. The B7-H3 fully human monoclonal antibody 13725 of the present invention is used as the antibody part, coupled by cysteine; DAR is 4; Linker is a tetrapeptide cleavable linker; positive control DS7300a. In order to test the efficacy of the ADC molecule in inhibiting tumor cell growth in vivo, the in vivo drug-drug interaction of the ADC molecule was performed on the mouse CDX model of human ovarian teratoma cells (PA-1) and human lung adenocarcinoma cells (NCI-H1975) that highly express human B7-H3. The efficacy was investigated and a positive control DS7300a was set up.
CDX模型考察体内药效的实验过程简述如下：使用高度免疫缺陷小鼠在第0天皮下注射约1～5×106个人PA-1或NCI-H1975细胞，在肿瘤长至100～200(mm3)左右时开始分组给药，均是单次剂量注射给药，针对PA-1的注射剂量是4.5mg/kg，针对NCI-H1975的注射剂量是5mg/kg，从给药开始连续2-3周观察测量肿瘤体积均值。The experimental process of investigating the in vivo efficacy of the CDX model is briefly described as follows: using highly immunodeficient mice, approximately 1 to 5×10 6 human PA-1 or NCI-H1975 cells were subcutaneously injected on day 0. When the tumor grew to about 100 to 200 (mm 3 ), the drugs were divided into groups and administered in a single dose. The injection dose for PA-1 was 4.5 mg/kg, and the injection dose for NCI-H1975 was 5 mg/kg. The mean tumor volume was measured and observed for 2-3 consecutive weeks from the start of administration.
动物分为3组(n＝6)：(1)Normal Saline；(2)ADC(基于13725制备的ADC分子)；(3)DS7300a阳性对照。The animals were divided into 3 groups (n=6): (1) Normal Saline; (2) ADC (ADC molecule prepared based on 13725); (3) DS7300a positive control.
结果图14A与图14B所示，在PA-1的CDX模型上单次给药后21天，基于13725的ADC分子对肿瘤的抑制作用明显优于DS7300a；在NCI-H1975的CDX模型上单次给药后11天，基于13725的ADC分子对肿瘤的生长抑制作用明显优于DS7300a。As shown in the results in Figures 14A and 14B, 21 days after a single administration in the CDX model of PA-1, the ADC molecule based on 13725 had a significantly better inhibitory effect on tumor growth than DS7300a; 11 days after a single administration in the CDX model of NCI-H1975, the ADC molecule based on 13725 had a significantly better inhibitory effect on tumor growth than DS7300a.
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。同时，在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。 The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims (17)

  1. 结合免疫调节分子B7-H3的抗体，其具有显著改善的抑制免疫调节分子B7-H3与其受体分子的结合的作用。An antibody that binds to the immunomodulatory molecule B7-H3 has a significantly improved effect of inhibiting the binding of the immunomodulatory molecule B7-H3 to its receptor molecule.
  2. 如权利要求1所述的抗体，其特征在于，该抗体具有轻链可变区和重链可变区，其中，重链CDR1的氨基酸序列如SEQ ID NO：13所示，CDR2的氨基酸序列如SEQ ID NO：14所示，CDR3的氨基酸序列如SEQ ID NO：15所示；轻链CDR1的氨基酸序列如SEQ ID NO：16所示，CDR2的氨基酸序列如SEQ ID NO：17所示，CDR3的氨基酸序列如SEQ ID NO：18所示；或The antibody according to claim 1, characterized in that the antibody has a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 13, the amino acid sequence of CDR2 is shown in SEQ ID NO: 14, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 15; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 16, the amino acid sequence of CDR2 is shown in SEQ ID NO: 17, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 18; or
    重链CDR1的氨基酸序列如SEQ ID NO：19所示，CDR2的氨基酸序列如SEQ ID NO：20所示，CDR3的氨基酸序列如SEQ ID NO：21所示；轻链CDR1的氨基酸序列如SEQ ID NO：22所示，CDR2的氨基酸序列如SEQ ID NO：23所示，CDR3的氨基酸序列如SEQ ID NO：24所示。The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 19, the amino acid sequence of CDR2 is shown in SEQ ID NO: 20, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 21; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 22, the amino acid sequence of CDR2 is shown in SEQ ID NO: 23, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 24.
  3. 如权利要求2所述的抗体，其特征在于，所述抗体的重链可变区具有SEQ ID NO：1所示氨基酸序列，轻链可变区具有SEQ ID NO：3所示氨基酸序列；或，The antibody according to claim 2, characterized in that the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 3; or,
    所述抗体的重链可变区具有SEQ ID NO：5所示氨基酸序列，轻链可变区具有SEQ ID NO：6所示氨基酸序列。The heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 5, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 6.
  4. 如权利要求1所述的抗体，其特征在于，所述抗体包含重链可变区和轻链可变区：The antibody according to claim 1, characterized in that the antibody comprises a heavy chain variable region and a light chain variable region:
    其重链可变区具有SEQ ID NO：7所示的氨基酸序列，轻链可变区具有SEQ ID NO：8所示的氨基酸序列；或，Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7, and its light chain variable region has the amino acid sequence shown in SEQ ID NO: 8; or,
    其重链可变区具有SEQ ID NO：9所示的氨基酸序列，轻链可变区具有SEQ ID NO：10所示的氨基酸序列；或，Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 9, and its light chain variable region has the amino acid sequence shown in SEQ ID NO: 10; or,
    其重链可变区具有SEQ ID NO：11所示的氨基酸序列，轻链可变区具有SEQ ID NO：12所示的氨基酸序列。Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 11, and its light chain variable region has the amino acid sequence shown in SEQ ID NO: 12.
  5. 如权利要求1-4任一所述的抗体，其特征在于，所述抗体包括：单克隆抗体、人类抗体、人源化抗体或嵌合抗体；或The antibody according to any one of claims 1 to 4, characterized in that the antibody comprises: a monoclonal antibody, a human antibody, a humanized antibody or a chimeric antibody; or
    所述抗体包括：Fab、Fab’-SH、Fv、Fd、scFv或(Fab’)2片段；或The antibody comprises: Fab, Fab'-SH, Fv, Fd, scFv or (Fab') 2 fragment; or
    所述抗体为IgG抗体。The antibody is an IgG antibody.
  6. 一种分离的多核苷酸或含有该多核苷酸的构建体，所述多核苷酸编码权利要求1-5任一所述的结合免疫调节分子B7-H3的抗体；较佳地，所述的构建体为表达载体。 An isolated polynucleotide or a construct containing the polynucleotide, wherein the polynucleotide encodes the antibody binding to the immunomodulatory molecule B7-H3 as claimed in any one of claims 1 to 5; preferably, the construct is an expression vector.
  7. 一种抗体表达系统，所述表达系统含有权利要求6中所述的构建体或基因组中整合有外源的权利要求6中所述的多核苷酸；较佳地所述表达系统为细胞。An antibody expression system, said expression system comprising the construct as claimed in claim 6 or the polynucleotide as claimed in claim 6 integrated with an exogenous source in its genome; preferably, said expression system is a cell.
  8. 一种制备权利要求1-5任一所述的抗体的方法，包括：在适合表达所述抗体的条件下，利用权利要求7所述的抗体表达系统进行表达，从而表达出所述的抗体；较佳地，还包括纯化分离出所述抗体。A method for preparing the antibody according to any one of claims 1 to 5, comprising: expressing the antibody using the antibody expression system according to claim 7 under conditions suitable for expressing the antibody, thereby expressing the antibody; preferably, further comprising purifying and isolating the antibody.
  9. 权利要求1-5任一所述的抗体的用途，用于：Use of the antibody according to any one of claims 1 to 5, for:
    制备特异性靶向表达免疫调节分子B7-H3的细胞的抗肿瘤药物，所述药物通过刺激抗原特异性免疫细胞应答抑制肿瘤；Preparation of an anti-tumor drug that specifically targets cells expressing the immunomodulatory molecule B7-H3, wherein the drug inhibits tumors by stimulating antigen-specific immune cell responses;
    制备抗体药物偶联物或免疫缀合物；preparing antibody drug conjugates or immunoconjugates;
    制备双功能或多功能抗体；Preparation of bifunctional or multifunctional antibodies;
    制备嵌合抗原受体修饰的免疫细胞。Preparation of chimeric antigen receptor modified immune cells.
  10. 一种融合蛋白或免疫缀合物，其包括权利要求1-5任一所述的抗体，以及与之操作性连接的功能性分子。A fusion protein or immunoconjugate, comprising the antibody according to any one of claims 1 to 5, and a functional molecule operably linked thereto.
  11. 如权利要求10所述的融合蛋白或免疫缀合物，其特征在于，所述功能性分子包括：抑制肿瘤的分子，靶向肿瘤表面标志物的分子，靶向免疫细胞的表面标志物的分子或可检测标记物，细胞毒素，放射性同位素，生物活性蛋白质，融合伴侣，基于嵌合抗原受体技术的胞外铰链区、跨膜区及胞内信号区，或其组合。The fusion protein or immunoconjugate according to claim 10, characterized in that the functional molecules include: tumor-suppressing molecules, molecules targeting tumor surface markers, molecules or detectable markers targeting surface markers of immune cells, cytotoxins, radioactive isotopes, biologically active proteins, fusion partners, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or a combination thereof.
  12. 如权利要求11所述的免疫缀合物，其特征在于，所述靶向免疫细胞的表面标志物的分子是结合免疫细胞表面标志物的抗体或配体；或The immunoconjugate of claim 11, wherein the molecule targeting the surface marker of an immune cell is an antibody or a ligand that binds to the surface marker of an immune cell; or
    所述的抑制肿瘤的分子是抗肿瘤毒素或抗肿瘤细胞因子；或The tumor-suppressing molecule is an anti-tumor toxin or an anti-tumor cytokine; or
    所述靶向肿瘤表面标志物的分子是结合肿瘤表面标志物的抗体或配体；或The molecule targeting the tumor surface marker is an antibody or a ligand that binds to the tumor surface marker; or
    所述的融合伴侣包括：具有延长体内半衰期作用的蛋白或活性结构域。The fusion partner includes: a protein or an active domain having the function of prolonging the half-life in vivo.
  13. 如权利要求12所述的免疫缀合物，其特征在于，所述结合免疫细胞表面标志物的抗体结合的抗原包括：CD3、CD20、FcRH5、HER2、LYPD1、LY6G6D、PMEL17、LY6E、CD19、CD33、CD22、CD79A、CD79B、EDAR、GFRA1、MRP4、RET、Steap1或TenB2；或The immunoconjugate of claim 12, wherein the antigen bound by the antibody that binds to the immune cell surface marker comprises: CD3, CD20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1 or TenB2; or
    所述结合肿瘤表面标志物的抗体是识别免疫调节分子B7-H3以外的其它抗原的抗体，所述的其它抗原包括：EGFR，EGFRvIII，mesothelin，HER2，EphA2，cMet，EpCAM，MUC1， MUC16，CEA，Claudin 18.2，Claudin 6，WT1，NY-ESO-1，MAGE 3，CD47，ASGPR1或CDH16；或The antibody binding to tumor surface markers is an antibody that recognizes other antigens besides the immunomodulatory molecule B7-H3, and the other antigens include: EGFR, EGFRvIII, mesothelin, HER2, EphA2, cMet, EpCAM, MUC1, MUC16, CEA, Claudin 18.2, Claudin 6, WT1, NY-ESO-1, MAGE 3, CD47, ASGPR1, or CDH16; or
    所述的抗肿瘤细胞因子包括：IL-2、IL-12、IL-15、IFN-beta、TNF-alpha；或The anti-tumor cytokines include: IL-2, IL-12, IL-15, IFN-beta, TNF-alpha; or
    所述抗肿瘤毒素包括：美登素，奥利司他汀，单甲基澳瑞他汀，美登木素生物碱，多拉司他汀，卡奇霉素，氨甲蝶呤，长春地辛，紫杉烷如多烯紫杉醇、紫杉醇、拉洛紫杉醇、特西紫杉醇或欧塔紫杉醇，单端孢霉毒素，CC1065，DXd，Duocarmycin，Calicheamicin，Pyrrolobenzodiazepines或SN-38；或The anti-tumor toxins include: Maytansine, Aulistatin, Monomethyl Auristatin, Maytansinoid, Dolastatin, Calichthyomycin, Methotrexate, Vindesine, Taxanes such as Docetaxel, Paclitaxel, Lalopaclitaxel, Tecitaxel or Otataxel, Trichothecenes, CC1065, DXd, Duocarmycin, Calicheaamicin, Pyrrolobenzodiazepines or SN-38; or
    所述具有延长体内半衰期作用的蛋白或活性结构域包括：免疫球蛋白Fc区、优选人免疫球蛋白Fc区，血清白蛋白或其片段。The protein or active domain having the effect of prolonging the half-life in vivo includes: immunoglobulin Fc region, preferably human immunoglobulin Fc region, serum albumin or a fragment thereof.
  14. 一种药物组合物，包括权利要求1-5任一所述的抗体、权利要求9-13任一所述的融合蛋白或免疫缀合物；较佳地，所述的药物组合物中还包括药学上可接受的载体。A pharmaceutical composition comprising the antibody according to any one of claims 1 to 5, the fusion protein or the immunoconjugate according to any one of claims 9 to 13; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  15. 权利要求1-5任一所述的抗体、权利要求9-13任一所述的融合蛋白或免疫缀合物、或含有它们的药物组合物在制备用于抑制肿瘤或刺激抗原特异性免疫细胞应答的制剂、试剂盒或药盒中的用途。Use of the antibody according to any one of claims 1 to 5, the fusion protein or immunoconjugate according to any one of claims 9 to 13, or a pharmaceutical composition containing them in the preparation of a preparation, a kit or a drug kit for inhibiting tumors or stimulating antigen-specific immune cell responses.
  16. 如权利要求15所述的用途，其特征在于，所述肿瘤包括实体瘤；较佳地，所述肿瘤包括表达免疫调节分子B7-H3的肿瘤；较佳地，所述肿瘤包括：卵巢癌，乳腺癌，中枢神经肿瘤，头颈部肿瘤，膀胱癌，直肠癌，胰腺癌，非小细胞肺癌，肾脏肿瘤。The use as claimed in claim 15, characterized in that the tumor includes a solid tumor; preferably, the tumor includes a tumor expressing the immunomodulatory molecule B7-H3; preferably, the tumor includes: ovarian cancer, breast cancer, central nervous system tumors, head and neck tumors, bladder cancer, rectal cancer, pancreatic cancer, non-small cell lung cancer, and kidney tumors.
  17. 一种试剂盒或药盒，包括权利要求1-5任一所述的抗体、权利要求9-13任一所述的融合蛋白或免疫缀合物、或含有它们的药物组合物。 A test kit or a medicine kit, comprising the antibody according to any one of claims 1 to 5, the fusion protein or the immunoconjugate according to any one of claims 9 to 13, or a pharmaceutical composition containing them.
PCT/CN2024/071909 2023-01-12 2024-01-12 Binding protein of immunoregulation protein molecule b7-h3 and use thereof WO2024149346A1 (en)

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