WO2023236844A1 - 靶向her2和pd-l1的双特异性抗体及其制备方法和应用 - Google Patents

靶向her2和pd-l1的双特异性抗体及其制备方法和应用 Download PDF

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WO2023236844A1
WO2023236844A1 PCT/CN2023/097835 CN2023097835W WO2023236844A1 WO 2023236844 A1 WO2023236844 A1 WO 2023236844A1 CN 2023097835 W CN2023097835 W CN 2023097835W WO 2023236844 A1 WO2023236844 A1 WO 2023236844A1
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seq
bispecific antibody
sequence shown
polypeptide chain
antibody
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PCT/CN2023/097835
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French (fr)
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刘婵娟
郎国竣
符智祥
许彩云
司远青
曹静丽
张震
闫闰
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三优生物医药(上海)有限公司
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    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • 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
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    • 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
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    • 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
    • A61K47/6851—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 determinant of a tumour cell
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • 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
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    • C12N15/09—Recombinant DNA-technology
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a bispecific antibody targeting HER2 and PD-L1 and its preparation method and application.
  • Human epidermal growth factor receptor 2 (human epidermal growth factor receptor 2, HER2) belongs to the epidermal growth factor receptor (Epidermal growth factor receptor, EGFR) protein family, which can be expressed through heterodimerization with other members of the EGFR family or due to its Homodimerization caused by its own overexpression causes autophosphorylation of tyrosine residues on the cytoplasmic domain and initiates a series of signaling pathways for cell proliferation and tumorigenesis (Oh, D.Y. and Y.J.Bang. (2020). "HER2-targeted therapeutics-a role beyond breast cancer.” Nat Rev Clin Oncol 17(1):33-48.). HER2 is only expressed at low levels in a very small number of normal tissues.
  • EGFR epidermal growth factor receptor
  • HER2 is overexpressed in cancer types such as breast cancer, gastric cancer, ovarian cancer, and endometrial cancer, and its overexpression is related to tumor malignancy and poor prognosis (Normanno, N., et al. (2005). "The ErbB receptors and their ligands in cancer:an overview.” Curr Drug Targets 6(3):243-257.).
  • HER2 Based on the expression characteristics and biological functions of HER2 in tumor development and development, it has become a widely verified target for tumor treatment, and multiple monoclonal antibody drugs targeting HER2 have been approved for marketing.
  • less than 30% of patients respond to it, and most patients who respond initially develop treatment resistance or relapse within a year (Gu, C.L., et al. (2021). "Bispecific antibody simultaneously targeting PD1 and HER2 inhibits tumor growth via direct tumor cell killing in combination with PD1/PDL1 blockade and HER2 inhibition.”Acta Pharmacol Sin.).
  • PD-L1 is a member of the immunoglobulin superfamily and a type I transmembrane glycoprotein expressed in macrophages, some activated T cells and B cells, DCs and some epithelial cells (Sharpe, A.H., et al. ( 2007). "The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection.” Nat Immunol 8(3):239-245.).
  • PD-L1 is also highly expressed by tumor cells to escape adaptive anti-tumor immune responses (Ohaegbulam, K.C., et al. (2015). "Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway.” Trends Mol Med 21(1):24-33.).
  • up-regulation of PD-L1 expression can directly inhibit the anti-tumor response of T cells through the PD-1 signaling pathway and mediate immune escape of tumor cells.
  • blocking antibody drugs targeting the PD-1/PD-L1 signaling pathway has attracted widespread attention.
  • Multiple monoclonal antibody drugs targeting PD-1 or PD-L1 have been approved for marketing and are in clinical use. Achieved exciting results.
  • most patients do not respond to immunotherapy with PD-1/PD-L1 blockade, and some patients who initially respond also develop treatment tolerance.
  • Bispecific antibodies are recombinant antibodies that can simultaneously bind to two antigens or different epitopes of the same antigen. They can not only redirect immune effector cells to tumor tissue to achieve specific killing of tumors by immune cells, but can also inhibit the same tumor at the same time. Different tumor-promoting signaling pathways on cells then exert synergistic tumor-suppressing functions (Labrijn, A.F., et al. (2019). "Bispecific antibodies: a mechanistic review of the pipeline.” Nat Rev Drug Discov 18(8):585-608 .). Therefore, in order to improve the treatment response rate of HER2-positive tumors and overcome tumor treatment resistance, there is an urgent need to develop bispecific antibodies targeting HER2 and PD-L1.
  • the present invention provides a bispecific antibody targeting HER2 and PD-L1 and its preparation method and application.
  • the bispecific antibody of the present invention has better physical and chemical properties, better purity and thermal stability; it can retain the functional activity against dual targets, and its binding activity for both targets is close to that of its corresponding monoclonal antibody ( That is, it binds to both PD-L1 and HER2 and has a binding ability that is equivalent to or better than that of a single monoclonal antibody), the activity of blocking PD-1/PD-L1 is equivalent to that of the corresponding monoclonal antibody, and the activity of inhibiting SK-BR-3 cell proliferation is equivalent to that of the corresponding monoclonal antibody.
  • the corresponding monoclonal antibodies are comparable; at the same time, it shows good ADCC activity, which is significantly better than its corresponding monoclonal antibodies.
  • One aspect of the present invention provides a bispecific antibody, which includes a first protein functional region targeting HER2 and a second protein functional region targeting PD-L1; wherein the first protein functional region includes a light chain variable Region (VL) and heavy chain variable region (VH), and the VL includes three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the sequence shown in SEQ ID NO:3, and the VH includes the three complementarity determining regions LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:3 The three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the sequence shown in NO:4;
  • VL light chain variable Region
  • VH heavy chain variable region
  • the second protein functional region includes VHH, wherein the VHH includes three complementarity determining regions CDR1, CDR2 and CDR3 contained in the sequence shown in SEQ ID NO:2.
  • the VL comprises the sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity with it ;
  • the VH includes the sequence shown in SEQ ID NO: 4, or an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.
  • the VHH comprises the sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto .
  • the first protein functional region preferably includes Fab
  • the Fab is operably connected to the VHH, for example, the VHH is connected to the N-terminal of the light chain variable region, or the N-terminal of the heavy chain variable region, or the C-terminal of the light chain constant region in the Fab. end, or the Fab and the VHH are respectively operably connected to the N-termini of the two hinge regions of the Fc region.
  • the first protein functional region is preferably IgG, and in a certain preferred embodiment, the VHH is operably connected to the C-terminus of the IgG heavy chain constant region.
  • the bispecific antibody as described above preferably contains two first polypeptide chains and two second polypeptide chains.
  • the first polypeptide chain is represented by the following formula:
  • VH-CH1-hinge region-CH2-CH3 preferably comprising the sequence shown in SEQ ID NO:9,
  • VHH-linker-VL-CL preferably contains the sequence shown in SEQ ID NO:10.
  • the first polypeptide chain is represented by the following formula:
  • VH-CH1-hinge region-CH2-CH3-linker-VHH preferably comprising the sequence shown in SEQ ID NO:6,
  • VL-CL preferably contains the sequence shown in SEQ ID NO:7.
  • the first polypeptide chain is represented by the following formula:
  • VHH-linker-VH-CH1-hinge region-CH2-CH3 preferably comprising the sequence shown in SEQ ID NO:8,
  • VL-CL preferably contains the sequence shown in SEQ ID NO:7.
  • the first polypeptide chain is represented by the following formula:
  • VH-CH1-hinge region-CH2-CH3 preferably comprising the sequence shown in SEQ ID NO:9,
  • VL-CL-linker-VHH preferably contains the sequence shown in SEQ ID NO:11.
  • the bispecific antibody as described above preferably contains a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein the first polypeptide chain is represented by the following formula:
  • the second polypeptide chain is represented by the following formula:
  • the third polypeptide chain is represented by the following formula:
  • the first polypeptide chain includes the sequence shown in SEQ ID NO:12
  • the second polypeptide chain includes the sequence shown in SEQ ID NO:13
  • the third polypeptide chain includes the sequence shown in SEQ ID NO:7.
  • the first polypeptide chain includes the sequence shown in SEQ ID NO:14
  • the second polypeptide chain includes the sequence shown in SEQ ID NO:15
  • the third polypeptide chain includes the sequence shown in SEQ ID NO:7.
  • the invention also provides an isolated nucleic acid encoding a bispecific antibody according to the invention.
  • the present invention also provides a recombinant expression vector comprising the isolated nucleic acid according to the present invention.
  • the present invention also provides a transformant comprising the isolated nucleic acid according to the present invention or the recombinant expression vector according to the present invention.
  • the host cell of the transformant is preferably a prokaryotic cell or a eukaryotic cell
  • the prokaryotic cell is preferably E.coli cells such as TG1, BL21
  • the eukaryotic cell is preferably HEK293 cells or CHO cells .
  • the expression vector can be transfected or introduced into a suitable host cell.
  • a variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques.
  • protoplast fusion cells are grown in culture medium and screened for appropriate activity. Methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on the present description and methods known from the prior art, depending on the specific expression vector used and Mammalian host cell changes or optimizations.
  • cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers may, for example, provide prototrophy, biocidal resistance (eg, antibiotics), or heavy metal (eg, copper) resistance to an auxotrophic host, etc.
  • the selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional components may also be required for optimal synthesis of mRNA. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.
  • the present invention also provides a method for preparing a bispecific antibody, which includes culturing the transformant according to the present invention and obtaining the bispecific antibody from the culture.
  • the present invention also provides a pharmaceutical composition, which includes the bispecific antibody according to the present invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition also includes other anti-tumor antibodies as active ingredients, and/or consists of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents , one or more from the group consisting of oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
  • the pharmaceutical composition or pharmaceutical preparation of the present invention includes a suitable pharmaceutically acceptable carrier such as a pharmaceutical excipient.
  • a suitable pharmaceutically acceptable carrier such as a pharmaceutical excipient.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • Pharmaceutical carriers suitable for use in the present invention can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When administering pharmaceutical compositions intravenously, water is the preferred carrier.
  • Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dry skim milk, glycerin , propylene, glycol, water, ethanol, etc.
  • excipients and their uses see also "Handbook of Pharmaceutical Excipients", 5th edition, RC Rowe, PJ Seskey and SCOwen, Pharmaceutical Press, London, Chicago.
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. It can be prepared by mixing the antibody of the invention or its antigen-binding fragment with the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980))
  • a pharmaceutical preparation or pharmaceutical composition comprising the invention is preferably in the form of a lyophilized preparation or aqueous solution.
  • compositions or preparations of the present invention may also contain more than one active ingredient required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other.
  • active ingredients such as other antibodies, anti-infective active agents, small molecule drugs or immunomodulators, etc.
  • the active ingredients are suitably present in combination in amounts effective for the intended use.
  • Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibodies of the invention or antigen-binding fragments thereof in the form of shaped articles, such as films or microcapsules.
  • the present invention also provides an antibody drug conjugate, which includes a cytotoxic agent or a label, and the bispecific antibody of the present invention; the cytotoxic agent is preferably MMAF or MMAE; the label is preferably As a fluorescent agent.
  • the present invention also provides a kit, which includes the bispecific antibody according to the present invention, the pharmaceutical composition according to the present invention, or the antibody-drug conjugate according to the present invention.
  • the kit preferably further includes (i) a device for administering the bispecific antibody or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.
  • the invention also provides a medicine box set, which includes medicine box A and medicine box B, wherein:
  • the kit A contains the bispecific antibody according to the present invention, the pharmaceutical composition according to the present invention, or the antibody-drug conjugate according to the present invention.
  • the kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and/or consists of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapy agents, One or more from the group consisting of oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
  • the present invention also provides a method for detecting specific antigens for non-diagnostic purposes, which includes using the bispecific antibody of the present invention for detection.
  • the application scenarios for non-diagnostic purposes may be routine in the art, such as detecting antigens or studying competitive binding of antibodies in a laboratory.
  • the invention also provides a bispecific antibody according to the invention, a nucleic acid according to the invention, a recombinant expression vector according to the invention, a transformant according to the invention, and Application of pharmaceutical compositions, antibody-drug conjugates according to the present invention, kits according to the present invention or kits according to the present invention in the preparation of drugs for diagnosing, preventing and/or treating tumors.
  • the tumor is preferably a tumor associated with HER2 and/or PD-L1.
  • Such tumors include, for example, breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, squamous cell carcinoma of the head and neck, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction Adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing's sarcoma, rhabdomyosarcoma, neuroblastoma, or small cell lung cancer.
  • the present invention also provides a method for diagnosing, treating and/or preventing HER2 and/or PD-L1 mediated tumors, the method comprising administering to a patient in need a therapeutically effective amount of a bispecific as described in the present invention.
  • Antibody, nucleic acid according to the present invention, recombinant expression vector according to the present invention, transformant according to the present invention, pharmaceutical composition according to the present invention or antibody drug conjugate according to the present invention Treat patients in need.
  • the tumor is preferably breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, squamous cell carcinoma of the head and neck, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, Gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing's sarcoma, rhabdomyosarcoma, neuroblastoma, or small cell lung cancer.
  • bispecific antibodies Compared with monotherapy, the bispecific antibodies provided by the present invention target HER2 and PD-L1.
  • Bispecific antibodies have multiple advantages: on the one hand, they can synergistically exert the dual anti-tumor activities of ADCC and PD-1/PD-L1, improve On the other hand, it can improve the targeting of tumor treatment and reduce the toxic and side effects on HER2 or PD-L1 positive normal tissues.
  • the bispecific antibody of the present invention has better physical and chemical properties, better purity and thermal stability; it can retain the functional activity against dual targets and has equal binding activity against both targets. It is close to its corresponding monoclonal antibody (that is, it binds to PD-L1 and binds to HER2 and has the same or better binding ability as a single monoclonal antibody), the activity of blocking PD-1/PD-L1 is equivalent to that of the corresponding monoclonal antibody, and it inhibits SK -BR-3 cell proliferation activity is comparable to that of the corresponding monoclonal antibody; it also exhibits good ADCC activity and in vivo tumor inhibitory activity.
  • Figures 1A-1E show schematic structures of candidate bispecific antibodies.
  • Figure 2A- Figure 2F are SEC-HPLC monomer detection profiles of candidate bispecific antibodies.
  • Figures 3A-3B show the binding activity of candidate bispecific antibodies to recombinant human PD-L1-His protein.
  • Figure 4 shows the binding activity of candidate bispecific antibodies to huPD-L1-CHO-S cells.
  • Figures 5A-5B show the binding activity of candidate bispecific antibodies to recombinant human HER2-His protein.
  • Figure 6 shows the binding activity of candidate bispecific antibodies to SK-BR-3 cells.
  • Figure 7 shows the blocking activity of candidate bispecific antibodies on the PD-1/PD-L1 signaling pathway detected by FACS method.
  • Figures 8A-8C show the blocking activity of candidate bispecific antibodies on the PD-1/PD-L1 pathway detected by luciferase reporter gene method.
  • Figure 9 shows antibody-dependent cell-mediated cytotoxicity (ADCC) of candidate bispecific antibodies on SK-BR-3 cells.
  • Figure 10 shows the proliferation inhibitory effect of candidate bispecific antibodies on SK-BR-3 cells.
  • Figures 11A-11B show the inhibitory effect of candidate bispecific antibodies on tumor growth in a mouse subcutaneous xenograft tumor model.
  • the amino acid sequences of the CDRs listed above are all shown in accordance with the AbM definition rules.
  • the CDRs of an antibody can be defined by a variety of methods in the art, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al.
  • CDR complementarity determining region
  • the protein functional region can be in the form of full-length antibody, scFv, Fab, VHH, etc., and can be selected according to the actual context.
  • full-length antibody is used interchangeably to refer to a glycoprotein containing at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH in the present invention) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region (abbreviated as CL in the present invention).
  • the light chain constant region consists of one domain, CL.
  • Mammalian heavy chains are classified as alpha, delta, epsilon, gamma and mu.
  • Mammalian light chains are classified as lambda or kappa.
  • Immunoglobulins containing alpha, delta, epsilon, gamma and mu heavy chains are classified as immunoglobulins IgA, IgD, IgE, IgG and IgM.
  • Complete antibodies form a "Y" shape.
  • the stem of Y consists of the second and third constant regions (and for IgE and IgM, the fourth constant region) of the two heavy chains held together, with disulfide bonds (interchain) formed in the hinge.
  • Heavy chains ⁇ , ⁇ , and ⁇ have a constant region composed of three tandem (in a row) Ig domains, and a hinge region for increased flexibility; heavy chains ⁇ and ⁇ have a constant region composed of four immunoglobulin domains. district.
  • the second and third constant regions are called "CH2 domain” and "CH3 domain” respectively.
  • Each arm of Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • "Fab fragment” consists of the CH1 and variable regions of a light chain and a heavy chain.
  • the heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.
  • the "Fc region” contains two heavy chain fragments comprising the CH2 and CH3 domains of the antibody.
  • the two heavy chain segments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • a "Fab' fragment” contains a light chain and a portion of a heavy chain including the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby between the two heavy chains of two Fab' fragments Interchain disulfide bonds are formed to form F(ab')2 molecules.
  • An "F(ab')2 fragment” contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains.
  • the F(ab')2 fragment therefore consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • Single chain antibody fragment is an antibody composed of an antibody heavy chain variable region and a light chain variable region connected by a short peptide (linker) of 15 to 20 amino acids.
  • the term "VHH” is also known as a single domain antibody, which is an antibody consisting of only one heavy chain variable region, and contains only one chain from the C-terminus to the N-terminus, FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1 , also known as "nanobody”.
  • Single domain antibodies are the smallest units currently known that can bind to a target antigen.
  • vector means a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression encapsulated in liposomes vectors as well as certain eukaryotic cells, such as producer cells.
  • host cell in the present invention may include cells into which exogenous nucleic acid has been introduced, including progeny of these cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cells and progeny derived therefrom, regardless of passage number.
  • the progeny may not be identical in nucleic acid content to the parent cells, but may contain mutations.
  • the present invention includes in the initially transformed cells Cells are screened or selected for mutant progeny that have the same function or biological activity.
  • the DNA coding sequences of recombinant human PD-L1 (UniProtKB-Q9NZQ7), PD-1 (UniProtKB-Q15116) and HER2 (UniProt NO-P04626) proteins were obtained through total gene synthesis by General Biotechnology Co., Ltd.
  • the target fragment was amplified by PCR and a His tag was introduced at the C-terminus of the coding sequence through primers, and the target fragment was constructed into the eukaryotic expression vector pcDNA3.4-TOPO (Invitrogen) using homologous recombination.
  • the constructed recombinant protein expression plasmid was transformed into Escherichia coli DH5 ⁇ competent cells, cultured at 37°C overnight, and the plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01).
  • Recombinant human PD-L1-His protein, recombinant human PD-1-His protein and recombinant human HER2-His protein were all prepared through the Expi293 transient expression system (ThermoFisher, A14635).
  • the transient expression method please refer to the Expi293 TM Expression System USER GUIDE. Seven days after transfection, the cell suspension was collected and centrifuged at 15,000 g for 10 min. The expression supernatant was affinity purified using Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050). The target protein was eluted with a gradient concentration of imidazole solution. . Each eluted protein was replaced into PBS buffer through ultrafiltration concentration tubes (Millipore, UFC901096). After passing the SDS-PAGE identification and activity test, aliquot and freeze at -80°C.
  • the anti-human PD-L1 antibody D21-4 (VHHPD-L1-huFc, the amino acid sequence is shown in SEQ ID NO: 1) is derived from patent application WO2021083335A1; the anti-human PD-L1 antibody Avelumab is derived from DrugBank ( Query No. DB11945); the anti-human HER2 antibody is Trastuzumab from DrugBank (Query No. DB00072).
  • Control antibodies were expressed using the transient transfer system (ExpiCHO).
  • ExpiCHO transient transfer method
  • the cell suspension was centrifuged at high speed and the supernatant was collected.
  • the supernatant was filtered through a 0.22 ⁇ m filter membrane and purified by affinity chromatography using a Protein A/G column.
  • the target protein is eluted with 100mM glycine hydrochloric acid (pH 3.0), concentrated, buffer exchanged, aliquoted, identified by SDS-PAGE and activity tested, and then stored in storage for cryopreservation.
  • This example describes the structure of an exemplary anti-HER2 & PD-L1 bispecific antibody (BsAb) and the construction of an expression vector.
  • VHH domain (VHH PD-L1 ) amino acid sequence of the anti-human PD-L1 antibody is from D21-4, and its variable region amino acid sequence is shown in SEQ ID NO: 2; anti-HER2
  • the antibody amino acid sequence comes from trastuzumab, and its light and heavy chain variable region amino acid sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively; the linker amino acid sequence is GGGGSGGGGSGGGGS (SEQ ID NO: 5).
  • Exemplary BsAb constructs are shown in Table 1 and the corresponding amino acid sequences are provided in Table 2.
  • Construct BsAb1 Contains two identical first polypeptide chains, which comprise the heavy chain variable region of trastuzumab, the human IgG1 heavy chain constant region, and the linker from the N terminus to the C terminus. and the VHH domain of an anti-human PD-L1 antibody; containing two identical second polypeptide chains, which comprise the light chain variable region and kappa of trastuzumab from the N-terminus to the C-terminus. Light chain constant region.
  • BsAb1 has the form shown in Figure 1A.
  • Construct BsAb2 Contains two identical first polypeptide chains, which comprise the VHH domain of the anti-human PD-L1 antibody and the heavy chain variable of trastuzumab from the N-terminus to the C-terminus. Region, human IgG1 heavy chain constant region; contains two identical second polypeptides chain, the second polypeptide chain comprising the light chain variable region and the kappa light chain constant region of trastuzumab from the N terminus to the C terminus.
  • BsAb2 has the form shown in Figure 1B.
  • Construct BsAb3 contains two identical first polypeptide chains, which comprise the heavy chain variable region of trastuzumab and the human IgG1 heavy chain constant region domain from the N terminus to the C terminus; Contains two identical second polypeptide chains, which comprise the VHH domain of the anti-human PD-L1 antibody, the linker, and the light chain variable region of trastuzumab from the N-terminus to the C-terminus. and kappa light chain constant region.
  • BsAb3 has the form shown in Figure 1C.
  • Construct BsAb4 contains two identical first polypeptide chains, which comprise the heavy chain variable region of trastuzumab and the human IgG1 heavy chain constant region domain from the N terminus to the C terminus; Containing two identical second polypeptide chains, the second polypeptide chain includes the light chain variable region of trastuzumab, the kappa light chain constant region, the linker and the anti-human PD- VHH domain of L1 antibody.
  • BsAb4 has the form shown in Figure ID.
  • Construct BsAb5 Contains three different polypeptide chains, namely the first polypeptide chain, the second polypeptide chain and the third polypeptide chain; the first polypeptide chain contains anti-human PD-L1 from the N terminus to the C terminus.
  • BsAb5 has the form shown in Figure 1E.
  • Construct BsAb6 Contains three different polypeptide chains, namely the first polypeptide chain, the second polypeptide chain and the third polypeptide chain; the first polypeptide chain contains anti-human PD-L1 antibody from N-terminus to C-terminus VHH domain, human IgG1 heavy chain hinge region and human IgG1 heavy chain Fc (Hole); wherein the second polypeptide chain contains the heavy chain variable region of trastuzumab and human IgG1 heavy chain from N terminus to C terminus Constant region (Knob); the third polypeptide chain contains the light chain variable region and the kappa light chain constant region of trastuzumab from the N-terminus to the C-terminus.
  • BsAb6 has the form shown in Figure 1E.
  • fragments of each antibody variable region and constant region were amplified by PCR, connected by overlap extension PCR, and then constructed into modified eukaryotic expression vector plasmids through homologous recombination.
  • pcDNA3.4-TOPO Invitrogen
  • a complete construct polypeptide chain expression vector was formed.
  • the constructed vectors containing the polypeptide chain coding sequence of the construct were transformed into E. coli DH5 ⁇ and cultured at 37°C overnight.
  • the endotoxin-free plasmid extraction kit (OMEGA, D6950-01) was used to extract the plasmid, and the endotoxin-free construct polypeptide chain expression plasmid was obtained for eukaryotic expression.
  • Example 2 The construct of Example 2 was expressed through the ExpiCHO transient expression system (Thermo Fisher, A29133). The specific operation was as follows: On the day of transfection, it was confirmed that the cell density was approximately 7 ⁇ 10 6 to 1 ⁇ 10 7 viable cells/mL. The survival rate is >98%. At this time, use fresh ExpiCHO expression medium pre-warmed at 37°C to adjust the cells to a final concentration of 6 ⁇ 10 cells/mL.
  • OptiPROTM SFM Dilute the target plasmid with OptiPROTM SFM pre-cooled at 4°C (add 1 ⁇ g plasmid to 1 mL of the medium), and at the same time, dilute ExpiFectamineTMCHO with OptiPROTMSFM, mix the two in equal volumes and mix gently by pipetting to prepare ExpiFectamineTMCHO/plasmid DNA.
  • the mixed solution was incubated at room temperature for 5 minutes, slowly added to the prepared cell suspension while shaking gently, and finally placed in a cell culture shaker and cultured at 37°C and 8% CO2 .
  • the concentration of the purified bispecific antibody obtained in Example 3.1 was measured using an ultra-trace spectrophotometer (Hangzhou Aosheng Instrument Co., Ltd., Nano-300), and the measured A280 reading was divided by the theoretical extinction coefficient of the antibody. As the antibody concentration for subsequent research, after passing the quality inspection, aliquot and store at -80°C.
  • Material preparation 1. Mobile phase: 150mmol/L phosphate buffer, pH 7.4; 2. Sample preparation: Candidate bispecific antibodies are diluted to 0.5mg/mL with mobile phase solution.
  • the Agilent HPLC 1100 column (XBridge BEH SEC 3.5 ⁇ m.8mm I.D. ⁇ 30cm, Waters) flow rate was set to 0.8mL/min, the injection volume was 20 ⁇ L, and the VWD detector wavelengths were 280nm and 214nm.
  • the SEC-HPLC results of the candidate bispecific antibodies are as follows: The percentages of high molecular polymers, antibody monomers and low molecular substances in the sample were calculated according to the area normalization method. The results are shown in Figures 2A-2F and Table 3, from which it can be seen that the candidate bispecific antibodies The monomer purity of antibodies BsAb1, BsAb2, BsAb3 and BsAb4 was all greater than 90%.
  • Differential scanning fluorescence can provide information about the structural stability of a protein based on the fluorescence change process in the protein map, detect the conformational changes of the protein, and obtain the melting temperature (Tm) of the protein.
  • Tm melting temperature
  • the candidate bispecific antibody solution was prepared at 0.2 mg/mL. Each test product was added into a 96-well plate (Nunc) at 19 ⁇ L/well. Three parallel wells were set up, and PBS and trastuzumab were used as references. Then add 1 ⁇ L of SYPRO orange dye with a concentration of 100 ⁇ to each well, mix well, and prepare for use on the machine.
  • the thermal stability test of the sample uses the ABI 7500FAST RT-PCR instrument. The test type selects the melting curve and adopts the continuous mode. The scanning temperature range is 25 ⁇ 95°C, the heating rate is 1%, and the equilibrium is 5 minutes at 25°C.
  • the reaction volume is 20 ⁇ L, and the temperature corresponding to the first peak and valley of the first derivative of the melting curve is determined as the melting temperature Tm of the antibody.
  • the experimental results are shown in Table 3.
  • the Tm of the candidate bispecific antibodies are all greater than 60°C, so they have good thermal stability.
  • Recombinant human PD-L1-His protein was coated on a 96-well ELISA plate and kept overnight at 4°C. The next day, the well plate was washed three times with PBST and blocked with 5% skim milk for 2 hours. After washing the plate three times with PBST, candidate bispecific antibodies and control antibody D21-4 at different concentrations were added and incubated for 1 hour. Afterwards, the cells were washed three times with PBST and the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 hour.
  • TMB PurModics, TMBS-1000-01
  • 2M HCl was added to stop the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM TM (GraphPad Software, San Diego, CA), and EC50 values were calculated.
  • huPD-L1-CHO-S cells in the exponential growth phase (a stably transfected cell line overexpressing human PD-L1 obtained by stably transfecting PD-L1 (Gene ID: 29126) into CHO-S (Thermo, A1461801)), The cells were resuspended in FACS buffer (PBS containing 1% BSA), counted, and the density of the cell suspension was adjusted to 2 ⁇ 10 6 cells/mL. Subsequently, huPD-L1-CHO-S cells were added into a 96-well round-bottom plate at 100 ⁇ L per well, and the supernatant was removed by centrifugation.
  • FACS buffer PBS containing 1% BSA
  • the FACS binding assay results are shown in Figure 4 and Table 4.
  • the candidate bispecific antibodies all showed comparable binding ability to PD-L1 expressed on cells as the control antibody D21-4, and among them, the bispecific antibodies BsAb2 and BsAb3 has the strongest binding ability.
  • Recombinant human HER2-His protein was coated on a 96-well ELISA plate and kept overnight at 4°C. The next day, the well plate was washed three times with PBST and blocked with 5% skim milk for 2 h. After the plate was washed three times with PBST, different concentrations of candidate bispecific antibodies and control antibody trastuzumab were added and incubated for 1 h. Afterwards, wash with PBST three times, add the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab97225) and incubate for 1 h.
  • TMB PurModics, TMBS-1000-01
  • 2M HCl was added to stop the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM TM (GraphPad Software, San Diego, CA), and EC50 values were calculated.
  • the ELISA binding assay results are shown in Figures 5A-5B and Table 4.
  • the candidate bispecific antibodies all showed comparable binding ability to HER2 as the control antibody trastuzumab.
  • SK-BR-3 cells ATCC, HTB-30
  • FACS buffer PBS containing 1% BSA
  • SK-BR-3 cells were added into a 96-well round-bottom plate at 100 ⁇ L per well, and the supernatant was removed by centrifugation.
  • the results of the FACS binding assay are shown in Figure 6 and Table 4.
  • the bispecific antibodies BsAb1, BsAb2 and BsAb4 showed comparable binding ability to HER2 expressed on cells as the control antibody trastuzumab.
  • the recombinant human HER2-His or PD-L1-His protein as the antigen was diluted to 10 ⁇ g/mL in 10 ⁇ KB buffer (10 ⁇ PBS containing 1% BSA, 0.5% Tween 20), and the candidate bispecific antibody and control antibody were D21-4 or trastuzumab was diluted 2-fold in 10 ⁇ KB buffer, in order of 100, 50, 25, and 0 nM.
  • 10 ⁇ KB buffer 10 ⁇ PBS containing 1% BSA, 0.5% Tween 20
  • the candidate bispecific antibody and control antibody were D21-4 or trastuzumab was diluted 2-fold in 10 ⁇ KB buffer, in order of 100, 50, 25, and 0 nM.
  • pre-wet the sensor Anti-Penta-HIS, HIS1K, Fortebio, CA
  • start testing the sample plate (GreinerBio, PN655209) after at least 10 minutes. After the test is correct, proceed according to the preset program.
  • This example uses the FACS method to detect the blocking activity of candidate bispecific antibodies against PD-1/PD-L1.
  • the specific method is as follows: collect the cultured huPD-L1-CHO-S cells, and use the prepared FACS buffer to Resuspend the cell suspension, count and adjust the cell suspension density to 2 ⁇ 10 6 cells/mL. Add huPD-L1-CHO-S cells to a 96-well round bottom plate at 100 ⁇ L per well, centrifuge to remove the supernatant, and then add different concentrations of candidate bispecific antibodies, control antibody D21-4, and human IgG1 isotype antibody (isotype (Control), resuspend the cells and incubate at 4°C for 30 minutes.
  • the FACS binding assay results are shown in Figure 7 and Table 6.
  • the bispecific antibodies BsAb2 and BsAb3 showed comparable blocking activity to PD-1/PD-L1 as the control antibody D21-4.
  • This example uses a luciferase reporter gene system to detect the activity of candidate anti-HER2&PD-L1 bispecific antibodies in blocking the PD-1/PD-L1 signaling pathway.
  • the specific method is as follows: take the logarithmic phase PD-1-NF-AT- Jurkat cells (Jurkat cells Stably express PD-1 (UniProtKB-Q15116) and luciferase) and CD3L-PD-L1-CHO cells (stably express PD-L1 (UniProtKB-Q9NZQ7) and anti-CD3-scFv (sequence derived from OKT3) in CHO cells )), mix the two cells at a ratio of 5:1 and make their densities 4 ⁇ 10 7 cells/mL and 8 ⁇ 10 7 cells/mL respectively.
  • the blocking activity test results are as follows: As shown in Figures 8A-8C, the bispecific antibodies BsAb2 and BsAb3 both showed comparable PD-1/PD-L1 blocking activity to the control antibody D21-4.
  • This example uses a luciferase reporter gene system to detect the ADCC activity of candidate anti-HER2&PD-L1 bispecific antibodies.
  • the specific method is as follows: logarithmic phase SK-BR-3 cells and CD16a(V158)-NF-AT-Jurkat cells are taken (Stably transfected CD16a (V158) sequence (UniProtKB-P08637) and pGL4.30 plasmid (promega, #E8481) containing the NF-AT-re nucleic acid sequence into Jurkat cells
  • stably transduced cell lines please refer to the reference Parekh, BS, et al. (2012).
  • the ADCC test results are as follows: As shown in Figure 9, the bispecific antibody BsAb2 showed significantly better ADCC activity than the control antibody trastuzumab.
  • the results of the proliferation inhibitory activity test are as follows: As shown in Figure 10, the bispecific antibody BsAb2 showed comparable inhibitory activity to SK-BR-3 cell proliferation as the control antibody trastuzumab.
  • mice Female NCG mice (purchased from Jiangsu Jicui, strain: T001475) aged 6 to 8 weeks were used. The experimental mice were kept in independent ventilation boxes with constant temperature and humidity. The temperature of the breeding room was 21-24°C and the humidity was 30-53%. Stable transfection of overexpressed human PD-L1 by huPD-L1-NCI-N87 cells (stably transfected with PD-L1 (Gene ID: 29126) into NCI-N87 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu130) Cell line) was injected subcutaneously on the right back of each mouse at 1 ⁇ 10 7 cells (day 0).
  • each administration group is set at a dose of 35nM/kg (corresponding to a monoclonal antibody of 5mpk and a double antibody of 6.1mpk).
  • the bispecific antibody BsAb2 administration group is also set at a dose of 87.5nM/kg (corresponding to a double antibody). 15.2mpk).
  • each mouse was injected with 5 ⁇ 10 6 PBMC cells (C2106025) into the tail vein, and 4 hours later, the first administration was performed by intraperitoneal injection (ip), twice a week, for a total of 7 administrations. Observe and record the tumor length (mm) and width (mm) at any time, and calculate the tumor volume (V).
  • mice in the PBS-treated group had the fastest tumor growth.
  • all antibody groups had significant tumor inhibitory effects; among them, the high-dose bispecific antibody BsAb2 administration group had smaller The tumor volume of the mice was significantly lower than that of the Trastuzumab+Avelumab combined administration group, showing better tumor inhibitory effect.

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Abstract

本发明公开了一种靶向HER2和PD-L1的双特异性抗体及其制备方法和应用。所述双特异性抗体包含靶向HER2的第一蛋白功能区和靶向PD-L1的第二蛋白功能区；所述第一蛋白功能区包含VL和VH，且所述VL包含如SEQ ID NO:3所示序列中所含的三个互补决定区LCDR1、LCDR2和LCDR3，所述VH包含如SEQ ID NO:4所示序列中所含的三个互补决定区HCDR1、HCDR2和HCDR3；所述第二蛋白功能区包含VHH，所述VHH包含如SEQ ID NO:2所示序列中所含的三个互补决定区CDR1、CDR2和CDR3。本发明的双特异性抗体具有较好的纯度和热稳定性；对两个靶点的结合活性均与其相对应的单抗接近，阻断PD-1/PD-L1的活性与对应的单抗相当，且抑制SK-BR-3细胞增殖活性与对应的单抗相当；同时表现出很好的ADCC活性和体内抑瘤活性。

Description

靶向HER2和PD-L1的双特异性抗体及其制备方法和应用
本申请要求申请日为2022/6/10的中国专利申请2022106577252的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物医药领域，具体涉及一种靶向HER2和PD-L1的双特异性抗体及其制备方法和应用。
背景技术
人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)隶属表皮生长因子受体(Epidermal growth factor receptor，EGFR)蛋白家族，可通过与EGFR家族其他成员的异源二聚化或由于其自身的过表达所引起的同源二聚化，使胞质结构域上的酪氨酸残基发生自磷酸化并起始细胞增殖和肿瘤发生的一系列信号途径(Oh,D.Y.and Y.J.Bang.(2020)."HER2-targeted therapies-a role beyond breast cancer."Nat Rev Clin Oncol 17(1):33-48.)。HER2仅在极少数正常组织中低水平表达。但研究表明，HER2在乳腺癌、胃癌、卵巢癌和子宫内膜癌等癌症类型中过表达，且其过表达与肿瘤恶性程度和不良预后相关(Normanno,N.,et al.(2005)."The ErbB receptors and their ligands in cancer:an overview."Curr Drug Targets 6(3):243-257.)。基于HER2的表达特性和在肿瘤发生发展中的生物学功能，其已成为经过广泛验证的肿瘤治疗靶点，多个靶向HER2的单克隆抗体药物已获批上市。然而，仅有不到30％的病人对其产生应答，而且大部分初始应答的患者会在一年内产生治疗耐受或复发(Gu,C.L.,et al.(2021)."Bispecific antibody simultaneously targeting PD1 and HER2 inhibits tumor growth via direct tumor cell killing in combination with PD1/PDL1 blockade and HER2 inhibition."Acta Pharmacol Sin.)。
PD-L1为免疫球蛋白超家族成员，是I型跨膜糖蛋白，表达于巨噬细胞、某些激活的T细胞和B细胞、DCs和某些上皮细胞(Sharpe,A.H.,et al.(2007)."The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection."Nat Immunol 8(3):239-245.)。此外，PD-L1也被肿瘤细胞高表达以逃逸适应性抗肿瘤免疫应答(Ohaegbulam,K.C.,et al.(2015)."Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway."Trends Mol Med 21(1):24-33.)。在肿瘤微环境中，PD-L1的表达上调通过PD-1信号通路可直接抑制T细胞的抗肿瘤反应，介导肿瘤细胞的免疫逃逸。近年来，针对PD-1/PD-L1信号途径的阻断型抗体药物开发吸引了广泛的关注，多个靶向PD-1或PD-L1的单克隆抗体药物已获批上市并且在临床上取得了令人振奋的疗效。尽管如此，大部分患者对PD-1/PD-L1阻断的免疫治疗未产生应答，而且部分初始应答的患者也产生了治疗耐受。
双特异性抗体是可以同时结合两个抗原或同一抗原的不同表位的重组抗体，不仅可以将免疫效应细胞重定向至肿瘤组织以实现免疫细胞对肿瘤的特异性杀伤，而且能够同时抑制同一肿瘤细胞上的不同促肿瘤信号途径进而发挥协同抑瘤功能(Labrijn,A.F.,et al.(2019)."Bispecific antibodies:a mechanistic review of the pipeline."Nat Rev Drug Discov 18(8):585-608.)。因此，为了提高HER2阳性肿瘤的治疗应答率并克服肿瘤治疗耐受，亟需开发靶向HER2和PD-L1的双特异性抗体。
发明内容
为解决上述技术问题，本发明提供一种靶向HER2和PD-L1的双特异性抗体及其制备方法和应用。本发明的双特异性抗体的理化性质较佳，具有较好的纯度和热稳定性；能够保留针对双靶点的功能活性，对两个靶点的结合活性均与其相对应的单抗接近(即结合PD-L1又结合HER2且与单独单抗结合能力相当或更佳)，阻断PD-1/PD-L1的活性与对应的单抗相当，且抑制SK-BR-3细胞增殖活性与对应的单抗相当；同时表现出很好的ADCC活性，显著优于其对应的单抗。
本发明一方面提供一种双特异性抗体，其包含靶向HER2的第一蛋白功能区和靶向PD-L1的第二蛋白功能区；其中，所述第一蛋白功能区包含轻链可变区(VL)和重链可变区(VH)，且所述VL包含如SEQ ID NO:3所示序列中所含的三个互补决定区LCDR1、LCDR2和LCDR3，所述VH包含如SEQ ID NO:4所示序列中所含的三个互补决定区HCDR1、HCDR2和HCDR3；
所述第二蛋白功能区包含VHH，其中，所述VHH包含如SEQ ID NO:2所示序列中所含的三个互补决定区CDR1、CDR2和CDR3。
在某一较佳实施方案中，所述VL包含如SEQ ID NO:3所示序列，或与其具有至少90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列；且所述VH包含如SEQ ID NO:4所示序列，或与其具有至少90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列。
在某一较佳实施方案中，所述VHH包含如SEQ ID NO:2所示序列，或与其具有至少90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列。
在如上所述的双特异性抗体，所述第一蛋白功能区较佳地包含Fab；
其中，所述Fab与所述VHH可操作地连接，例如所述VHH连接于所述Fab中轻链可变区的N端、或重链可变区的N端、或轻链恒定区的C端，或者所述Fab与所述VHH分别可操作地连接于Fc区的两个铰链区的N端。
在如上所述的双特异性抗体，所述第一蛋白功能区较佳地为IgG，在某一优选实施方案中，所述VHH可操作地连接于所述IgG重链恒定区的C端。
如上所述的双特异性抗体较佳地含有两条第一多肽链和两条第二多肽链。
在某一较佳实施方案中，
所述第一多肽链如下式所示：
VH-CH1-铰链区-CH2-CH3，优选包含如SEQ ID NO:9所示的序列，
且所述第二多肽链如下式所示：
VHH-连接子-VL-CL，优选包含如SEQ ID NO:10所示的序列。
在某一较佳实施方案中，
所述第一多肽链如下式所示：
VH-CH1-铰链区-CH2-CH3-连接子-VHH，优选包含如SEQ ID NO:6所示的序列，
且所述第二多肽链如下式所示：
VL-CL，优选包含如SEQ ID NO:7所示的序列。
在某一较佳实施方案中，
所述第一多肽链如下式所示：
VHH-连接子-VH-CH1-铰链区-CH2-CH3，优选包含如SEQ ID NO:8所示的序列，
且所述第二多肽链如下式所示：
VL-CL，优选包含如SEQ ID NO:7所示的序列。
在某一较佳实施方案中，
所述第一多肽链如下式所示：
VH-CH1-铰链区-CH2-CH3，优选包含如SEQ ID NO:9所示的序列，
且所述第二多肽链如下式所示：
VL-CL-连接子-VHH，优选包含如SEQ ID NO:11所示的序列。
如上所述的双特异性抗体较佳地含有第一多肽链、第二多肽链和第三多肽链，其中，所述第一多肽链如下式所示：
VHH-铰链区-CH2-CH3；
所述第二多肽链如下式所示：
VH-CH1-铰链区-CH2-CH3；
所述第三多肽链如下式所示：
VL-CL。
在某一较佳实施方案中，所述第一多肽链包含如SEQ ID NO:12所示的序列，且所述第二多肽链包含如SEQ ID NO:13所示的序列，且所述第三多肽链包含如SEQ ID NO:7所示的序列。
在某一较佳实施方案中，所述第一多肽链包含如SEQ ID NO:14所示的序列，且所述第二多肽链包含如SEQ ID NO:15所示的序列，且所述第三多肽链包含如SEQ ID NO:7所示的序列。
本发明还提供一种分离的核酸，其编码如本发明所述的双特异性抗体。
本发明还提供一种包含如本发明所述的分离核酸的重组表达载体。
本发明还提供一种转化体，其包含如本发明所述的分离的核酸或如本发明所述的重组表达载体。
如上所述的转化体，所述转化体的宿主细胞较佳地为原核细胞或真核细胞，所述原核细胞优选E.coli细胞如TG1、BL21，所述真核细胞优选HEK293细胞或CHO细胞。
一旦已经制备了用于表达的表达载体或DNA序列，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质的转染或其他常规技术。在原生质体融合的情况下，将细胞在培养基中培育并且筛选适宜的活性。用于培养所产生的转染细胞和用于回收产生的抗体分子的方法和条件是本领域技术人员已知的并且可以基于本说明书和现有技术已知的方法，根据使用的特定表达载体和哺乳动物宿主细胞变动或优化。另外，可以通过引入允许选择已转染的宿主细胞的一个或多个标记物，选出已经稳定将DNA掺入至其染色体中的细胞。标记物可以例如向营养缺陷型宿主提供原养型、杀生物抗性(例如，抗生素)或重金属(如铜)抗性等。可选择标记基因可以与待表达的DNA序列直接连接或通过共转化引入相同的细胞中。也可能需要额外元件以便最佳合成mRNA。这些元件可以包括剪接信号，以及转录启动子、增强子和终止信号。
本发明还提供一种双特异性抗体的制备方法，其包含培养如本发明所述的转化体，从培养物中获得双特异性抗体。
本发明还提供一种药物组合物，其包含如本发明所述的双特异性抗体，以及药学上可接受的载体。
在某一较佳实施方案中，所述药物组合物还包括其他抗肿瘤抗体作为活性成分、和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
在一些实施方案中，本发明的药物组合物或药物制剂包含合适的药学上可接受的载体例如药用辅 料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。如本发明所用，“药学上可接受的载体”或“药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。适用于本发明的药用载体可以是无菌液体，如水和油，包括那些石油、动物、植物或合成来源的，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。合适的赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Handbook of Pharmaceutical Excipients”，第五版，R.C.Rowe、P.J.Seskey和S.C.Owen，Pharmaceutical Press，London，Chicago。若期望的话，所述组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。这些组合物可以采用溶液、悬浮液、乳剂、片剂、丸剂、胶囊剂、粉末、持续释放配制剂等的形式。可以通过将具有所需纯度的本发明的抗体或其抗原结合片段与一种或多种任选的药用辅料(Remington’s Pharmaceutical Sciences，第16版，Osol，A.编(1980))混合来制备包含本发明所述的药物制剂或药物组合物，优选地以冻干制剂或水溶液的形式。本发明的药物组合物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应症所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它抗感染活性成分，例如其它抗体、抗感染活性剂、小分子药物或免疫调节剂等。所述活性成分以对于目的用途有效的量合适地组合存在。可制备持续释放制剂。持续释放制剂的合适实例包括含有本发明的抗体或其抗原结合片段的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。
本发明还提供一种抗体药物偶联物，其包含细胞毒性剂或标签，以及本发明所述的双特异性抗体；所述细胞毒性剂较佳地为MMAF或MMAE；所述标签较佳地为荧光剂。
本发明还提供一种试剂盒，其包括如本发明所述的双特异性抗体、如本发明所述的药物组合物、或如本发明所述的抗体药物偶联物。
所述试剂盒较佳地还包括(i)施用双特异性抗体或抗体药物偶联物或药物组合物的装置；和/或(ii)使用说明。
本发明还提供一种套装药盒，其包含药盒A和药盒B，其中：
所述药盒A含有如本发明所述的双特异性抗体、如本发明所述的药物组合物、或如本发明所述的抗体药物偶联物。
所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
本发明还提供一种检测特异性抗原的非诊断目的的方法，其包括使用本发明所述的双特异性抗体进行检测。
所述非诊断目的的应用场景可为本领域常规，例如如在实验室中检测抗原或者研究抗体的竞争结合。
本发明还提供一种如本发明所述的双特异性抗体、如本发明所述的核酸、如本发明所述的重组表达载体、如本发明所述的转化体、如本发明所述的药物组合物、如本发明所述的抗体药物偶联物、如本发明所述的试剂盒或如本发明所述的套装药盒在制备诊断、预防和/或治疗肿瘤的药物中的应用。
所述肿瘤较佳地为与HER2和/或PD-L1有关的肿瘤。
所述肿瘤例如乳腺癌、胃癌、骨肉瘤、促纤维增生性小圆细胞癌、头颈癌的鳞状细胞癌、卵巢癌、前列腺癌、胰癌、多形性胶质母细胞瘤、胃结合部腺癌、胃食管结合部腺癌、宫颈癌、唾液腺癌、软组织肉瘤、白血病、黑素瘤、尤文氏肉瘤、横纹肌肉瘤、成神经细胞瘤、或小细胞肺癌。
本发明还提供一种诊断、治疗和/或预防HER2和/或PD-L1介导的肿瘤的方法，所述方法包括向有需要的患者施用治疗有效量的如本发明所述的双特异性抗体、如本发明所述的核酸、如本发明所述的重组表达载体、如本发明所述的转化体、如本发明所述的药物组合物或如本发明所述的抗体药物偶联物治疗有需要的患者。
所述肿瘤较佳地为乳腺癌、胃癌、骨肉瘤、促纤维增生性小圆细胞癌、头颈癌的鳞状细胞癌、卵巢癌、前列腺癌、胰癌、多形性胶质母细胞瘤、胃结合部腺癌、胃食管结合部腺癌、宫颈癌、唾液腺癌、软组织肉瘤、白血病、黑素瘤、尤文氏肉瘤、横纹肌肉瘤、成神经细胞瘤或小细胞肺癌。
本发明提供的双特异性抗体相比单一疗法，靶向HER2和PD-L1的双特异性抗体具有多重优势：一方面，可以协同发挥ADCC和PD-1/PD-L1双重抗肿瘤活性，提高肿瘤治疗效果；另一方面，可以提高肿瘤治疗的靶向性，减少对HER2或PD-L1阳性正常组织的毒副作用。
本发明的积极进步效果在于：本发明的双特异性抗体的理化性质较佳，具有较好的纯度和热稳定性；能够保留针对双靶点的功能活性，对两个靶点的结合活性均与其相对应的单抗接近(即结合PD-L1又结合HER2且与单独单抗结合能力相当或更佳)，阻断PD-1/PD-L1的活性与对应的单抗相当，且抑制SK-BR-3细胞增殖活性与对应的单抗相当；同时表现出很好的ADCC活性和体内抑瘤活性。
附图说明
图1A-图1E显示了候选双特异性抗体的示意性结构。
图2A-图2F为候选双特异性抗体SEC-HPLC单体检测图谱。
图3A-图3B显示了候选双特异性抗体与重组人PD-L1-His蛋白的结合活性。
图4显示了候选双特异性抗体与huPD-L1-CHO-S细胞结合活性。
图5A-图5B显示了候选双特异性抗体与重组人HER2-His蛋白的结合活性。
图6显示了候选双特异性抗体与SK-BR-3细胞的结合活性。
图7显示了通过FACS法检测的候选双特异性抗体对PD-1/PD-L1信号途径的阻断活性。
图8A-图8C显示了通过荧光素酶报告基因方法检测的候选双特异性抗体对PD-1/PD-L1途径的阻断活性。
图9显示了候选双特异性抗体在SK-BR-3细胞上的抗体依赖的细胞介导的细胞毒性作用(ADCC)。
图10显示了候选双特异性抗体对SK-BR-3细胞的增殖抑制效应。
图11A-图11B显示了候选双特异性抗体在小鼠皮下移植瘤模型中对肿瘤生长的抑制作用。
具体实施方式
定义
在本发明中，上述所列CDR的氨基酸序列均是按照AbM定义规则所示出的。但是，本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人，Journal of Molecular Biology，273，927-948(1997))、基于抗体序列可变性的Kabat(Kabat等人，U.S.Department of Health and Human  Services，National Institutes of Health(1987))、AbM(University of Bath)，Contact(University College London)、国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应理解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。
本发明中，所述的蛋白功能区可为全长抗体、scFv、Fab、VHH等形式，可根据实际上下文进行选择。其中，“全长抗体”可互换地用来指包含由二硫键相互连接的至少两条重链(HC)和两条轻链(LC)的糖蛋白。每条重链由重链可变区(本发明中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本发明中缩写为VL)和轻链恒定区(本发明中缩写为CL)组成。轻链恒定区由一个结构域CL组成。哺乳动物重链分类为α、δ、ε、γ和μ。哺乳动物轻链分类为λ或κ。包含α、δ、ε、γ和μ重链的免疫球蛋白分类为免疫球蛋白IgA、IgD、IgE、IgG和IgM。完全抗体形成“Y”形状。Y的茎由两条重链的第二和第三恒定区(并且对于IgE和IgM，第四恒定区)结合在一起组成，并且二硫键(链间)在铰链中形成。重链γ、α和δ具有由三个串联(成一行)Ig结构域构成的恒定区，和用于增加柔性的铰链区；重链μ和ε具有由四个免疫球蛋白结构域构成的恒定区。第二和第三恒定区分别称为“CH2结构域”和“CH3结构域”。Y的每个臂包括结合到单个轻链的可变和恒定区的单个重链的可变区和第一恒定区。轻链和重链的可变区负责抗原结合。本发明中，“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。“Fc区”含有包含抗体的CH2和CH3结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。“Fab’片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’)2分子。“F(ab’)2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此F(ab’)2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。单链抗体(single chain antibody fragment，scFv)是由抗体重链可变区和轻链可变区通过15～20个氨基酸的短肽(linker)连接而成的抗体。术语“VHH”也称为单结构域抗体，其为仅由一个重链可变区组成，是从C端到N端仅包含一条链FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1的抗体，也称为“纳米抗体(nanobody)”。单结构域抗体是目前已知的可结合目标抗原的最小单位。
本发明所用氨基酸三字母代码和单字母代码如本领域技术人员知晓，或参见文献(J.Biol.Chem，243，p3558(1968))中所述。
如本发明使用的，术语“包括”或“包含”旨在表示组合物和方法包括所述的元素但不排除其他元素，但根据上下文的理解，也包括“由……组成”的情况。
如本发明所用，“载体”表示构建体，其能够将一种或多种所关注的基因或序列递送入宿主细胞并且优选在宿主细胞中表达所述基因或序列。载体的实例包括但不限于病毒载体、裸DNA或RNA表达载体、质粒、粘粒或噬菌体载体、与阳离子凝聚剂相关的DNA或RNA表达载体、包囊化于脂质体中的DNA或RNA表达载体以及某些真核细胞，例如生产细胞。
在本发明中术语“宿主细胞”可包括已经引入外源性核酸的细胞，包括这些细胞的子代。宿主细胞包括“转化子”和“转化的细胞”，其包括原代转化细胞以及由此来源的子代，而不考虑传代次数。子代在核酸含量上与亲代细胞可能不完全相同，但可能含有突变。本发明包括与在初始转化的细胞中 筛选或选择的细胞具有相同功能或生物学活性的突变子代。
以下实施例旨在仅对本发明进行举例说明，因此并不应被视为以任何方式限制本发明。
实施例1原材料制备
1.1抗原制备
重组人PD-L1(UniProtKB-Q9NZQ7)、PD-1(UniProtKB-Q15116)和HER2(UniProt NO-P04626)蛋白的DNA编码序列由通用生物科技股份有限公司通过全基因合成获得。PCR扩增目的片段并通过引物在编码序列C端引入His标签，采用同源重组法将目的片段构建至真核表达载体pcDNA3.4-TOPO(Invitrogen)。将构建好的重组蛋白表达质粒转化大肠杆菌DH5α感受态细胞，37℃过夜培养，使用无内毒素质粒提取试剂盒(OMEGA，D6950-01)抽提质粒。
重组人PD-L1-His蛋白、重组人PD-1-His蛋白和重组人HER2-His蛋白均通过Expi293瞬转表达系统(ThermoFisher，A14635)制备，瞬转方法参见Expi293TM Expression System USER GUIDE。转染7天后，收集细胞悬液于15000g离心10min，表达上清使用Ni Smart Beads 6FF(常州天地人和生物科技有限公司，SA036050)进行亲和纯化，目的蛋白由梯度浓度的咪唑溶液进行洗脱。洗脱获得的各蛋白分别通过超滤浓缩管(Millipore，UFC901096)置换至PBS缓冲液中。经SDS-PAGE鉴定和活性检测合格后分装并冻存于-80℃。
1.2对照抗体制备
在本实施例中，抗人PD-L1抗体D21-4(VHHPD-L1-huFc，氨基酸序列如SEQ ID NO:1所示)源自专利申请WO2021083335A1；抗人PD-L1抗体Avelumab源自DrugBank(查询号DB11945)；抗人HER2抗体为曲妥珠单抗(Trastuzumab)源自DrugBank(查询号DB00072)。
对照抗体都采用瞬转系统(ExpiCHO)进行表达，瞬转方法参见ExpiCHOTM Expression System Kit User Guide。培养结束后将细胞混悬液进行高速离心并收集上清，所得上清经0.22μm滤膜过滤后，采用Protein A/G柱进行亲和层析纯化。目的蛋白使用100mM甘氨酸盐酸(pH 3.0)进行洗脱，经浓缩，缓冲液置换，分装，SDS-PAGE鉴定和活性检测后入库冻存。
实施例2抗HER2& PD-L1双特异性抗体的构建
本实施例描述了示例性抗HER2 & PD-L1双特异性抗体(BsAb)的结构和表达载体的构建。设计并构建了6种构建体：其中抗人PD-L1抗体的VHH结构域(VHHPD-L1)氨基酸序列来自D21-4，其可变区氨基酸序列如SEQ ID NO:2所示；抗HER2抗体氨基酸序列来自曲妥珠单抗，其轻重链可变区氨基酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示；连接子氨基酸序列为GGGGSGGGGSGGGGS(SEQ ID NO:5)。示例性BsAb构建体如表1所示，对应的氨基酸序列提供于表2中。
构建体BsAb1：含有两条相同的第一多肽链，所述第一多肽链从N末端到C末端包含曲妥珠单抗的重链可变区、人IgG1重链恒定区、连接子和抗人PD-L1抗体的VHH结构域；含有两条相同的第二多肽链，所述第二多肽链从N末端到C末端包含曲妥珠单抗的轻链可变区和κ轻链恒定区。BsAb1具有如图1A所示的形式。
构建体BsAb2：含有两条相同的第一多肽链，所述第一多肽链从N末端到C末端包含抗人PD-L1抗体的VHH结构域、曲妥珠单抗的重链可变区、人IgG1重链恒定区；含有两条相同的第二多肽 链，所述第二多肽链从N末端到C末端包含曲妥珠单抗的轻链可变区和κ轻链恒定区。BsAb2具有如图1B所示的形式。
构建体BsAb3：含有两条相同的第一多肽链，所述第一多肽链从N末端到C末端包含曲妥珠单抗的重链可变区和人IgG1重链恒定区结构域；含有两条相同的第二多肽链，所述第二多肽链从N末端到C末端包含抗人PD-L1抗体的VHH结构域、连接子、曲妥珠单抗的轻链可变区和κ轻链恒定区。BsAb3具有如图1C所示的形式。
构建体BsAb4：含有两条相同的第一多肽链，所述第一多肽链从N末端到C末端包含曲妥珠单抗的重链可变区和人IgG1重链恒定区结构域；含有两条相同的第二多肽链，所述第二多肽链从N末端到C末端包含曲妥珠单抗的轻链可变区、κ轻链恒定区、连接子和抗人PD-L1抗体的VHH结构域。BsAb4具有如图1D所示的形式。
构建体BsAb5：含有3条不同的多肽链，分别为第一多肽链、第二多肽链和第三多肽链；其中第一多肽链从N末端到C末端包含抗人PD-L1抗体的VHH结构域、人IgG1重链铰链区和人IgG1重链Fc(Knob)；其中第二多肽链从N末端到C末端包含曲妥珠单抗的重链可变区和人IgG1重链恒定区(Hole)；其中第三多肽链从N末端到C末端包含曲妥珠单抗的轻链可变区和κ轻链恒定区。BsAb5具有如图1E所示的形式。
构建体BsAb6：含有3条不同多肽链，分别为第一多肽链、第二多肽链和第三多肽链；其中第一多肽链从N末端到C末端包含抗人PD-L1抗体的VHH结构域、人IgG1重链铰链区和人IgG1重链Fc(Hole)；其中第二多肽链从N末端到C末端包含曲妥珠单抗的重链可变区和人IgG1重链恒定区(Knob)；第三多肽链从N末端到C末端包含曲妥珠单抗的轻链可变区和κ轻链恒定区。BsAb6具有如图1E所示的形式。
根据构建体的结构，通过PCR方法扩增获取各个抗体可变区及恒定区的片段，通过重叠延伸PCR法将各个片段连接起来，再通过同源重组分别构建至经过改造的真核表达载体质粒pcDNA3.4-TOPO(Invitrogen)上，组成完整的构建体多肽链表达载体。将构建好的含构建体多肽链编码序列的载体分别转化到大肠杆菌DH5α中，37℃过夜培养。利用无内毒素质粒提取试剂盒(OMEGA，D6950-01)进行质粒提取，得到无内毒素的构建体多肽链表达质粒以供真核表达使用。
表1抗HER2 & PD-L1双特异性抗体的构建体
表2抗HER2 & PD-L1双特异性抗体构建体的氨基酸序列

实施例3抗HER2& PD-L1双特异性抗体的表达、纯化和理化性质分析
3.1抗HER2 & PD-L1双特异性抗体的表达、纯化
实施例2的构建体通过ExpiCHO瞬转表达系统(Thermo Fisher，A29133)进行表达，具体操作如下：转染当天，确认细胞密度约为7×106至1×107个活细胞/mL，细胞存活率>98％，此时，用37℃预温的新鲜ExpiCHO表达培养基将细胞调整到终浓度为6×106个细胞/mL。用4℃预冷的OptiPROTM SFM稀释目的质粒(向1mL所述培养基中加入1μg质粒)，同时，用OptiPROTMSFM稀释ExpiFectamineTMCHO，再将两者等体积混合并轻轻吹打混匀制备成ExpiFectamineTMCHO/质粒DNA混合液，室温孵育5min，缓慢加入到准备好的细胞悬液中并同时轻轻摇晃，最后置于细胞培养摇床中，在37℃、8％CO2条件下培养。在转染后18-22h，向培养液中添加ExpiCHOTMEnhancer和ExpiCHOTMFeed，摇瓶放置于32℃摇床和5％CO2条件下继续培养。在转染后第5天，添加相同体积的ExpiCHOTM Feed，缓慢加入的同时轻轻混匀细胞混悬液。在转染10天后，将表达有目的蛋白的细胞培养上清于15000g高速离心10min，所得上清用COLUMN XK16/20(购自Cytiva)进行亲和纯化，然后用100mM乙酸钠(pH3.0)洗脱目的蛋白，接着用1M Tris-HCl中和，最后通过超滤浓缩管(Millipore，UFC901096)将所得蛋白置换至PBS缓冲液中。
3.2抗HER2 & PD-L1双特异性抗体的浓度测定
将实施例3.1经纯化获得的双特异性抗体使用超微量分光光度计(杭州奥盛仪器有限公司，Nano-300)进行浓度测定，将经测定的A280读值除以抗体理论消光系数后所得数值作为后续研究的抗体浓度，质检合格后，分装并保存于-80℃。
3.3抗HER2 & PD-L1双特异性抗体的SEC-HPLC单体纯度鉴定
材料准备：1、流动相：150mmol/L磷酸缓冲液，pH 7.4；2、样品制备：候选双特异性抗体均用流动相溶液稀释到0.5mg/mL。Agilent HPLC 1100色谱柱(XBridge BEH SEC 3.5μm.8mm I.D.×30cm，Waters)流速设为0.8mL/min，进样体积20μL，VWD检测器波长为280nm和214nm。
候选双特异性抗体的SEC-HPLC结果如下：按照面积归一法计算样品中高分子聚合物，抗体单体和低分子物质百分比，结果显示在图2A-2F和表3，从中可知候选双特异性抗体BsAb1、BsAb2、BsAb3和BsAb4的单体纯度都大于90％。
3.4抗HER2 & PD-L1双特异性抗体的热稳定性研究
差示扫描荧光法(differential scanning fluorimetry；DSF)能够根据蛋白质图谱中的荧光变化过程提供有关蛋白质结构稳定性的信息，检测蛋白的构型变化，获得蛋白质的熔解温度(Tm)。在本实施例中，采用DSF法检测了三功能抗体的Tm值。
候选双特异性抗体溶液制备成0.2mg/mL，每个供试品以19μL/孔加入96孔板(Nunc)中，设置三个平行孔，并以PBS和曲妥珠单抗作为参比，然后在每个孔中加入1μL浓度为100×的SYPRO orange染料，混匀后准备上机。样品热稳定测试采用ABI 7500FAST RT-PCR仪器，试验类型选择熔解曲线，采用连续模式，扫描温度范围为25～95℃，升温速率为1％，25℃平衡5min，在升温过程中 采集数据，报告基团选择“ROX”，淬灭基团选择“None”，反应体积20μL，以熔解曲线一阶导数的第一个峰谷对应的温度确定为抗体的熔解温度Tm。
实验结果显示在表3中，候选双特异性抗体的Tm均大于60℃，因此具有较好的热稳定性。
表3抗HER2 & PD-L1双特异性抗体的制备、理化数据
实施例4抗HER2 & PD-L1双特异性抗体的亲和活性分析
4.1 ELISA法检测候选双特异性抗体对重组人PD-L1-His蛋白的结合能力
在96孔ELISA板上包被重组人PD-L1-His蛋白，4℃过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭2h，用PBST洗板3次后，加入不同浓度的候选双特异性抗体、对照抗体D21-4后孵育1h。之后，用PBST清洗3次后加入二抗Anti-human-IgG-Fc-HRP(abcam，ab79225)并孵育1h。孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色。根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板。利用PRISMTM(GraphPad Software，San Diego，CA)分析数据，并且计算EC50值。
ELISA结合测定结果如图3A-3B和表4所示，双特异性抗体BsAb2和BsAb3对PD-L1的结合能力显著优于对照抗体D21-4。
4.2基于FACS法检测候选双特异性抗体对huPD-L1-CHO-S细胞的结合能力
收集指数生长期的huPD-L1-CHO-S细胞(稳定转染PD-L1(Gene ID:29126)至CHO-S(Thermo，A1461801)获得的过表达人PD-L1的稳转细胞株)，将细胞用FACS缓冲液(含有1％BSA的PBS)重悬，计数并将细胞悬液密度调整为2×106个/mL。随后，将huPD-L1-CHO-S细胞以每孔100μL加入96孔圆底板中，离心去上清。向对应孔中加入不同浓度的候选双特异性抗体、对照抗体D21-4稀释液和人IgG1同型抗体(作为同型对照)，将细胞重悬后放置于4℃孵育30min。将孵育后的细胞混合液洗涤3次后加入PE标记的anti-human-IgG-Fc流式抗体(Abcam，98596)，重悬后放置于4℃孵育30min。将孵育后的细胞混合液洗涤3次后加入200μL的FACS缓冲液重悬细胞，通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)上机检测分析。利用PRISMTM(GraphPad Software，San Diego，CA)分析数据，并且计算EC50值。
FACS结合测定结果如图4和表4所示，候选双特异性抗体都表现出与对照抗体D21-4相当的对细胞上表达的PD-L1的结合能力，且其中双特异性抗体BsAb2和BsAb3的结合能力最强。
4.3 ELISA法检测候选双特异性抗体对重组人HER2-His蛋白的结合能力
在96孔ELISA板上包被重组人HER2-His蛋白，4℃过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭2h，用PBST洗板3次后，加入不同浓度的候选双特异性抗体、对照抗体曲妥珠单抗孵育1h。之后，用PBST清洗3次后加入二抗Anti-human-IgG-Fc-HRP(abcam，ab97225)并孵育1 h。孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色。根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板。利用PRISMTM(GraphPad Software，San Diego，CA)分析数据，并且计算EC50值。
ELISA结合测定结果如图5A-5B和表4所示，候选双特异性抗体都表现出对照抗体曲妥珠单抗相当的对HER2的结合能力。
4.4基于FACS法检测候选双特异性抗体对SK-BR-3细胞的结合能力
收集指数生长期的内源表达HER2的SK-BR-3细胞(ATCC，HTB-30)，将细胞用FACS缓冲液(含有1％BSA的PBS)重悬，计数并将细胞悬液密度调整为2×106个/mL。随后，将SK-BR-3细胞以每孔100μL加入96孔圆底板中，离心去上清。向对应孔中加入不同浓度的候选双特异性抗体、对照抗体曲妥珠单抗和人IgG1同型抗体(作为同型对照)，将细胞重悬后放置于4℃孵育30min。将孵育后的细胞混合液洗涤3次后加入PE标记的anti-human-IgG-Fc流式抗体(Abcam，98596)，重悬后放置于4℃孵育30min。将孵育后的细胞混合液洗涤3次后加入200μL的FACS缓冲液重悬细胞，最后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)上机检测分析。利用PRISMTM(GraphPad Software，San Diego，CA)分析数据，并且计算EC50值。
FACS结合测定结果如图6和表4所示，双特异性抗体BsAb1、BsAb2和BsAb4表现出与对照抗体曲妥珠单抗相当的对细胞上表达的HER2的结合能力。
表4抗HER2& PD-L1双特异性抗体的抗原结合力数据
4.5基于Fortebio检测候选双特异性抗体对HER2和PD-L1的亲和力
采用Fortebio BLItz仪器，检测了候选双特异性抗体对重组人HER2-His和PD-L1-His蛋白的亲和力。
作为抗原的重组人HER2-His或PD-L1-His蛋白以10×KB缓冲液(含有1％BSA、0.5％Tween 20的10×PBS)稀释成10μg/mL，候选双特异性抗体及对照抗体D21-4或曲妥珠单抗以10×KB缓冲液进行2倍梯度稀释，依次为100、50、25、0nM。避光条件下，采用10×KB缓冲液预湿传感器(Anti-Penta-HIS，HIS1K，Fortebio，CA)，至少10min后开始测试样品板(GreinerBio，PN655209)，测试无误后按预设程序进行。首先将抗原和传感器结合300s，结合完毕后在10×KB缓冲液中继续平衡30s后，将结合有抗原的传感器转移至不同浓度抗体稀释液中结合300s，待信号稳定后，再转移到10×KB缓冲液中，解离时间为900s，最后通过不同浓度抗体的结合解离数据拟合得到KD、Kon和Koff，结果显示在表5中，双特异性抗体BsAb2表现出与对照抗体D21-4相当的对PD-L1的亲和力，且双特异性抗体BsAb2对HER2的亲和力显著优于对照抗体曲妥珠单抗。
表5抗HER2 & PD-L1双特异性抗体的亲和力测定数据表
实施例5基于FACS法检测抗HER2 & PD-L1双特异性抗体阻断活性
本实施例采用FACS方法检测候选双特异性抗体对PD-1/PD-L1的阻断活性，具体方法如下：收集培养好的huPD-L1-CHO-S细胞，将细胞用配制好的FACS缓冲液重悬，计数并将细胞悬液密度调整为2×106个/mL。将huPD-L1-CHO-S细胞以100μL每孔加入96孔圆底板，离心去上清后向对应孔中加入不同浓度的候选双特异性抗体、对照抗体D21-4和人IgG1同型抗体(同型对照)，重悬细胞后放置于4℃孵育30min。将孵育后的细胞混合液洗涤3次后加入100μL生物素标记的重组人PD-1-His蛋白稀释液(1μg/mL)，4℃孵育30min。洗涤3次后加入PE标记的streptavidin(eBioscience，12-4317-87)，4℃孵育30min，将孵育后的细胞混合液洗涤3次后加入200μL的FACS缓冲液重悬细胞，最后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)上机检测。利用PRISMTM(GraphPad Software，San Diego，CA)分析数据，并且计算IC50值。
FACS结合测定结果如图7和表6所示，双特异性抗体BsAb2和BsAb3表现出与对照抗体D21-4相当的对PD-1/PD-L1的阻断活性。
表6抗HER2 & PD-L1双特异性抗体的阻断活性
实施例6基于报告基因法检测抗HER2&PD-L1双特异性抗体的阻断PD-1/PD-L1的活性
本实施例采用荧光素酶报告基因系统检测候选抗HER2&PD-L1双特异性抗体阻断PD-1/PD-L1信号途径的活性，具体方法如下：取对数期PD-1-NF-AT-Jurkat细胞(Jurkat细胞稳定表达PD-1(UniProtKB-Q15116)和荧光素酶)和CD3L-PD-L1-CHO细胞(在CHO细胞中稳定表达PD-L1(UniProtKB-Q9NZQ7)和anti-CD3-scFv(序列来源于OKT3))，将两种细胞按5:1混合并使其密度分别为4×107个/mL和8×107个/mL。梯度稀释待检抗体，向96孔透明底白板(Corning，3610)每孔加入50μL抗体稀释液，随后以50μL/孔加入混合细胞悬液，放置于37℃细胞培养箱中静置培养6h。每孔加入50μL Bright-Lite(Vazyme，DD1204-03)，避光孵育10min，检测荧光信号。
阻断活性检测结果如下：如图8A-8C所示，双特异性抗体BsAb2、BsAb3均表现出与对照抗体D21-4相当的阻断PD-1/PD-L1活性。
实施例7抗HER2&PD-L1双特异性抗体的ADCC活性分析
本实施例采用荧光素酶报告基因系统检测候选抗HER2&PD-L1双特异性抗体的ADCC活性，具体方法如下：取对数期SK-BR-3细胞和CD16a(V158)-NF-AT-Jurkat细胞(稳定转染CD16a(V158)序列(UniProtKB-P08637)和含有NF-AT-re核酸序列的pGL4.30质粒(promega，#E8481)至Jurkat细胞获得的稳转细胞株，具体见参考文献Parekh,B.S.,et al.(2012)."Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay."MAbs.4(3):310-318.)，将两种细胞按10:1混合并使其密度分别为4×105个/mL和4×106个/mL。梯度稀释待检抗体，向96孔透明底白板(Corning，3610)每孔加入50μL抗体稀释液，随后以50μL/孔加入混合细胞悬液，放置于37℃细胞培养箱中静置培养6h。向每孔加入50μL Bright-Lite(Vazyme，DD1204-03)，避光孵育10min，检测荧光信号。
ADCC检测结果如下：如图9所示，双特异性抗体BsAb2表现出显著优于对照抗体曲妥珠单抗的ADCC活性。
实施例8抗HER2&PD-L1双特异性抗体抑制SK-BR-3细胞增殖活性分析
取对数生长期SK-BR-3细胞，使用RPMI1640(含1％FBS)培养基将细胞密度调整为2×104个/mL，以100μL/孔接种至96孔平底细胞培养板，于37℃细胞培养箱中培养过夜。使用RPMI 1640(含1％FBS)培养基梯度稀释待检抗体，以50μL/孔加至过夜培养的SK-BR-3的96孔细胞培养板中并将其放置于37℃培养箱中培养72h。提前将MTS检测试剂(Promega，G3581)放置于室温化冻并平衡至室温。将细胞培养板放置于室温15min使其平衡至室温，以30μL/孔向96孔细胞培养板加入MTS检测试剂，于酶标仪上振荡1min后，然后37℃避光孵育3h，取出细胞培养板并平衡至室温，读取OD492。
增殖抑制活性检测结果如下：如图10所示，双特异性抗体BsAb2表现出与对照抗体曲妥珠单抗相当抑制SK-BR-3细胞增殖活性。
实施例9抗HER2&PD-L1双特异性抗体的体内抑瘤实验
使用6-8周雌性NCG小鼠(购于江苏集萃，品系：T001475)，实验小鼠饲养在恒温恒湿的独立通风盒内，饲养室温度21-24℃，湿度30-53％。将huPD-L1-NCI-N87细胞(稳定转染PD-L1(Gene ID:29126)至NCI-N87细胞(购于中科院细胞库，目录号：TCHu130)获得的过表达人PD-L1的稳转细胞株)以1×107个/每只小鼠进行右侧背部皮下注射(第0天)，待小鼠皮下荷瘤体积达到100～150mm3左右时(第7天)，剔除肿瘤体积差异较大的小鼠样本，然后依据肿瘤体积进行随机分组(每组8只小鼠)：分别是PBS处理组、Trastuzumab给药组、Avelumab给药组、Trastuzumab+Avelumab联合给药组和双特异性抗体BsAb2给药组，每个给药组均设置35nM/kg剂量(对应单抗5mpk和双抗6.1mpk)，其中双特异性抗体BsAb2给药组还设置了87.5nM/kg剂量(对应双抗15.2mpk)。随后每只小鼠尾静脉注射5×106PBMC细胞(C2106025)，4h后通过腹腔注射方式(i.p.)进行首次给药，每个星期两次给药，共给药7次。随时观察和记录肿瘤长(mm)和宽(mm)，计算其肿瘤体积(V)，计算方式为V＝(长×宽2)/2，抑瘤率TGI(％)＝(1-给药组肿瘤平均体积/PBS处理组肿瘤平均体积)×100％。
结果显示在图11A-11B和表7。
表7不同给药组的抑瘤率TGI(％)
从图11A可以看出，各组小鼠体重之间无明显差异，并且治疗期间各组小鼠体重均无明显变化，表明小鼠对抗体的耐受性良好。从图11B和表7可以看出，PBS处理组小鼠肿瘤增长最快，和PBS组相比，所有抗体组都具有显著的抑瘤效果；其中，高剂量双特异性抗体BsAb2给药组小鼠的瘤体积显著低于Trastuzumab+Avelumab联合给药组，显示出较好抑瘤效果。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (15)

  1. 一种双特异性抗体，其包含靶向HER2的第一蛋白功能区和靶向PD-L1的第二蛋白功能区；其中：
    所述第一蛋白功能区包含VL和VH，其中，所述VL包含如SEQ ID NO:3所示序列中所含的三个互补决定区LCDR1、LCDR2和LCDR3，所述VH包含如SEQ ID NO:4所示序列中所含的三个互补决定区HCDR1、HCDR2和HCDR3；
    所述第二蛋白功能区包含VHH，其中，所述VHH包含如SEQ ID NO:2所示序列中所含的三个互补决定区CDR1、CDR2和CDR3。
  2. 如权利要求1所述的双特异性抗体，所述VL包含如SEQ ID NO:3所示的氨基酸序列，或与其具有至少90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列；且所述VH包含如SEQ ID NO:4所示的氨基酸序列，或与其具有至少90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列；和/或，所述VHH包含如SEQ ID NO:2所示的氨基酸序列，或与其具有至少90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列。
  3. 如权利要求2所述的双特异性抗体，其特征在于，所述第一蛋白功能区包含Fab；
    其中，所述Fab与所述VHH可操作地连接，所述VHH优选连接于所述Fab中轻链可变区或重链可变区的N端、或轻链恒定区的C端；
    或，所述Fab与所述VHH分别可操作地连接于Fc区的两个铰链区的N端。
  4. 如权利要求2所述的双特异性抗体，所述第一蛋白功能区为IgG；所述VHH可操作地连接于所述IgG重链恒定区的C端。
  5. 如权利要求1-4任一项所述的双特异性抗体，其满足以下条件中的任意一种：
    (i)所述的双特异性抗体含有两条第一多肽链和两条第二多肽链，其中：
    所述第一多肽链如下式所示：
    VH-CH1-铰链区-CH2-CH3，优选包含如SEQ ID NO:9所示的序列，
    且所述第二多肽链如下式所示：
    VHH-连接子-VL-CL，优选包含如SEQ ID NO:10所示的序列；
    或，所述第一多肽链如下式所示：
    VH-CH1-铰链区-CH2-CH3-连接子-VHH，优选包含如SEQ ID NO:6所示的序列，
    且所述第二多肽链如下式所示：
    VL-CL，优选包含如SEQ ID NO:7所示的序列；
    或，所述第一多肽链如下式所示：
    VHH-连接子-VH-CH1-铰链区-CH2-CH3，优选包含如SEQ ID NO:8所示的序列，
    且所述第二多肽链如下式所示：
    VL-CL，优选包含如SEQ ID NO:7所示的序列；
    或，所述第一多肽链如下式所示：
    VH-CH1-铰链区-CH2-CH3，优选包含如SEQ ID NO:9所示的序列，
    且所述第二多肽链如下式所示：
    VL-CL-连接子-VHH，优选包含如SEQ ID NO:11所示的序列；
    (ii)所述的双特异性抗体含有第一多肽链、第二多肽链和第三多肽链，所述第一多肽链如下式所示：
    VHH-铰链区-CH2-CH3；
    所述第二多肽链如下式所示：
    VH-CH1-铰链区-CH2-CH3；
    所述第三多肽链如下式所示：
    VL-CL；
    较佳地，所述第一多肽链包含如SEQ ID NO:12所示的序列，且所述第二多肽链包含如SEQ ID NO:13所示的序列，且所述第三多肽链包含如SEQ ID NO:7所示的序列，
    或者所述第一多肽链包含如SEQ ID NO:14所示的序列，且所述第二多肽链包含如SEQ ID NO:15所示的序列，且所述第三多肽链包含如SEQ ID NO:7所示的序列。
  6. 一种分离的核酸，其编码如权利要求1-5任一项所述的双特异性抗体。
  7. 一种重组表达载体，其包含如权利要求6所述的分离的核酸。
  8. 一种转化体，其包含如权利要求6所述的分离的核酸或如权利要求7所述的重组表达载体；
    较佳地，所述转化体的宿主细胞为原核细胞或真核细胞，所述原核细胞优选E.coli细胞如TG1、BL21，所述真核细胞优选HEK293细胞或CHO细胞。
  9. 一种双特异性抗体的制备方法，其包含以下步骤：培养如权利要求8中所述的转化体，并从培养物中获得所述双特异性抗体。
  10. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1-5任一项所述的双特异性抗体，以及药学上可接受的载体；
    较佳地，所述药物组合物还包括其他抗肿瘤抗体作为活性成分、和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  11. 一种抗体药物偶联物，其包含细胞毒性剂或标签，以及如权利要求1-5任一项所述的双特异性抗体；
    较佳地，所述细胞毒性剂为MMAF或MMAE，所述标签为荧光剂。
  12. 一种试剂盒，其包括如权利要求1-5任一项所述的双特异性抗体、如权利要求10所述的药物组合物或如权利要求11所述的抗体药物偶联物；
    较佳地，所述试剂盒还包括(i)施用双特异性抗体或抗体药物偶联物或药物组合物的装置；和/或(ii)使用说明。
  13. 一种套装药盒，其包含药盒A和药盒B，其中：
    所述药盒A含有如权利要求1-5任一项所述的双特异性抗体、如权利要求10所述的药物组合物或如权利要求11所述的抗体药物偶联物；
    所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  14. 一种如权利要求1-5任一项所述的双特异性抗体、如权利要求6所述的核酸、如权利要求7所述的重组表达载体、如权利要求8所述的转化体、如权利要求10所述的药物组合物、如权利要求 11所述的抗体药物偶联物、如权利要求12所述的试剂盒或如权利要求13所述的套装药盒在制备诊断、预防和/或治疗肿瘤的药物中的应用；较佳地，所述肿瘤为与HER2和/或PD-L1有关的肿瘤，更佳地，所述肿瘤为乳腺癌、胃癌、骨肉瘤、促纤维增生性小圆细胞癌、头颈癌的鳞状细胞癌、卵巢癌、前列腺癌、胰癌、多形性胶质母细胞瘤、胃结合部腺癌、胃食管结合部腺癌、宫颈癌、唾液腺癌、软组织肉瘤、白血病、黑素瘤、尤文氏肉瘤、横纹肌肉瘤、成神经细胞瘤或小细胞肺癌。
  15. 一种检测特异性抗原的非诊断目的的方法，其包括使用如权利要求1-5任一项所述的双特异性抗体进行检测。
PCT/CN2023/097835 2022-06-10 2023-06-01 靶向her2和pd-l1的双特异性抗体及其制备方法和应用 WO2023236844A1 (zh)

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