WO2021023267A1 - 包含抗pd-1/her2双特异性抗体的制剂及其制备方法和用途 - Google Patents

包含抗pd-1/her2双特异性抗体的制剂及其制备方法和用途 Download PDF

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WO2021023267A1
WO2021023267A1 PCT/CN2020/107441 CN2020107441W WO2021023267A1 WO 2021023267 A1 WO2021023267 A1 WO 2021023267A1 CN 2020107441 W CN2020107441 W CN 2020107441W WO 2021023267 A1 WO2021023267 A1 WO 2021023267A1
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antibody
preparation
liquid
her2
seq
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PCT/CN2020/107441
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English (en)
French (fr)
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刘洋涵
马一冬
汪音爵
周凯松
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信达生物制药(苏州)有限公司
北京韩美药品有限公司
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Priority to EP20849224.9A priority Critical patent/EP4011390A4/en
Priority to JP2022506961A priority patent/JP2022543422A/ja
Priority to CN202080055615.8A priority patent/CN114206382B/zh
Priority to AU2020325415A priority patent/AU2020325415A1/en
Priority to US17/631,641 priority patent/US20220281972A1/en
Priority to KR1020227004778A priority patent/KR20220044286A/ko
Priority to BR112022001775A priority patent/BR112022001775A2/pt
Priority to CA3146138A priority patent/CA3146138A1/en
Publication of WO2021023267A1 publication Critical patent/WO2021023267A1/zh

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • 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
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—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 CD28 or CD152
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to the field of antibody preparations. More specifically, the present invention relates to a bispecific antibody comprising recombinant anti-programmed death receptor 1 (PD-1) and anti-human epidermal growth factor receptor 2 (HER2) (also known as anti-PD-1/HER2 dual Specific antibody) pharmaceutical preparations, especially stable liquid preparations, and methods for preparing the pharmaceutical preparations, as well as the therapeutic and/or preventive uses of the pharmaceutical preparations.
  • PD-1 anti-programmed death receptor 1
  • HER2 anti-human epidermal growth factor receptor 2
  • HER2 human epidermal growth factor receptor 2
  • NEU nuclear epidermal growth factor receptor 2
  • ERBB-2 also known as NEU, ERBB-2, CD340, or p185
  • HER2 overexpression has been observed in invasive and metastatic breast cancers, in breast cancers with high recurrence rates and/or poor patient prognosis.
  • trastuzumab is a therapeutic anti-HER2 antibody that blocks intracellular signal transduction mediated by HER2 and is widely used to treat HER2 overexpressing tumors.
  • trastuzumab is a therapeutic anti-HER2 antibody that blocks intracellular signal transduction mediated by HER2 and is widely used to treat HER2 overexpressing tumors.
  • anti-HER2 antibodies are usually used in combination with chemotherapeutics (Slamon DJ et al., N Engl J Med, 344:783-792, 2001).
  • immune checkpoint immune checkpoint
  • Programmed death protein-1 is an important immune checkpoint protein. It is a 55kDa type I transmembrane protein. It is mainly expressed inductively on the surface of activated T cells, and also expressed on B cells, NK cells, and single cells. Nuclear cells, DC cells and other cells. Two cell surface glycoprotein ligands for PD-1 have been identified, namely programmed death protein ligand 1 (PD-L1) and programmed death protein ligand 2 (PD-L2). PD-1 ligand is highly expressed on many cancer cells. The binding of PD-1 and PD-1 ligand can lead to T cell apoptosis, immune non-response, T cell "exhaustion” and secretion of IL-10, etc.
  • BMS Bristol-Myers Squibb
  • Nivolumab Nivolumab
  • Merck Merck
  • Pembrolizumab Pembrolizumab
  • the anti-PD-1 monoclonal antibody can inhibit the binding of PD-1 and its ligands PD-L1 and PD-L2 after binding to PD-1 on T lymphocytes, thereby promoting T lymphocyte activation, proliferation and immunity Activated cytokines such as IL-2, and relieve the inhibition of PD-1 on the immune surveillance of T lymphocytes with anti-tumor activity.
  • the inventors have obtained an anti-PD-1/HER2 bispecific antibody targeting PD-1 and targeting HER2 through intensive research. It can simultaneously target HER2 on tumor cells and activate T lymphocytes, and has the advantage of enhancing anti-tumor effects while reducing side effects.
  • the patent application number of the anti-PD-1/HER2 bispecific antibody is PCT/CN2018/075851 (application date: February 8, 2018), in which the anti-PD-1 half antibody and the anti-HER2 half antibody are constructed and expressed Anti-PD-1/HER2 bispecific antibody composed of antibodies.
  • the anti-PD-1/HER2 bispecific antibody was administered to tumor-bearing mice produced by using HCC1954 human breast cancer cells to inoculate immunodeficient NCG mice. The results showed that it was similar to the administration of anti-HER2 monoclonal antibody or anti-PD-1 monoclonal antibody. In comparison, the anti-PD-1/HER2 bispecific antibody has significantly improved anti-tumor activity and can significantly reduce tumor volume.
  • anti-PD-1/HER2 bispecific antibody preparations that can be used to treat, prevent or delay various diseases related to the HER2 signaling pathway and the PD-1 signaling pathway, and the preparations have good stability,
  • the anti-PD-1/HER2 bispecific antibody in the liquid solution is not easy to decompose, aggregate, or undergo undesired chemical modification.
  • the present invention meets the above-mentioned needs by providing a pharmaceutical preparation containing an anti-PD-1/HER2 bispecific antibody protein that specifically binds PD-1 and HER2.
  • the antibody preparation of the present invention not only enables the antibody to be formulated in a manner suitable for administration to a subject, but also maintains its stability during storage and subsequent use.
  • the present invention provides a liquid antibody preparation comprising (i) anti-PD-1/HER2 bispecific antibody protein; (ii) buffer, (iii) stabilizer, and (iv) surfactant .
  • the anti-PD-1/HER2 bispecific antibody protein in the antibody preparation of the present invention comprises a first half antibody and a second half antibody, wherein the first half antibody comprises a first VH/VL unit that specifically binds PD-1 and a second half antibody
  • the half antibody contains a second VH/VL unit that specifically binds HER2.
  • the anti-PD-1/HER2 bispecific antibody protein can be at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or more
  • the affinity constant binds to PD-1 on the surface of T lymphocytes, thereby blocking the binding of PD-1 to its ligand, promoting T lymphocyte activation, proliferation and production of immune-activated cytokines such as IL-2; and at least about An affinity constant of 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or stronger binds to HER2 on the surface of tumor cells, thereby blocking intracellular signals mediated by HER2 Conduct and play an anti-tumor effect.
  • the anti-PD-1/HER2 bispecific antibody protein is the recombinant anti-PD-1/HER2 double disclosed in PCT application number PCT/CN2018/075851 (application date: February 8, 2018) Specific antibody protein.
  • PCT application number PCT/CN2018/075851 application date: February 8, 2018
  • Specific antibody protein for the purposes of this application, the entire content of the PCT application is hereby incorporated by reference.
  • the anti-PD-1/HER2 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises a first VH/VL unit that specifically binds PD-1 and a second half antibody
  • the half-antibody comprises a second VH/VL unit that specifically binds HER2, wherein the first VH/VL unit comprises the paired heavy chain variable region sequence/light chain variable of SEQ ID NO: 12/SEQ ID NO: 10 All the heavy chain CDRs and light chain CDRs contained in the region sequence, and wherein the second VH/VL unit includes the paired heavy chain variable region sequence/light chain sequence of SEQ ID NO: 6/SEQ ID NO: 2 All heavy chain CDRs and light chain CDRs contained in the variable region sequence.
  • the anti-PD-1/HER2 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises a first VH/VL unit that specifically binds PD-1 and a second half antibody
  • the half-antibody comprises a second VH/VL unit that specifically binds HER2, wherein the first VH/VL unit comprises the paired heavy chain variable region sequence/light chain variable of SEQ ID NO: 12/SEQ ID NO: 10 Region sequence, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the paired heavy chain variable region sequence/light chain variable region sequence , A sequence of 99% or more sequence identity
  • the second VH/VL unit includes the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 6/SEQ ID NO: 2 , Or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the paired heavy
  • the anti-PD-1/HER2 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises SEQ ID NO: 12 and SEQ ID NO: from N to C direction. 14 heavy chain sequence or heavy chain sequence with at least 90%, 95%, 98% or 99% identity with it, and light chain sequence of SEQ ID NO: 10 and SEQ ID NO: 4 from N to C direction Or a light chain sequence with at least 90%, 95%, 98%, or 99% identity with the light chain, and wherein the second half antibody comprises the heavy chain of SEQ ID NO: 6 and SEQ ID NO: 8 from the N to C direction The sequence or the heavy chain sequence with at least 90%, 95%, 98% or 99% identity, and the light chain sequence of SEQ ID NO: 2 and SEQ ID NO: 4 from N to C direction or with Light chain sequences that are at least 90%, 95%, 98%, or 99% identical.
  • the anti-PD-1/HER2 bispecific antibody protein is HEK293T, HEK293F, or HEK293E cells modified on the basis of HEK293 cells or HEK293 cells; CHO cells or modified on the basis of CHO cells the resulting CHO-S, CHO-dhfr - , CHO / DG44, ExpiCHO recombinantly expressed anti-PD-1 / HER2 bispecific antibody proteins.
  • the concentration of the anti-PD-1/HER2 bispecific antibody protein in the liquid antibody preparation of the present invention is about 1-150 mg/ml. In another embodiment, the concentration of the anti-PD-1/HER2 bispecific antibody protein in the liquid antibody preparation of the present invention is about 10-100 mg/mL. In other embodiments, the concentration of the anti-PD-1/HER2 bispecific antibody protein in the liquid antibody preparation of the present invention is about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg/ml .
  • the concentration of the buffer in the liquid antibody formulation of the present invention is about 5-50 mM. In one embodiment, the concentration of the buffer in the liquid antibody preparation of the present invention is about 10-30 mM, for example, about 10, 15, 20, 25, 30 mM.
  • the buffer is selected from histidine, histidine hydrochloride and combinations thereof.
  • the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 50-500 mM. In one embodiment, the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 100-400 mM, for example, about 100, 150, 200, 250, 300, 350, 400 mM.
  • the stabilizer is selected from polyols (for example, sorbitol), sugars (for example, sucrose, trehalose), and any combination thereof.
  • polyols for example, sorbitol
  • sugars for example, sucrose, trehalose
  • the stabilizer is selected from polyols (e.g., sorbitol), sugars (e.g., sucrose, trehalose), and combinations of any combination thereof and antioxidants.
  • the total concentration of the stabilizer in the liquid antibody preparation is about 50-500 mM, preferably the total concentration is about 100-400 mM, for example, about 100, 150, 200, 250, 300, 350, 400 mM
  • the concentration of the antioxidant is about 1-50 mM, preferably about 5-40 mM, for example about 5, 10, 20, 30, 40 mM.
  • the antioxidant is methionine.
  • the concentration of the surfactant in the liquid antibody preparation of the present invention is about 0.1-1 mg/ml. In one embodiment, the concentration of the surfactant in the liquid antibody preparation of the present invention is about 0.2-0.8 mg/ml, such as about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml.
  • the surfactant is a nonionic surfactant. In one embodiment, the surfactant is selected from polysorbate surfactants. In a specific embodiment, the surfactant in the liquid antibody formulation of the present invention is polysorbate-80.
  • the pH of the liquid formulation is about 5.0-6.5. In some embodiments, the pH of the liquid formulation is about any value from 5.0 to 6.5, for example, about 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4.
  • the liquid preparation is a pharmaceutical preparation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection. In one embodiment, the liquid formulation is an intravenous infusion.
  • liquid antibody formulation of the invention comprises:
  • the pH of the liquid formulation is about 5.0-6.5, preferably about 5.5.
  • liquid antibody preparation of the present invention comprises:
  • the pH of the liquid formulation is about 5.0-6.5, preferably about 5.5.
  • liquid antibody preparation of the invention comprises
  • the pH of the liquid formulation is about 5.0-6.5, preferably about 5.5.
  • liquid antibody preparation of the invention comprises
  • the pH of the liquid formulation is about 5.0-6.5, preferably about 5.5.
  • liquid antibody preparation of the invention comprises
  • the pH of the liquid formulation is about 5.0-6.5, preferably about 5.5.
  • liquid antibody preparation of the invention comprises
  • pH of the liquid formulation is about 5.0-6.5, preferably about 5.5
  • liquid antibody preparation of the invention comprises
  • pH of the liquid formulation is about 5.0-6.5, preferably about 5.5
  • liquid antibody preparation of the invention comprises
  • pH of the liquid formulation is about 5.0-6.5, preferably about 5.5
  • the present invention provides a solid antibody preparation, which is obtained by subjecting the liquid antibody preparation of the present invention to curing treatment.
  • the curing treatment is performed by, for example, a crystallization method, a spray drying method, or a freeze drying method.
  • the solid antibody preparation is, for example, in the form of a lyophilized powder injection.
  • the solid antibody preparation can be reconstituted in a suitable solvent before use to form the reconstituted preparation of the present invention.
  • the reconstituted preparation is also a liquid antibody preparation of the present invention.
  • the appropriate solvent is selected from water for injection, organic solvent for injection, including but not limited to oil for injection, ethanol, propylene glycol, etc., or a combination thereof.
  • the liquid preparation of the present invention can be stored stably for a long time, for example, at least 24 months or more.
  • the liquid formulation of the present invention can be heated at about -80°C to about 45°C, for example -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about Store at 35°C, about 38°C, about 40°C, about 42°C or about 45°C for at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months , At least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months , At least 36 months, or longer, and stable.
  • the liquid formulation of the present invention can be stored stably for at least 24 months.
  • the liquid formulation of the invention is stable at at least 40°C.
  • the liquid formulation of the present invention is stable at about 2°C-8°C for at least 3 months, preferably at least 12 months, and more preferably at least 24 months.
  • the liquid formulation of the present invention is stable at room temperature or, for example, about 25°C for at least 2 months, preferably at least 3 months, more preferably at least 6 months.
  • the liquid formulation of the invention remains stable at about 40°C for at least 2 weeks, preferably at least 1 month.
  • the stability of the preparation after storage can be indicated by detecting changes in the appearance, visible foreign matter, protein content, turbidity, purity, and/or charge variants of the preparation. In one embodiment, it can be in a forced experiment of high temperature stress, for example after storage at 40°C ⁇ 2°C for at least 1 week, 2 weeks or preferably 1 month, or in an accelerated experiment, for example at 25°C ⁇ 2°C After storage for at least 1 month or 2 months, or in a long-term experiment, for example, after storage at 5°C ⁇ 3°C for at least 2 months or 3 months, the stability of the liquid preparation of the present invention is tested.
  • the stability of the liquid formulation of the present invention is visually inspected, wherein the liquid formulation of the present invention remains clear to slightly opalescent in appearance, a colorless to pale yellow liquid, and no foreign matter. In one embodiment, by visual inspection under a clarity detector, no visible foreign matter exists in the preparation. In one embodiment, after storage, the stability of the liquid formulation of the present invention is checked by measuring the change in protein content, for example, by ultraviolet spectrophotometry (UV) method, relative to the initial value on day 0 of storage, the rate of change in protein content is Not more than 20%, preferably not more than 10%, for example 7-8%, more preferably not more than 5%.
  • UV ultraviolet spectrophotometry
  • the stability of the liquid preparation of the present invention is checked by measuring the change in turbidity of the liquid preparation of the present invention, for example, by the OD 350mm method, the change value is relative to the initial value on the 0th day of storage. Not more than 0.06, preferably not more than 0.05, more preferably not more than 0.04. In one embodiment, after storage, the stability of the liquid preparation of the present invention is checked by measuring the change in purity of the liquid preparation of the present invention, wherein size exclusion high performance liquid chromatography (SEC-HPLC) is used to check the stability of the liquid preparation of the present invention.
  • SEC-HPLC size exclusion high performance liquid chromatography
  • the change value of monomer purity does not exceed 10%, such as 5%, 4%, 3%, for example, the change value does not exceed 1-2%, and preferably does not exceed 1%.
  • the stability of the liquid preparation of the invention is checked by measuring the purity change of the liquid preparation of the invention, wherein the stability of the liquid preparation of the invention is checked by non-reduced and/or reduced sodium lauryl sulfate capillary electrophoresis (CE- In the SDS method, the change value of monomer purity does not exceed 10%, for example, does not exceed 5%, 4%, or 3%.
  • the stability of the liquid preparation of the present invention is tested by imaging capillary isoelectric focusing electrophoresis (iCIEF), wherein the charge variant of the antibody (principal component, acidic) is compared to the initial value on day 0 of storage.
  • iCIEF capillary isoelectric focusing electrophoresis
  • the sum of the change values of the components and the alkaline components does not exceed 50%, for example, does not exceed 40%, 30%, 20%, 10%, 5%.
  • the stability of the liquid preparation of the present invention is tested by cation exchange high performance liquid chromatography (CEX-HPLC method), wherein the charge variant of the antibody (The total change value of the main component, acidic component and alkaline component) does not exceed 40%, for example, does not exceed 38%, 36%, 34%, 32%, 30%.
  • CEX-HPLC method cation exchange high performance liquid chromatography
  • the formulation is stable after storage, such as after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 3 months, or after storage at 40°C ⁇ 2°C for 1 month ,
  • the formulation is stable after storage, such as after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 3 months, or after storage at 40°C ⁇ 2°C for 1 month ,
  • the preparation has a purity greater than 90%, preferably greater than 95%, 96%, 97%, 98%, 99% purity;
  • the preparation has a purity greater than 90%, preferably greater than 92%, 94%, 96%, 98% purity;
  • the relative binding activity of the anti-PD-1/HER2 bispecific antibody protein in the preparation is 70%-130%, for example, 70%, 80% , 90%, 100%, 110%, 120%, 130%.
  • the present invention provides a delivery device comprising the liquid antibody preparation or solid antibody preparation of the present invention.
  • the delivery device of the present invention is provided in the form of a pre-filled syringe containing the liquid antibody preparation or solid antibody preparation of the present invention, for example for intravenous, subcutaneous, intradermal or intramuscular injection, intravenous infusion .
  • the present invention provides a method for delivering anti-PD-1/HER2 bispecific antibody protein to a subject, such as a mammal, comprising the step of administering the liquid antibody preparation or solid antibody preparation of the present invention to the subject
  • the delivery is carried out, for example, by a delivery device using a pre-filled syringe.
  • the present invention provides the use of the liquid antibody preparation or solid antibody preparation of the present invention for preparing a drug for treating, preventing or delaying diseases related to the HER2 signaling pathway and PD-1 signaling pathway in a subject Delivery devices (eg, pre-filled syringes) or drugs, such as various blood diseases and solid tumors, including but not limited to leukemia, lymphoma, myeloma, brain tumors, head and neck squamous cell carcinoma, non-small Cell lung cancer, nasopharyngeal cancer, esophageal cancer, stomach cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, Renal cell carcinoma, melanoma.
  • Delivery devices eg, pre-filled syringes
  • drugs such as various blood diseases and solid tumors, including but not limited
  • Figure 1 illustrates the structure of an anti-PD-1/HER2 bispecific antibody, including an anti-PD-1 half antibody molecule and an anti-HER2 half antibody molecule.
  • Figure 2 shows the turbidity change trend graph of each sample measured by the OD 350nm method after anti-PD-1/HER2 bispecific antibody preparations are placed at pH 5.0, 5.5, 6.0, 6.5 at 40°C ⁇ 2°C for different times.
  • T0 on the abscissa in the figure represents 0 days; 1W represents 1 week; 2W represents 2 weeks; 1M represents 1 month.
  • Figure 3 shows the trends of protein purity in each sample measured by SEC-HPLC after anti-PD-1/HER2 bispecific antibody preparations are placed at pH 5.0, 5.5, 6.0, 6.5 at 40°C ⁇ 2°C for different times .
  • T0 on the abscissa in the figure represents 0 days; 1W represents 1 week; 2W represents 2 weeks; 1M represents 1 month.
  • Figure 4 shows the protein purity of each sample measured by non-reducing CE-SDS after anti-PD-1/HER2 bispecific antibody preparations are placed at pH 5.0, 5.5, 6.0, 6.5 at 40°C ⁇ 2°C for different time Change trend graph.
  • T0 on the abscissa in the figure represents 0 days; 1W represents 1 week; 2W represents 2 weeks; 1M represents 1 month.
  • Figure 5 shows the changes in protein purity of each sample measured by the reduced CE-SDS method after anti-PD-1/HER2 bispecific antibody preparations are placed at pH 5.0, 5.5, 6.0, 6.5 at 40°C ⁇ 2°C for different times Trend.
  • T0 on the abscissa in the figure represents 0 days; 1W represents 1 week; 2W represents 2 weeks; 1M represents 1 month.
  • Figure 6 shows the changes in the principal components of charge variants in each sample measured by the iCIEF method after anti-PD-1/HER2 bispecific antibody preparations are placed at pH 5.0, 5.5, 6.0, 6.5 at 40°C ⁇ 2°C for different times Trend.
  • T0 on the abscissa in the figure represents 0 days; 1W represents 1 week; 2W represents 2 weeks; 1M represents 1 month.
  • Figure 7 shows that anti-PD-1/HER2 bispecific antibody preparations (prescriptions 1-4) with different stabilizers are stored at about 40°C for 0 days, 1 week, 2 weeks and 4 weeks, and then measured by iCIEF method The main component of the charge variant of the graph over time.
  • T0 on the abscissa represents 0 day; 1W represents 1 week; 2W represents 2 weeks; 4W represents 4 weeks; F1 represents prescription 1, F2 represents prescription 2, F3 represents prescription 3, and F4 represents prescription 4.
  • Figure 8 shows that anti-PD-1/HER2 bispecific antibody preparations (prescriptions 1-4) with different stabilizers were stored at 25°C ⁇ 2°C for 0 days, 1 week, 2 weeks and 4 weeks, and passed iCIEF The main component of the charge variant measured by the method over time.
  • T0 on the abscissa indicates 0 days; 1M indicates 1 month; 2M indicates 2 months; F1 indicates prescription 1, F2 indicates prescription 2, F3 indicates prescription 3, and F4 indicates prescription 4.
  • the term “comprising” or “including” means to include the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprises” or “includes” when used, unless otherwise specified, it also covers the situation consisting of the stated elements, integers or steps.
  • an antibody variable region that "comprises” a specific sequence when referring to an antibody variable region that "comprises” a specific sequence, it is also intended to encompass the antibody variable region composed of the specific sequence.
  • antibody is used in the broadest sense, and refers to a protein containing an antigen binding site, encompassing natural antibodies and artificial antibodies of various structures, including but not limited to whole antibodies and antigen-binding fragments of antibodies.
  • the terms “whole antibody”, “full-length antibody”, “full antibody” and “whole antibody” are used interchangeably herein to refer to at least two heavy chains (H) and two Light chain (L) glycoprotein.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region.
  • the heavy chain constant region is composed of three structural domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • the VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDR)), with more conservative regions (framework regions (FR)) interposed between them.
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four
  • the FR composition is arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant region does not directly participate in the binding of the antibody to the antigen, but displays a variety of effector functions.
  • humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human HVR and amino acid residues from human FR.
  • the humanized antibody comprises all or substantially all of the HVR (eg, CDR) corresponding to those of the non-human antibody and all or substantially all of the FR regions corresponding to those of the human antibody.
  • a humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.
  • half antibody refers to a monovalent antigen binding polypeptide.
  • the half-antibody or semimer comprises a VH/VL unit and optionally at least a portion of an immunoglobulin constant domain.
  • the half-antibody or semimer comprises an immunoglobulin heavy chain associated with an immunoglobulin light chain, or an antigen-binding fragment thereof.
  • the half-antibody or semimer is monospecific, that is, binds to a single antigen or epitope. In some specific embodiments, the half-antibody binds to HER2 and does not bind to PD-1.
  • the half-antibody binds to PD-1 and does not bind to HER2.
  • a half antibody may have an antigen binding domain composed of a single variable domain (for example, derived from Camelidae).
  • VH/VL unit refers to an antigen binding region in an antibody that includes at least one VH CDR and at least one VL CDR.
  • the VH/VL unit comprises at least one, at least two, or all three VH CDRs and at least one, at least two, or all three VL CDRs.
  • the VH/VL unit also includes at least a portion of the framework region (FR).
  • the VH/VL unit includes three VH CDRs and three VL CDRs.
  • the VH/VL unit comprises at least one, at least two, at least three, or all four VH FRs and at least one, at least two, at least three, or all four VL FRs.
  • the term "bispecific antibody” includes antigen binding domains that specifically bind to epitopes on two different biomolecules. Unless otherwise specified, the order of the antigens bound by the bispecific antibodies in the listed names of the bispecific antibodies is arbitrary. That is, in some embodiments, the terms “anti-PD-1/HER2 bispecific antibody” and “anti-HER2/PD-1 bispecific antibody” can be used interchangeably.
  • the bispecific antibody comprises two half antibodies, wherein each half antibody comprises a single heavy chain variable region and optionally at least a portion of a heavy chain constant region and a single light chain variable region and optionally At least part of the constant region of the light chain.
  • the bispecific antibody comprises two half antibodies, wherein each half antibody comprises a single heavy chain variable region and a single light chain variable region and does not contain more than one single heavy chain variable region and does not contain More than one single light chain variable region. In some embodiments, the bispecific antibody comprises two half antibodies, wherein each half antibody comprises a single heavy chain variable region and a single light chain variable region, and wherein the first half antibody binds to the first antigen and does not bind to The second antigen binds and the second half antibody binds to the second antigen and does not bind to the first antigen.
  • antibody preparation refers to a preparation in a form that allows the biological activity of the antibody as an active ingredient to be effectively exerted, and does not contain unacceptable toxicity to the subject to which the preparation is to be administered. Other components. Such antibody preparations are usually sterile. Generally, pharmaceutically acceptable excipients are included in antibody preparations.
  • a "pharmaceutically acceptable" excipient is an agent that can be reasonably administered to a test mammal so that an effective dose of the active ingredient used in the formulation can be delivered to the subject. The concentration of the excipient is adapted to the mode of administration, for example, it may be acceptable for injection.
  • anti-PD-1/HER2 bispecific antibody preparation is also abbreviated as "antibody preparation of the present invention” herein, meaning that it contains an anti-PD-1/HER2 bispecific antibody protein as an active ingredient and contains pharmaceutically acceptable Preparation of excipients. After combining the anti-PD-1/HER2 bispecific antibody protein with pharmaceutically acceptable excipients, the anti-PD-1/HER2 bispecific antibody protein as the active ingredient is suitable for therapeutic or prophylactic administration to humans or non-humans animal.
  • the antibody preparation of the present invention can be prepared, for example, as an aqueous liquid preparation, for example, a ready-to-use pre-filled syringe, or prepared as a lyophilized preparation, which is carried out by dissolving and/or suspending in a physiologically acceptable solution immediately before use Reconstitution (ie, reconstitution).
  • the anti-PD-1/HER2 bispecific antibody protein preparation is in the form of a liquid preparation.
  • a “stable” antibody preparation is when the antibody in the preparation retains an acceptable degree of physical and/or chemical stability after storage under specific conditions. Although the antibody contained in the antibody preparation may not maintain 100% of its chemical structure after storage for a specific time, it usually maintains about 90%, about 95%, about 96%, about 97%, about 98% after storage for a specific time.
  • the antibody preparation is considered “stable.”
  • the anti-PD-1/HER2 bispecific antibody protein preparations of the present invention exhibit low to detectable antibody aggregation or degradation or chemical modification during the manufacturing, preparation, transportation and long-term storage processes, As a result, there is little or no loss of the biological activity of the anti-PD-1/HER2 bispecific antibody protein, showing high stability.
  • the anti-PD-1/HER2 bispecific antibody protein formulation of the present invention substantially retains its physical and chemical stability after storage.
  • the liquid formulation of the present invention can be stable at room temperature or at 40°C for at least 2 weeks, and/or at 25°C for at least 2 months, and/or at 2-8°C for at least 24 months.
  • the stability can be measured at a selected temperature and selected storage time. For example, the storage time can be selected based on the expected shelf life of the formulation. Alternatively, an accelerated stability test can be used. In some embodiments, stability testing is performed by performing various stress tests on antibody preparations.
  • the formulated anti-PD-1/HER2 bispecific antibody protein preparation can be filled into glass vials to test the antibody stability under high temperature stress.
  • the preparation After a period of storage, the preparation does not show aggregation, precipitation, turbidity and/or denaturation; or shows very little aggregation, precipitation, turbidity and/or denaturation, it can be considered that the antibody "maintains its physical stability" in the preparation.
  • the accumulation of antibodies in the preparation can potentially lead to an increased immune response in the patient, leading to safety issues. Therefore, there is a need to minimize or prevent aggregation of antibodies in the formulation.
  • Light scattering methods can be used to determine visible aggregates in the formulation.
  • SEC can be used to determine soluble aggregates in the formulation.
  • the stability of the preparation can be indicated by visually inspecting the appearance, color, and/or clarity of the preparation, or detecting the turbidity of the preparation by the OD 350nm method, or measuring the purity of the preparation by the non-reducing CE-SDS method.
  • the stability of the formulation is measured by determining the percentage of antibody monomers in the formulation after storage at a specific temperature for a specific time, wherein the higher the percentage of antibody monomers in the formulation, the higher the stability of the formulation .
  • an "acceptable degree" of physical stability can mean that at least about 92% of the anti-PD-1/HER2 bispecific antibody protein monomer is detected in the preparation after storage at a specific temperature for a specific time.
  • an acceptable degree of physical stability indicates At least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-PD-1/HER2 bispecific antibody protein list body.
  • the specific temperature at which the pharmaceutical preparation is stored can be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C, about -20°C, about 0°C, About 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C.
  • the pharmaceutical preparation is considered stable.
  • the anti-PD-1/HER2 bispecific antibody protein monomer is considered stable. If stored at about 5°C for 9 months, at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% are detected The anti-PD-1/HER2 bispecific antibody protein monomer is considered stable. If stored at about 5°C for 9 months, at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% are detected The anti-PD-1/HER2 bispecific antibody protein monomer is considered stable.
  • the antibody in the preparation After a period of storage, if the antibody in the preparation does not show significant chemical changes, it can be considered that the antibody "maintains its chemical stability" in the preparation.
  • Most chemical instabilities result from the formation of covalently modified forms of antibodies (for example, charge variants of antibodies).
  • charge variants of antibodies for example, by aspartic acid isomerization, N and C terminal modification, basic variants can be formed; by deamidation, sialylation and saccharification, acidic variants can be generated.
  • Chemical stability can be assessed by detecting and/or quantifying the chemically modified form of the antibody.
  • the charge variant of the antibody in the preparation can be detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing electrophoresis (iCIEF).
  • CEX cation exchange chromatography
  • iCIEF imaging capillary isoelectric focusing electrophoresis
  • the stability of the formulation is measured by determining the percentage change in the charge variant of the antibody in
  • the "acceptable degree" of chemical stability can mean that the percentage change of charge variants (such as the main component or acidic component or alkaline component) in the preparation after storage at a specific temperature for a specific time does not exceed 50%, for example, does not exceed 30%, not more than 20%.
  • an acceptable degree of chemical stability can be The percentage change value of the main component charge variant does not exceed about 50%, 40%, 30%, 20%, 15%.
  • the temperature for storing the pharmaceutical preparation can be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, or about 45°C.
  • the pharmaceutical preparation can be considered stable.
  • the pharmaceutical preparation can also be regarded as stable. If the percentage change value of the principal component charge variant is less than about 50%, 40%, 30%, 20%, 10%, 5% or 4% after storage at 40°C for 1 month, the pharmaceutical preparation can also be Considered to be stable.
  • lyophilized preparation refers to a composition obtained or obtainable by freeze-drying a liquid preparation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.
  • reconstituted preparation refers to a liquid preparation obtained by dissolving and/or suspending a solid preparation (for example, a lyophilized preparation) in a physiologically acceptable solution.
  • room temperature refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, more preferably 25°C.
  • Stress conditions refer to environments that are chemically and/or physically unfavorable to the antibody protein, which can lead to unacceptable instability of the antibody protein.
  • High temperature stress refers to storing the antibody preparation at room temperature or even higher temperature (for example, 40°C ⁇ 2°C) for a period of time. Through the accelerated test of high temperature stress, the stability of the antibody preparation can be checked.
  • parenteral administration means administration methods other than enteral and local administration, usually by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, and intrathecal , Intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspine, epidural and intrasternal injections and infusions .
  • the stabilized anti-PD-1/HER2 bispecific antibody protein formulation of the present invention is administered to a subject parenterally.
  • the anti-PD-1/HER2 bispecific antibody protein formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular or intravenous injection.
  • the present invention provides a stable liquid antibody preparation comprising (i) an anti-PD-1/HER2 bispecific antibody protein, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, the antibody preparation
  • the pH is about 5.0-6.5.
  • the liquid antibody preparation of the present invention is in the form of an injection preparation.
  • the "anti-PD-1/HER2 bispecific antibody protein" in the antibody preparation of the present invention includes a first half antibody and a second half antibody, wherein the first half antibody includes a first VH/VL unit that specifically binds PD-1 and The second half antibody contains a second VH/VL unit that specifically binds HER2.
  • the anti-PD-1/HER2 bispecific antibody protein can be at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or more
  • the affinity constant of is bound to PD-1 on the surface of T lymphocytes, and can be at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or stronger affinity constant
  • the HER2 on the surface of tumor cells binds, so that the antibody can be used as a therapeutic and/or preventive agent for bispecific targeting of PD-1 molecules and HER2 molecules.
  • VH/VL unit that specifically binds PD-1 or HER2
  • VH/VL unit that specifically binds PD-1 or HER2
  • it contains 6 VH/VL units derived from any anti-PD-1 antibody reported in the prior art and an anti-PD-1 antibody developed in the future
  • a sequence of one, four, five, six or more amino acid changes e.g., amino acid substitutions or deletions).
  • the first VH/VL unit of the anti-PD-1/HER2 bispecific antibody protein that specifically binds to PD-1 comprises SEQ ID NO: 12/SEQ ID NO derived from the anti-PD-1 half antibody : All 6 heavy chain CDRs and light chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequence of 10, or one or more of the 6 CDRs, A sequence of two, three, four, five, six or more amino acid changes (for example, amino acid substitutions or deletions).
  • the second VH/VL unit of the anti-PD-1/HER2 bispecific antibody protein that specifically binds to HER2 comprises the composition of SEQ ID NO: 6/SEQ ID NO: 2 derived from the anti-HER2 half antibody
  • SEQ ID NO: 6/SEQ ID NO: 2 derived from the anti-HER2 half antibody
  • CDR or “complementarity determining region” or “CDR region” (used interchangeably with hypervariable region “HVR” herein) is an amino acid region in the variable region of an antibody that is mainly responsible for binding to an epitope.
  • the CDRs of the heavy and light chains are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • CDR1, CDR2, and CDR3 are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • Various schemes for determining the CDR sequence of a given VH or VL or VHH amino acid sequence are known in the art. For example, the Kabat complementarity determining region (CDR) is determined based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
  • amino acid changes for example, amino acid substitutions are preferably conservative amino acid substitutions.
  • conservative amino acid substitution refers to an amino acid change that results in the substitution of a certain amino acid with a chemically similar amino acid. It is well known in the art to provide conservative substitution tables of functionally similar amino acids.
  • the conservatively substituted residues are from conservative substitution table A below, preferably the preferred substituted residues shown in Table A.
  • the anti-PD-1/HER2 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises a first VH/VL unit that specifically binds PD-1 and a second half antibody
  • the half-antibody comprises a second VH/VL unit that specifically binds HER2, wherein the first VH/VL unit comprises the paired heavy chain variable region sequence/light chain variable of SEQ ID NO: 12/SEQ ID NO: 10 Region sequence, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the paired heavy chain variable region sequence/light chain variable region sequence , A sequence of 99% or more sequence identity
  • the second VH/VL unit includes the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 6/SEQ ID NO: 2 , Or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the paired heavy
  • the type of the heavy chain constant region of the first half antibody and the second half antibody in the anti-PD-1/HER2 bispecific antibody protein is not particularly limited, and is preferably the heavy chain constant region of an IgG1, IgG2 or IgG4 immunoglobulin, Or a sequence that is substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical). More preferably, the heavy chain constant region is the heavy chain constant region of human IgG1 immunoglobulin, or is substantially the same (for example, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) sequence.
  • the anti-PD-1/HER2 bispecific antibody protein comprises the heavy chain constant region used in IgG1 (e.g., human IgG1). In yet another embodiment, the anti-PD-1/HER2 bispecific antibody protein comprises a heavy chain constant region for IgG4 (e.g., human IgG4).
  • the Fc domains of the two heavy chains of the anti-PD-1/HER2 bispecific antibody each contain a hinge region with "CPPC" amino acid residues, and/or each contains Y349C and S354C (according to Kabat's "EU numbering "), thus, the anti-PD-1 half antibody and the anti-HER2 half antibody form an interchain disulfide bond in the Fc region, thereby stabilizing the correct pairing of the anti-PD-1 half antibody and the anti-HER2 half antibody.
  • the anti-PD-1 half antibody and/or anti-HER2 half antibody of the anti-PD-1/HER2 bispecific antibody protein contains amino acid mutations in the Fc domain that affect the effector function of the antibody.
  • the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the amino acid mutation is present in the CH2 domain of the Fc region, for example, the anti-PD-1/HER2 bispecific antibody protein is contained in the anti-PD-1 half antibody and/or the anti-HER2 half antibody Fc Amino acid substitutions at positions 234 and 235 (EU numbering) of the region.
  • the amino acid substitutions are L234A and L235A (also referred to as "LALA mutations").
  • the light chain of the anti-PD-1/HER2 bispecific antibody protein comprises a kappa light chain constant region or a lambda light chain constant region, for example, a human kappa light chain constant region or a human lambda light chain constant region.
  • the Fc domains of the two heavy chains of the anti-PD-1/HER2 bispecific antibody protein respectively contain bumps ("knobs") or holes ("holes”). ), and the bumps or holes in the Fc domain of one heavy chain can be respectively placed in the holes or bumps in the Fc domain of the other heavy chain, whereby the two heavy chains form each other Stable association of "knob-in-hole”.
  • the amino acid substitution T366W is included in one of the two heavy chains, and the amino acid substitutions T366S, L368A, and Y407V (EU numbering) are included in the other of the two heavy chains. In this way, the bulge in one chain can be placed in the cavity in the other chain to promote the correct pairing of the two heavy chains of the anti-PD-1/HER2 bispecific antibody protein.
  • the immunoglobulin CH1 domain and CL domain of the heavy chain and light chain in each half-antibody of the anti-PD-1/HER2 bispecific antibody protein contain bumps or cavities, respectively, and CH1
  • the protrusions or holes in the domain can be placed in the holes or protrusions in the CL domain, respectively, so that the heavy and light chains in each half of the antibody also form a stable "knot-in" each other Associate.
  • the anti-PD-1/HER2 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises SEQ ID NO: 12 and SEQ ID NO from N to C direction
  • the second half of the antibody comprises SEQ ID NO: 6 and SEQ ID NO: 8 from N to C direction.
  • sequence identity refers to the degree of sequence identity on a nucleotide-by-nucleotide or amino acid-by-amino-acid basis in the comparison window.
  • the “percentage of sequence identity” can be calculated in the following way: the two best aligned sequences are compared in the comparison window, and it is determined that the same nucleic acid base (for example, A, T, C, G, I) exists in the two sequences.
  • sequence identity percentage e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met
  • the optimal alignment to determine the percent sequence identity can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithm needed to achieve the maximum alignment within the full-length sequence being compared or within the target sequence region.
  • the anti-PD-1/HER2 bispecific antibody protein in the antibody preparation of the present invention can bind to PD-1 and HER2 proteins at the same time, and maintains the affinity constant of each parent antibody, thereby blocking the HER2 signal transduction pathway and blocking Cut off the PD-1 signal transduction pathway, so as to treat, prevent or delay various diseases or disorders related to the HER2 signal transduction pathway and/or PD-1 signal transduction pathway.
  • the anti-PD-1/HER2 bispecific antibody protein of the present invention is the recombinant anti-PD-1 disclosed in PCT application number PCT/CN2018/075851 (application date: February 8, 2018) /HER2 bispecific antibody protein, which comprises a fully human anti-PD-1 half antibody and a humanized anti-HER2 half antibody, wherein the heavy chain sequence of the fully human anti-PD-1 half antibody is SEQ ID from N to C direction NO: 12 and SEQ ID NO: 14, the light chain sequence from N to C direction is SEQ ID NO: 10 and SEQ ID NO: 4, and the heavy chain sequence of the humanized anti-HER2 half antibody is from N to C direction SEQ ID NO: 6 and SEQ ID NO: 8, the light chain sequence from N to C direction is SEQ ID NO: 2 and SEQ ID NO: 4.
  • the anti-PD-1/HER2 bispecific antibody protein is obtained from HEK293 cells or HEK293T, HEK293F, and HEK293E cells modified on the basis of HEK293 cells; CHO cells or modified on the basis of CHO cells CHO-S, CHO-dhfr - , CHO/DG44, ExpiCHO are recombinantly expressed and purified.
  • the antibody in the liquid formulation of the present invention exhibits significant anti-tumor activity.
  • the anti-PD-1/HER2 bispecific antibody was administered to tumor-bearing mice produced by inoculating immunodeficient NCG mice with HCC1954 human breast cancer cells.
  • the amount of the anti-PD-1/HER2 bispecific antibody protein contained in the antibody preparation of the present invention can be changed according to the specific purpose characteristics of the preparation, the specific environment, and the specific purpose of using the preparation.
  • the antibody preparation is a liquid preparation, which may contain about 1-150 mg/ml, preferably about 10-100 mg/mL, such as about 10, 20, 30, 40, 50, 60, 70, 80, Anti-PD-1/HER2 bispecific antibody protein at 90 or 100 mg/ml.
  • the buffer is an agent that can maintain the pH of the solution within an acceptable range.
  • the buffering agent used in the formulation of the present invention can control the pH of the formulation of the present invention in a pH range of about 5.0-6.5, such as a pH of about 5.5.
  • the antibody preparations of the invention have a pH of about 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4.
  • the buffer used in the formulation of the present invention is selected from histidine, histidine hydrochloride, and combinations thereof.
  • the concentration of the buffer in the liquid antibody formulation of the present invention is about 5-50 mM. In one embodiment, the concentration of the buffer in the liquid antibody preparation of the present invention is about 10-30 mM, for example, about 10, 15, 20, 25, 30 mM.
  • the buffer used in the formulation of the invention is about 10 mM histidine. In another embodiment, the buffer used in the formulation of the invention is about 20 mM histidine.
  • the buffer used in the formulation of the invention is a combination of about 5.5 mM histidine and about 15 mM histidine hydrochloride.
  • Suitable stabilizers for use in the present invention can be selected from sugars, polyols and combinations thereof. Further, the stabilizer of the present invention may also include an antioxidant.
  • the sugars used as stabilizers can be disaccharides, trisaccharides and polysaccharides, and the sugars can be selected from but not limited to: sucrose, dextrose, lactose, maltose, trehalose, cyclodextrin, maltodextrin and dextran .
  • the sugar as a stabilizer is sucrose and/or trehalose.
  • the polyol as a stabilizer can be selected from but not limited to: mannitol, sorbitol and xylitol. In one embodiment, the polyol used as a stabilizer is sorbitol.
  • sugars and/or polyols as stabilizers are in the liquid formulation of the present invention at about 50-500 mM, preferably about 100-400 mM, for example, about 100, 150, 200, 250, 300, Concentrations of 350 and 400mM exist.
  • the antioxidant that may be included in the stabilizer of the present invention is selected from but not limited to: homocysteine, cysteine, cystathionine, methionine, glutathione, and homocysteine , Cysteine, cystathionine, methionine, and glutathione.
  • the total concentration of stabilizers is about 50-500mM, preferably the total concentration is about 100-400mM, for example, about 100, 150, 200, 250, 300, 350, 400mM, wherein the The concentration is about 1-50 mM, preferably about 5-40 mM, for example about 5, 10, 20, 30, 40 mM.
  • the liquid formulation of the present invention contains sorbitol as a stabilizer.
  • the amount of sorbitol in the liquid formulation of the present invention may be about 50-400 mM, for example, about 50, 100, 150, 200, 250, 300, 350, 400 mM.
  • the liquid formulation of the present invention contains sucrose as a stabilizer.
  • the amount of sucrose in the liquid formulation of the present invention may be about 50-300 mM, for example, about 50, 100, 150, 200, 250, 300 mM.
  • the liquid formulation of the present invention contains trehalose as a stabilizer.
  • the amount of trehalose in the liquid formulation of the present invention may be about 50-300 mM, for example, about 50, 100, 150, 200, 250, 300 mM.
  • the liquid formulation of the present invention contains a combination of sucrose and methionine as a stabilizer.
  • the total concentration of the stabilizer is about 50-500 mM, preferably the total concentration is about 100-400 mM, for example, about 100, 150, 200, 250, 300, 350, 400 mM, wherein the concentration of methionine is About 1-50 mM, preferably about 5-40 mM, for example about 5, 10, 20, 30, 40 mM.
  • surfactant refers to an organic substance having an amphiphilic structure; that is, they are composed of groups with opposite solubility tendencies, usually oil-soluble hydrocarbon chains and water-soluble ionic groups. group.
  • the surfactant in the liquid formulation of the present invention is a nonionic surfactant, for example, alkyl poly(ethylene oxide).
  • specific nonionic surfactants that can be included in the formulation of the present invention include, for example, polysorbates such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; Nick waits.
  • the liquid formulation of the present invention contains polysorbate-80 as a surfactant.
  • the amount of the surfactant contained in the antibody preparation of the present invention can be changed according to the specific purpose characteristics of the preparation, the specific environment, and the specific purpose of using the preparation.
  • the formulation may contain about 0.1-1 mg/ml, preferably about 0.2-0.8 mg/ml, for example about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml of polysorbate Class surfactants (for example, polysorbate-80).
  • the antibody liquid preparation of the present invention may or may not contain other excipients.
  • the antibody liquid preparation of the present invention also contains a tonicity modifier.
  • the tonicity modifier can be selected from the group consisting of sodium acetate, sodium lactate, sodium chloride, potassium chloride and calcium chloride.
  • the present invention provides a stable formulation containing anti-PD-1/HER2 bispecific antibody protein.
  • the anti-PD-1/HER2 bispecific antibody protein used in the formulation of the present invention can be prepared using techniques known in the art for antibody production.
  • the anti-PD-1/HER2 bispecific antibody protein can be recombinantly prepared.
  • the anti-PD-1/HER2 bispecific antibody protein of the present invention is HEK293T, HEK293F, and HEK293E cells obtained by modifying HEK293 cells or HEK293 cells; CHO cells or CHO cells.
  • recombinantly produced antibodies can be purified by conventional purification methods to provide pharmaceutical substances with sufficient reproducibility and moderate purity for the preparation of antibody preparations.
  • a commercially available protein concentration filter such as Amicon's ultrafiltration device can be used to concentrate the supernatant from the expression system.
  • the antibody can be purified using methods such as chromatography, dialysis, and affinity purification.
  • Protein A is suitable as an affinity ligand for the purification of IgG1, IgG2 and IgG4 type antibodies.
  • Other antibody purification methods such as ion exchange chromatography, can also be used. After obtaining antibodies of sufficient purity, preparations containing antibodies can be prepared according to methods known in the art.
  • the following steps can be used for preparation: (1) After fermentation, the fermentation broth is centrifuged to clarify impurities such as cells to obtain the supernatant; (2) affinity chromatography (for example, specific for IgG1, IgG2, and IgG4 antibodies) Affinity protein A column) capture antibody; (3) virus inactivation; (4) purification and purification (usually CEX cation exchange chromatography can be used) to remove impurities in the protein; (4) virus filtration (to make the virus titer Reduce, for example, 4 log10 or more); (5) Ultrafiltration/diafiltration (which can be used to replace the protein in a formulation buffer that is conducive to its stability and concentrate it to a suitable concentration for injection). See, for example, B. Minow, P. Rogge, K. Thompson, BioProcess International, Vol. 10, No. 6, 2012, pp. 48-57.
  • affinity chromatography for example, specific for IgG1, IgG2, and IgG4 antibodies
  • Affinity protein A column capture antibody
  • antibodies may undergo aggregation, degradation or chemical modification, resulting in antibody heterogeneity (including size heterogeneity and charge heterogeneity), aggregates and fragments, etc., thereby affecting the quality of antibody preparations. Therefore, it is necessary to monitor the stability of antibody preparations.
  • Various methods are known in the art that can be used to test the stability of antibody preparations.
  • methods such as reduced CE-SDS, non-reduced CE-SDS, and SEC-HPLC can be used to analyze the purity of antibody preparations and evaluate the aggregation level of antibodies; capillary isoelectric focusing (cIEF), imaging capillary, etc.
  • Focused electrophoresis (iCIEF) and ion exchange chromatography (IEX) are used to analyze charge variants in antibody preparations.
  • the stability of the preparation can be quickly judged by visually inspecting the appearance of the preparation.
  • the OD 350nm method can also be used to detect changes in the turbidity of the preparation, which can give information about the amount of soluble and insoluble aggregates.
  • ultraviolet spectrophotometry UV method
  • UV method ultraviolet spectrophotometry
  • the non-reduced CE-SDS method is an antibody purity determination method that uses capillary as a separation channel.
  • CE-SDS protein migration is driven by the surface charge caused by SDS binding, and the surface charge is proportional to the molecular weight of the protein. Since all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on molecular size or hydrodynamic radius can be achieved in the molecular sieve gel matrix of the capillary. This method has been widely used to monitor the purity of denatured intact antibodies.
  • the test sample is mixed with the SDS sample buffer and iodoacetamide.
  • the mixture can be incubated at 68-72°C for about 10-15 minutes, and the supernatant centrifuged after cooling to room temperature for analysis.
  • a UV detector is used to detect the migration of the protein and obtain an electrophoresis spectrum.
  • the purity of the antibody preparation can be calculated as the percentage of the peak area of the main IgG peak to the sum of all peak areas.
  • Size exclusion high performance liquid chromatography is another important method for antibody standards and quality control. This method is mainly based on the molecular size or hydrodynamic radius difference to separate molecules.
  • SEC-HPLC antibodies can be separated into three main forms: high molecular weight form (HMMS), main peak (mainly antibody monomer), and low molecular weight form (LMMS).
  • HMMS high molecular weight form
  • LMMS low molecular weight form
  • Antibody purity can be calculated as the percentage of the main peak area on the chromatogram of the sum of all peak areas.
  • the SEC-HPLC method the percentage of antibody monomers in the preparation product can be measured, and the content information of soluble aggregates and shears can be given.
  • Imaging capillary isoelectric focusing electrophoresis can be used to analyze the charge heterogeneity of antibodies. This method can provide quantitative distribution of charge variants.
  • iCIEF achieves molecular separation based on the charge difference (apparent pI value) of molecules in the pH gradient.
  • the separation column is usually a short capillary (for example, a silica capillary with a length of 5 cm and an inner diameter of 100 ⁇ m).
  • the protein is focused in the capillary column under high voltage, and the focusing is performed by a whole column imaging detection system operating at 280 nM. Real-time online monitoring.
  • One advantage of this technology is that the whole column detection system can simultaneously record various charge variants of antibody samples.
  • icIEF the sample is mixed with urea and icIEF buffer, where the buffer contains methyl cellulose, pi molecular weight standards, and ampholytes.
  • an iCIEF column such as an iCIEF column assembled by ProtionSimple on an iCIEF analyzer such as iCE280 analyzer (Protein Simple, Santa Clara, CA).
  • iCE280 analyzer Protein Simple, Santa Clara, CA.
  • the protein-related peak eluted before the main peak ie, the main component
  • the protein-related peak eluted after the main peak is classified as the basic component.
  • the relative amounts of the main component, acidic component, and basic component can be expressed as a percentage of the total peak area.
  • the charge variant of the antibody in the antibody preparation can also be determined by cation exchange high performance liquid chromatography (CEX-HPLC).
  • CEX-HPLC cation exchange high performance liquid chromatography
  • Accelerated stability studies can be used to check the stability properties of products, which is conducive to the screening of stable pharmaceutical formulations.
  • the formulation sample can be placed at an elevated temperature, such as about 40°C ⁇ 2°C, 25°C ⁇ 2°C, for accelerated stability studies.
  • Detection indicators can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (iCIEF method, CEX-HPLC method).
  • the efficacy or biological activity of the antibody can be tested.
  • the binding ability of the antibody and its antigen molecule (HER2 molecule and PD-1 molecule) in the preparation can be tested.
  • HER2 molecule and PD-1 molecule a variety of methods can be used to quantify the specific binding of antibodies to antigens, such as immunoassay tests, ELISA, and the like.
  • the anti-PD-1/HER2 bispecific antibody protein preparation of the present invention is stable. In one embodiment, after storage at about 5°C, 25°C, 37°C, 40°C, or 45°C for at least 1 month, 2 months or 3 months, for example, storage at 5°C ⁇ 3°C for 3 months Afterwards, the anti-PD-1/HER2 bispecific antibody protein purity in the antibody preparation of the present invention is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Or more than 99%, as determined by size exclusion chromatography or by non-reduced CS-SDS.
  • the anti-PD-1/HER2 bispecific antibody protein in the antibody preparation of the present invention is in the non-basic and non-acidic form (that is, the main peak or the main charge form), such as by iCIEF method Measured.
  • the antibody preparation of the present invention comprising the anti-PD-1/HER2 bispecific antibody protein of the present invention can be used to treat, prevent or delay various diseases related to the HER2 signaling pathway and/or PD-1 signaling pathway or disease.
  • Disease or disorder related to the HER2 signaling pathway and/or “disease or disorder related to the PD-1 signaling pathway” herein means that the anti-PD-1/HER2 bispecific antibody protein preparation of the present invention can be used for treatment (E.g. to improve) or prevent a disease or condition. Any disease or condition that can benefit from the treatment of the antibody preparation of the present invention is applicable to the present invention.
  • the preparation of the present invention containing anti-PD-1/HER2 bispecific antibody protein can be used to prevent or treat various hematological diseases and solid tumors in subjects, including but not limited to leukemia, lymphoma, myeloma, brain tumor, head Neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, Uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma.
  • the present invention also provides the use of the preparation of the present invention in the preparation of a medicament, wherein the medicament is used to deliver anti-PD-1/HER2 bispecific antibody protein to mammals, or to treat, prevent or ameliorate the aforementioned diseases and disorders One or more of.
  • the mammal is a human.
  • the antibody formulation of the present invention can be administered to a subject or patient in various ways.
  • administration can be by infusion or by syringe.
  • the present invention provides a delivery device (e.g., a syringe) comprising the antibody formulation of the present invention (e.g., a pre-filled syringe).
  • the patient will receive an effective amount of anti-PD-1/HER2 bispecific antibody protein as the main active ingredient, that is, an amount sufficient to treat, ameliorate or prevent the target disease or condition.
  • the therapeutic effect may include reducing physical symptoms.
  • the optimal effective amount and concentration of antibodies for any particular subject will depend on many factors, including the patient’s age, weight, health and/or gender, the nature and extent of the disease, the activity of the particular antibody, and the body’s Its clearance rate, and also includes any possible other treatments administered in combination with the antibody formulation.
  • the effective amount delivered can be determined within the judgment of the clinician.
  • the effective dose may be about 0.005 mg/kg body weight to about 50 mg/kg body weight, or about 0.1 mg/kg body weight to about 20 mg/kg body weight.
  • the application of known antibody-based drugs can provide some guidance.
  • the dosage can be a single dose schedule or a multiple dose schedule.
  • CE-SDS Sodium Lauryl Sulfate Capillary Gel Electrophoresis
  • iCIEF Imaging Capillary Isoelectric Focusing Electrophoresis
  • N/A means "Not applicable”.
  • the mobile phase is phosphate buffer (weigh 3.12g sodium dihydrogen phosphate dihydrate, 8.77g sodium chloride and 34.84g arginine, after dissolving in ultrapure water, adjust the pH to 6.8 with hydrochloric acid And dilute to 1000ml), the column protection solution is 0.05% (w/v) NaN 3 , the injection volume is 50 ⁇ l, the flow rate is 0.5ml/min, the collection time is 30 minutes, the column temperature is 25°C, and the detection wavelength is 280nm. Dilute the sample to be tested with ultrapure water to 2mg/ml as the test solution. Take the preparation buffer solution and dilute it with the same treatment method as the blank solution. Take 50 ⁇ l each of the blank solution and the test solution into the liquid chromatograph to start the test.
  • phosphate buffer weigh 3.12g sodium dihydrogen phosphate dihydrate, 8.77g sodium chloride and 34.84g arginine, after dissolving in ultrapure water, adjust the pH to 6.8 with hydro
  • the capillary is an uncoated capillary with an inner diameter of 50 ⁇ m, a total length of 30.2cm, and an effective length of 20.2cm. Wash the capillary column with 0.1mol/L sodium hydroxide, 0.1mol/L hydrochloric acid, ultrapure water, and 70psi electrophoresis gel before electrophoresis.
  • Sample injection conditions -5kV for 20 seconds; separation voltage: -15kV for 35 minutes.
  • the capillary column temperature is controlled at 25°C, and the detection wavelength is 220nm.
  • the capillary is an uncoated capillary with an inner diameter of 50 ⁇ m, a total length of 30.2cm, and an effective length of 20.2cm. Wash the capillary column with 0.1mol/L sodium hydroxide, 0.1mol/L hydrochloric acid, ultrapure water, and 70psi electrophoresis gel before electrophoresis.
  • Sample injection conditions -5kV for 20 seconds; separation voltage: -15kV for 35 minutes.
  • the capillary column temperature is controlled at 25°C, and the detection wavelength is 220nm.
  • iCIEF method imaging capillary isoelectric focusing electrophoresis (iCIEF method) detection.
  • the inner diameter of the capillary is 100 ⁇ m and the total length is 5cm.
  • MC solution 0.5% methylcellulose solution
  • ultrapure water should be used to rinse the capillary column.
  • the vacuum sampling method is adopted, the pre-focusing voltage and time are 1.5kV for 1 minute, the focusing voltage and time are 3kV for 8 minutes, the sampling time is 55 seconds, the sample tray temperature is 10°C, and the detection wavelength is 280nm.
  • Cathodic Stabilizer is 500mmol/L arginine solution, 0.5% MC solution reduces the adhesion between protein and capillary.
  • the test product Dilute the test product to 1.0 mg/ml with water, take 20 ⁇ l of the diluted test product solution, and add 78 ⁇ l of the pre-mixed solution to it (the pre-mixed solution is as follows: 70 ⁇ l pI 0.5% MC solution, 4 ⁇ l amphoteric electrolyte (pH 3-10) ), 2 ⁇ l cathode stabilizer, 1 ⁇ l pI 5.85 marker, 1 ⁇ l pI 9.99 marker), mix well to prepare the sample solution to be tested.
  • Sample injection analysis according to the area normalization method, calculate the main component, acidic component and alkaline component content.
  • the anti-PD-1/HER2 bispecific antibody was prepared and purified.
  • the X0GC expression vector containing the heavy and light chains of the anti-human PD-1 antibody was constructed separately (for the construction of the X0GC expression vector, see Chinese Patent Application No. 200780038403.3), in which the nucleotide sequence of the light chain variable region is as SEQ ID As shown in NO.9, the amino acid sequence is shown in SEQ ID NO: 10; the nucleotide sequence of the light chain constant region is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO: 4; the heavy chain variable region
  • the nucleotide sequence is shown in SEQ ID NO. 11, the amino acid sequence is shown in SEQ ID NO: 12; the heavy chain constant region nucleotide sequence is shown in SEQ ID NO. 13, and the amino acid sequence is shown in SEQ ID NO: 14. Show.
  • the X0GC expression vector containing the heavy and light chains of the anti-human HER2 antibody was constructed respectively, wherein the nucleotide sequence of the light chain variable region is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO: 2; light chain The constant region nucleotide sequence is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO: 4; the heavy chain variable region nucleotide sequence is shown in SEQ ID NO. 5, and the amino acid sequence is shown in SEQ ID NO. The nucleotide sequence of the constant region of the heavy chain is shown in SEQ ID NO. 7, and the amino acid sequence is shown in SEQ ID NO: 8.
  • the expression vectors containing the heavy and light chains of the anti-human PD-1 antibody were respectively transfected into 293F cells (FreeStyle TM 293-F Cells, catalog number R79007, invitrogen), expressed, purified and reduced through a process to obtain a heavy chain Chain and one light chain anti-human PD-1 half antibody molecule.
  • the expression vectors containing the heavy and light chains of the anti-human HER2 antibody were transfected into 293F cells (FreeStyle TM 293-F Cells, catalog number R79007, invitrogen), respectively, and expressed, purified and reduced through a process to obtain Anti-human HER2 half-antibody molecule of heavy chain and one light chain.
  • the reduced anti-PD-1 half-antibody molecule and the reduced anti-HER2 half-antibody molecule were mixed in an equimolar ratio, and the recombination reaction was carried out at 4°C for 24 hours to obtain a molecule containing anti-PD-1 half-antibody and anti-HER2 half-antibody A solution of bispecific antibodies of heterodimers of molecules. After the solution was concentrated by ultrafiltration through an ultrafiltration concentration tube, it was purified by AKTA explorer 100 protein purification system (GE Healthcare) and ion chromatography column Source 15S (16mm ID, 17ml, GE Healthcare) at 4°C to obtain a purity of 99.96% anti-PD-1/HER2 bispecific antibody.
  • Example 2 One of the test of the influence of pH on the stability of the formulation
  • This example examines the stability of formulations containing anti-PD-1/HER2 bispecific antibodies at pH 5.0 to 6.5. A total of 4 pH values were designed, namely 5.0, 5.5, 6.0 and 6.5.
  • x means sampling at this point in time.
  • the obtained samples are first placed in an ultra-low temperature refrigerator at -80°C for inspection, and defrosted for inspection as needed.
  • the standard for determining whether the quality of the sample has not changed compared with the initial value is set to determine whether the sample has changed. See Table 2 for details.
  • N/A means that the detection item is not set.
  • the results of the above pH test on the stability of the preparation showed that the anti-PD-1/HER2 bispecific antibody was placed at pH 5.0-6.5 at 40°C ⁇ 2°C for 2 weeks.
  • the appearance of the sample and visible foreign matter were all qualified, and the protein content was not Significant changes occurred, and the relative binding activity to the HER2 antigen and PD-1 antigen did not change significantly; in addition, the anti-PD-1/HER2 bispecific antibody was placed at pH 5.0-6.5 at 40°C ⁇ 2°C for one month.
  • the appearance of the sample and the visible foreign matter are all qualified, the protein content has not changed significantly, and the relative binding activity to the PD-1 antigen has not changed significantly.
  • the anti-PD-1/HER2 bispecific antibody is only at pH 6.0 and pH 6.5. The relative binding activity of binding to HER2 antigen decreases, but is still higher than 50%.
  • pH 5.5 is selected from pH 5.0-6.5 for the experiment.
  • a total of 4 prescriptions were designed, and the detailed prescription information is shown in Table 10.
  • the stability of each sample was investigated under the conditions of 40°C, 25°C, and 5°C.
  • the specific scheme is shown in Table 11.
  • the detection indicators are appearance, visible foreign matter, protein content, purity (SEC-HPLC method and CE-SDS method) and charge variant (iCIEF method).
  • Charge variants (iCIEF method): The results are shown in Table 16. At 40°C and 25°C ⁇ 2°C, the change trends of the main components of the charge variants in each square are shown in Figure 7 and Figure 8, respectively.
  • Formula 5 is designed, and the detailed information of prescription 5 is shown in Table 17.
  • the most preferred formulation is: about 42.0mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and humanized anti-human epidermal growth factor receptor 2 (HER2) bispecific Antibody, 0.85mg/ml histidine, 3.17mg/ml histidine hydrochloride, 80.00mg/ml sucrose, 0.2mg/ml polysorbate 80, pH 5.5.
  • PD-1 fully human anti-programmed death receptor 1
  • HER2 humanized anti-human epidermal growth factor receptor 2

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Abstract

涉及包含抗PD-1/HER2双特异性抗体的制剂，尤其涉及包含抗PD-1/HER2双特异性抗体、缓冲剂、稳定剂和表面活性剂的药物制剂。此外，还涉及这些制剂的疾病治疗或预防用途。

Description

包含抗PD-1/HER2双特异性抗体的制剂及其制备方法和用途 技术领域
本发明涉及抗体制剂领域。更具体而言，本发明涉及包含重组的抗程序性死亡受体1(PD-1)和抗人表皮生长因子受体2(HER2)双特异性抗体(也称为抗PD-1/HER2双特异性抗体)的药物制剂，尤其是稳定的液体制剂，以及用于制备所述药物制剂的方法，以及所述药物制剂的治疗和/或预防用途。
背景技术
人表皮生长因子受体2(HER2)(也称为NEU、ERBB-2、CD340或p185)的过量表达与多种癌症相关联，这些癌症包括乳腺癌、卵巢癌、胃癌、子宫癌、黑色素瘤以及胆管癌等。例如，在侵袭性、转移性乳腺癌中、在具有高复发率和/或患者预后不良的乳腺癌中，均观察到了HER2过量表达。
治疗HER2过量表达癌症的一种方法是使用抑制HER2信号传导的抗HER2抗体。例如，曲妥珠单抗(trastuzumab)是阻断由HER2介导的细胞内信号传导的一种治疗性抗HER2抗体，并且被广泛用来治疗HER2过表达肿瘤。遗憾的是，其在临床应用中的抗肿瘤效果往往没有临床前实验那么好。在现有技术中，通常将抗HER2抗体与化疗药等联合用药(Slamon DJ等人，N Engl J Med,344:783-792,2001)。
近年来，随着对免疫检查点(immune checkpoint)分子的研究，发现免疫检查点的抑制性信号通路活化导致T淋巴细胞不能有效发挥对肿瘤的杀伤效应(Yao S,Zhu Y和Chen L.，Advances in targeting cell surface signaling molecules for immune modulation.Nat Rev Drug Discov,2013,12(2):130-146)，这从一个方面导致了仅靶向肿瘤细胞上的靶点的药物(例如曲妥珠单抗)的抗肿瘤效果不佳。
程序性死亡蛋白-1(PD-1)是一种重要的免疫检查点蛋白，为55kDa I型跨膜蛋白，主要诱导性表达于活化的T细胞表面，也表达于B细胞、NK细胞、单核细胞、DC细胞等细胞上。已鉴定到PD-1的两种细胞表面糖蛋白配体，分别为程序性死亡蛋白配体1(PD-L1)和程序性死亡蛋白配体2(PD-L2)。PD-1的配体在许多癌细胞上高表达。PD-1与PD-1的配体结合后可导致T细胞凋亡、免疫无应答、T细胞“耗竭”和分泌IL-10等，因此，阻断PD1途径能在癌症患者中恢复T细胞功能(Sheridan,Nature Biotechnology 30(2012),729-730)。针对PD-1的单克隆抗体已有记载，例如，百时美施贵宝(BMS)公司的纳武单抗(Nivolumab)和默克(Merck)公司的派姆单抗(Pembrolizumab)。纳武单抗(商品名
Figure PCTCN2020107441-appb-000001
)为完全人源化的IgG4抗体分子，派姆单抗(商品名
Figure PCTCN2020107441-appb-000002
)为人源化IgG4抗体分子。所述抗PD-1单克隆抗体与T淋巴细胞上的PD-1结合后能够抑制PD-1与其配体PD-L1和PD-L2的结合，由此促进T淋巴细胞活化、增殖和产生免疫活化型细胞因子如IL-2，并解除PD-1对具有抗肿瘤活性的T淋巴细胞免疫监视的抑制。
鉴于免疫检查点分子PD-1在调节免疫应答中的重要性，本发明人通过锐意研究，已获得了一种靶向PD-1且靶向HER2的抗PD-1/HER2双特异性抗体，它能够同时靶向肿瘤细胞上的 HER2且活化T淋巴细胞，具有在增强抗肿瘤作用的同时减少副作用的优势。所述抗PD-1/HER2双特异性抗体的专利申请号是PCT/CN2018/075851(申请日：2018年2月8日)，其中构建并表达了由抗PD-1半抗体和抗HER2半抗体组成的抗PD-1/HER2双特异性抗体。对使用HCC1954人乳腺癌细胞接种免疫缺陷NCG小鼠产生的荷瘤小鼠施用抗PD-1/HER2双特异性抗体，结果表明，与施用抗HER2单克隆抗体或抗PD-1单克隆抗体相比较，抗PD-1/HER2双特异性抗体具有显著提高的抗肿瘤活性，能够显著缩小肿瘤体积。
本领域需要能够用来治疗、预防或延缓各种与HER2信号传导通路和PD-1信号传导通路相关的疾病的抗PD-1/HER2双特异性抗体制剂，且所述制剂的稳定性好，当在液体中调配时，液体溶液中的抗PD-1/HER2双特异性抗体不易分解、聚集或发生不希望的化学修饰等。
发明概述
本发明通过提供含有特异结合PD-1和HER2的抗PD-1/HER2双特异性抗体蛋白的药物制剂来满足上述需求。本发明的抗体制剂除了能够使抗体以适于施用给受试者的方式调配之外，还能在储存以及后续使用期间维持其稳定性。
在一个方面，本发明提供了一种液体抗体制剂，其包含(i)抗PD-1/HER2双特异性抗体蛋白；(ii)缓冲剂，(iii)稳定剂，和(iv)表面活性剂。
本发明抗体制剂中的抗PD-1/HER2双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含特异性结合PD-1的第一VH/VL单元并且第二半抗体包含特异性结合HER2的第二VH/VL单元。在一些实施方案中，所述抗PD-1/HER2双特异性抗体蛋白能够以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与T淋巴细胞表面的PD-1结合，由此阻断PD-1与其配体的结合，促进T淋巴细胞活化、增殖和产生免疫活化型细胞因子如IL-2；并以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与肿瘤细胞表面的HER2结合，由此阻断由HER2介导的细胞内信号传导并发挥抗肿瘤作用。
在一个实施方案中，所述抗PD-1/HER2双特异性抗体蛋白为PCT申请号PCT/CN2018/075851(申请日：2018年2月8日)中公开的重组抗PD-1/HER2双特异性抗体蛋白。为了本申请的目的，该PCT申请的全部内容特此并入本文作为参考。在一个实施方案中，抗PD-1/HER2双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含特异性结合PD-1的第一VH/VL单元并且第二半抗体包含特异性结合HER2的第二VH/VL单元，其中所述第一VH/VL单元包含SEQ ID NO:12/SEQ ID NO:10的成对重链可变区序列/轻链可变区序列中所含的全部重链CDR与轻链CDR，并且其中所述第二VH/VL单元包含SEQ ID NO:6/SEQ ID NO:2的成对重链可变区序列/轻链可变区序列中所含的全部重链CDR与轻链CDR。
在一个实施方案中，抗PD-1/HER2双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含特异性结合PD-1的第一VH/VL单元并且第二半抗体包含特异性结合HER2的第二VH/VL单元，其中所述第一VH/VL单元包含SEQ ID NO:12/SEQ ID NO:10的 成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，并且其中所述第二VH/VL单元包含SEQ ID NO:6/SEQ ID NO:2的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列。
在一个实施方案中，抗PD-1/HER2双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含从N至C方向的SEQ ID NO:12和SEQ ID NO:14的重链序列或与之具有至少90％,95％,98％或99％同一性的重链序列，和从N至C方向的SEQ ID NO:10和SEQ ID NO:4的轻链序列或与之具有至少90％,95％,98％或99％同一性的轻链序列，并且其中第二半抗体包含从N至C方向的SEQ ID NO:6和SEQ ID NO:8的重链序列或与之具有至少90％,95％,98％或99％同一性的重链序列，和从N至C方向的SEQ ID NO:2和SEQ ID NO:4的轻链序列或与之具有至少90％,95％,98％或99％同一性的轻链序列。
在一个实施方案中，所述抗PD-1/HER2双特异性抗体蛋白是在HEK293细胞或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293E细胞；CHO细胞或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO中重组表达的抗PD-1/HER2双特异性抗体蛋白。
在一个实施方案中，本发明的液体抗体制剂中的抗PD-1/HER2双特异性抗体蛋白的浓度为约1-150mg/ml。在另一个实施方案中，本发明的液体抗体制剂中的抗PD-1/HER2双特异性抗体蛋白的浓度为约10-100mg/mL。在其他实施方案中，本发明的液体抗体制剂中的抗PD-1/HER2双特异性抗体蛋白的浓度为约10、20、30、40、50、60、70、80、90或100mg/ml。
在一个实施方案中，本发明的液体抗体制剂中的缓冲剂的浓度为约5-50mM。在一个实施方案中，本发明的液体抗体制剂中的缓冲剂的浓度为约10-30mM，例如，约10、15、20、25、30mM。
在一个实施方案中，所述缓冲剂选自组氨酸、盐酸组氨酸和它们的组合。
在一个实施方案中，本发明的液体抗体制剂中的稳定剂的浓度为约50-500mM。在一个实施方案中，本发明的液体抗体制剂中的稳定剂的浓度为约100-400mM，例如，约100、150、200、250、300、350、400mM。
在一个实施方案中，所述稳定剂选自多元醇(例如，山梨醇)、糖类(例如，蔗糖、海藻糖)和它们的任意组合。
在又一个实施方案中，所述稳定剂选自多元醇(例如，山梨醇)、糖类(例如，蔗糖、海藻糖)和它们的任意组合与抗氧化剂的组合。在一个实施方案中，所述液体抗体制剂中稳定剂的总浓度为约50-500mM，优选地总浓度为约100-400mM，例如，约100、150、200、250、300、350、400mM，其中抗氧化剂的浓度为约1-50mM，优选地约5-40mM，例如约5、10、20、30、40mM。在一个实施方案中，所述抗氧化剂是甲硫氨酸。
在一个实施方案中，本发明的液体抗体制剂中的表面活性剂的浓度为约0.1-1mg/ml。在一个实施方案中，本发明的液体抗体制剂中的表面活性剂的浓度为约0.2-0.8mg/ml，例如约 0.2、0.3、0.4、0.5、0.6、0.7、0.8mg/ml。
在一个实施方案中，所述表面活性剂是非离子型表面活性剂。在一个实施方案中，所述表面活性剂选自聚山梨酯类表面活性剂。在一个具体实施方案中，本发明的液体抗体制剂中的表面活性剂是聚山梨酯-80。
在一个实施方案中，所述液体制剂的pH值为约5.0-6.5。在一些实施方案中，所述液体制剂的pH值为约5.0-6.5中的任意值，例如约5.0、5.2、5.4、5.6、5.8、6.0、6.2、6.4。
在一个实施方案中，所述液体制剂为药物制剂，优选为注射剂，更优选为皮下注射剂或静脉内注射剂。在一个实施方案中，所述液体制剂为静脉输注剂。
在一个实施方案中，本发明的液体抗体制剂包含：
(i)约1-150mg/ml的抗PD-1/HER2双特异性抗体蛋白；
(ii)约5-50mM的组氨酸和/或盐酸组氨酸；
(iii)约50-500mM的山梨醇、蔗糖、海藻糖和它们的任意组合；或者
总浓度为约50-500mM的山梨醇、蔗糖、海藻糖和它们的任意组合与甲硫氨酸的组合，其中甲硫氨酸的浓度为约1-50mM，和
(iv)约0.1-1mg/ml聚山梨醇酯80；
其中所述液体制剂的pH为约5.0-6.5，优选地约5.5。
在一个优选的实施方案中，本发明的液体抗体制剂包含：
(i)约10-100mg/ml的抗PD-1/HER2双特异性抗体蛋白；
(ii)约10-30mM的组氨酸和/或盐酸组氨酸；
(iii)约100-400mM的山梨醇、蔗糖、和/或海藻糖；或者
总浓度为约100-400mM的山梨醇、蔗糖、和/或海藻糖与甲硫氨酸的组合，其中甲硫氨酸的浓度为约5-40mM，和
(iv)约0.2-0.8mg/ml聚山梨醇酯80；
其中所述液体制剂的pH为约5.0-6.5，优选地约5.5。
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约20mg/ml的抗PD-1/HER2双特异性抗体蛋白；
(ii)约10mM组氨酸；
(iii)约50mg/ml山梨醇，和
(iv)约0.3mg/ml聚山梨醇酯80；
其中所述液体制剂的pH为约5.0-6.5，优选地约5.5。
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约50mg/ml的抗PD-1/HER2双特异性抗体蛋白；
(ii)约20mM组氨酸；
(iii)约50mg/ml山梨醇，和
(iv)约0.2mg/ml聚山梨醇酯80；
其中所述液体制剂的pH为约5.0-6.5，优选地约5.5。
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约50mg/ml的抗PD-1/HER2双特异性抗体蛋白；
(ii)约20mM组氨酸；
(iii)约80mg/ml蔗糖，和
(iv)约0.2mg/ml聚山梨醇酯80；
其中所述液体制剂的pH为约5.0-6.5，优选地约5.5。
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约50mg/ml的抗PD-1/HER2双特异性抗体蛋白；
(ii)约20mM组氨酸；
(iii)约80mg/ml海藻糖，和
(iv)约0.2mg/ml聚山梨醇酯80；
其中所述液体制剂的pH为约5.0-6.5，优选地约5.5
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约50mg/ml的抗PD-1/HER2双特异性抗体蛋白；
(ii)约20mM组氨酸；
(iii)约80mg/ml蔗糖和约1.49mg/ml甲硫氨酸，和
(iv)约0.2mg/ml聚山梨醇酯80；
其中所述液体制剂的pH为约5.0-6.5，优选地约5.5
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约42mg/ml的抗PD-1/HER2双特异性抗体蛋白；
(ii)约0.85mg/ml组氨酸和约3.17mg/ml盐酸组氨酸；
(iii)约80mg/ml蔗糖，和
(iv)约0.2mg/ml聚山梨醇酯80；
其中所述液体制剂的pH为约5.0-6.5，优选地约5.5
另一方面，本发明提供了ー种固体抗体制剂，其是通过将本发明的液体抗体制剂经固化处理而获得的。所述固化处理是通过例如结晶法、喷雾干燥法、冷冻干燥法实施的。在一个优选的实施方案中，所述固体抗体制剂例如是冻干粉针剂形式。固体抗体制剂可在使用前，通过重构于适当的溶媒中，形成本发明的重构制剂。所述重构制剂也是一种本发明的液体抗体制剂。在一个实施方案中，所述适当的溶媒选自注射用水、注射用有机溶剂，包括但不限于注射用油、乙醇、丙二醇等，或其组合。
本发明的液体制剂可以长期稳定储存，例如至少24个月或更长时间。在一个实施方案中，本发明的液体制剂可以在约-80℃至约45℃，例如-80℃、约-30℃、约-20℃、约0℃、约5℃、约25℃、约35℃、约38℃、约40℃、约42℃或约45℃的条件下，储存至少10天、至少20天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，至少36个月，或更长时间，且是稳定的。
在一个实施方案中，本发明的液体制剂可以稳定储存至少24个月。在再一实施方案中，本发明的液体制剂在至少40℃是稳定的。在再一实施方案中，本发明的液体制剂在约2℃-8℃保持稳定至少3个月，优选至少12个月，更优选至少24个月。在一个实施方案中，本发明的液体制剂在室温或例如约25℃保持稳定至少2个月，优选至少3个月，更优选至少6个月。在再一实施方案中，本发明的液体制剂在约40℃保持稳定至少2周、优选至少1个月。
在一个实施方案中，可以通过检测制剂的外观、可见异物、蛋白含量、浊度、纯度、和/或电荷变异体的变化，来指示储存后制剂的稳定性。在一个实施方案中，可以在高温胁迫的强制实验中，例如在40℃±2℃储存至少1周、2周或优选地1个月后，或在加速实验中，例如在25℃±2℃储存至少1个月或2个月后，或在长期实验中，例如在5℃±3℃储存至少2个月或3个月后，检测本发明液体制剂的稳定性。
在一个实施方案中，在储存后，通过目视检查本发明液体制剂的稳定性，其中本发明液体制剂在外观上保持为澄明至微乳光，为无色至淡黄色液体，且无异物。在一个实施方案中，在澄明度检测仪下目视检查，制剂中无可见异物存在。在一个实施方案中，在储存后，通过测定蛋白含量变化，检查本发明液体制剂的稳定性，其中例如通过紫外分光光度(UV)法，相对于储存第0天的初始值，蛋白含量变化率不超过20％，优选不超过10％，例如7-8％，更优选不超过5％。在一个实施方案中，在储存后，通过测定本发明液体制剂的浊度变化，检查本发明液体制剂的稳定性，其中例如通过OD 350mm法检测，相对于储存第0天的初始值，变化值不超过0.06，优选地不超过0.05，更优选地不超过0.04。在一个实施方案中，在储存后，通过测定本发明液体制剂的纯度变化，检查本发明液体制剂的稳定性，其中通过尺寸排阻高效液相色谱法(SEC-HPLC)，相对于储存第0天的初始值，单体纯度的变化值不超过10％，例如不超过5％、4％、3％、例如变化值不超过1-2％，优选不超过1％。在一个实施方案中，在储存后，通过测定本发明液体制剂的纯度变化，检查本发明液体制剂的稳定性，其中通过非还原型和/或还原型十二烷基硫酸钠毛细管电泳(CE-SDS)法，单体纯度的变化值下降不超过10％，例如不超过5％、4％、3％。在一个实施方案中，在储存后，通过成像毛细管等电聚焦电泳(iCIEF)检测本发明液体制剂的稳定性，其中相对于储存第0天的初始值，抗体的电荷变异体(主成分、酸性组分和碱性组分)的变化值总和不超过50％，例如不超过40％、30％、20％、10％、5％。在一个实施方案中，在储存后，通过阳离子交换高效液相色谱法(CEX-HPLC法)检测本发明液体制剂的稳定性，其中相对于储存第0天的初始值，抗体的电荷变异体(主成分、酸性组分和碱性组分)的变化值总和不超过40％，例如不超过38％、36％、34％、32％、30％。
在一个实施方案中，制剂在储存后，例如在2-8℃储存至少24个月后，或在室温储存至少3个月后，或在40℃±2℃储存1个月后，是稳定的，优选地具有如下特征之一或多项：
(i)通过SEC-HPLC法测量，制剂具有大于90％的纯度，优选大于95％、96％、97％、98％、99％的纯度；
(ii)通过还原型或非还原型CE-SDS法测量，制剂具有大于90％的纯度，优选大于92％、94％、96％、98％的纯度；
(iii)通过iCIEF法测量，相对于储存第0天的初始值，制剂中抗PD-1/HER2双特异性抗体蛋白的各组分(主成分、酸性组分和碱性组分)的变化值总和不超过50％，例如不超过40％、30％、20％、10％、5％；
(iv)通过ELISA法测量，相对于储存第0天的初始值，制剂中抗PD-1/HER2双特异性抗体蛋白的相对结合活性为70％-130％，例如，为70％、80％、90％、100％、110％、120％、130％。
在一个方面，本发明提供了一种递送装置，其包含本发明的液体抗体制剂或固体抗体制剂。在一个实施方案中，本发明的递送装置以包含本发明的液体抗体制剂或固体抗体制剂的预填装注射器形式提供，例如用于静脉内、皮下、皮内或者肌内注射、静脉内输注。
在又一方面，本发明提供向受试者，例如哺乳动物递送抗PD-1/HER2双特异性抗体蛋白的方法，包括给予所述受试者本发明的液体抗体制剂或固体抗体制剂的步骤，所述递送是例如通过使用预填装注射器的递送装置实施的。
在又一方面，本发明提供本发明的液体抗体制剂或固体抗体制剂的用途，用于制备在受试者中治疗、预防或延缓与HER2信号传导通路和PD-1信号传导通路相关的病症的递送装置(如，预填装注射器)或药物，所述病症例如各种血液病和实体瘤，包括但不限于白血病、淋巴瘤、骨髓瘤、脑肿瘤、头颈部鳞状细胞癌、非小细胞肺癌、鼻咽癌、食道癌、胃癌、胰腺癌、胆囊癌、胆管癌、肝癌、结直肠癌、乳腺癌、卵巢癌、宫颈癌、子宫内膜癌、子宫肉瘤、前列腺癌、膀胱癌、肾细胞癌、黑色素瘤。
本发明的其它实施方案将通过参阅此后的详细说明而清楚明了。
附图简述
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1例示了一种抗PD-1/HER2双特异性抗体的结构，包含抗PD-1半抗体分子和抗HER2半抗体分子。
图2显示了抗PD-1/HER2双特异性抗体制剂在pH 5.0、5.5、6.0、6.5于40℃±2℃放置不同时间后，通过OD 350nm法测定的各样品浊度变化趋势图。图中横坐标上的T0表示0天；1W表示1周；2W表示2周；1M表示1个月。
图3显示了抗PD-1/HER2双特异性抗体制剂在pH 5.0、5.5、6.0、6.5于40℃±2℃放置不同时间后，通过SEC-HPLC法测定的各样品中蛋白纯度变化趋势图。图中横坐标上的T0表示0天；1W表示1周；2W表示2周；1M表示1个月。
图4显示了抗PD-1/HER2双特异性抗体制剂在pH 5.0、5.5、6.0、6.5于40℃±2℃放置不同时间后，通过非还原型CE-SDS法测定的各样品中蛋白纯度变化趋势图。图中横坐标上的T0表示0天；1W表示1周；2W表示2周；1M表示1个月。
图5显示了抗PD-1/HER2双特异性抗体制剂在pH 5.0、5.5、6.0、6.5于40℃±2℃放置不同时间后，通过还原型CE-SDS法测定的各样品中蛋白纯度变化趋势图。图中横坐标上的T0表示0天；1W表示1周；2W表示2周；1M表示1个月。
图6显示了抗PD-1/HER2双特异性抗体制剂在pH 5.0、5.5、6.0、6.5于40℃±2℃放置不同时间后，通过iCIEF法测定的各样品中电荷变异体主成分的变化趋势图。图中横坐标上的T0表示0天；1W表示1周；2W表示2周；1M表示1个月。
图7显示了将具有不同稳定剂的抗PD-1/HER2双特异性抗体制剂(处方1-4)置于约40℃储存0天、1周、2周和4周后，通过iCIEF法测定的电荷变异体主成分随时间的变化图。图中横坐标上的T0表示0天；1W表示1周；2W表示2周；4W表示4周；F1表示处方1，F2表示处方2，F3表示处方3，F4表示处方4。
图8显示了将具有不同稳定剂的抗PD-1/HER2双特异性抗体制剂(处方1-4)置于25℃±2℃储存0天、1周、2周和4周后，通过iCIEF法测定的电荷变异体主成分随时间的变化图。图中横坐标上的T0表示0天；1M表示1个月；2M表示2个月；F1表示处方1，F2表示处方2，F3表示处方3，F4表示处方4。
发明详述
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说明特定实施方案之用，而不意欲为限制性的。
定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
在本文中，术语“抗体”以最广意义使用，指包含抗原结合位点的蛋白质，涵盖各种结构的天然抗体和人工抗体，包括但不限于完整抗体和抗体的抗原结合片段。
术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换地用来指包含由二硫键相互连接的至少两条重链(H)和两条轻链(L)的糖蛋白。每条重链由重链可变区(本文中缩写为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(本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH区和VL区可以进一步再划分为超变区(为互补决定区(CDR)，其间插有较保守的区域(为构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。
术语“人源化”抗体指包含来自非人类HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在一些实施方案中，人源化抗体包含全部或基本上全部的HVR(例如，CDR)与非人抗体的那些HVR对应并且全部或基本上全部的FR区与人抗体的那些FR对应。人源化抗体任选地可以包含从人抗体衍生的抗体恒定区的至少一部分。抗体(例如非人抗体)的“人源化形式”指已经历过人源化的抗体。
术语“半抗体”或“半聚物”指单价抗原结合多肽。在一些实施方案中，半抗体或半聚物包含VH/VL单元和任选地免疫球蛋白恒定结构域的至少一部分。在一些实施方案中，半抗体或半聚物包含与一条免疫球蛋白轻链缔合的一条免疫球蛋白重链，或其抗原结合片段。在一些实施方案中，半抗体或半聚物是单特异的，即，与单个抗原或表位结合。在一些具体的实施方案中，半抗体与HER2结合并且不与PD-1结合。在一些具体的实施方案中，半抗体与PD-1结合并且不与HER2结合。本领域技术人员将会轻易地理解，半抗体可以具有由单一可变结构域组成(例如，源自骆驼属(Camelidae))的抗原结合结构域。
术语“VH/VL单元”指抗体中包含至少一个VH CDR和至少一个VL CDR的抗原结合区。在一些实施方案中，VH/VL单元包含至少一个、至少两个或全部三个VH CDR和至少一个、至少两个或全部三个VL CDR。在某些实施方案中，VH/VL单元还包含构架区(FR)的至少一部分。在一些实施方案中，VH/VL单元包含三个VH CDR和三个VL CDR。在一些实施方案中，VH/VL单元包含至少一个、至少两个、至少三个或全部四个VH FR和至少一个、至少两个、至少三个或全部四个VL FR。
在本文中，术语“双特异性抗体”包含与两种不同生物分子上的表位特异性结合的抗原结合结构域。除非另外说明，否则列出的双特异性抗体名称中双特异性抗体结合的抗原的顺序是任意的。即，在一些实施方案中，术语“抗PD-1/HER2双特异性抗体”和“抗HER2/PD-1双特异性抗体”可以互换使用。在一些实施方案中，双特异性抗体包含两个半抗体，其中每个半抗体包含单个重链可变区和任选地重链恒定区的至少一部分以及单个轻链可变区和任选地轻链恒定区的至少一部分。在一些实施方案中，双特异性抗体包含两个半抗体，其中每个半抗体包含单个重链可变区和单个轻链可变区并且不包含多于一个单个重链可变区且不包含多于一个单个轻链可变区。在一些实施方案中，双特异性抗体包含两个半抗体，其中每个半抗体包含单个重链可变区和单个轻链可变区，并且其中第一半抗体与第一抗原结合且不与第二抗原结合并且第二半抗体与第二抗原结合且不与第一抗原结合。
术语“抗体制剂”指一种制备物，所述制备物处于允许作为活性成分的抗体的生物活性可以有效发挥的形式，并且不含有对于待施用该制剂的受试者而言具有不可接受毒性的其它组分。这类抗体制剂通常是无菌的。通常，抗体制剂中包含可药用赋形剂。“可药用”赋形剂是 可以合理地施用至受试哺乳动物以便制剂中所用活性成分的有效剂量可以递送至受试者的试剂。赋形剂的浓度与施用模式相适应，例如可以是注射可接受的。
术语“抗PD-1/HER2双特异性抗体制剂”在本文中也简称为“本发明的抗体制剂”，意指包含抗PD-1/HER2双特异性抗体蛋白作为活性成分并包含可药用赋形剂的制备物。将抗PD-1/HER2双特异性抗体蛋白与可药用赋形剂组合后，作为活性成分的抗PD-1/HER2双特异性抗体蛋白适于治疗性或预防性施与人类或非人类动物。本发明的抗体制剂可以例如制备成水性形式的液体制剂，例如，即用式预填装注射器，或者制备成冻干制剂，在即将使用前通过溶解和/或悬浮于生理可接受的溶液中进行重构(即，复溶)。在一些实施方案中，抗PD-1/HER2双特异性抗体蛋白制剂是液体制剂形式。
“稳定的”抗体制剂是制剂中的抗体在储存于特定条件下之后保有可接受程度的物理稳定性和/或化学稳定性。尽管抗体制剂中所含的抗体在储存特定时间之后可能不会100％维持其化学结构，但通常在储存特定时间之后维持约90％、约95％、约96％、约97％、约98％或约99％的抗体结构或功能，则认为抗体制剂是“稳定的”。在一些具体的实施方案中，本发明的抗PD-1/HER2双特异性抗体蛋白制剂在制造、制备、运输和长期储存过程中表现出低至检测不到的抗体聚集或降解或化学修饰，从而极少或甚至是没有抗PD-1/HER2双特异性抗体蛋白的生物活性损失，表现出高度稳定性。在一些实施方案中，本发明的抗PD-1/HER2双特异性抗体蛋白制剂在储存后，基本上保留其物理和化学稳定性。优选地，本发明液体制剂可以在室温或在40℃稳定至少2周，和/或在25℃稳定至少2个月，和/或在2-8℃稳定至少24个月。
本领域已知多种分析技术可以用于测定蛋白质的稳定性，参见例如Peptide and Protein Drug Delivery,247-301,Vincent Lee Ed.,Marcel Dekker,Inc.,New York,N.Y.,Pubs(1991)and Jones,A.Adv.Drug Delivery Rev.10:29-90(1993)。可以在选定的温度和选定的储存时间测量稳定性。例如，可以基于预期的制剂货架期来选择储存时间。备选地，可以使用加速稳定性试验。在一些实施方案中，通过对抗体制剂进行各种胁迫测试来进行稳定性测试。这些测试可以代表调配的抗体制剂在制造、储存或运输期间可能遭遇到的极端条件，也可以代表在非制造、储存或运输期间可能使抗体制剂中的抗体的不稳定性加速的条件。例如，可以将经调配的抗PD-1/HER2双特异性抗体蛋白制剂充填至玻璃小瓶中以检验在高温胁迫下的抗体稳定性。
经一段储存时间后，制剂不显示聚集、沉淀、混浊和/或变性；或显示非常少的聚集、沉淀、混浊和/或变性，则可以认为抗体在制剂中“保持其物理稳定性”。由于制剂中抗体的聚集可以潜在地导致患者增加的免疫反应，从而导致安全性问题。因此，需要使在制剂中的抗体聚集最小化或防止聚集。光散射法可以用于测定制剂中的可见聚集物。SEC可以用于测定制剂中的可溶性聚集物。此外，可以通过目视检查制剂的外观、颜色和/或澄清度、或者通过OD 350nm法检测制剂的浊度、或者通过非还原型CE-SDS法测定制剂的纯度，来指示制剂的稳定性。在一个实施方案中，通过测定在特定温度下储存特定时间之后制剂中的抗体单体的百分比来测量制剂的稳定性，其中制剂中的抗体单体的百分比越高，则制剂的稳定性越高。
“可接受程度的”物理稳定性可以表示于特定温度下储存特定时间之后，在制剂中检测到至少约92％的抗PD-1/HER2双特异性抗体蛋白单体。在一些实施方案中，在特定温度储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月或更久后，可接受程度的物理稳定性表示至少约88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗PD-1/HER2双特异性抗体蛋白单体。当评估物理稳定性时，药物制剂储存的特定温度可为约-80℃至约45℃的任一温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约4℃-8℃、约5℃、约25℃、约35℃、约37℃、约40℃、约42℃或约45℃。例如，若储存于约40℃±2℃1个月或4周之后，检测到至少约88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗PD-1/HER2双特异性抗体蛋白单体，则药物制剂视为是稳定的。若储存于约25℃2个月之后，检测到至少约88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗PD-1/HER2双特异性抗体蛋白单体，则药物制剂视为是稳定的。若储存于约5℃9个月之后，检测到至少约88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗PD-1/HER2双特异性抗体蛋白单体，则药物制剂视为是稳定的。
经一段储存时间后，如果制剂中的抗体不显示显著的化学改变，则可以认为抗体在制剂中“保持其化学稳定性”。大多数化学不稳定性源自于形成了抗体的共价修饰形式(例如，抗体的电荷变异体)。例如由天冬氨酸异构化、N和C末端修饰，可以形成碱性变异体；由脱酰胺化、唾液酸化和糖化，可以产生酸性变异体。化学稳定性可以通过检测和/或定量抗体的化学改变形式来评估。例如，可以通过阳离子交换色谱(CEX)或成像毛细管等电聚焦电泳(iCIEF)检测制剂中抗体的电荷变异体。在一个实施方案中，通过测定在特定温度下储存特定时间之后制剂中抗体的电荷变异体百分比变化值来测量制剂的稳定性，其中该变化值越小，则制剂的稳定性越高。
“可接受程度”的化学稳定性可以表示于特定温度下储存特定时间之后制剂中电荷变异体(例如主成分或酸性组分或碱性组分)的百分比变化值不超过50％，例如不超过30％、不超过20％。在一些实施方案中，在特定温度储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月或更久后，可接受程度的化学稳定性可以表现为主成分电荷变异体的百分比变化值不超过约50％、40％、30％、20％、15％。当评估化学稳定性时，储存药物制剂的温度可为约-80℃至约45℃的任一温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约4℃-8℃、约5℃、约25℃或约45℃。例如，若在储存于5℃24个月之后，主成分电荷变异体的百分比变化值少于约25％、24％、23％、22％、21％、20％、19％、18％、17％、16％、15％、14％、13％、12％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、0.5％或0.1％，则药物制剂可被视为是稳定的。若在储存于25℃2个月后，主成分电荷变异体的百分比变化值少于约20％、19％、18％、17％、16％、15％、14％、13％、12％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、0.5％或0.1％，则药物制剂亦可 被视为是稳定的。若在储存于40℃1个月之后，主成分电荷变异体的百分比变化值少于约50％、40％、30％、20％、10％、5％或4％，则药物制剂亦可被视为是稳定的。
术语“冻干制剂”是指通过液体制剂的冷冻干燥处理得到或能够得到的组合物。优选地，其为具有少于5％、优选少于3％水含量的固体组合物。
术语“重构制剂”是指将固体制剂(例如冻干制剂)溶解和/或悬浮于生理可接受的溶液中得到的液体制剂。
文中使用的术语“室温”是指15℃至30℃、优选20℃至27℃、更优选25℃的温度。
“胁迫条件”是指在化学和/或物理上不利于抗体蛋白的环境，所述环境可以导致不可接受的抗体蛋白失稳定。“高温胁迫”是指，将抗体制剂置于室温或甚至于更高温度(例如40℃±2℃)储存一段时间。通过高温胁迫加速试验，可以检查抗体制剂的稳定性。
如本文所使用，术语“肠胃外施用”意指肠内和局部给药以外的给药方式，通常通过注射或输注方式，并且包括但不限于，静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下(subcuticular)、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射以及输注。在一些实施方案中，本发明的稳定抗PD-1/HER2双特异性抗体蛋白制剂肠胃外施用于受试者。在一个实施方案中，本发明的抗PD-1/HER2双特异性抗体蛋白制剂以皮下、皮内、肌内或静脉内注射方式施用于受试者。
I.抗体制剂
本发明提供稳定的液体抗体制剂，其包含(i)抗PD-1/HER2双特异性抗体蛋白，(ii)缓冲剂，(iii)稳定剂，和(iv)表面活性剂，所述抗体制剂的pH为约5.0-6.5。在一个优选方案中，本发明的液体抗体制剂是注射制剂形式。
(i)抗PD-1/HER2双特异性抗体蛋白
本发明抗体制剂中的“抗PD-1/HER2双特异性抗体蛋白”包含第一半抗体和第二半抗体，其中第一半抗体包含特异性结合PD-1的第一VH/VL单元并且第二半抗体包含特异性结合HER2的第二VH/VL单元。在一些实施方案中，所述抗PD-1/HER2双特异性抗体蛋白能够以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与T淋巴细胞表面的PD-1结合，且能够以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与肿瘤细胞表面的HER2结合，以致所述抗体可以用作双特异性靶向PD-1分子和HER2分子的治疗剂和/或预防剂。
对于所述特异性结合PD-1或HER2的VH/VL单元，其包含衍生自任何现有技术中报导的抗PD-1抗体和将来研发出的抗PD-1抗体VH/VL单元的6个CDR或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；或者包含衍生自任何现有技术中报导的抗HER2抗体和将来研发出的抗HER2抗体VH/VL单元的6个CDR或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。
在一个实施方案中，抗PD-1/HER2双特异性抗体蛋白的特异性结合PD-1的第一VH/VL 单元包含衍生自抗PD-1半抗体的SEQ ID NO:12/SEQ ID NO:10的成对重链可变区序列/轻链可变区序列中所含的全部6个重链CDR与轻链CDR，或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。
在一个实施方案中，抗PD-1/HER2双特异性抗体蛋白的特异性结合HER2的第二VH/VL单元包含衍生自抗HER2半抗体的SEQ ID NO:6/SEQ ID NO:2的成对重链可变区序列/轻链可变区序列中所含的全部6个重链CDR与轻链CDR，或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。
术语“CDR”或“互补决定区”或“CDR区”(在本文中与超变区“HVR”可以互换使用)，是抗体可变区中主要负责与抗原表位结合的氨基酸区域。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。本领域公知多种用于在一个给定的VH或VL或VHH氨基酸序列中确定其CDR序列的方案。例如，Kabat互补决定区(CDR)是基于序列变异性确定的并且是最常用的(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))。而Chothia指的是结构环的位置(Chothia和Lesk，J.Mol.Biol.196:901-917(1987))。AbM HVR是Kabat HVR和Chothia结构环之间的折中，并且由Oxford Molecular的AbM抗体建模软件使用。“接触性”(Contact)HVR基于对可获得的复杂晶体结构的分析。
所述氨基酸变化，例如，氨基酸置换优选地是保守氨基酸取代。“保守氨基酸取代”是指导致某个氨基酸置换为化学上相似的氨基酸的氨基酸改变。提供功能上相似氨基酸的保守性置换表是本领域熟知的。在本发明任一实施方案中，在一个优选的方面，保守取代残基来自以下的保守替代表A，优选地为表A中所示优选置换残基。
表A
原始残基 示例性取代 优选的保守氨基酸取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp；Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；正亮氨酸 Leu
Leu(L) 正亮氨酸；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸 Leu
在一个实施方案中，抗PD-1/HER2双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含特异性结合PD-1的第一VH/VL单元并且第二半抗体包含特异性结合HER2的第二VH/VL单元，其中所述第一VH/VL单元包含SEQ ID NO:12/SEQ ID NO:10的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，并且其中所述第二VH/VL单元包含SEQ ID NO:6/SEQ ID NO:2的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列。
不特别地限制抗PD-1/HER2双特异性抗体蛋白中第一半抗体和第二半抗体的重链恒定区的类型，优选地是IgG1、IgG2或IgG4免疫球蛋白的重链恒定区，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。更优选地，所述重链恒定区是人IgG1免疫球蛋白的重链恒定区，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。
在一个实施方案中，抗PD-1/HER2双特异性抗体蛋白包含IgG1(例如，人IgG1)中使用的重链恒定区。在又一个实施方案中，抗PD-1/HER2双特异性抗体蛋白包含用于IgG4(例如，人IgG4)的重链恒定区。例如，抗PD-1/HER2双特异性抗体的两条重链的Fc结构域中分别包含具有“CPPC”氨基酸残基的铰链区，和/或分别包含Y349C和S354C(根据Kabat的“EU编号”)，由此，抗PD-1半抗体和抗HER2半抗体在Fc区形成链间二硫键，由此，稳定抗PD-1半抗体和抗HER2半抗体的正确配对。
在一个实施方案中，抗PD-1/HER2双特异性抗体蛋白的抗PD-1半抗体和/或抗HER2半抗体在Fc结构域中包含影响抗体效应子功能的氨基酸突变。在一个具体实施方案中，所述效应子功能是抗体依赖的细胞介导的细胞毒性(ADCC)。在一个实施方案中，所述氨基酸突变存在于Fc区的CH2结构域，例如，所述抗PD-1/HER2双特异性抗体蛋白包含在抗PD-1半抗体和/或抗HER2半抗体Fc区第234和235位置(EU编号)处的氨基酸置换。在一个具体实施方案中，所述氨基酸置换是L234A和L235A(也称为“LALA突变”)。
在又一个实施方案中，抗PD-1/HER2双特异性抗体蛋白的轻链包含κ轻链恒定区或者λ轻链恒定区，例如，人κ轻链恒定区或者人λ轻链恒定区。
在一个实施方案中，抗PD-1/HER2双特异性抗体蛋白的两条重链各自的Fc结构域中分 别包含凸起(“结(knob)”)或空穴(“扣(hole)”)，并且一条重链Fc结构域中的所述凸起或空穴可分别置于另一条重链Fc结构域中的所述空穴或凸起中，由此所述两条重链彼此形成“结入扣(knob-in-hole)”的稳定缔合。在一个实施方案中，在所述两条重链之一条链中包含氨基酸置换T366W，并且在所述两条重链之另一条链中包含氨基酸置换T366S、L368A和Y407V(EU编号)。由此一条链中的凸起能够置于另一条链中的空穴中，促进抗PD-1/HER2双特异性抗体蛋白的两条重链的正确配对。
在一个实施方案中，抗PD-1/HER2双特异性抗体蛋白的各半抗体中的重链和轻链的免疫球蛋白CH1结构域和CL结构域中分别包含凸起或空穴，并且CH1结构域中的所述凸起或空穴可分别置于CL结构域中的所述空穴或凸起中，从而各半抗体中的重链和轻链彼此也形成“结入扣”的稳定缔合。
在一个实施方案中，抗PD-1/HER2双特异性抗体蛋白包含包含第一半抗体和第二半抗体，其中第一半抗体包含从N至C方向的SEQ ID NO:12和SEQ ID NO:14的重链序列或与之具有至少90％,95％,98％或99％同一性的重链序列，和从N至C方向的SEQ ID NO:10和SEQ ID NO:4的轻链序列或与之具有至少90％,95％,98％或99％同一性的轻链序列，并且其中第二半抗体包含从N至C方向的SEQ ID NO:6和SEQ ID NO:8的重链序列或与之具有至少90％,95％,98％或99％同一性的重链序列，和从N至C方向的SEQ ID NO:2和SEQ ID NO:4的轻链序列或与之具有至少90％,95％,98％或99％同一性的轻链序列。
在本文中，“序列同一性”是指在比较窗中以逐个核苷酸或逐个氨基酸为基础的序列相同的程度。可以通过以下方式计算“序列同一性百分比”：将两条最佳比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同一性百分比。为了确定序列同一性百分数而进行的最佳比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。
本发明抗体制剂中的抗PD-1/HER2双特异性抗体蛋白能够同时与PD-1和HER2蛋白结合，且维持了各亲本抗体的亲和力常数，由此，能够阻断HER2信号传导通路和阻断PD-1信号传导通路，从而用于治疗、预防或延缓各种与HER2信号传导通路和/或与PD-1信号传导通路相关的疾病或病症。
在一个优选的实施方案中，本发明的抗PD-1/HER2双特异性抗体蛋白是PCT申请号PCT/CN2018/075851(申请日：2018年2月8日)中公开的重组抗PD-1/HER2双特异性抗体蛋白，其包含全人源抗PD-1半抗体和人源化抗HER2半抗体，其中全人源抗PD-1半抗体的重链序列从N至C方向为SEQ ID NO:12和SEQ ID NO:14，轻链序列从N至C方向为SEQ ID NO:10和SEQ ID NO:4，并且其中人源化抗HER2半抗体的重链序列从N至C方向为SEQ  ID NO:6和SEQ ID NO:8，轻链序列从N至C方向为SEQ ID NO:2和SEQ ID NO:4。
在一个实施方案中，该抗PD-1/HER2双特异性抗体蛋白由HEK293细胞或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293E细胞；CHO细胞或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO重组表达产生并经纯化。优选地，在本发明液体制剂中的所述抗体表现出显著的抗肿瘤活性。对使用HCC1954人乳腺癌细胞接种免疫缺陷NCG小鼠产生的荷瘤小鼠施用抗PD-1/HER2双特异性抗体，结果表明，与施用抗PD-1单克隆抗体或抗HER2单克隆抗体相比较，施用抗PD-1/HER2双特异性抗体具有显著提高的抗肿瘤活性，可以导致肿瘤体积的显著缩小。
本发明的抗体制剂中所包含的抗PD-1/HER2双特异性抗体蛋白的量可随着制剂的特定目的特性、特定环境、和使用制剂的特定目的而改变。在一些实施方案中，抗体制剂为液体制剂，其可含有约1-150mg/ml，优选地为约10-100mg/mL，例如约10、20、30、40、50、60、70、80、90或100mg/ml的抗PD-1/HER2双特异性抗体蛋白。
(ii)缓冲剂
缓冲剂是可以将溶液的pH维持在可接受范围的试剂。在一些实施方案中，用于本发明制剂中的缓冲剂可以将本发明制剂的pH控制在大约5.0-6.5的pH范围，例如约5.5的pH。在一些具体的实施方案中，本发明的抗体制剂具有约5.0、5.2、5.4、5.6、5.8、6.0、6.2、6.4的pH。
在一些实施方案中，用于本发明制剂中的缓冲剂选自组氨酸、盐酸组氨酸和它们的组合。在一个实施方案中，本发明的液体抗体制剂中的缓冲剂的浓度为约5-50mM。在一个实施方案中，本发明的液体抗体制剂中的缓冲剂的浓度为约10-30mM，例如，约10、15、20、25、30mM。
在一个实施方案中，用于本发明制剂中的缓冲剂是约10mM组氨酸。在另一个实施方案中，用于本发明制剂中的缓冲剂是约20mM组氨酸。
又在一个实施方案中，用于本发明制剂中的缓冲剂是约5.5mM组氨酸和约15mM盐酸组氨酸的组合。
(iii)稳定剂
用于本发明的合适的稳定剂可以选自糖类、多元醇及其组合。进一步地，本发明的稳定剂还可以包含抗氧化剂。
作为稳定剂的糖类可以是二糖、三糖和多糖，而且糖类可以选自但不限于：蔗糖、右旋糖、乳糖、麦芽糖、海藻糖、环糊精、麦芽糖糊精和葡聚糖。在一个实施方案中，作为稳定剂的糖类是蔗糖和/或海藻糖。
作为稳定剂的多元醇可以选自但不限于：甘露醇、山梨醇和木糖醇。在一个实施方案中，作为稳定剂的多元醇是山梨醇。
在一些实施方案中，作为稳定剂的糖类和/或多元醇在本发明的液体制剂中以约50-500mM，优选地约100-400mM，例如，约100、150、200、250、300、350、400mM的浓度存在。
本发明的稳定剂中还可以包含的抗氧化剂选自但不限于：同型半胱氨酸、半胱氨酸、胱硫醚、甲硫氨酸、谷胱甘肽、以及包含同型半胱氨酸、半胱氨酸、胱硫醚、甲硫氨酸和谷胱甘肽中任意一种的肽。在包含抗氧化剂的情形下，稳定剂的总浓度为约50-500mM，优选地总浓度为约100-400mM，例如，约100、150、200、250、300、350、400mM，其中抗氧化剂的浓度为约1-50mM，优选地约5-40mM，例如约5、10、20、30、40mM。
在一个实施方案中，本发明液体制剂包含山梨醇作为稳定剂。山梨醇在本发明液体制剂中的量可以是约50-400mM，例如，约50、100、150、200、250、300、350、400mM。
在一个实施方案中，本发明液体制剂包含蔗糖作为稳定剂。蔗糖在本发明液体制剂中的量可以是约50-300mM，例如，约50、100、150、200、250、300mM。
在一个实施方案中，本发明液体制剂包含海藻糖作为稳定剂。海藻糖在本发明液体制剂中的量可以是约50-300mM，例如，约50、100、150、200、250、300mM。
在一个实施方案中，本发明液体制剂包含蔗糖和甲硫氨酸的组合作为稳定剂。该组合中，稳定剂的总浓度为约50-500mM，优选地总浓度为约100-400mM，例如，约100、150、200、250、300、350、400mM，其中甲硫氨酸的浓度为约1-50mM，优选地约5-40mM，例如约5、10、20、30、40mM。
(iv)表面活性剂
如本文所使用的，术语“表面活性剂”是指具有两亲结构的有机物质；即，它们由相反的溶解性倾向的基团所组成，通常是油溶性的烃链和水溶性的离子基团。
在一个实施方案中，本发明的液体制剂中的表面活性剂是非离子型表面活性剂，例如，烷基聚(环氧乙烯)。可包括在本发明制剂中的特定非离子型表面活性剂包括，例如聚山梨酯，诸如聚山梨酯-20、聚山梨酯-80、聚山梨酯-60、或聚山梨酯-40；普洛尼克等。在一个优选实施方案中，本发明的液体制剂中包含聚山梨酯-80作为表面活性剂。
本发明抗体制剂中所含的表面活性剂的量可随制剂的特定目的特性、特定环境、和使用制剂的特定目的而改变。在优选的一些实施方案中，制剂可含有约0.1-1mg/ml，优选地约0.2-0.8mg/ml，例如约0.2、0.3、0.4、0.5、0.6、0.7、0.8mg/ml的聚山梨酯类表面活性剂(例如，聚山梨酯-80)。
(v)其它赋形剂
本发明的抗体液体制剂中可以包含或不包含其它赋形剂。例如，本发明的抗体液体制剂还包含张力调节剂。张力调节剂可以选自下组：醋酸钠、乳酸钠、氯化钠、氯化钾和氯化钙。
这些和另外已知的药物赋形剂和/或适用于本发明制剂的添加剂是本领域公知的，例如，列出于“The Handbook of Pharmaceutical Excipients，第4版，Rowe等人编，American Pharmaceuticals Association(2003)；和Remington:the Science and Practice of Pharmacy，第21版，Gennaro编，Lippincott Williams&Wilkins(2005)”。
II.制剂的制备
本发明提供了包含抗PD-1/HER2双特异性抗体蛋白的稳定制剂。在本发明制剂中使用的抗PD-1/HER2双特异性抗体蛋白可以使用本领域已知的用于生产抗体的技术进行制备。例如， 可以重组制备抗PD-1/HER2双特异性抗体蛋白。在一个优选的实施方案中，本发明的抗PD-1/HER2双特异性抗体蛋白通过在HEK293细胞或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293E细胞；CHO细胞或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO中重组表达而制备，例如，如PCT申请号PCT/CN2018/075851中所述，重组制备抗PD-1/HER2双特异性抗体蛋白。
抗体作为药物的活性成分的应用现在已经很广泛。用于将治疗性抗体纯化至药用级的技术是本领域公知的。例如，Tugcu等(Maximizing productivity of chromatography steps for purification of monoclonal antibodies,Biotechnology and Bioengineering 99(2008)599–613)描述了在蛋白A捕获步骤后使用离子交换色谱(阴离子IEX和/或阳离子CEX色谱)的抗体三柱纯化方法。Kelley等(Weak partitioning chromatography for anion exchange purification of monoclonal antibodies,Biotechnology and Bioengineering 101(2008)553–566)描述了两柱纯化法，其中在蛋白A亲和色谱后使用弱分配阴离子交换树脂。
一般地，重组产生的抗体可以利用常规的纯化方法纯化，以提供具有足够的可重复性和适度纯度的药物物质用于抗体制剂的配制。例如，在抗体从重组表达细胞分泌至培养基中后，可以使用商业可得的蛋白浓缩过滤器例如Amicon的超滤装置，浓缩来自该表达系统的上清液。之后，可以使用例如色谱、透析和亲和纯化等方式进行抗体的纯化。蛋白A适应于作为亲和配体用于纯化IgG1、IgG2和IgG4型抗体。也可以使用其它抗体纯化方法，例如离子交换色谱。在获得足够纯度的抗体后，可以按照本领域已知的方法，制备包含抗体的制剂。
例如，可以采用如下步骤进行制备：(1)在发酵结束后将发酵液离心澄清去除细胞等杂质以获得上清；(2)使用亲和层析(例如对IgG1、IgG2和IgG4型抗体具有特异亲和力的蛋白A柱)捕获抗体；(3)进行病毒灭活；(4)精制纯化(一般可以采用CEX阳离子交换层析)，以去除蛋白中的杂质；(4)病毒过滤(使病毒滴度降低例如4log10以上)；(5)超滤/渗滤(可以用于将蛋白置换于利于其稳定的制剂缓冲液中并浓缩至合适的浓度供注射用)。参见例如，B.Minow,P.Rogge,K.Thompson,BioProcess International,Vol.10,No.6,2012,pp.48–57。
III.制剂的分析方法
在抗体制剂的储存过程中，抗体可能会发生聚集、降解或化学修饰，导致抗体异质性(包括大小异质性和电荷异质性)以及聚集物和片段等，从而影响抗体制剂的质量。因此，有必要进行抗体制剂稳定性的监测。
在本领域中已知多种方法可以用于检测抗体制剂的稳定性。例如，可以通过还原型CE-SDS、非还原型CE-SDS和SEC-HPLC等方法，分析抗体制剂的纯度和评估抗体的聚集水平；可以通过毛细管等电聚焦电泳(cIEF)、成像毛细管等电聚焦电泳(iCIEF)和离子交换色谱(IEX)等，分析抗体制剂中的电荷变异体。此外，可以通过目视检测制剂外观，快速地判断制剂的稳定性。也可以使用OD 350nm法检测制剂的浊度改变，该方法可以给出有关可溶性和不溶性聚集物量的信息。此外，可以使用紫外分光光度法(UV法)检测制剂中的蛋白质含量变化。
非还原型CE-SDS法是一种以毛细管为分离通道进行的抗体纯度测定方法。在CE-SDS 中，蛋白迁移由SDS结合引起的表面电荷来驱动，而该表面电荷与蛋白质的分子量成正比。由于所有的SDS-蛋白质复合物都具有相似的质量-电荷比，故可以在毛细管的分子筛凝胶基质中，实现基于分子的大小或流体动力学半径的电泳分离。该方法已经被广泛地用于监测变性的完整抗体的纯度。一般，在非还原型CE-SDS法中，供试样品与SDS样品缓冲液和碘乙酰胺混合。之后，混合物可以于68-72℃孵育约10-15分钟，冷却至室温后离心的上清液用于分析。采用紫外检测器检测蛋白的迁移，获得电泳谱图。抗体制剂纯度可以计算为IgG主峰的峰面积占所有峰面积之和的百分比。关于CE-SDS法的进一步描述，可以参见例如Richard R.等,Application of CE SDS gel in development of biopharmaceutical antibody-based products,Electrophoresis,2008,29,3612-3620。
尺寸排阻高效液相色谱法，即SEC-HPLC法，是用于抗体标准和质控的另一重要方法。该方法主要依据分子的尺寸大小或流体动力学半径差异来进行分子的分离。通过SEC-HPLC，抗体可以分离出三种主要形式：高分子量形式(HMMS)、主峰(主要是抗体单体)、和低分子量形式(LMMS)。抗体纯度可以计算为色谱图上主峰面积占所有峰面积之和的百分比。通过SEC-HPLC法，可以测量制剂产品中抗体单体的百分数，给出可溶性聚集物和剪切物的含量信息。关于SEC-HPLC法的进一步描述，可以参见例如，J.Pharm.Scien.,83:1645-1650,(1994)；Pharm.Res.,11:485(1994)；J.Pharm.Bio.Anal.,15:1928(1997)；J.Pharm.Bio.Anal.,14:1133-1140(1986)。此外，也可以参见例如，R.Yang等，High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography(SE-UHPLC),Journal of Pharmaceutical and Biomedical Analysis(2015),http://dx.doi.org/10.1016/j.jpba.2015.02.032；和Alexandre Goyon等,Protocols for the analytical characterization of therapeutic monoclonal antibodies.I–Non-denaturing chromatographic techniques,Journal of Chromatography，http://dx.doi.org/10.1016/j.jchromb.2017.05.010。
成像毛细管等电聚焦电泳(iCIEF)可以用于分析抗体的电荷异质性。该方法可以提供电荷变异体的定量分布情况。iCIEF基于分子在pH梯度中的电荷差异(表观pI值)来实现分子分离的目的。在iCIEF中，分离柱通常是短毛细管(例如,5cm长,100μm内径的二氧化硅毛细管)，蛋白质在高电压下在毛细管柱中聚焦，并通过在280nM操作的全柱成像检测系统对聚焦进行实时在线监测。该技术的一个优点是，可以通过该全柱检测系统同时记录抗体样品的各种电荷变异体。一般而言，在icIEF中，将样品与尿素和icIEF缓冲液混合，其中所述缓冲液含有甲基纤维素、pI分子量标准和两性电解质。然后，可以在iCIEF分析仪例如iCE280分析仪(Protein Simple,Santa Clara,CA)上，使用iCIEF柱例如ProtionSimple组装的iCIEF柱，在样品聚焦一定时间后，测定280nm的吸光度，获得聚焦抗体电荷变异体的谱图。在iCEIF谱图中，在主峰(即主成分)之前洗脱的蛋白相关峰被分类为酸性组分；相对地，在主峰之后洗脱的蛋白相关峰被分类为碱性组分。主成分、酸性组分和碱性组分的相对量可以表示为占总峰面积的百分数。关于iCIEF的进一步描述，可以参见例如，Salas-Solano O等,Robustness of iCIEF methodology for the analysis of monoclonal antibodies:an interlaboratory study,J Sep Sci. 2012 Nov；35(22):3124-9.doi:10.1002/jssc.201200633.Epub 2012 Oct 15；和Dada OO等,Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation,Electrophoresis.2015 Nov；36(21-22):2695-2702.doi:10.1002/elps.201500219.Epub 2015 Sep 18.
也可以通过阳离子交换高效液相色谱法(CEX-HPLC)测定抗体制剂中抗体的电荷变异体。在该测定法中，以比主峰的保留时间更早从CEX-HPLC柱洗脱出的峰被标记为“酸性峰”，而那些以比主峰的保留时间更晚从CEX-HPLC柱洗脱出的峰被标记为“碱性峰”。
加速稳定性研究可以用于检查产品的稳定性性质，有利于筛选稳定药物制剂形式。例如，可以将制剂样品放置于升高的温度，例如约40℃±2℃、25℃±2℃条件下进行加速稳定性研究。检测指标可以包括外观、可见异物、蛋白含量、浊度、纯度(SEC-HPLC法、非还原型CE-SDS法)和电荷变异体(iCIEF法、CEX-HPLC法)。
此外，可以检测抗体的功效或生物活性。例如，可以检测制剂中抗体与其抗原分子(HER2分子和PD-1分子)的结合能力。本领域技术人员已知多种方法可以用于定量抗体与抗原的特异性结合，例如免疫测定试验，ELISA等。
本发明的抗PD-1/HER2双特异性抗体蛋白制剂是稳定的。在一个实施方案中，于约5℃、25℃、37℃、40℃、或45℃储存至少1个月、2个月或3个月后，例如，在5℃±3℃储存3个月后，本发明的抗体制剂中的抗PD-1/HER2双特异性抗体蛋白纯度是至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％以上，如通过尺寸排阻色谱法或通过非还原型CS-SDS所测定。在一个实施方案中，于约5℃、25℃、37℃、40℃、或45℃储存至少1个月、2个月或3个月后，例如，在5℃±3℃储存3个月后，本发明的抗体制剂中抗PD-1/HER2双特异性抗体蛋白的至少60％，优选至少65％是非碱性及非酸性形式(亦即，主峰或主要电荷形式)，如通过iCIEF法所测定。
IV.制剂的用途
本发明的包含抗PD-1/HER2双特异性抗体蛋白的本发明的抗体制剂可以用于治疗、预防或延缓各种与HER2信号传导通路和/或与PD-1信号传导通路相关的疾病或病症。“与HER2信号传导通路相关的疾病或病症”和/或“与PD-1信号传导通路相关的疾病或病症”在本文中指可以用本发明抗PD-1/HER2双特异性抗体蛋白制剂进行治疗(例如改善)或预防的疾病或病症。任何可以得益于本发明抗体制剂治疗的疾病或病症都适用于本发明。
包含抗PD-1/HER2双特异性抗体蛋白的本发明制剂能够用于预防或治疗受试者的各种血液病和实体瘤，包括但不限于白血病、淋巴瘤、骨髓瘤、脑肿瘤、头颈部鳞状细胞癌、非小细胞肺癌、鼻咽癌、食道癌、胃癌、胰腺癌、胆囊癌、胆管癌、肝癌、结直肠癌、乳腺癌、卵巢癌、宫颈癌、子宫内膜癌、子宫肉瘤、前列腺癌、膀胱癌、肾细胞癌、黑色素瘤。
本发明也提供本发明的制剂在制备药物中的用途，其中所述药物用于向哺乳动物递送抗PD-1/HER2双特异性抗体蛋白，或用于治疗、预防或改善上述疾病和病症中的一种或多种。优选地，哺乳动物是人。
可以以多种途径将本发明的抗体制剂施用于受试者或患者。例如，施用可以通过输注或 通过注射器进行。因此，在一个方面，本发明提供了一种递送装置(例如注射器)，其包含本发明的抗体制剂(例如，预填装注射器)。患者将接受有效量的抗PD-1/HER2双特异性抗体蛋白作为主要活性成分，即足以治疗、改善或预防目的疾病或病症的量。
治疗效果可包括减少生理症状。用于任何特定受试者的抗体的最佳有效量和浓度将取决于多种因素，包括患者的年龄、体重、健康状况和/或性别、疾病的性质和程度、特定抗体的活性，身体对其清除率，并且也包括与所述抗体制剂组合施用的任何可能的其它治疗。对于具体的情况，所递送的有效量可以在临床医师的判断范围内来确定。取决于待治疗的适应症，有效剂量可为约0.005mg/kg体重至约50mg/kg体重，或约0.1mg/kg体重至约20mg/kg体重。在这方面，已知的基于抗体的药物的应用可以提供一定的指导。剂量可以是单剂量方案或多剂量方案。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
缩略词描述
CE-SDS：十二烷基硫酸钠毛细管凝胶电泳
ELISA：酶联免疫吸附测定法
iCIEF：成像毛细管等电聚焦电泳
SEC-HPLC：尺寸排阻高效液相色谱法
实施例
为了开发出重组抗程序性死亡受体1(PD-1)和抗人表皮生长因子受体2(HER2)双特异性抗体注射液长期稳定储存的制剂处方，确保产品在有效期内(至少24个月)的质量可控，设计了处方筛选试验，考察了不同辅料对于抗PD-1/HER2双特异性抗体制剂稳定性的影响。试验所用材料和方法如下：
材料和方法
1.1.本发明的制剂研究中使用的材料
名称 级别 产地及品牌 货号 符合标准
组氨酸 药用级 上海味之素 N/A Ch.P(2015年版)
盐酸组氨酸 药用级 上海味之素 N/A Ch.P(2015年版)
山梨醇 药用级 法国 罗盖特 H20110265 EP、BP、NF、USP
蔗糖 药用级 德国 Merck 1.00892.9050 Ch.P(2015年版),USP
海藻糖 药用级 美国 Pfanstiehl T-104-4 USP/NF、EP、JP
甲硫氨酸 药用级 湖北八峰 N/A Ch.P(2015年版)
聚山梨酯80 药用级 南京威尔 苏药准字F15423203 Ch.P(2015年版)
盐酸 药用级 德国 Merck 1.00314.2508 Ph Eur、BP、JP、NF
注：N/A表示“不适用”(Not applicable)。
1.2.本发明的制剂研究中使用的仪器设备
设备名称 产地及品牌 型号 编号
恒温恒湿箱 德国 BINDER KBF P 720 PD-A1-069
生化培养箱 上海 精宏 SHP-150 PD-A1-200
漩涡混合器 美国 VWR DVX-2500 PD-A1-140
医用冷藏箱 青岛 海尔 HYC-360 PD-A1-166
超低温冰箱 美国 Thermo 907 PD-A1-175
澄明度检测仪 天津 天大天发 YB-2 PD-A1-033
紫外可见分光光度计 日本 岛津 UV-1800 AS-A1-037
pH计 瑞士 梅特勒 S220/FE20 PD-A1-002
多通道微量分光光度计 美国 Thermo Nanodrop8000 PD-A1-052
台式冷冻离心机 美国 Thermo SL16R PD-A1-082
洁净工作台 苏州 苏净安泰 SW-CJ-2FD QC-A1-011
不溶性微粒检测仪 天津 天大天发 GWJ-8 QC-A1-094
1.3.制剂稳定性的检测项目和检测方法
对抗体制剂检测了以下项目：(1)检测外观以及是否存在可见异物；(2)通过紫外法(UV法)测定制剂中的蛋白质含量；(3)通过检测在350nm处的吸光度，测定浊度；(4)通过尺寸排阻色谱法，例如，尺寸排阻高效液相色谱法(size-exclusion chromatography-HPLC；SEC-HPLC)测定抗体制剂的纯度，表示为单体的面积占所有峰面积之和的百分数；(5)通过还原型十二烷基硫酸钠毛细管电泳(还原型CE-SDS)和/或非还原型十二烷基硫酸钠毛细管电泳(非还原型CE-SDS)测定抗体制剂的纯度，表示为单体的面积占所有峰面积之和的百分数；(6)通过成像毛细管等电聚焦电泳法(iCIEF法)测定抗体制剂中电荷变异体，表示为主成分、酸性组分和碱性组分的百分数；(7)通过免疫测定法，例如，直接ELISA法测定抗体制剂中抗PD-1/HER2双特异性抗体对PD-1抗原和HER2抗原的相对结合活性。
可见异物检测
按照国家药典委员会，中华人民共和国药典(2015年版，四部通则0904“可见异物检查法”，北京：中国医药科技出版社.2015)中所记载的方法，采用澄明度检测仪(天津天大天发生产，型号YB-2)，检查样品中的可见异物。
蛋白含量测定
使用紫外分光光度计(日本岛津生产，型号UV-1800)测定样品中的蛋白质含量。
浊度测定
使用紫外分光光度计(日本岛津生产，型号UV-1800)，测定样品在350nm的吸光度，确定样品浊度。
纯度(SEC-HPLC法)
使用体积排阻色谱柱分离，流动相为磷酸盐缓冲液(称取3.12g二水合磷酸二氢钠，8.77g氯化钠和34.84g精氨酸，超纯水溶解后用盐酸调节pH至6.8并定容至1000ml)，色谱柱保护液为0.05％(w/v)NaN 3，进样量50μl，流速0.5ml/分钟，采集时间30分钟，柱温25℃，检测波长280nm。取待测样品用超纯水稀释至2mg/ml，作为供试品溶液。取制剂缓冲液用上述相同处理方式稀释后做为空白溶液。取空白溶液、供试品溶液各50μl注入液相色谱仪，开始检测。
纯度(还原型CE-SDS法)
采用毛细管凝胶电泳法检测。毛细管为无涂层毛细管，内径50μm，总长30.2cm，有效长度20.2cm。电泳前分别使用0.1mol/L氢氧化钠、0.1mol/L盐酸、超纯水、电泳胶70psi冲洗毛细管柱。将待测样品用适量超纯水稀释至2.0mg/ml，取以上稀释后的样品50μl于1.5ml离心管中，分别向其中加入45μl pH 6.5的样品缓冲液(称取一水柠檬酸0.32g，十二水合磷酸氢二钠2.45g，溶于45ml超纯水中，定容至50ml，制得柠檬酸-磷酸盐缓冲液，精密量取该缓冲液200μl，加10％(w/v)十二烷基硫酸钠溶液80μl，加水至1ml，混匀，即得)、1μl内标(10kDa蛋白质，5mg/mL)(Beckman Coulter，货号：390953)和5μlβ-巯基乙醇，充分混匀后70±2℃加热10±2分钟，冷却至室温后转移至样品瓶作为供试品溶液。取与供试品相同体积的制剂缓冲液，按上述方法同样操作，制得空白溶液。样品进样条件：-5kV 20秒；分离电压：-15kV 35分钟。毛细管柱温控制在25℃，检测波长为220nm。
纯度(非还原型CE-SDS法)
采用毛细管凝胶电泳法检测。毛细管为无涂层毛细管，内径50μm，总长30.2cm，有效长度20.2cm。电泳前分别使用0.1mol/L氢氧化钠、0.1mol/L盐酸、超纯水、电泳胶70psi冲洗毛细管柱。将待测样品用适量超纯水稀释至2.0mg/ml，取以上稀释后的样品50μl于1.5ml离心管中，分别向其中加入45μl pH 6.5的样品缓冲液(称取一水柠檬酸0.32g，十二水合磷酸氢二钠2.45g，溶于45ml超纯水中，定容至50ml，制得柠檬酸-磷酸盐缓冲液，精密量取该缓冲液200μl，加10％(w/v)十二烷基硫酸钠溶液80μl，加水至1ml，混匀，即得)、1μl内标(10kDa蛋白质，5mg/mL)(Beckman Coulter，货号：390953)和5μl 250mmol/L NEM溶液(称取N-乙基顺丁稀二酰亚胺62mg，溶于2ml超纯水中)，充分混匀后70±2℃加热10±2分钟，冷却至室温后转移至样品瓶作为供试品溶液。取与供试品相同体积的制剂缓冲液，按上述方法同样操作，制得空白溶液。样品进样条件：-5kV 20秒；分离电压：-15kV 35分钟。毛细管柱温控制在25℃，检测波长为220nm。
电荷变异体(iCIEF法)
采用成像毛细管等电聚焦电泳(iCIEF法)检测。毛细管内径100μm，总长5cm。样品电泳前需分别使用0.5％甲基纤维素溶液(下文中也缩写为MC溶液)、超纯水冲洗毛细管柱。采用真空进样方式，预聚焦电压及时间为1.5kV 1分钟，聚焦电压及时间为3kV 8分钟，进样时间55秒，样品盘温度为10℃，检测波长为280nm。阴极稳定剂(Cathodic Stabilizer)为500mmol/L精氨酸溶液，0.5％MC溶液降低蛋白与毛细管之间的粘附。将供试品用水稀释至1.0 mg/ml，取稀释后的供试品溶液20μl，向其中加入78μl预混液(预混液配比如下：70μl pI 0.5％MC溶液，4μl两性电解质(pH 3-10)，2μl阴极稳定剂，1μl pI 5.85标志物，1μl pI 9.99标志物)，充分混匀制得待测样品溶液。进样分析，根据面积归一化法，计算主成分、酸性组分及碱性组分含量。
相对结合活性(直接ELISA法)
用CBS稀释抗原(检测抗PD-1/HER2双特异性抗体的抗PD-1端对PD-1的相对结合活性时，使用购自Sinobiological的重组人PD-1，货号：10377-H08H；检测抗PD-1/HER2双特异性抗体的抗HER2端对HER2的相对结合活性时，使用购自Sinobiological的Human HER2/ErbB2Protein(His Tag)，货号：10004-H08H-100)至0.5μg/ml，100μl/孔，4℃过夜包被于96孔酶标板上。洗板后加封闭液(2％BSA-PBST，300μl/孔)37℃封闭2h。以2％BSA-PBST稀释抗PD-1/HER2双特异性抗体至3μg/ml，3倍梯度稀释至第11个浓度(0.05～3000ng/ml)。将梯度稀释的供试品以100μl/孔加入到弃去封闭液的酶标板中，设置阴性对照每孔只加100μl稀释液(2％BSA-PBST)，37℃恒温培养箱中孵育60min。洗板后加入以2％BSA-PBST稀释的HRP缀合山羊抗人IgG-Fc片段(美国BETHYL，货号A80-104P)作为二抗(100000倍稀释，100μl/孔)，37℃反应30min。洗板后每孔加入100μl TMB显色液，显色10min后，每孔加入100μl的1mol/L H 2SO 4终止反应。以620nm为参比波长，测450nm处的OD值。以各浓度梯度样品的浓度值作为横坐标，各梯度样品的OD450nm-OD620nm值为纵坐标，应用Prism四参数拟合计算EC 50反映抗体与各抗原的结合活性。
实施例1.制备和纯化抗PD-1/HER2双特异性抗体
根据PCT申请号PCT/CN2018/075851所述，制备和纯化抗PD-1/HER2双特异性抗体。
具体而言，分别构建含抗人PD-1的抗体重链和轻链的X0GC表达载体(X0GC表达载体的构建参见中国专利申请号200780038403.3)，其中轻链可变区核苷酸序列如SEQ ID NO.9所示，氨基酸序列如SEQ ID NO:10所示；轻链恒定区核苷酸序列如SEQ ID NO.3所示，氨基酸序列如SEQ ID NO:4所示；重链可变区核苷酸序列如SEQ ID NO.11所示，氨基酸序列如SEQ ID NO:12所示；重链恒定区核苷酸序列如SEQ ID NO.13所示，氨基酸序列如SEQ ID NO:14所示。
分别构建含抗人HER2的抗体重链和轻链的X0GC表达载体，其中轻链可变区核苷酸序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO:2所示；轻链恒定区核苷酸序列如SEQ ID NO.3所示，氨基酸序列如SEQ ID NO:4所示；重链可变区核苷酸序列如SEQ ID NO.5所示，氨基酸序列如SEQ ID NO:6所示；重链恒定区核苷酸序列如SEQ ID NO.7所示，氨基酸序列如SEQ ID NO:8所示。
分别将所述含抗人PD-1抗体的重链和轻链的表达载体转染293F细胞(FreeStyle TM293-F Cells,货号R79007,invitrogen)，表达、纯化和通过还原过程，得到含有一条重链和一条轻链的抗人PD-1半抗体分子。
类似地，分别将所述含抗人HER2抗体的重链和轻链的表达载体转染293F细胞(FreeStyle TM293-F Cells,货号R79007,invitrogen)，表达、纯化和通过还原过程，得到含有 一条重链和一条轻链的抗人HER2半抗体分子。
将还原的抗PD-1半抗体分子以及还原的抗HER2半抗体分子以等摩尔比例混合，在4℃条件下进行重组反应24小时，获得了含有抗PD-1半抗体分子以及抗HER2半抗体分子的异源二聚体的双特异性抗体的溶液。对所述溶液通过超滤浓缩管超滤浓缩后，采用AKTA explorer 100型蛋白纯化系统(GE Healthcare)以及离子色谱柱Source 15S(16mm I.D.，17ml，GE Healthcare)于4℃纯化，获得了纯度为99.96％的抗PD-1/HER2双特异性抗体。
实施例2.pH对制剂的稳定性影响试验之一
本实施例考察了包含抗PD-1/HER2双特异性抗体的制剂在pH 5.0至6.5的稳定性。共设计了4个pH值，分别为5.0、5.5、6.0和6.5。
2.1实验步骤
配制10mM组氨酸，5％(w/v)山梨醇缓冲液，用稀盐酸将pH分别调节为5.0、5.5、6.0和6.5，将实施例1的经纯化的抗PD-1/HER2双特异性抗体超滤置换到所述不同pH值的溶液中。置换完成后，调节样品中的双特异性抗体蛋白含量至约20mg/ml；然后加入聚山梨酯80，使聚山梨酯80的终浓度为0.30mg/ml；过滤分装至西林瓶中，加塞、轧盖。将各样品于40℃±2℃条件下进行稳定性考察，具体实验方案见表1。
表1.实验方案
Figure PCTCN2020107441-appb-000003
注：(1)x表示在该时间点取样。(2)在所述时间点取样后，均将取得的样品先放入超低温冰箱中－80℃冻存待检，按需要化冻送检。
2.2判断标准
根据对产品的认识以及仪器和方法的精密度，设定了样品检测指标数值与初始值相比质量未发生变化的判定标准，用以判断样品是否发生了变化，具体见表2。
表2.质量未发生变化的判断标准
Figure PCTCN2020107441-appb-000004
Figure PCTCN2020107441-appb-000005
2.3处方筛选试验之一的实验结果
(1)外观和可见异物
在40℃±2℃条件下放置一个月后，pH 5.0、pH 5.5、pH 6.0和pH 6.5样品外观和可见异物均合格。
(2)蛋白含量
在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，各样品的蛋白含量检测结果见表3。结果表明，在40℃±2℃条件下放置1个月，各pH值样品均未发生显著变化。
表3.在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，各样品的蛋白含量(UV法，mg/ml)
Figure PCTCN2020107441-appb-000006
(3)浊度
在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，各样品的浊度检测结果见表4，其变化趋势见图2。结果表明，在40℃±2℃条件下放置1个月，各pH值样品的浊度均升高，且pH越高，浊度变化速率越快。
表4.在各pH下于40℃±2℃放置不同时间后，各样品的浊度结果(OD 350nm法)
Figure PCTCN2020107441-appb-000007
(4)纯度
在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，通过SEC-HPLC法测定各样品的蛋白纯度。结果见表5，其变化趋势见图3。结果表明，在40℃±2℃条件下考察1个月，不同pH值的样品纯度均未发生明显变化。
表5.通过SEC-HPLC法对各样品测定的蛋白纯度(％)
Figure PCTCN2020107441-appb-000008
在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，通过非还原型CE-SDS法分别测定各样品的蛋白纯度。结果见表6，其变化趋势见图4。结果表明，在40℃±2℃条件下考察1个月，各pH值样品纯度均下降，与0天的样品纯度比较分别下降了2.6％、2.5％、2.5％和3.2％。
表6.通过非还原型CE-SDS法对各样品测定的蛋白纯度(％)
Figure PCTCN2020107441-appb-000009
在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，通过还原型CE-SDS法分别测定各样品的蛋白纯度。结果见表7，其变化趋势见图5。结果表明，在40℃±2℃条件下考察1个月，各pH值样品纯度与0天的样品纯度比较分别下降了0.4％、0.7％、0.6％和1.4％。
表7.通过还原型CE-SDS法对各样品测定的蛋白纯度(％)
Figure PCTCN2020107441-appb-000010
(5)电荷变异体
在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，通过iCIEF法测定各样品的电荷变异体。结果见表8，其变化趋势见图6。结果表明，在40℃±2℃条件下考察1个月，各pH值样品主成分和酸碱各组分均发生明显变化。pH值越高，样品的主成分下降越快，酸性组分上升越快。
表8.通过iCIEF法测定的各样品的电荷变异体(％)
Figure PCTCN2020107441-appb-000011
Figure PCTCN2020107441-appb-000012
(6)相对结合活性
在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，通过直接ELISA法测定各样品的相对结合活性。结果见表9。结果表明，在40℃±2℃条件下考察2周时，各样品结合PD-1抗原和HER2抗原的相对结合活性均高于70％；pH 6.0和pH 6.5的样品抗HER2端相对结合活性有明显下降，均低于70％；在40℃±2℃条件下考察1个月时，各样品结合PD-1抗原的相对结合活性仍高于70％，仅pH 6.0和pH 6.5的样品结合HER2抗原的相对结合活性低于70％，但仍高于50％。
表9.通过直接ELISA法测定的样品的相对结合活性(％)
Figure PCTCN2020107441-appb-000013
注：N/A表示未设置该检测项。
以上进行的pH对制剂的稳定性影响试验结果表明，抗PD-1/HER2双特异性抗体在pH 5.0－6.5于40℃±2℃放置2周，样品外观和可见异物均合格，蛋白含量未发生显著变化，且对 HER2抗原和PD-1抗原的相对结合活性也未发生明显变化；另外，抗PD-1/HER2双特异性抗体在pH 5.0－6.5于40℃±2℃放置一个月，样品外观和可见异物均合格，蛋白含量未发生显著变化，且对PD-1抗原的相对结合活性也未发生明显变化，抗PD-1/HER2双特异性抗体仅在pH 6.0和pH 6.5的情形下，结合HER2抗原的相对结合活性下降，但仍高于50％。在随后的实施例中，从pH 5.0－6.5中选择pH 5.5进行实验。
实施例3.处方筛选试验
3.1稳定剂筛选试验
考察了不同稳定剂：作为多元醇的山梨醇；作为糖类的蔗糖、海藻糖；作为抗氧化剂的甲硫氨酸等对包含抗PD-1/HER2双特异性抗体制剂稳定性的影响。
3.1.1稳定剂筛选试验步骤
共设计了4个处方，详细处方信息见表10。按照表10配制各个处方的缓冲液，将抗PD-1/HER2双特异性抗体超滤置换至各自的处方溶液中。置换完成后，调节各处方的蛋白含量至约50.0mg/ml；加入聚山梨酯80，使聚山梨酯80的终浓度为0.20mg/ml；过滤分装至西林瓶，加塞、轧盖。将各样品于40℃、25℃、5℃条件下进行稳定性考察，具体方案见表11。检测指标为外观、可见异物、蛋白含量、纯度(SEC-HPLC法和CE-SDS法)和电荷变异体(iCIEF法)。
表10.稳定剂筛选试验备选处方信息表
Figure PCTCN2020107441-appb-000014
注：表中％是指％w/v，下同。
表11.稳定性考察方案
Figure PCTCN2020107441-appb-000015
Figure PCTCN2020107441-appb-000016
3.1.2判定标准
判定标准具体参见实施例2中的表2。
3.1.3稳定剂筛选试验
(1)外观、可见异物
在40℃条件下观察至1个月，25℃±2℃条件下观察至2个月，5℃±3℃条件下观察至3个月，结果表明：各处方样品外观、可见异物均合格。
(2)蛋白含量
在40℃条件下观察至1个月，25℃±2℃条件下观察至2个月，5℃±3℃条件下观察至3个月，各处方样品的蛋白含量测定结果见表12。结果表明，在40℃、25℃±2℃和5℃±3℃这三个不同温度条件下，四个处方中的蛋白含量均未发生变化。
表12.稳定剂筛选试验蛋白含量结果(UV法，mg/ml)
Figure PCTCN2020107441-appb-000017
(3)纯度
纯度(SEC-HPLC法)：结果见表13。结果表明，在40℃条件下考察4周，各处方样品纯度均未发生显著变化；在25℃±2℃条件下2个月，各处方样品纯度均未发生明显变化；在5℃±3℃条件下3个月，各处方样品纯度也未发生明显变化。
表13.稳定剂筛选试验的纯度结果(SEC-HPLC法，％)
Figure PCTCN2020107441-appb-000018
纯度(非还原型CE-SDS法)：结果见表14。结果表明，在40℃条件下考察4周，各处方样品纯度均未发生显著变化；在25℃±2℃条件下2个月，各处方样品纯度均未发生明显变 化；在5℃±3℃条件下3个月，各处方样品纯度均未发生显著变化。
表14.稳定剂筛选试验的纯度结果(非还原型CE-SDS法，％)
Figure PCTCN2020107441-appb-000019
纯度(还原型CE-SDS法)：结果见表15。结果表明，在40℃条件下考察4周，各处方样品纯度均未发生显著变化；在25℃±2℃条件下2个月，各处方样品纯度均未发生明显变化；在5℃±3℃条件下3个月，各处方样品纯度也均未发生显著变化。
表15.稳定剂筛选试验的纯度结果(还原型CE-SDS法，％)
Figure PCTCN2020107441-appb-000020
(4)电荷变异体(iCIEF法)
电荷变异体(iCIEF法)：结果见表16。40℃和25℃±2℃时，各处方电荷变异体主成分的变化趋势分别见图7和图8。
结果表明，40℃条件下考察4周，各处方电荷变异体主成分及酸碱组分均发生明显变化，主成分下降，酸性组分上升，其变化趋势基本一致，处方1－4之间无明显差异。25℃±2℃条件下加速2个月，各处方电荷变异体主成分及酸碱组分均发生明显变化，主成分下降，酸性组分上升，其变化趋势基本一致，处方1－4之间无明显差异。5℃±3℃条件下考察3个月，各处方电荷变异体主成分及酸碱组分均无明显变化。
表16.稳定剂筛选试验的电荷变异体结果(iCIEF法，％)
Figure PCTCN2020107441-appb-000021
Figure PCTCN2020107441-appb-000022
处方确定实验结果表明，处方1－4之间有比较一致的变化趋势，它们之间无明显差异。从处方简单性方面考虑，处方1、处方2和处方3中使用单一稳定剂对蛋白的保护无明显差异，考虑到后期冻干制剂的开发，选用处方2。从后期生产安全性考虑，将处方2的缓冲体系由浓盐酸调节pH调整为组氨酸和盐酸组氨酸。为确保调整后的制剂处方的稳定性，进一步实施以下实验。
实施例4：处方确认实验
4.1处方设计和实验方案
设计了处方5，处方5的详细信息见表17。
表17.处方信息表
Figure PCTCN2020107441-appb-000023
处方确认实验方案见表18。
表18.处方确认实验方案
Figure PCTCN2020107441-appb-000024
4.2实验结果
40℃强制实验结果见表19。在40℃条件下放置4周，外观、可见异物和生物学活性均合格，蛋白含量、纯度(SEC-HPLC法和CE-SDS法)均未发生显著变化，仅电荷变异体(iCIEF法)主成分下降16.0％，酸性组分上升14.0％，碱性组分上升2.0％。
表19.稳定性实验结果
Figure PCTCN2020107441-appb-000025
结果表明，42.0mg/ml本发明的重组全人源抗程序性死亡受体1(PD-1)和人源化抗人表皮生长因子受体2(HER2)双特异性抗体在处方5(0.85mg/ml组氨酸、3.17mg/ml盐酸组氨酸、80.00mg/ml蔗糖、0.2mg/ml聚山梨酯80，pH 5.5)中与实施例3中的处方2有比较一致的变化趋势。
由此，确定最优选的制剂方案为：约42.0mg/ml重组全人源抗程序性死亡受体1(PD-1)和人源化抗人表皮生长因子受体2(HER2)双特异性抗体、0.85mg/ml组氨酸、3.17mg/ml盐酸组氨酸、80.00mg/ml蔗糖、0.2mg/ml聚山梨酯80，pH 5.5。
以上描述了本发明的示例性实施方案，本领域技术人员应当理解的是，这些公开内容仅是示例性的，在本发明的范围内可以进行各种其它替换、适应和修改。因此，本发明不限于文中列举的具体实施方案。

Claims (16)

  1. 一种液体抗体制剂，包含
    (i)抗PD-1/HER2双特异性抗体蛋白；
    (ii)缓冲剂，
    (iii)稳定剂，和
    (iv)表面活性剂，
    其中所述抗PD-1/HER2双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含特异性结合PD-1的第一VH/VL单元并且第二半抗体包含特异性结合HER2的第二VH/VL单元，其中所述第一VH/VL单元包含SEQ ID NO:12/SEQ ID NO:10的成对重链可变区序列/轻链可变区序列中所含的全部重链CDR与轻链CDR，并且其中所述第二VH/VL单元包含SEQ ID NO:6/SEQ ID NO:2的成对重链可变区序列/轻链可变区序列中所含的全部重链CDR与轻链CDR，
    优选地，所述液体抗体制剂的pH约为5.0-6.5，例如，pH约为5.0、5.5、6.0、6.5。
  2. 权利要求1的液体抗体制剂，特征在于所述液体抗体制剂中的抗PD-1/HER2双特异性抗体蛋白的浓度为约1-150mg/ml，优选地为约10-100mg/mL，例如约10、20、30、40、50、60、70、80、90或100mg/ml。
  3. 根据权利要求1或2所述的液体抗体制剂，特征在于所述液体抗体制剂中的缓冲剂选自组氨酸、盐酸组氨酸和它们的组合，优选地，所述缓冲剂的浓度为约5-50mM，优选地为约10-30mM，例如，约10、15、20、25、30mM。
  4. 根据权利要求1-3中任何一项所述的液体抗体制剂，特征在于所述稳定剂选自多元醇(例如，山梨醇)、糖类(例如，蔗糖、海藻糖)和它们的任意组合；优选地，所述稳定剂的浓度为约50-500mM，优选地为约100-400mM，例如，约100、150、200、250、300、350、400mM。
  5. 根据权利要求1-3中任何一项所述的液体抗体制剂，特征在于所述稳定剂选自多元醇(例如，山梨醇)、糖类(例如，蔗糖、海藻糖)和它们的任意组合与抗氧化剂的组合，优选地，所述稳定剂的总浓度为约50-500mM，优选地总浓度为约100-400mM，例如，约100、150、200、250、300、350、400mM，其中抗氧化剂的浓度为约1-50mM，优选地约5-40mM，例如约5、10、20、30、40mM，例如，所述抗氧化剂是甲硫氨酸。
  6. 根据权利要求1-5中任何一项所述的液体抗体制剂，特征在于所述液体抗体制剂中的表面活性剂选自聚山梨酯类表面活性剂，优选为聚山梨酯-80。
  7. 根据权利要求1-6中任何一项所述的液体抗体制剂，特征在于所述表面活性剂的浓度为约0.1-1mg/ml，优选地为约0.2-0.8mg/ml，例如约0.2、0.3、0.4、0.5、0.6、0.7、0.8mg/ml。
  8. 根据权利要求1所述的液体抗体制剂，特征在于所述抗PD-1/HER2双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含特异性结合PD-1的第一VH/VL单元并且第二半抗体包含特异性结合HER2的第二VH/VL单元，其中所述第一VH/VL单元包含SEQ ID NO:12/SEQ ID NO:10的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区 序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，并且其中所述第二VH/VL单元包含SEQ ID NO:6/SEQ ID NO:2的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，
    优选地，其中第一半抗体包含从N至C方向的SEQ ID NO:12和SEQ ID NO:14的重链序列或与之具有至少90％,95％,98％或99％同一性的重链序列，和从N至C方向的SEQ ID NO:10和SEQ ID NO:4的轻链序列或与之具有至少90％,95％,98％或99％同一性的轻链序列，并且其中第二半抗体包含从N至C方向的SEQ ID NO:6和SEQ ID NO:8的重链序列或与之具有至少90％,95％,98％或99％同一性的重链序列，和从N至C方向的SEQ ID NO:2和SEQ ID NO:4的轻链序列或与之具有至少90％,95％,98％或99％同一性的轻链序列。
  9. 根据权利要求1-8中任一项所述的液体抗体制剂，特征在于所述抗PD-1/HER2双特异性抗体蛋白在HEK293细胞或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293E细胞；CHO细胞或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO中重组表达。
  10. 根据权利要求1-9中任一项所述的液体抗体制剂，特征在于所述液体制剂为注射剂，优选用于皮下注射或静脉内注射，或者为输注剂，例如用于静脉内输注。
  11. 根据权利要求1-10中任一项所述的液体抗体制剂，其包含：
    (i)约1-150mg/ml的抗PD-1/HER2双特异性抗体蛋白；
    (ii)约5-50mM的组氨酸和/或盐酸组氨酸；
    (iii)约50-500mM的山梨醇、蔗糖、海藻糖和它们的任意组合；或者
    总浓度为约50-500mM的山梨醇、蔗糖、海藻糖和它们的任意组合与甲硫氨酸的组合，其中甲硫氨酸的浓度为约1-50mM，和
    (iv)约0.1-1mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH为约5.0-6.5，优选地约5.5；
    例如，所述液体抗体制剂包含
    (i)约10-100mg/ml的抗PD-1/HER2双特异性抗体蛋白；
    (ii)约10-30mM的组氨酸和/或盐酸组氨酸；
    (iii)约100-400mM的山梨醇、蔗糖、和/或海藻糖；或者
    总浓度为约100-400mM的山梨醇、蔗糖、和/或海藻糖与甲硫氨酸的组合，其中甲硫氨酸的浓度为约5-40mM，和
    (iv)约0.2-0.8mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH为约5.0-6.5，优选地约5.5；
    或者，所述液体抗体制剂包含
    (i)约20mg/ml的抗PD-1/HER2双特异性抗体蛋白；
    (ii)约10mM组氨酸；
    (iii)约50mg/ml山梨醇，和
    (iv)约0.3mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH为约5.0-6.5，优选地约5.5；
    或者，所述液体抗体制剂包含
    (i)约50mg/ml的抗PD-1/HER2双特异性抗体蛋白；
    (ii)约20mM组氨酸；
    (iii)约50mg/ml山梨醇，和
    (iv)约0.2mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH为约5.0-6.5，优选地约5.5；
    或者，所述液体抗体制剂包含
    (i)约50mg/ml的抗PD-1/HER2双特异性抗体蛋白；
    (ii)约20mM组氨酸；
    (iii)约80mg/ml蔗糖，和
    (iv)约0.2mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH为约5.0-6.5，优选地约5.5；
    或者，所述液体抗体制剂包含
    (i)约50mg/ml的抗PD-1/HER2双特异性抗体蛋白；
    (ii)约20mM组氨酸；
    (iii)约80mg/ml海藻糖，和
    (iv)约0.2mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH为约5.0-6.5，优选地约5.5；
    或者，所述液体抗体制剂包含
    (i)约50mg/ml的抗PD-1/HER2双特异性抗体蛋白；
    (ii)约20mM组氨酸；
    (iii)约80mg/ml蔗糖和约1.49mg/ml甲硫氨酸，和
    (iv)约0.2mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH为约5.0-6.5，优选地约5.5；
    或者，所述液体抗体制剂包含
    (i)约42mg/ml的抗PD-1/HER2双特异性抗体蛋白；
    (ii)约0.85mg/ml组氨酸和约3.17mg/ml盐酸组氨酸；
    (iii)约80mg/ml蔗糖，和
    (iv)约0.2mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH为约5.0-6.5，优选地约5.5。
  12. 根据权利要求1-11中任何一项所述的液体抗体制剂，其特征在于，该制剂在储存后，例如在2-8℃储存至少24个月后，或在室温储存至少3个月后，或在40℃±2℃储存1个月后，是稳定的，优选地具有如下特征之一或多项：
    (i)通过SEC-HPLC法测量，制剂具有大于90％的纯度，优选大于95％、96％、97％、98％、99％的纯度；
    (ii)通过还原型或非还原型CE-SDS法测量，制剂具有大于90％的纯度，优选大于92％、94％、96％、98％的纯度；
    (iii)通过iCIEF法测量，相对于储存第0天的初始值，制剂中抗PD-1/HER2双特异性抗体蛋白的各组分(主成分、酸性组分和碱性组分)的变化值总和不超过50％，例如不超过40％、30％、20％、10％、5％；
    (iv)通过ELISA法测量，相对于储存第0天的初始值，制剂中抗PD-1/HER2双特异性抗体蛋白的相对结合活性为70％-130％，例如，为70％、80％、90％、100％、110％、120％、130％。
  13. ー种固体抗体制剂，其通过固化权利要求1-12中任何一项所述的液体抗体制剂而获得，所述固体抗体制剂例如是冻干粉针剂形式。
  14. 递送装置，其包含权利要求1-12中任何一项的液体抗体制剂或权利要求13的固体抗体制剂。
  15. 预填装注射器，其包含权利要求1-12中任何一项的液体抗体制剂或权利要求13的固体抗体制剂，用于静脉内注射或者肌内注射。
  16. 根据权利要求1-12中任何一项的液体抗体制剂或权利要求13的固体抗体制剂的用途，用于制备治疗、预防或延缓与HER2信号传导通路和PD-1信号传导通路相关的病症的药物，所述病症例如各种血液病和实体瘤，包括但不限于白血病、淋巴瘤、骨髓瘤、脑肿瘤、头颈部鳞状细胞癌、非小细胞肺癌、鼻咽癌、食道癌、胃癌、胰腺癌、胆囊癌、胆管癌、肝癌、结直肠癌、乳腺癌、卵巢癌、宫颈癌、子宫内膜癌、子宫肉瘤、前列腺癌、膀胱癌、肾细胞癌、黑色素瘤。
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