WO2022017468A1 - Pd-l1/lag-3双特异性抗体制剂及其制备方法和用途 - Google Patents

Pd-l1/lag-3双特异性抗体制剂及其制备方法和用途 Download PDF

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WO2022017468A1
WO2022017468A1 PCT/CN2021/107878 CN2021107878W WO2022017468A1 WO 2022017468 A1 WO2022017468 A1 WO 2022017468A1 CN 2021107878 W CN2021107878 W CN 2021107878W WO 2022017468 A1 WO2022017468 A1 WO 2022017468A1
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antibody
formulation
lag
arginine
liquid
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PCT/CN2021/107878
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English (en)
French (fr)
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姚天怡
马一冬
汪音爵
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信达生物制药(苏州)有限公司
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Priority to JP2023504418A priority Critical patent/JP2023535433A/ja
Priority to AU2021311673A priority patent/AU2021311673A1/en
Priority to CA3189631A priority patent/CA3189631A1/en
Priority to EP21845945.1A priority patent/EP4186522A1/en
Priority to US18/005,903 priority patent/US20230287124A1/en
Publication of WO2022017468A1 publication Critical patent/WO2022017468A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to the field of antibody formulations. More specifically, the present invention relates to pharmaceutical formulations comprising bispecific antibodies that bind both PD-L1 and LAG-3, in particular stable liquid formulations, lyophilized formulations and reconstituted stable liquid formulations, and for use in the preparation of such formulations. Methods of said pharmaceutical formulations, and therapeutic and/or prophylactic uses of said pharmaceutical formulations.
  • Drug stability is one of the important indicators to ensure the effectiveness and safety of drugs. Obtaining a good formulation is a key condition to ensure that the drug product maintains its efficacy and safety during the shelf life.
  • Due to the complexity of the antibodies themselves and their degradation pathways it is currently impossible to predict the formulation conditions required to optimize antibody stability. Especially considering that different antibodies often have very different CDR sequences, antibody structures, and these sequence and structure differences can lead to different stability properties of different antibodies in solution. Therefore, based on the strict requirements on the safety and efficacy of human antibodies, it is necessary to optimize the optimal formulation for each antibody individually.
  • LAG-3 is mainly expressed on the cell membrane surface of activated T cells and NK cells, and plays a role in inhibiting the activity of immune cells to kill tumor cells.
  • the PD-1/PD-L1 signaling pathway is one of the main mechanisms by which the immune system suppresses tumor clearance, which has been widely proven in clinical practice. Simultaneous targeting of the LAG-3/MHC II signaling pathway and the PD-1/PD-L1 signaling pathway has also been shown to have a certain synergistic effect in clinical applications. Therefore, designing a bispecific antibody that can block PD-1/PD-L1 signaling pathway and LAG-3/MHC II at the same time has great clinical application value.
  • the present invention addresses the above needs by providing pharmaceutical formulations containing bispecific antibodies that specifically bind to PD-L1 and LAG-3.
  • the antibody preparation of the present invention exhibits excellent stability against various stability influencing factors.
  • the present invention provides a liquid antibody formulation comprising (i) a PD-L1/LAG-3 bispecific antibody, (ii) a buffer, (iii) a stabilizer, and (iv) Surfactant.
  • the liquid antibody formulation further comprises (v) a chelating agent.
  • the bispecific antibody structure of PD-L1/LAG-3 of the present invention is shown in Figure 1, wherein antigen A is LAG-3, antigen B is PD-L1, and its structure comprises:
  • VH represents heavy chain variable region
  • CH represents heavy chain constant region
  • Fc comprises CH2, CH3, and optionally CH4;
  • CH1, CH2, CH3 and CH4 represent domains 1, 2, 3 and 4 of the heavy chain constant region, respectively;
  • X may be absent, or, when present, represent a linker
  • VHH represents a single domain antigen binding site
  • VL represents light chain variable region
  • CL represents the light chain constant region
  • the PD-L1/LAG-3 bispecific antibody of the present invention comprises the sequences shown in the following table:
  • Anti-LAG-3 antibody HCDR3 (HCDR3 of an exemplary VH of formula (I)) SEQ ID NO: 15 Anti-LAG-3 antibody LCDR1 (LCDR1 of exemplary VL of formula (II)) SEQ ID NO: 16 Anti-LAG-3 antibody LCDR2 (LCDR2 of VL of exemplary formula (II)) SEQ ID NO: 17 Anti-LAG-3 antibody LCDR3 (LCDR3 of exemplary VL of formula (II)) SEQ ID NO: 18 Anti-LAG-3 antibody heavy chain (exemplary VH-CH1-Fc of formula (I)) SEQ ID NO: 19
  • the PD-L1/LAG-3 bispecific antibody is the bispecific antibody IGN-LP disclosed in PCT Application No. PCT/CN2020/073964 (International Application Date: January 23, 2020).
  • the PD-L1/LAG-3 bispecific antibody is recombinantly expressed in HEK 293 cells or CHO cells.
  • the concentration of PD-L1/LAG-3 diabody in the liquid antibody formulation of the present invention is about 10-200 mg/ml. In another embodiment, the concentration of PD-L1/LAG-3 diabody in the liquid antibody formulation of the present invention is about 20-100 mg/ml, eg, about 20, 25, 30, 35, 40, 45, 50 , 55, 60, 70, 80, 90 or 100 mg/ml, preferably the concentration of PD-L1/LAG-3 double antibody is about 20-60 mg/ml, more preferably about 20-30 mg/ml.
  • the concentration of buffer in the liquid antibody formulation of the invention is about 5-50 mM. In one embodiment, the concentration of buffer in the liquid antibody formulation of the invention is about 5-30 mM, eg, about 5, 10, 15, 20, 25, 30 mM.
  • the buffer is histidine buffer, citrate buffer, acetate buffer, phosphate buffer, preferably, the buffer is histidine buffer. In one embodiment, the buffer is a histidine buffer, preferably, the buffer consists of histidine and histidine hydrochloride. In a preferred embodiment, the buffer is about 5-30 mM histidine buffer, eg about 5-15 mM, such as about 10 mM histidine buffer. In another embodiment, the histidine buffer used in the formulations of the present invention consists of, for example, about 0.85 mg/ml histidine and about 0.97 mg/ml histidine hydrochloride.
  • the stabilizer in the liquid antibody formulation of the present invention is selected from the group consisting of polyols (eg, sorbitol, mannitol, or combinations thereof), carbohydrates (eg, sucrose, trehalose, maltose, or combinations thereof) , amino acids (eg, arginine hydrochloride, methionine, glycine, proline, and combinations or salts thereof), and any combination thereof.
  • the stabilizer comprises about 10-80 mg/ml sorbitol, such as 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80 mg/ml sorbitol, Preferably about 15-25 mg/ml.
  • the stabilizer comprises about 20-100 mg/ml sucrose, such as 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/ml sucrose, preferably about 40-80 mg/ml sucrose.
  • the stabilizer comprises arginine, eg, about 50 mM-200 mM, eg, 60-180 mM, preferably about 70-170 mM.
  • the arginine is arginine hydrochloride.
  • the stabilizer has arginine as a single component, the arginine being about 120-200 mM, preferably about 150-180 mM, such as 150, 155, 160, 165, 170, 175, 180 mM , more preferably about 160-170 mM.
  • the arginine is arginine hydrochloride.
  • the stabilizer comprises a combination of sorbitol and arginine, the arginine being about 60-100 mM, preferably about 70-90 mM, such as 70, 75, 80, 85, 90 mM; the sorbitol
  • the alcohol is about 10-30 mg/ml, preferably about 15-25 mg/ml, eg 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mg/ml.
  • the arginine is arginine hydrochloride.
  • the stabilizer has sucrose as a single component, the sucrose being about 60-100 mg/ml, preferably about 70-90 mg/ml, eg, 70, 75, 80, 85, 90 mg/ml.
  • the stabilizer comprises a combination of sucrose and arginine, the arginine being about 60-100 mM, preferably about 70-90 mM, such as 70, 75, 80, 85, 90 mM; the sucrose About 20-60 mg/ml, preferably about 35-45 mg/ml, eg 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 mg/ml.
  • the arginine is arginine hydrochloride.
  • the surfactant is a nonionic surfactant.
  • the surfactant is selected from the group consisting of polysorbate surfactants, poloxamers, polyethylene glycols.
  • the surfactant is selected from the group of polysorbate surfactants.
  • the surfactant in the liquid antibody formulation of the present invention is polysorbate-80.
  • the concentration of surfactant in the liquid antibody formulation of the invention is about 0.1-1 mg/ml. In one embodiment, the concentration of surfactant in the liquid antibody formulation of the invention is about 0.2-0.8 mg/ml, eg, about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml.
  • the chelating compound in the liquid formulation of the present invention is a carboxylic acid type chelating agent.
  • the chelating agent is selected from the group consisting of disodium edetate, aminotriacetic acid, diethylenetriaminepentaacetic acid, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, diethylenediamine Hydroxyethylglycine.
  • the chelating agent is selected from disodium edetate.
  • the concentration of the chelating agent in the liquid antibody formulation of the invention is about 0.005-0.05 mg/ml. In one embodiment, the concentration of the chelating agent in the liquid antibody formulation of the invention is about 0.008-0.018 mg/ml, eg, about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018 mg/ml.
  • the pH of the liquid formulation is about 5.5-6.5. In some embodiments, the pH of the liquid formulation is any of about 5.5-6.5, eg, about 5.6, 5.8, 6.0, 6.2, 6.4. Preferably, the pH of the formulation is 5.8-6.4, eg, pH is 6.0 ⁇ 0.2 or 6.2 ⁇ 0.2, preferably pH is 6.0.
  • liquid antibody formulation of the present invention comprises:
  • PD-L1/LAG-3 diabody protein e.g. 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or 100 mg/ml, preferably 20-60 mg/ml, more preferably 20-30 mg/ml;
  • arginine such as 150, 155, 160, 165, 170, 175, 180 mM, preferably about 160-170 mM; or a combination of sorbitol and arginine, said arginine being about 60 - 100 mM, eg 60, 65, 70, 75, 80, 85, 90, 100 mM, preferably about 70-90 mM
  • the sorbitol is about 10-30 mg/ml, preferably about 15-25 mg/ml, eg 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25 mg/ml; or about 60-100 mg/ml of sucrose, preferably about 70-90 mg/ml, such as 70, 75, 80, 85, 90 mg /ml; or a combination of sucrose and arginine, the arginine being about 60-100 mM, such as 60, 65, 70, 75, 80, 85, 90, 100 mM, preferably about 70-90 mM
  • the liquid antibody formulation further comprises (v) about 0.008-0.018 mg/ml, such as about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018 mg/ml disodium edetate;
  • the pH of the liquid formulation is about 5.5-6.5, preferably the pH is about 5.8-6.4, eg, the pH is 6.0 ⁇ 0.2 or 6.2 ⁇ 0.2, eg, about 6.0.
  • the liquid antibody preparation comprises:
  • sorbitol a combination of sorbitol and arginine, said arginine being about 60-100 mM and said sorbitol being about 10-30 mg/ml; or
  • a combination of sucrose and arginine is about 60-100 mM, and the sucrose is about 20-60 mg/ml;
  • pH of the liquid formulation is about 5.8-6.4;
  • the liquid antibody preparation comprises:
  • sorbitol a combination of sorbitol and arginine, said arginine being about 70-90 mM and said sorbitol being about 15-25 mg/ml; or
  • a combination of sucrose and arginine is about 70-90 mM, and the sucrose is about 35-45 mg/ml;
  • pH of the liquid formulation is about 5.8-6.4;
  • the liquid antibody preparation comprises:
  • the liquid formulations of the present invention are stable for long-term storage, eg, at least 24 months or more.
  • the liquid formulation of the present invention may be at about -80°C to about 45°C, eg, -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about Store 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 35°C, about 38°C, about 40°C, about 42°C, or about 45°C , 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 is stable.
  • the liquid formulations of the present invention are storage stable for at least 24 months. In yet another embodiment, the liquid formulations of the present invention are stable at at least 40°C. In yet another embodiment, the liquid formulations of the present invention are stable at about 2°C-8°C for at least 3 months, preferably at least 12 months, more preferably at least 24 months. In one embodiment, the liquid formulations of the present invention are 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. In yet another embodiment, the liquid formulations of the present invention are stable at about 40°C for at least 2 weeks, preferably at least 1 month.
  • the stability of the formulation can be indicated by detecting changes in the formulation's appearance, visible foreign matter, protein content, turbidity, purity, surfactant content, relative binding activity, and/or charge variants. In one embodiment, it may be in a forced experiment under high temperature stress, eg after storage at 40°C ⁇ 2°C for at least 1 week, 2 weeks or preferably 1 month, or in an accelerated experiment, eg at 25°C ⁇ 2°C After storage for at least 1 month or 2 months, or in long-term experiments, such as after storage at 5°C ⁇ 3°C for at least 2 months or 3 months, or in shaking experiments (eg, at room temperature, protected from light, 650 r/min)
  • the stability of the liquid formulation of the present invention was tested in freeze-thaw experiments (eg, repeated freeze-thaw cycles at -30°C/room temperature for 5 days).
  • the stability of the liquid formulations of the invention is tested relative to an initial value, eg, an initial value on day 0
  • the stability of the liquid formulations of the present invention is visually inspected for stability, wherein the liquid formulations of the present invention remain clear to slightly opalescent in appearance , is a colorless to slightly yellow liquid with no foreign matter. In one embodiment, no visible foreign matter is present in the formulation upon visual inspection under a clarity tester.
  • the stability of the liquid formulations of the present invention is checked by measuring changes in protein content after storage, or after shaking experiments, or after freeze-thaw experiments, wherein, for example, by ultraviolet spectrophotometry (UV) methods, relative to From the initial value, the rate of change in protein content is not more than 20%, preferably not more than 10%, eg 7-8%, more preferably not more than 5%, 2% or 1%.
  • UV ultraviolet spectrophotometry
  • the stability of the liquid formulation of the present invention is checked by measuring the turbidity change of the liquid formulation of the present invention after storage, or after a shaking experiment, or after a freeze-thaw experiment, wherein, for example, by the OD350nm method, Relative to the initial value, the change value is not more than 0.06, preferably not more than 0.05, more preferably not more than 0.04, not more than 0.02.
  • the stability of the liquid formulations of the present invention is checked for stability after storage, or after shaking experiments, or after freeze-thaw experiments, by measuring changes in the purity of the liquid formulations of the present invention, wherein size exclusion HPLC Chromatography (SEC-HPLC), the change in monomer purity (or the main peak change) by no more than 10% relative to the initial value, e.g. no more than 5%, 4%, 3%, e.g. no more than 2%, Preferably not more than 1%.
  • SEC-HPLC size exclusion HPLC Chromatography
  • the stability of the liquid formulation of the present invention is examined by measuring the change in purity of the liquid formulation of the present invention after storage, or after a shaking experiment, or after a freeze-thaw experiment, wherein by non-reduced dodecane Sodium sulfate capillary electrophoresis (CE-SDS) method, relative to the initial value, the change value (or the main peak change value) of the monomer purity decreases by not more than 10%, such as not more than 5%, 4%, 3%, 2% or 1%.
  • CE-SDS dodecane Sodium sulfate capillary electrophoresis
  • the stability of the liquid formulations of the present invention is tested by imaging capillary isoelectric focusing electrophoresis (iCIEF) after storage, or after shaking experiments, or after freeze-thaw experiments, wherein the antibody relative to the initial value is tested for stability
  • the sum of the change values of the charge variants (principal component, acidic component and basic component) of the The change value does not exceed 20%, 15%, 10%, 8%, 5%.
  • the stability of the liquid formulation of the present invention is tested by direct ELISA after storage, or after shaking experiments, or after freeze-thaw experiments, wherein the relative binding activity of the antibody is 70 relative to the initial value.
  • the polysorbate 80 content is detected by high performance liquid chromatography-fluorescence detection method (HPLC-FLD method), polysorbate 80 content It should be 0.2 to 0.8 mg/ml, preferably 0.3 to 0.7 mg/ml.
  • the liquid formulation of the present invention is stable after storage, for example at 25°C for at least 2 months, or at 40°C ⁇ 2°C for 1 month, preferably having one of the following characteristics or multinomial: relative to the initial value stored on day 0,
  • the variation of the main peak is less than 1% as measured by SEC-HPLC method, and/or the preparation has a purity greater than 96%, preferably greater than 97%, 98%;
  • the main peak change value is less than 2% as measured by non-reducing CE-SDS method, and/or the preparation has a purity of more than 96%, preferably a purity of more than 97%, 98%;
  • the total change value does not exceed about 40% (for example, not more than 35%, 30%, 25%, 20%, 15%, 10%) or the principal component change value does not exceed 20% (eg, no more than 15%, 12%, 10%, 8%), or
  • the sum of the change values does not exceed about 20% (eg, not more than 15%, 14%, 13%, 12%) or the principal component change value does not exceed about 15% (eg, not more than 10%) %, 8%, 7%, 6%, 5%);
  • the relative binding activity of PD-L1/LAG-3 diabody protein in the preparation is 70%-130% as measured by ELISA, for example, 90, 93, 95, 98, 100, 103, 105, 108, 110, 115, 120%;
  • liquid formulations of the present invention are stable after storage, for example at 5°C ⁇ 3°C for at least 6 months, preferably having one or more of the following characteristics:
  • the liquid preparation of the present invention is a pharmaceutical preparation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection.
  • the liquid formulation is an intravenous infusion.
  • the present invention provides a solid antibody preparation obtained by subjecting the liquid antibody preparation of the present invention to a solidification treatment.
  • the solidification treatment is carried out by, for example, a crystallization method, a spray drying method or a freeze drying method.
  • the solid antibody formulation is, for example, in the form of a lyophilized powder for injection.
  • Solid antibody formulations can be reconstituted in a suitable vehicle prior to use to form a reconstituted formulation of the invention.
  • the reconstituted formulation is also a liquid antibody formulation of the present invention.
  • the appropriate vehicle 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 invention provides a delivery device comprising a liquid antibody formulation or a solid antibody formulation of the invention.
  • the delivery device of the invention is provided in the form of a prefilled syringe containing the liquid antibody formulation or solid antibody formulation of the invention, eg, for intravenous, subcutaneous, intradermal or intramuscular injection, intravenous infusion .
  • the present invention provides a method of delivering a PD-L1/LAG-3 diabody protein to a subject, such as a mammal, comprising the step of administering to said subject a liquid antibody formulation or solid antibody formulation of the present invention,
  • the delivery is effected, for example, by a delivery device using a prefilled syringe.
  • the present invention provides use of the liquid antibody formulation or solid antibody formulation of the present invention for the preparation of a delivery device or prefilled syringe or a prefilled syringe that simultaneously targets LAG-3 and PD-L1 signaling pathways to treat or prevent tumors.
  • a drug wherein the tumor is cancer, including but not limited to gastrointestinal cancers such as colon cancer or colorectal cancer or rectal cancer.
  • the present invention also provides a method by administering to a subject a liquid antibody formulation or solid antibody formulation of the invention or a delivery device (eg, a prefilled syringe) or medicament comprising the liquid antibody formulation or solid antibody formulation, in a subject Methods of blocking the LAG-3 and/or PD-L1 signaling pathway in patients to reduce or eliminate the immunosuppressive effects of LAG-3 and/or PD-L1.
  • a delivery device eg, a prefilled syringe
  • medicament comprising the liquid antibody formulation or solid antibody formulation
  • the invention also provides a method for treating a subject by administering to a subject a liquid antibody formulation or solid antibody formulation of the invention or a delivery device (eg, a prefilled syringe) or drug comprising the liquid antibody formulation or solid antibody formulation disease of the patient, such as the method for the above-mentioned tumor.
  • a delivery device eg, a prefilled syringe
  • drug comprising the liquid antibody formulation or solid antibody formulation disease of the patient, such as the method for the above-mentioned tumor.
  • Fig. 1 Structure of bispecific antibody of PD-L1/LAG-3 of the present invention
  • the term “comprising” or “comprising” means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.
  • the term “comprising” or “comprising” is used, unless otherwise indicated, it also encompasses situations consisting of the recited elements, integers or steps.
  • reference to an antibody variable region that "comprises” a particular sequence is also intended to encompass antibody variable regions that consist of that particular sequence.
  • antibody is used in the broadest sense to refer to a protein comprising an antigen-binding site, encompassing natural 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”, “complete antibody” and “intact antibody” are used interchangeably herein to refer to a naturally occurring heavy chain (H) comprising at least two heavy chains (H) interconnected by disulfide bonds and two light chain (L) glycoproteins.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions (complementarity determining regions (CDRs), with more conserved regions (framework regions (FR)) interposed therebetween.
  • CDRs complementarity determining regions
  • FR frame regions
  • Each VH and VL consists of three CDRs and four
  • the FRs are composed, from the amino terminus to the carboxy terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions.
  • antibody preparation refers to a preparation that is in a form that allows the biological activity of the antibody as the active ingredient to be effectively exerted, and that does not contain unacceptable toxicity to the subject to which the preparation is to be administered. other components. Such antibody preparations are generally sterile.
  • pharmaceutically acceptable excipients are included in the antibody formulation.
  • a "pharmaceutically acceptable" excipient is an agent that can reasonably be administered to a subject mammal so that an effective dose of the active ingredient used in the formulation can be delivered to the subject.
  • concentration of the excipient is adapted to the mode of administration, eg, may be acceptable for injection.
  • PD-L1/LAG-3 diabody preparation is also abbreviated as "antibody preparation of the present invention” herein, which means comprising PD-L1/LAG-3 diabody protein as an active ingredient and pharmaceutically acceptable excipients preparation of the agent.
  • antibody preparation of the present invention means comprising PD-L1/LAG-3 diabody protein as an active ingredient and pharmaceutically acceptable excipients preparation of the agent.
  • the PD-L1/LAG-3 diabody protein as an active ingredient is suitable for therapeutic or prophylactic administration to human or non-human animals.
  • Antibody formulations of the invention can be prepared, for example, as liquid formulations in aqueous form, eg, ready-to-use prefilled syringes, or as lyophilized formulations by dissolving and/or suspending in a physiologically acceptable solution immediately before use Reconstitute (ie, reconstitute).
  • the PD-L1/LAG-3 diabody protein formulation is in the form of a liquid formulation.
  • a “stable” antibody formulation is one in which the antibody in the formulation retains an acceptable degree of physical and/or chemical stability after storage under specified conditions, or after shaking, or after repeated freeze-thaw cycles.
  • antibodies contained in antibody formulations may not maintain 100% of their chemical structure after storage, shaking, or repeated freeze-thaw cycles, typically if maintained about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of the structure or function of the antibody, an antibody preparation is considered “stable.”
  • the PD-L1/LAG-3 diabody protein formulations of the invention exhibit low to undetectable antibody aggregation or degradation or chemical modification during manufacture, preparation, shipping, and long-term storage, thereby There is little or even no loss of biological activity of the PD-L1/LAG-3 diabody protein, showing a high degree of stability.
  • the PD-L1/LAG-3 diabody protein formulation of the present invention substantially retains its physical and chemical stability after storage, shaking and/or repeated freezing and thawing.
  • the liquid formulation of the present invention is 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 6 months.
  • Stability can be measured at selected temperatures and selected storage times. For example, the storage time can be selected based on the expected shelf life of the formulation. Alternatively, accelerated stability testing can be used. In some embodiments, stability testing is performed by subjecting antibody formulations to various stress tests.
  • formulated PD-L1/LAG-3 diabody protein formulations can be filled into glass vials to test antibody stability under high temperature stress.
  • Antibodies can be considered to "retain their physical stability" in the formulation. Safety concerns arise as the aggregation of antibodies in the formulation can potentially lead to an increased immune response in the patient. Therefore, there is a need to minimize or prevent aggregation of antibodies in formulations.
  • Light scattering methods can be used to determine visible aggregates in formulations.
  • SEC-HPLC can be used to determine soluble aggregates in formulations.
  • the stability of the formulation can be indicated by visually inspecting the appearance, color and/or clarity of the formulation, or by measuring the turbidity of the formulation by the OD350nm method, or by measuring the purity of the formulation by the non-reducing CE-SDS method.
  • the stability of the formulation is measured by determining the percentage of antibody monomer in the formulation after storage at a specific temperature for a specific time or after shaking or after repeated freeze-thaw cycles, wherein the percentage of antibody monomer in the formulation The higher, the higher the stability of the formulation.
  • an "acceptable level" of physical stability may mean that at least about 90% of the PD-L1/LAG-3 diabody monoclonal antibody is detected in the formulation after storage at a specified temperature for a specified period of time, after shaking or after repeated freezing and thawing body.
  • An acceptable degree of physical stability indicates an acceptable degree of physical stability after 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 or more At least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of PD-L1/LAG-3 double-antibody protein monomers.
  • the particular temperature at which the pharmaceutical formulation is stored can be any temperature from about -80°C to about 45°C, eg, 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 drug formulation is considered stable.
  • the pharmaceutical preparation is considered to be stable. If stored at about 5°C for 6 months, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% PD-L1/LAG detected -3 double antibody protein monomer, the pharmaceutical preparation is considered to be stable. If stored at about 5°C for 6 months, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% PD-L1/LAG detected -3 double antibody protein monomer, the pharmaceutical preparation is considered to be stable.
  • An antibody in a formulation may be considered to "retain its chemical stability" if the antibody in the formulation does not show significant chemical changes after a period of storage, or after a period of shaking, or after repeated freeze-thaw cycles.
  • Most chemical instability results from the formation of covalently modified forms of the antibody (eg, charge variants of the antibody).
  • charge variants of the antibody e.g., charge variants of the antibody.
  • chemical stability can be assessed by detecting and/or quantifying chemically altered forms of the antibody.
  • charge variants 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 percent change in the charge variant of the antibody in the formulation after storage at a specific temperature for a specific time or after shaking or multiple freeze-thaw cycles, wherein the smaller the change , the higher the stability of the formulation.
  • An "acceptable level" of chemical stability can be expressed in terms of charge variants (such as principal or acidic components or bases) in a formulation after storage at a specified temperature for a specified period of time, or after a period of shaking, or after repeated freeze-thaw cycles.
  • the percentage change value of the chemical component does not exceed 40%, such as not more than 30%, not more than 20%; or the sum of the percentage change value of the charge variants (main component, acidic component and basic component) does not exceed 60% , such as not more than 50%, not more than 30%; or the main component content is not less than 50%, such as not less than 60%, not less than 70%.
  • an acceptable level of chemical stability may Indicates that the percent change in charge variants of the principal component does not exceed about 50%, 40%, 30%, 20%, or 15%; or the sum of the percent change in charge variants does not exceed about 60%, 50%, or 30 %.
  • the temperature at which the pharmaceutical formulation is stored can be any temperature from about -80°C to about 45°C, eg, 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 formulation can be considered stable.
  • the pharmaceutical formulation can also be considered stable. If after 1 month of storage at 40°C, the percent change in principal component charge variants is less than about 50%, 40%, 30%, 20%, 16%, 15%, 14%, 13%, 12%, 10%, 5% or 4%, the pharmaceutical formulation can also be considered stable.
  • lyophilized formulation refers to a composition obtained or obtainable by lyophilization of a liquid formulation. Preferably, it is a solid composition with a water content of less than 5%, preferably less than 3%.
  • reconstituted formulation refers to a liquid formulation obtained by dissolving and/or suspending a solid formulation (eg, a lyophilized formulation) 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 refers to chemically and/or physically unfavorable environments for an antibody protein that can lead to unacceptable destabilization of the antibody protein, eg, high temperature, shaking, freezing and thawing.
  • High temperature stress refers to the storage of antibody preparations at room temperature or even at higher temperatures (eg, 40°C ⁇ 2°C) for a period of time. The stability of the antibody preparation can be checked by a high temperature stress accelerated test.
  • parenteral administration means administration other than enteral and topical administration, usually by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal , intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion .
  • the stable PD-L1/LAG-3 dual antibody protein formulations of the invention are administered parenterally to a subject.
  • the PD-L1/LAG-3 dual antibody protein formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular or intravenous injection.
  • the present invention provides stable liquid antibody formulations comprising (i) a PD-L1/LAG-3 diabody, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant.
  • the pH of the antibody formulation is about 5.5-6.5.
  • the liquid antibody formulation further comprises (v) a chelating agent.
  • the liquid antibody formulation of the present invention is in the form of an injectable formulation.
  • the PD-L1/LAG-3 diabody in the antibody preparation of the present invention is the bispecific antibody IGN-LP disclosed in PCT Application No. PCT/CN2020/073964 (International Application Date: January 23, 2020).
  • the PD-L1/LAG-3 double antibody is produced by recombinant expression in CHO cells or HEK293 cells.
  • the antibody in the liquid formulation of the present invention exhibits significant anti-tumor activity.
  • administration of the antibody formulations of the present invention can produce significant tumor suppressive effects.
  • the amount of antibody or antigen-binding fragment thereof contained in the antibody formulation of the invention can vary depending on the specific intended properties of the formulation, the specific environment, and the specific purpose for which the formulation is used.
  • the antibody formulation is a liquid formulation, which may contain about 10-200 mg/ml, preferably about 20-100 mg/mL, eg, about 20, 25, 30, 35, 40, 45, 50, 55, 60 , 70, 80, 90 or 100 mg/ml PD-L1/LAG-3 double antibody, preferably the concentration of PD-L1/LAG-3 double antibody is about 20-60 mg/ml, more preferably about 20-30 mg/ml.
  • a buffer is an agent that can maintain the pH of a solution within an acceptable range.
  • the buffers used in the formulations of the present invention can control the pH of the formulations of the present invention in a pH range of about 5.5-6.5.
  • the formulations of the present invention comprise a buffer system selected from the group consisting of: histidine-histidine hydrochloride buffer system, citric acid-sodium citrate buffer system, acetic acid-sodium acetate buffer system, phosphate buffer system , preferably histidine-histidine hydrochloride buffer system.
  • the buffer used in the formulations of the present invention is a histidine buffer, especially a buffer system consisting of histidine and histidine hydrochloride.
  • Suitable stabilizers for use in the present invention may be selected from sugars, polyols and amino acids and combinations thereof.
  • Sugars for use as stabilizers include, but are not limited to, sucrose, trehalose, maltose, and combinations.
  • Polyols for use as stabilizers include, but are not limited to, sorbitol, mannitol, or combinations thereof.
  • Amino acids for use as stabilizers include, but are not limited to, arginine, arginine hydrochloride, methionine, glycine, proline, and combinations.
  • the liquid formulation of the present invention contains arginine as a stabilizer.
  • the liquid formulation of the present invention comprises a combination of arginine and sorbitol as a stabilizer.
  • the liquid formulation of the present invention contains sucrose as a stabilizer.
  • the liquid formulation of the present invention comprises a combination of sucrose and arginine as a stabilizer.
  • the arginine is arginine hydrochloride.
  • surfactant refers to organic substances that have an amphiphilic structure; that is, they are composed of groups of opposite solubility tendencies, usually an oil-soluble hydrocarbon chain and a water-soluble ionic group group.
  • the surfactant in the liquid formulation of the present invention is a nonionic surfactant, eg, an alkyl poly(ethylene oxide).
  • Particular nonionic surfactants that can be included in the formulations of the present invention include, for example, polysorbates such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; polox Sham et al.
  • polysorbate-80 is included as a surfactant in the liquid formulation of the present invention.
  • surfactants that can be used in the liquid formulations of the present invention include, but are not limited to, polysorbate surfactants (eg, polysorbate 80, polysorbate 20), poloxamers, and polyethylene glycol.
  • the amount of surfactant contained in the antibody formulation of the invention can vary depending on the specific intended nature of the formulation, the specific environment, and the specific purpose for which the formulation is used.
  • excipients are optionally included in the antibody liquid formulation of the present invention.
  • excipients include, for example, antimicrobial agents, antistatic agents, antioxidants, chelating agents, gelatin, and the like.
  • excipients and/or additives suitable for use in the formulations of the present invention are well known in the art, and are listed, for example, in "The Handbook of Pharmaceutical Excipients, 4th Ed., Rowe et al., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st Ed. Gennaro ed. Lippincott Williams & Wilkins (2005)”.
  • chelating agent refers to the ability to form a chelate with a central atom, the stability of the complex being greatly increased due to the formation of the chelate.
  • the chelating compound in the liquid formulation of the present invention is a carboxylic acid type chelating agent.
  • the chelating agent is selected from the group consisting of disodium edetate, aminotriacetic acid, diethylenetriaminepentaacetic acid, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, diethylenediamine Hydroxyethylglycine.
  • the chelating agent is selected from disodium edetate.
  • the present invention provides a stable formulation comprising PD-L1/LAG-3 diabody protein.
  • the PD-L1/LAG-3 diabody proteins used in the formulations of the present invention can be prepared using techniques known in the art for producing antibodies.
  • antibodies can be produced recombinantly.
  • the antibodies of the invention are recombinantly produced in 293 cells or CHO cells.
  • recombinantly produced monoclonal antibodies can be purified using conventional purification methods to provide drug substance with sufficient reproducibility and modest purity for formulation of antibody preparations.
  • the supernatant from the expression system can be concentrated using a commercially available protein concentration filter such as Amicon's ultrafiltration device.
  • purification of the antibody can be performed using, for example, 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 the antibody of sufficient purity, preparations comprising the antibody can be prepared according to methods known in the art.
  • the fermentation broth is centrifuged to remove impurities such as cells to obtain a supernatant;
  • affinity chromatography for example, specific for IgG1, IgG2 and IgG4 antibodies
  • virus inactivation can be used.
  • purification generally, CEX cation exchange chromatography can be used
  • virus filtration to make the virus titer
  • ultrafiltration/diafiltration can be used to replace the protein in a formulation buffer that is conducive to its stability and concentrate to a suitable concentration for injection). See, eg, B. Minow, P. Rogge, K. Thompson, BioProcess International, Vol. 10, No. 6, 2012, pp. 48-57.
  • Biological product stability studies generally include real-time stability studies (long-term stability studies) under actual storage conditions, accelerated stability studies, and forced-condition test studies. Stability research should explore and optimize the research conditions according to the research purpose and the characteristics of the product itself; for various influencing factors (such as temperature, repeated freezing and thawing, vibration, etc.), formulate long-term, accelerated and/or forced condition tests and other stability studies plan. Accelerated and forced condition tests are useful for understanding product stability under short-term deviations from storage conditions and extreme conditions, and to provide supporting data for the determination of expiration dates and storage conditions.
  • antibodies may aggregate, degrade, or chemically modify, resulting in antibody heterogeneity (including size and charge heterogeneity), aggregates and fragments, etc. Affect the quality of antibody preparations. Therefore, it is necessary to monitor the stability of antibody preparations.
  • the purity of antibody preparations and the level of antibody aggregation can be assessed by methods such as reduced CE-SDS, non-reduced CE-SDS, and SEC-HPLC; capillary isoelectric focusing (cIEF), imaging capillary isoelectric Analysis of charge variants in antibody preparations by focused electrophoresis (iCIEF) and ion exchange chromatography (IEX), among others.
  • the stability of the formulation can be quickly judged by visually inspecting the appearance of the formulation.
  • the turbidity change of the formulation can also be detected using the OD350nm method, which can give information on the amount of soluble and insoluble aggregates.
  • ultraviolet spectrophotometry UV method
  • UV method ultraviolet spectrophotometry
  • the antibody preparation of the present invention comprising the PD-L1/LAG-3 double anti-protein of the present invention is used for preventing or treating diseases, such as autoimmune diseases, inflammatory diseases, infections, tumors and the like.
  • diseases such as autoimmune diseases, inflammatory diseases, infections, tumors and the like.
  • the disease is a tumor (eg, cancer) or an infection.
  • the tumor is tumor immune escape.
  • a tumor such as colon cancer or colorectal cancer or rectal cancer.
  • the present invention also provides the use of the formulation of the present invention in the preparation of a medicament, wherein the medicament is used to deliver PD-L1/LAG-3 double antibody protein to mammals.
  • the present invention also provides a method of using the formulations of the present invention for the treatment or prevention of one or more of the aforementioned diseases and disorders.
  • the mammal is a human.
  • the antibody formulations of the invention can be administered to a subject or patient in a variety of ways.
  • administration can be by infusion or by syringe.
  • the present invention provides a delivery device (eg, a syringe) comprising an antibody formulation of the present invention (eg, a prefilled syringe).
  • the patient will receive an effective amount of the PD-L1/LAG-3 diabody protein as the main active ingredient, ie, an amount sufficient to treat, ameliorate or prevent the disease or disorder of interest.
  • N/A means not applicable.
  • the detection items during the whole research process mainly include: (1) detection of appearance and presence of visible foreign matter; (2) determination of protein content in preparations by ultraviolet method (UV method); (3) detection of turbidity of preparations by OD350nm method; ( 4) Determine the purity of the antibody preparation by size exclusion high performance liquid chromatography (SEC-HPLC), expressed as the percentage of the area of the main peak accounting for the sum of all peak areas; (5) through a non-reducing sodium dodecyl sulfate capillary The purity of the antibody preparation was determined by electrophoresis (non-reducing CE-SDS), expressed as the percentage of the area of the main peak to the sum of all peak areas; (6) The charge variant in the antibody preparation was determined by the iCIEF method, expressed as the main component, the acid group (7) Determination of the relative binding activity of PD-L1/LAG-3 double antibody to PD-L1 and LAG-3 antigens in antibody preparations by direct ELISA assay or luciferase reporter gene cells Assays
  • the protein content in the samples was determined using an ultraviolet spectrophotometer (manufactured by Shimadzu, Japan, model UV-1800) or a multi-channel microspectrophotometer (manufactured by Thermo, USA, model Nanodrop 8000).
  • the mobile phase is phosphate buffer (weigh 3.12g sodium dihydrogen phosphate dihydrate, 8.77g sodium chloride and 34.84g arginine, dissolve in ultrapure water and adjust the pH to 6.8 with hydrochloric acid and dilute to 1000ml), the column protection solution is 0.05% (w/v) NaN3, 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.
  • phosphate buffer weigh 3.12g sodium dihydrogen phosphate dihydrate, 8.77g sodium chloride and 34.84g arginine, dissolve in ultrapure water and adjust the pH to 6.8 with hydrochloric acid and dilute to 1000ml
  • the column protection solution is 0.05% (w/v) NaN3
  • 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
  • the detection wavelength is 280
  • sample diluent (0.85mg/ml histidine, 0.97mg/ml histidine hydrochloride, 35.11mg/ml arginine hydrochloride, 0.01mg/ml disodium edetate, 0.10mg/ml polyamide Sorbitan 80) diluted to 2mg/ml, as the test solution.
  • the preparation buffer was diluted in the same way as above and used as blank solution. Take 50 ⁇ l of blank solution and 50 ⁇ l of test solution and inject into liquid chromatograph to start detection.
  • the capillary is an uncoated capillary with an inner diameter of 50 ⁇ m, a total length of 30.2 cm, and an effective length of 20.2 cm.
  • the capillary column was washed with 0.1 mol/L sodium hydroxide, 0.1 mol/L hydrochloric acid, ultrapure water, and electrophoresis gel at 70 psi, respectively.
  • the sample to be tested was diluted to 0.6 mg/ml with a pH 6.5 diluent (200 ⁇ l of pH 6.5 citric acid-phosphate buffer was added to 400 ⁇ l of 10% sodium dodecyl sulfate, and water was added to make up to 1 ml).
  • Imaging capillary isoelectric focusing electrophoresis was used for detection.
  • the inner diameter of the capillary is 100 ⁇ m and the total length is 5 cm.
  • the sample was diluted to 1.0mg/ml with 3mol/L urea-0.5%MC (hydroxymethyl cellulose) solution, 70 ⁇ l of 3mol/L urea-0.5%MC solution, 3 ⁇ l of ampholyte (pH 3 ⁇ 10), pI 5.85, 9.46 Maker (isoelectric point marker) 0.3 ⁇ l each, 5mol/L NDSB (3-[(2-hydroxyethyl)dimethylamino]propane-1-sulfonate) 5 ⁇ l, 0.5mol/L Taurine (bovine) sulfonic acid) 2 ⁇ l, final sample concentration 0.2 mg/ml, focusing voltage 3 kV, focusing time 5 minutes.
  • the blank solution was prepared by the same method using the preparation buffer.
  • the SA protein was diluted with PBS (phosphate buffered saline) to 1.0 ⁇ g/ml, 100 ⁇ l/well, incubated at 37° C. for 2 hours, and the protein was coated on a 96-well microtiter plate. After washing the plate, add blocking solution (2% BSA-PBST, 300 ⁇ l/well), and block at 37° C. for 2 hours. After washing the plate, Biotin LAG-3 was diluted to 0.5 ⁇ g/ml with 2% BSA-PBST (PBST: phosphate buffer + Tween 20; BSA: bovine serum albumin), and 100 ⁇ l/well was put into the ELISA plate, Wash the plate after 30 min incubation at 37°C.
  • PBS phosphate buffered saline
  • test sample was diluted to 40 ⁇ g/ml with 2% BSA-PBST, and the 4-fold gradient was diluted to the 12th concentration.
  • HRP horseradish peroxidase
  • the SA protein was diluted with PBS to 1.0 ⁇ g/ml, 100 ⁇ l/well, incubated at 37°C for 2 hours, and the protein was coated on a 96-well microtiter plate. After washing the plate, add blocking solution (2% BSA-PBST, 300 ⁇ l/well), and block at 37° C. for 2 hours. Wash the plate, dilute Biotin PD-L1 with 2% BSA-PBST to 0.5 ⁇ g/ml, put 100 ⁇ l/well into the ELISA plate, and wash the plate after incubating at 37°C for 30 minutes. The test sample was diluted to 60 ⁇ g/ml with 2% BSA-PBST, and the 4-fold gradient was diluted to the 12th concentration.
  • the polysorbate 80 content of the samples to be tested was determined by HPLC-FLD method (fluorescence method).
  • the chromatographic column was Knitted Reactor coil column (5m ⁇ 0.5mm ID) from SUPELCO, mobile phase: 0.15mol/L sodium chloride, 0.05mol/L Tris, pH 8.0, 5% acetonitrile, 5.0 ⁇ mol/L NPN ( N-phenyl-1-naphthylamine), 15 ppm Brij solution (polyoxyethylene (23) lauryl ether solution).
  • Detection conditions flow rate 1.5ml/min, excitation wavelength 350nm, emission wavelength 420nm, column temperature 30°C, elution time 3 minutes, injection volume 10 ⁇ l.
  • the content of polysorbate 80 in the finished product was calculated by the standard curve method.
  • MHCII APC cells Adjust MHCII APC cells to 0.4 ⁇ 10 6 cells/ml with MHCII APC cells Assay Buffer (1% FBS (Fetal Bovine Serum, Cat. No. 10099-141), 99% DMEM-High Glucose Medium (Cat. No. 11995-040)) , dilute HA-peptide (Cat. No.: PP-1901-13) to 40 ⁇ g/ml, mix the two in equal volumes 1:1, inoculate 100 ⁇ l/well in a 96-well cell culture plate according to the experimental layout, and culture overnight (18-22h). ).
  • Jurkat-LAG-3-NFAT-Luc2 was adjusted with LAG-3 Effector cells Assay Buffer (1% FBS (Fetal Bovine Serum, Cat. No.
  • DMEM-High Glucose Medium (Cat. No. 11995-040)) (Cat. No. CS194812) to 3.0 ⁇ 10 6 cells/ml; dilute the sample to 80.00 ⁇ g/ml, and then dilute 4-fold gradient, with a total of 10 concentration points (0.3ng/ml ⁇ 80000ng/ml).
  • the medium in the 96-well plate was aspirated, and the treated samples and Jurkat-LAG-3-NFAT-Luc2 cells (40 ⁇ l/well, respectively) were added, and the culture was continued for 7 h.
  • CHOK1-PDL1 (Cat. No. CS187108) cells to 0.3 ⁇ 10 6 cells/ml with Assay Buffer (10% fetal bovine serum, 90% RPMI1640 medium (Cat. No.: SH30809.01)), and seed 100 ⁇ l/well in 96 cells according to the experimental layout Well culture plate, adherent culture for 16 ⁇ 20h.
  • Assay Buffer 10% fetal bovine serum, 90% RPMI1640 medium (Cat. No.: SH30809.01)
  • Jurkat-NFAT-Luc2/PD1 (Cat. No. CS187102) cells to 0.6 ⁇ 10 6 cells/ml with Assay Buffer, dilute the sample to 12.00 ⁇ g/ml, 2.6-fold gradient dilution, a total of 10 concentration points (2.2ng/ml ⁇ 12000ng /ml).
  • the medium in the 96-well plate was aspirated, and the treated samples and Jurkat-NFAT-Luc2/PD1 cells (40 ⁇ l/well, respectively) were added, and the culture was continued for 6 h. After the cell culture plate was equilibrated to room temperature, 80 ⁇ l of Bio-Glo TM Luciferase Assay System chromogenic solution (equilibrated to room temperature in advance) was added to each well, and the reaction was carried out for 10 min. Using the software of the Max i3 microplate reader, according to the established layout, set the reading program to read the chemiluminescence value of the full wavelength.
  • the four-parameter fitting curve of the GraphPad Prism software was used to calculate the ratio of the EC50 of the reference product to the EC50 of the sample, reflecting the biological activity of the anti-PD-L1 end of the sample to be tested.
  • PCT/CN2020/073964 the bispecific antibody IGN-LP that binds both PD-L1 and LAG-3 was obtained.
  • PCT Application No. PCT/CN2020/073964 is hereby incorporated by reference in its entirety.
  • antibodies were recombinantly expressed in HEK293 cells and purified by filtration, chromatography, virus inactivation, filtration, and the like.
  • a total of 4 prescriptions were designed, and the detailed prescription information is shown in Table 1.
  • the buffers for each formulation were prepared, and the purified bispecific antibody protein of the invention of Example 1 was ultrafiltered into the respective formulation solution. After displacement was complete, each formula protein concentration was diluted to about 20 mg/ml and polysorbate 80 was added. Filter and pack into vials, stoppered and capped, and conduct stability inspection.
  • the test items are appearance, visible foreign matter, protein content (UV method), turbidity (OD350nm method), polysorbate 80 content (HPLC-FLD method), purity (SEC-HPLC method and non-reducing CE-SDS method), Charge variants (iCIEF method) and relative binding activity (direct ELISA method).
  • the prescription screening experiment used the criteria shown in Table 3 to judge the quality
  • formula 1 was selected to conduct a pilot-scale long-term stability investigation.
  • prescription 1 (20.0mg/ml recombinant anti-lymphocyte activation gene-3 (LAG-3) and anti-programmed death ligand 1 (PD-L1) bispecific antibody, 0.85mg/ml histidine acid, 0.97mg/ml histidine hydrochloride, 35.11mg/ml arginine hydrochloride, 0.01mg/ml disodium edetate, 0.50mg/ml polysorbate 80, pH 5.8-6.4) to produce 3 batches of finished preparations,
  • the long-term stability was investigated at 5°C ⁇ 3°C, and the samples were sampled at 0, 3, and 6 months. The results are shown in Table 6. The results showed that the protein in the formulation was stable, and there was no significant difference between batches, which was in line with the actual production process quality standards.
  • PD-L1 transgenic mice (MC38-huPD-L1 KI tumor-bearing mouse model, referred to as MC38/PD-L1 model) inoculated with MC38-huPD-L1 KI (MJ-1) colon cancer cells were used to determine the anti-bacterial effect of the present invention.
  • Antitumor effect of LAG-3/PD-L1 bispecific antibody IGNLP ie, IGN-LP prepared as described in Example 1).
  • the MC38-huPD-L1 KI tumor-bearing mouse model was established by subcutaneous inoculation. After tumor formation, the mice were divided into groups and treated with different antibodies. The tumor volume and body weight of the mice in each group were monitored during the administration period. The administration frequency was 2 times/day. Week, 2 weeks of administration, a total of 5 doses. The monitoring frequency was 2 times/week, and the monitoring was performed continuously for 4 weeks.
  • the dosage and method of administration were as follows.
  • TGI% 100%*(tumor volume in the h-IgG control group – tumor volume in the treatment group)/(tumor volume in the h-IgG control group – h- The tumor volume of the IgG control group before administration), wherein the average tumor volume of the h-IgG control group before administration was 80 mm 3 .
  • mice PD-L1 transgenic mice, female, 7-8 weeks (the mouse age at the time of tumor cell inoculation), body weight 18-20 g, purchased from Shanghai Southern Model Biotechnology Co., Ltd. Mice were acclimated for 7 days after arrival before the study began.
  • Mouse colon cancer cell MC38 (Shanghai Heyuan Biotechnology, HYC0116) was purchased from Shanghai Heyuan Biotechnology Co., Ltd. and knocked in the human PD-L1 gene (Nanjing Galaxy Biomedical Co., Ltd.) to obtain MC38 cells containing human PD-L1 , cultured in RPMI 1640 medium (Gibco, 22400-071), and routinely subcultured in strict accordance with MC38 culture requirements for subsequent in vivo experiments. Centrifugation (400g / min, 5 min) cells were harvested, the cells were resuspended in sterile RPMI 1640 basal medium and cell density was adjusted to 5 ⁇ 10 6 cells / ml.
  • mice with an average tumor volume in the range of 76-80 mm 3 were selected and randomly grouped according to tumor volume.
  • the anti-tumor activity of the anti-LAG-3/PD-L1 antibody alone was tested as follows.
  • Anti-PD-L1 antibody humanized anti-PD-L1 antibody Nb-Fc was obtained according to the method in patent ZL201710657665.3.
  • Anti-LAG-3 antibody (ADI-31853) was obtained according to the method in patent application WO2019/129137.
  • mice were divided into 11 groups (6 mice in each group), and each group was subcutaneously injected with the following doses of antibodies:
  • Anti-PD-L1 antibody humanized anti-PD-L1 antibody Nb-Fc), 1.25mg/kg;
  • Anti-PD-L1 antibody humanized anti-PD-L1 antibody Nb-Fc, 2.5mg/kg;
  • Anti-PD-L1 antibody humanized anti-PD-L1 antibody Nb-Fc, 5mg/kg;
  • LAG-3 (ADI-31853), 10mg/kg + anti-PD-L1 antibody (humanized anti-PD-L1 antibody Nb-Fc), 2.5mg/kg;
  • LAG-3 (ADI-31853), 10mg/kg + anti-PD-L1 antibody (humanized anti-PD-L1 antibody Nb-Fc), 5mg/kg;
  • Human IgG was a human IgG preparation obtained from Equitech-Bio.
  • mice in each group were dosed as above with the above antibodies, respectively.
  • Tumor inhibition rate (%) Complete tumor disappearance human IgG 2411 - 0/6 Nb-Fc, 1.25mg/kg 829 68 0/6 Nb-Fc, 2.5mg/kg 1065 58 0/6 Nb-Fc, 5mg/kg 902 65 0/6

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Abstract

提供了一种包含PD-L1/LAG-3双抗、缓冲剂、稳定剂、表面活性剂和螯合剂的药物制剂，及其相应的治疗或预防用途。

Description

PD-L1/LAG-3双特异性抗体制剂及其制备方法和用途
本申请要求申请日为2020/7/23的中国专利申请2020107202480的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及抗体制剂领域。更具体而言，本发明涉及包含同时结合PD-L1和LAG-3的双特异性抗体的药物制剂，尤其是稳定的液体制剂、冻干制剂和重构的稳定液体制剂，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
药品稳定性是保证药品有效性和安全性的重要指标之一。获得良好的制剂处方是保证药品在货架期内保持其有效性和安全性的关键条件。然而，由于抗体本身及其降解途径的复杂性，目前尚不可能对优化抗体稳定性所需的制剂条件作出预测。尤其是考虑到，不同抗体通常具有十分不同的CDR序列、抗体结构，而这些序列和结构的差异会导致不同抗体在溶液中具有不同的稳定性性质。因此，基于对人用抗体在安全性和有效性上的严格要求，有必要针对每个抗体单独进行最佳制剂配方的优化。
LAG-3主要表达在激活的T细胞和NK细胞细胞膜表面，起到抑制免疫细胞杀伤肿瘤细胞活性的作用。PD-1/PD-L1信号通路是已经在临床上被广泛证实的抑制免疫系统清除肿瘤的主要机制之一。而同时靶向LAG-3/MHC II信号通路和PD-1/PD-L1信号通路也在临床应用中被证明在具有一定的协同作用。因此，设计一种可以同时阻断PD-1/PD-L1信号通路和LAG-3/MHC II的双特异抗体具有巨大的临床应用价值。
发明概述
本发明通过提供含有特异结合至PD-L1和LAG-3的双特异性抗体的药物制剂来满足上述需求。本发明的抗体制剂针对多种稳定性影响因素，均表现出了优良的稳定性。
在一个方面，因此，本发明提供了一种液体抗体制剂，其包含(i)PD-L1/LAG-3的双特异性抗体，(ii)缓冲剂，(iii)稳定剂，和(iv)表面活性剂。优选的，该液体抗体制剂进一步包括(v)螯合剂。
在一个实施方案中，本发明PD-L1/LAG-3的双特异性抗体结构如图1所示，其中抗原A为LAG-3，抗原B为PD-L1，其结构包含：
(i)式(I)的多肽链：
VH-CH1-Fc-X-VHH；和
(ii)式(II)的多肽链：
VL-CL；
其中：
VH表示重链可变区；
CH表示重链恒定区；
Fc包含CH2、CH3，以及任选的CH4；
CH1、CH2、CH3和CH4分别表示重链恒定区的结构域1、2、3和4；
X可以不存在，或者在存在时表示接头；
VHH表示单结构域抗原结合位点；
VL表示轻链可变区；
CL表示轻链恒定区；
任选地，CH1和Fc之间存在铰链区。
在一个实施例中，本发明PD-L1/LAG-3双特异性抗体包含下表所示序列：
名称 序列号
VH-CH1-Fc-X-VHH(示例性式(I)的多肽链) SEQ ID NO:1
抗LAG-3抗体VH(示例性式(I)的VH) SEQ ID NO:2
CH1(示例性式(I)的CH1) SEQ ID NO:3
Fc(示例性式(I)的Fc) SEQ ID NO:4
接头(示例性式(I)的X) SEQ ID NO:5
抗PD-L1单结构域抗体(示例性式(I)的VHH) SEQ ID NO:6
VL-CL(示例性式(II)的多肽链) SEQ ID NO:7
抗LAG-3抗体VL(示例性式(II)的VL) SEQ ID NO:8
CL(示例性式(II)的CL) SEQ ID NO:9
抗PD-L1单结构域抗体CDR1(示例性式(I)的VHH CDR1) SEQ ID NO:10
抗PD-L1单结构域抗体CDR2(示例性式(I)的VHH CDR2) SEQ ID NO:11
抗PD-L1单结构域抗体CDR3(示例性式(I)的VHH CDR3) SEQ ID NO:12
抗LAG-3抗体HCDR1(示例性式(I)的VH的HCDR1) SEQ ID NO:13
抗LAG-3抗体HCDR2(示例性式(I)的VH的HCDR2) SEQ ID NO:14
抗LAG-3抗体HCDR3(示例性式(I)的VH的HCDR3) SEQ ID NO:15
抗LAG-3抗体LCDR1(示例性式(II)的VL的LCDR1) SEQ ID NO:16
抗LAG-3抗体LCDR2(示例性式(II)的VL的LCDR2) SEQ ID NO:17
抗LAG-3抗体LCDR3(示例性式(II)的VL的LCDR3) SEQ ID NO:18
抗LAG-3抗体重链(示例性式(I)的VH-CH1-Fc) SEQ ID NO:19
Figure PCTCN2021107878-appb-000001
Figure PCTCN2021107878-appb-000002
所述PD-L1/LAG-3双特异性抗体是PCT申请号PCT/CN2020/073964(国际申请日： 2020年1月23日)中公开的双特异性抗体IGN-LP。
在一个实施方案中，所述PD-L1/LAG-3双特异性抗体是在HEK 293细胞或CHO细胞中重组表达的。
在一个实施方案中，本发明的液体抗体制剂中的PD-L1/LAG-3双抗的浓度为约10-200mg/ml。在另一个实施方案中，本发明的液体抗体制剂中的PD-L1/LAG-3双抗的浓度为约20-100mg/ml，例如为约20、25、30、35、40、45、50、55、60、70、80、90或100mg/ml，优选PD-L1/LAG-3双抗的浓度为约20-60mg/ml，更优选约20-30mg/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5-30mM，例如，约5、10、15、20、25、30mM。在一个实施方案中，所述缓冲剂是组氨酸缓冲剂、枸橼酸缓冲剂、醋酸缓冲剂、磷酸缓冲剂，优选地，所述缓冲剂是组氨酸缓冲剂。在一个实施方案中，所述缓冲剂是组氨酸缓冲剂，优选地，所述缓冲剂由组氨酸和盐酸组氨酸组成。在一个优选的实施方案中，所述缓冲剂是大约5-30mM组氨酸缓冲剂，例如约5-15mM，如大约10mM组氨酸缓冲剂。在另一个实施方案中，用于本发明制剂的组氨酸缓冲剂由例如约0.85mg/ml的组氨酸和约0.97mg/ml的盐酸组氨酸组成。
在一个实施方案中，本发明的液体抗体制剂中的稳定剂选自多元醇(例如，山梨醇、甘露醇、或其组合)、糖类(例如，蔗糖、海藻糖、麦芽糖、或其组合)、氨基酸(例如盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸及组合或其盐)、和它们的任意组合。在一个实施方案中，所述稳定剂包含约10-80mg/ml的山梨醇，例如10、20、25、30、35、40、45、50、55、60、70、80mg/ml山梨醇，优选约15-25mg/ml。又在一个实施方案中，所述稳定剂包含约20-100mg/ml的蔗糖，例如20、30、40、50、60、70、80、90、100mg/ml蔗糖，优选约40-80mg/ml蔗糖。在一个实施方案中，所述稳定剂包含精氨酸，例如约50mM-200mM,例如60-180mM，优选地为约70-170mM。优选地，所述精氨酸为盐酸精氨酸。
在一个实施方案中，所述稳定剂以精氨酸作为单一组分，所述精氨酸约为120-200mM，优选约150-180mM，例如150、155、160、165、170、175、180mM，更优选约160-170mM。优选地，所述精氨酸为盐酸精氨酸。
在一个实施方案中，所述稳定剂包含山梨醇和精氨酸的组合，所述精氨酸约为60-100mM，优选约70-90mM，例如70、75、80、85、90mM；所述山梨醇约为10-30mg/ml，优选约15-25mg/ml，例如15、16、17、18、19、20、21、22、23、24、25mg/ml。优选地，所述精氨酸为盐酸精氨酸。
在一个实施方案中，所述稳定剂以蔗糖作为单一组分，所述蔗糖约为60-100mg/ml，优选约70-90mg/ml，例如70,75,80,85,90mg/ml。
在一个实施方案中，所述稳定剂包含蔗糖和精氨酸的组合，所述精氨酸约为60-100mM，优选约70-90mM，例如70、75、80、85、90mM；所述蔗糖约为20-60mg/ml，优选约35-45mg/ml，例如35、36、37、38、39、40、41、42、43、44、45mg/ml。优选地，所述精氨酸为盐酸精氨酸。
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、泊洛沙姆、聚乙二醇。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，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，本发明的液体制剂中的螯合物是羧酸型螯合剂。在一个实施方案中，所述的螯合剂选自依地酸二钠、氨基三乙酸、二亚乙基三胺五乙酸、柠檬酸、酒石酸、葡萄糖酸、羟乙基乙二胺三乙酸、二羟乙基甘氨酸。在一个实施方案中，所述螯合剂选自依地酸二钠。
在一个实施方案中，本发明的液体抗体制剂中的螯合剂的浓度为约0.005-0.05mg/ml。在一个实施方案中，本发明的液体抗体制剂中的螯合剂的浓度为约0.008-0.018mg/ml，例如约0.008、0.009、0.010、0.012、0.014、0.018mg/ml。
在一个实施方案中，所述液体制剂的pH值为约5.5-6.5。在一些实施方案中，所述液体制剂的pH值为约5.5-6.5中的任意值，例如约5.6、5.8、6.0、6.2、6.4。优选地，所述制剂的pH为5.8-6.4，例如，pH为6.0±0.2或6.2±0.2，优选地pH为6.0。
在一个实施方案中，本发明的液体抗体制剂包含：
(i)约20-100mg/ml PD-L1/LAG-3双抗蛋白，例如20、25、30、35、40、45、50、55、60、70、80、90或100mg/ml，优选20-60mg/ml，更优选20-30mg/ml；
(ii)约5-30mM的组氨酸缓冲剂，优选约5-15mM，更优选约10mM的组氨酸缓冲剂；
(iii)约150-180mM的精氨酸，例如150、155、160、165、170、175、180mM，优选约160-170mM；或山梨醇和精氨酸的组合，所述精氨酸约为60-100mM，例如60、65、70、75、80、85、90、100mM，优选约70-90mM，所述山梨醇约为10-30mg/ml， 优选约15-25mg/ml，例如15、16、17、18、19、20、21、22、23、24、25mg/ml；或约为60-100mg/ml的蔗糖，优选约70-90mg/ml，例如70,75,80,85,90mg/ml；或蔗糖和精氨酸的组合，所述精氨酸约为60-100mM，例如60、65、70、75、80、85、90、100mM，优选约70-90mM，所述蔗糖约为20-60mg/ml，优选约35-45mg/ml，例如35、36、37、38、39、40、41、42、43、44、45mg/ml；和
(iv)约0.2-0.8mg/ml，例如0.2,0.3,0.4,0.5,0.6,0.7,0.8mg/ml，例如0.3-0.6mg/ml聚山梨酯80；
优选地，所述液体抗体制剂进一步包含(v)约0.008-0.018mg/ml，例如约0.008、0.009、0.010、0.012、0.014、0.018mg/ml的依地酸二钠；
其中所述液体制剂的pH为约5.5-6.5，优选pH为约5.8-6.4，例如，pH为6.0±0.2或6.2±0.2，例如约6.0。
在一个优选方案中，所述液体抗体制剂包含：
(i)约20-100mg/ml的PD-L1/LAG-3双抗蛋白；
(ii)约5-30mM的组氨酸缓冲剂；
(iii)约150-180mM的精氨酸；或
山梨醇和精氨酸的组合，所述精氨酸约为60-100mM，所述山梨醇约为10-30mg/ml；或
约60-100mg/ml的蔗糖；或
蔗糖和精氨酸的组合，所述精氨酸约为60-100mM，所述蔗糖约为20-60mg/ml；
(iv)约0.2-0.8mg/ml聚山梨酯80；和
(v)约0.008-0.018mg/ml依地酸二钠，
其中所述液体制剂的pH为约5.8-6.4；
在一个优选方案中，所述液体抗体制剂包含：
(i)约20-60mg/ml的PD-L1/LAG-3双抗蛋白；
(ii)约5-15mM的组氨酸缓冲剂；
(iii)约160-170mM的精氨酸；或
山梨醇和精氨酸的组合，所述精氨酸约为70-90mM，所述山梨醇约为15-25mg/ml；或
约70-90mg/ml的蔗糖；或
蔗糖和精氨酸的组合，所述精氨酸约为70-90mM，所述蔗糖约为35-45mg/ml；
(iv)约0.3-0.6mg/ml聚山梨酯80；和
(v)约0.008-0.018mg/ml依地酸二钠，
其中所述液体制剂的pH为约5.8-6.4；
在一个优选方案中，所述液体抗体制剂包含：
(i)约20mg/ml的PD-L1/LAG-3双抗蛋白，约10mM组氨酸缓冲剂，约165mM盐酸精氨酸，约0.50mg/ml聚山梨酯80，约0.01mg/ml依地酸二钠，pH约6.0；或
(ii)约20mg/ml的PD-L1/LAG-3双抗蛋白，约10mM组氨酸缓冲剂，约80mM盐酸精氨酸，约23.66mg/ml山梨醇，约0.50mg/ml聚山梨酯80，约0.01mg/ml依地酸二钠，pH约6.0；或
(iii)约20mg/ml的PD-L1/LAG-3双抗蛋白，约10mM组氨酸缓冲剂，约80.00mg/ml蔗糖，约0.50mg/ml聚山梨酯80，约0.01mg/ml依地酸二钠，pH约6.0；或
(iv)约20mg/ml的PD-L1/LAG-3双抗蛋白，约10mM组氨酸缓冲剂，约80mM盐酸精氨酸，约42.00mg/ml蔗糖，约0.50mg/ml聚山梨酯80，约0.01mg/ml依地酸二钠，pH约6.0。
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、表面活性剂含量、相对结合活性、和/或电荷变异体的变化，来指示制剂的稳定性。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，或在振荡实验中(例如在室温、避光650r/min条件下振荡5天)，和/或在冻融实验中(例如，在-30℃/室温反复冻融 6次)，检测本发明液体制剂的稳定性。在一个实施方案中，相对于初始值，例如储存第0天的初始值、或振荡或冻融实验前的初始值，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，或在振荡实验后，或在冻融实验后，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，为无色至微黄色液体，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，或在振荡实验后，或在冻融实验后，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％，2％或1％。在一个实施方案中，在储存后，或在振荡实验后，或在冻融实验后，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OD350nm法检测，相对于初始值，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，不超过0.02。在一个实施方案中，在储存后，或在振荡实验后，或在冻融实验后，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，其中通过体积排阻高效液相色谱法(SEC-HPLC)，相对于初始值，单体纯度的变化值(或主峰变化值)不超过10％，例如不超过5％、4％、3％、例如变化值不超过2％，优选不超过1％。在一个实施方案中，在储存后，或在振荡实验后，或在冻融实验后，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，其中通过非还原型十二烷基硫酸钠毛细管电泳(CE-SDS)法，相对于初始值，单体纯度的变化值(或主峰变化值)下降不超过10％，例如不超过5％,4％,3％,2％或1％。在一个实施方案中，在储存后，或在振荡实验后，或在冻融实验后，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％，和/或主成分的变化值不超过20％,15％,10％,8％,5％。在一个实施方案中，在储存后，或在振荡实验后，或在冻融实验后，通过直接ELISA法，检测本发明液体制剂的稳定性，其中相对于初始值，抗体的相对结合活性为70-130％，例如，70,80,90,93,95,98,100,103,105,108,110,115,120,125,130％,优选90-110％。在一个实施方案中，在储存后，或在振荡实验后，或在冻融实验后，通过高效液相色谱-荧光检测法(HPLC-FLD法)检测聚山梨酯80含量，聚山梨酯80含量应为0.2～0.8mg/ml，优选0.3～0.7mg/ml。
在一个实施方案中，本发明液体制剂在储存后，例如在25℃储存至少2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：相对于储存第0天的初始值，
(i)通过SEC-HPLC法测量，主峰变化值小于1％，和/或制剂具有大于96％的纯度，优选大于97％、98％的纯度；
(ii)通过非还原型CE-SDS法测量，主峰变化值小于2％，和/或制剂具有大于96％的纯度，优选大于97％、98％的纯度；
(iii)通过iCIEF法测量，制剂中PD-L1/LAG-3双抗蛋白的各组分(主成分、酸性组分和碱性组分)的变化值总和不超过40％和/或主成分的变化值不超过20％，
例如在40℃±2℃储存1个月后变化值总和不超过约40％(例如，不超过35％，30％，25％,20％,15％,10％)或主成分变化值不超过20％(例如，不超过15％,12％,10％,8％)，或
例如在25℃储存2个月后变化值总和不超过约20％(例如，不超过15％，14％,13％,12％)或主成分变化值不超过约15％(例如，不超过10％,8％,7％,6％,5％)；
(iv)通过ELISA法测量，制剂中PD-L1/LAG-3双抗蛋白的相对结合活性为70％-130％，例如，90,93,95,98,100,103,105,108,110,115,120％；
在一个实施方案中，本发明液体制剂在储存后，例如在5℃±3℃储存至少6个月后，是稳定的，优选地具有如下特征之一或多项：
(i)纯度，通过SEC-HPLC法测量，主峰应≥95.0％；
(ii)纯度，通过非还原型CE-SDS法测量，主峰应≥90.0％；
(iii)电荷变异体，通过iCIEF法测量，主成分应≥58.0％；
(iv)制剂中PD-L1/LAG-3双抗蛋白的生物学活性，通过荧光素酶报告基因细胞测定法测定，生物学活性应为70-130％；
(v)pH值，应为5.8～6.4
在一个方面中，本发明的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。
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、喷雾干燥法或冷冻干燥法实施的。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。
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PD-L1/LAG-3双抗蛋白的方法，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，用于制备同时靶向LAG-3和PD-L1信号通路以治疗或预防肿瘤的递送装置或预填装注射器或药物，所述肿瘤为癌症，包括但不限于胃肠道癌症，例如结肠癌或结直肠癌或直肠癌。
本发明还提供了一种通过向受试者施用本发明的液体抗体制剂或固体抗体制剂或包含该液体抗体制剂或固体抗体制剂的递送装置(如，预填装注射器)或药物，在受试者中阻断LAG-3和/或PD-L1信号通路以降低或消除LAG-3和/或PD-L1的免疫抑制作用的方法。
本发明还提供了一种通过向受试者施用本发明的液体抗体制剂或固体抗体制剂或包含该液体抗体制剂或固体抗体制剂的递送装置(如，预填装注射器)或药物,治疗受试者的疾病，例如上述肿瘤的方法。
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本发明PD-L1/LAG-3的双特异性抗体结构
图2处方筛选实验纯度(SEC-HPLC法)变化趋势图(40℃±2℃)
图3处方筛选实验浊度(OD350nm法)变化趋势图(40℃±2℃)
图4处方筛选实验电荷变异体-酸性组分(iCIEF法)变化趋势图(40℃±2℃)
图5处方筛选实验电荷变异体-主成分(iCIEF法)变化趋势图(40℃±2℃)
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天然存在的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“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“PD-L1/LAG-3双抗制剂”在本文中也简称为“本发明的抗体制剂”，意指包含PD-L1/LAG-3双抗蛋白作为活性成分并包含可药用赋形剂的制备物。将PD-L1/LAG-3双抗蛋白与可药用赋形剂组合后，作为活性成分的PD-L1/LAG-3双抗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，即用预填装式注射器，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，PD-L1/LAG-3双抗蛋白制剂是液体制剂形式。
“稳定的”抗体制剂是指，制剂中的抗体在储存于特定条件下之后、或在振荡后、或在反复冻融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、振荡或反复冻融之后可能不会100％维持其化学结构，但通常如果维持约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PD-L1/LAG-3双抗蛋白制剂在制造、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PD-L1/LAG-3双抗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PD-L1/LAG-3双抗蛋白制剂在储存、振荡和/或反复冻融后，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6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PD-L1/LAG-3双抗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、或经振荡一段时间后或经反复冻融多次后，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-HPLC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OD350nm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或在振荡后或在反复冻融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，振荡后或反复冻融后，在制剂中检测到至少约90％的PD-L1/LAG-3双抗蛋白单体。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90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的PD-L1/LAG-3双抗蛋白单体。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90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的PD-L1/LAG-3双抗蛋白单体，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90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的PD-L1/LAG-3双抗蛋白单体，则药物制剂视为是稳定的。若储存于约5℃6个月之后，检测到至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的PD-L1/LAG-3双抗蛋白单体，则药物制剂视为是稳定的。
经一段储存时间后、或经振荡一段时间后或经反复冻融多次后，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或经振荡后或反复冻融多次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、或经振荡一段时间后或经反复冻融多次后，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40％，例如不超过30％、不超过20％；或电荷变异体(主成分、酸性组分和碱性组分)的百分比变化值总和不超过60％，例如不超过50％、不超过30％；或主成分含量不低于50％，例如不低于60％、不低于70％。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％、或15％；或电荷变异体的百分比变化值总和不超过约60％、50％、或30％。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％、16％、15％、14％、13％、12％、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，例如，高温、振荡、冻融。“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PD-L1/LAG-3双抗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PD-L1/LAG-3双抗蛋白制剂以皮下、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)PD-L1/LAG-3双抗，(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5.5-6.5。优选的，该液体抗体制剂进一步包括(v)螯合剂。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)PD-L1/LAG-3双抗
本发明抗体制剂中的PD-L1/LAG-3双抗是PCT申请号PCT/CN2020/073964(国际申请日：2020年1月23日)中公开的双特异性抗体IGN-LP。在一个实施方案中，该PD-L1/LAG-3双抗是由CHO细胞或HEK293细胞重组表达产生。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。例如，在MC38-huPD-L1 KI荷瘤小鼠模型中，施用本发明的抗体制剂可以产生显著的肿瘤抑制效应。
本发明的抗体制剂中所包含的抗体或其抗原结合片段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10-200mg/ml，优选地约20-100mg/mL，例如约20、25、30、35、40、45、50、55、60、70、80、90或100mg/mlPD-L1/LAG-3双抗，优选PD-L1/LAG-3双抗的浓度为约20-60mg/ml，更优选约20-30mg/ml。
(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5.5-6.5的pH范围。
在一些实施方案中，本发明制剂包含选自以下的缓冲体系：组氨酸-盐酸组氨酸缓冲体系，枸橼酸-枸橼酸钠缓冲体系，醋酸-醋酸钠缓冲体系，磷酸盐缓冲体系，优选组氨酸-盐酸组氨酸缓冲体系。
在一些实施方案中，用于本发明制剂中的缓冲剂为组氨酸缓冲剂，尤其是由组氨酸和盐酸组氨酸组成的缓冲体系。
(iii)稳定剂
用于本发明的合适的稳定剂可以选自糖、多元醇和氨基酸及其组合。对于作为稳定剂的糖包括但不限于蔗糖，海藻糖、麦芽糖及组合。对于作为稳定剂的多元醇包括但不限于山梨醇、甘露醇、或其组合。对于作为稳定剂的氨基酸包括但不限于精氨酸、盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸及组合。
在一个实施方案中，本发明液体制剂包含精氨酸作为稳定剂。
在一个实施方案中，本发明液体制剂包含精氨酸和山梨醇的组合作为稳定剂。
在一个实施方案中，本发明液体制剂包含蔗糖作为稳定剂。
在一个实施方案中，本发明液体制剂包含蔗糖和精氨酸的组合作为稳定剂。
在一个实施方案中，所述精氨酸为盐酸精氨酸。
(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；泊洛沙姆等。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。
在一些实施方案中，可以用于本发明液体制剂中的表面活性剂包括但不限于，聚山梨酯类表面活性剂(例如聚山梨酯80，聚山梨酯20)、泊洛沙姆和聚乙二醇。
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。
(v)其它赋形剂
本发明的抗体液体制剂中任选地包含其它赋形剂。所述其它赋形剂包括，例如，抗微生物剂、抗静电剂、抗氧化剂、螯合剂、明胶等等。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)”。
如本文所使用的，术语“螯合剂”是指能与中心原子形成螯合物，由于螯合物的生成而是配合物的稳定性大大增加。
在一个实施方案中，本发明的液体制剂中的螯合物是羧酸型螯合剂。在一个实施方案中，所述的螯合剂选自依地酸二钠、氨基三乙酸、二亚乙基三胺五乙酸、柠檬酸、酒石酸、葡萄糖酸、羟乙基乙二胺三乙酸、二羟乙基甘氨酸。在一个实施方案中，所述螯合剂选自依地酸二钠。
II.制剂的制备
本发明提供了包含PD-L1/LAG-3双抗蛋白的稳定制剂。在本发明制剂中使用的PD-L1/LAG-3双抗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，可以重组制备抗体。在一个优选的实施方案中，本发明的抗体在293细胞或CHO细胞中重组制备。
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)的单克隆抗体三柱纯化方法。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，重组产生的单克隆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；(5)病毒过滤(使病毒滴度降低例如4log10以上)；(6)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
生物制品稳定性研究一般包括实际贮存条件下的实时稳定性研究(长期稳定性研究)、加速稳定性研究和强制条件试验研究。稳定性研究应根据研究目的和产品自身特性对研究条件进行摸索和优化；针对各种影响因素(如温度、反复冻融、振动等)，制定长期、加速和/或强制条件试验等稳定性研究方案。加速和强制条件试验有利于了解产品在短期偏离保存条件和极端情况下产品的稳定性情况，并为有效期和保存条件的确定提供支持性数据。
在抗体制剂的储存、振荡或反复冻融过程中，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OD350nm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。
IV.制剂的用途
本发明的包含PD-L1/LAG-3双抗蛋白的本发明抗体制剂用于预防或治疗疾病，如自身免疫病、炎性疾病、感染、肿瘤等。例如，所述疾病是肿瘤(例如癌症)或感染。在一些实施方案中，肿瘤是肿瘤免疫逃逸。优选地，肿瘤例如结肠癌或结直肠癌或直肠癌。
本发明也提供了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PD-L1/LAG-3双抗蛋白。本发明还提供了本发明的制剂用于治疗或预防一种或多种上述疾病和病症的方法。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PD-L1/LAG-3双抗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。
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
缩略词 全称
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
FLD 荧光检测器
HPLC 高效液相色谱仪
iCIEF 成像毛细管等电聚焦电泳
SEC-HPLC 体积排阻高效液相色谱
具体实施方式
为了开发出适用于该全人源抗体的长期稳定储存且简单易用的注射剂制剂处方，通过40℃强制和25℃加速稳定性实验，考察了不同制剂处方对于该抗体蛋白质量的影响，最终筛选出有利于其稳定的制剂处方。并在中试规模下通过长期稳定性研究(5℃±3℃)证明处方有效性。整个研究过程中所用的材料和方法如下：
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),USP
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
依地酸二钠 药用级 美国 Avantor 8995-01 EP,BP,JP,USP
蔗糖 药用级 德国 Merck 1.00892.9050 Ch.P(2015年版),USP
盐酸精氨酸 药用级 上海味之素 N/A Ch.P(2015年版),USP
聚山梨酯80 药用级 南京威尔 苏药准字F15423203 Ch.P(2015年版)
囊式滤器H4 N/A 德国 Sartorius 5441307H4-OO N/A
白金硅胶管 N/A 美国 Nalgene 8600-0080 N/A
6R西林瓶 N/A 苏州 Schott 1142196 N/A
20mm胶塞 N/A 新加坡 West 7002-2354 N/A
20mm铝塑盖 N/A 新加坡 West 5420-1035 N/A
注：N/A表示不适用。
1.2.本发明的制剂研究中使用的仪器设备
名称 产地及品牌 型号 编号
电子天平 德国 Sartorius BSA3202S PD-A1-186
电子天平 瑞士 梅特勒 XPE3003S PD-A1-247
恒温恒湿箱 德国 BINDER KBF P 720 PD-A1-070
生化培养箱 上海 精宏 SHP-150 PD-A1-200
医用冷藏箱 青岛 海尔 HYC-360 PD-A1-166
医用冷藏箱 青岛 海尔 HYC-360 PD-A1-165
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
名称 产地及品牌 型号 编号
pH计 瑞士 梅特勒 FE20 PD-A1-161
多通道微量分光光度计 美国 Thermo Nanodrop 8000 PD-A1-052
台式冷冻离心机 美国 Thermo SL16R PD-A1-082
洁净工作台 苏州 Airtech SW-CJ-2FD QC-A1-011
中流量手动蠕动泵 英国 Watson Marlow 520S/R2 PD-A1-235
灌装机 丹麦 Watson Marlow FP50 PD-C14-115
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
整个研究过程中检测项目主要包括：(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OD350nm法检测制剂的浊度；(4)通过体积排阻高效液相色谱法(SEC-HPLC)测定抗体制剂的纯度，表示为主峰的面积占所有峰面积之和的百分数；(5)通过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，表示为主峰的面积占所有峰面积之和的百分数；(6)通过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)通过直接ELISA测定法测定抗体制剂中PD-L1/LAG-3双抗对PD-L1和LAG-3抗原的相对结合活性或荧光素酶报告基因细胞测定法测定抗体制剂中PD-L1/LAG-3双抗对PD-L1和LAG-3抗原的生物学活性；(8)通过HPLC-FLD法检测聚山梨酯80含量。
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)和不溶性微粒检测仪(天津天大天发生产，型号GWJ-8)，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)或多通道微量分光光度计(美国Thermo生产，型号Nanodrop 8000)，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)NaN3，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。取待测样品用样品稀释剂(0.85mg/ml组氨酸、0.97mg/ml盐酸组氨酸、35.11mg/ml盐酸精氨酸、0.01mg/ml依地酸二钠、0.10mg/ml聚山梨酯80)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(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pH 6.5的稀释剂(吸取200μl pH 6.5柠檬酸-磷酸盐缓冲液加入400μl 10％十二烷基硫酸钠，加水定容到1ml)稀释至0.6mg/ml。取稀释后样品95μl，其中还原型样品加5μlβ-巯基乙醇，非还原型样品加5μl 250mmol/L NEM(N-乙基马来酰亚胺)，充分混匀后于70℃条件下加热10分钟，待冷却后转移至样品管。取样电压均为-5kV，取样时间均20秒，分离电压均为-12kV，分析时间均为25分钟。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。样品用3mol/L尿素-0.5％MC(羟甲基纤维素)溶液稀释至1.0mg/ml，3mol/L尿素-0.5％MC溶液70μl，两性电解质(pH 3～10)3μl，pI 5.85、9.46Maker(等电点标记物)各0.3μl，5mol/L NDSB(3-[(2-羟乙基)二甲基胺基]丙烷-1-磺酸盐)5μl，0.5mol/L Taurine(牛磺酸)2μl，样品终浓度0.2mg/ml，聚焦电压3kV，聚焦时间5分钟。使用制剂缓冲液同法操作，制得空白溶液。
相对结合活性(直接ELISA法)
抗LAG-3端结合活性
用PBS(磷酸缓冲盐溶液)稀释SA蛋白至1.0μg/ml，100μl/孔，37℃孵育2小时，将蛋白包被于96孔酶标板上。洗板后加封闭液(2％BSA-PBST，300μl/孔)，37℃封闭2小时。洗板，将Biotin LAG-3用2％BSA-PBST(PBST：磷酸盐缓冲液+吐温20；BSA：牛血清白蛋白)稀释至0.5μg/ml，100μl/孔入到酶标板中，37℃孵育30分钟后洗板。以2％BSA-PBST稀释待测样品至40μg/ml，4倍梯度稀释至第12个浓度。将梯度稀释的供试品以100μl/孔加入到洗过的酶标板中，设置阴性孔只加入稀释液，37℃恒温培养 箱中孵育1小时。洗板后加入2％BSA-PBST稀释HRP(辣根过氧化物酶)标记的二抗(130000倍稀释，100μl/孔)，37℃反应30分钟。洗板后加入100μl TMB显色液，显色15分钟后，每孔加入100μl的1mol/L H2SO4终止反应。以620nm为参比波长，检测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GraphPad Prism软件四参数拟合曲线，计算参照品EC50与样品EC50的比值，反映待测样品与LAG-3的相对结合活性。
抗PD-L1端结合活性
用PBS稀释SA蛋白至1.0μg/ml，100μl/孔，37℃孵育2小时，将蛋白包被于96孔酶标板上。洗板后加封闭液(2％BSA-PBST，300μl/孔)，37℃封闭2小时。洗板，将Biotin PD-L1用2％BSA-PBST稀释至0.5μg/ml，100μl/孔入到酶标板中，37℃孵育30分钟后洗板。以2％BSA-PBST稀释待测样品至60μg/ml，4倍梯度稀释至第12个浓度。将梯度稀释的供试品以100μl/孔加入到洗过的酶标板中，设置阴性孔只加入稀释液，37℃恒温培养箱中孵育1小时。洗板后加入2％BSA-PBST稀释HRP标记的二抗(100000倍稀释，100μl/孔)，37℃反应30分钟。洗板后加入100μl TMB显色液，显色15分钟后，每孔加入100μl的1mol/L H2SO4终止反应。以620nm为参比波长，检测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GraphPad Prism软件四参数拟合曲线，计算参照品EC50与样品EC50的比值，反映待测样品与PD-L1的相对结合活性。
HPLC-FLD法
采用HPLC-FLD法(荧光法)测定待测样品的聚山梨酯80含量。色谱柱为SUPELCO厂家Knitted Reactor coil色谱柱(5m×0.5mm ID)，流动相：0.15mol/L的氯化钠，0.05mol/L的Tris，pH 8.0，5％乙腈，5.0μmol/L NPN(N-苯基-1-萘胺)，15ppm Brij溶液(聚氧乙烯(23)月桂醚溶液)。检测条件：流速1.5ml/min，激发波长350nm，发射波长420nm，柱温30℃，洗脱时间3分钟，进样体积10μl。运行完成后用标准曲线法计算成品中聚山梨酯80的含量。
荧光素酶报告基因细胞测定法
抗LAG-3端生物学活性
用MHCII APC cells Assay Buffer(1％FBS(胎牛血清,货号：10099-141)，99％DMEM-高糖培养基(货号：11995-040))调整MHCII APC细胞至0.4×10 6个/ml，稀释HA-peptide(货号：PP-1901-13)至40μg/ml，二者1:1等体积混匀，按实验布局100μl/孔接种于 96孔细胞培养板中，过夜培养(18～22h)。用LAG-3 Effector cells Assay Buffer(1％FBS(胎牛血清,货号：10099-141)，99％DMEM-高糖培养基(货号：11995-040))调整Jurkat-LAG-3-NFAT-Luc2(货号CS194812)至3.0×10 6个/ml；稀释样品至80.00μg/ml，然后4倍梯度稀释，共10个浓度点(0.3ng/ml～80000ng/ml)。吸去96孔板中的培养基，加入处理好的样品和Jurkat-LAG-3-NFAT-Luc2细胞(分别40μl/孔)，继续培养7h。待细胞培养板平衡至室温后，每孔加入80μl Bio-Glo TM Luciferase Assay System显色液(提前平衡至室温)，反应10min。采用Max i3型酶标仪自带软件，根据既定布局，设置读数程序读取全波长的化学发光值。以样品浓度为横坐标，以Fold值为纵坐标，应用GraphPad Prism软件四参数拟合曲线，计算参照品EC50与样品EC50的比值，反映待测样品抗LAG-3端的生物学活性。
抗PD-L1端结合活性
用Assay Buffer(10％胎牛血清，90％RPMI1640培养基(货号：SH30809.01))调整CHOK1-PDL1(货号CS187108)细胞至0.3×10 6个/ml，按实验布局100μl/孔种于96孔培养板，贴壁培养16～20h。用Assay Buffer调整Jurkat-NFAT-Luc2/PD1(货号CS187102)细胞至0.6×10 6个/ml，稀释样品至12.00μg/ml，2.6倍梯度稀释，共10个浓度点(2.2ng/ml～12000ng/ml)。吸去96孔板中的培养基，加入处理好的样品和Jurkat-NFAT-Luc2/PD1细胞(分别40μl/孔)，继续培养6h。待细胞培养板平衡至室温后，每孔加入80μl Bio-Glo TM Luciferase Assay System显色液(提前平衡至室温)，反应10min。采用Max i3型酶标仪自带软件，根据既定布局，设置读数程序读取全波长的化学发光值。以样品浓度为横坐标，以Fold值为纵坐标，应用GraphPad Prism软件四参数拟合曲线，计算参照品EC50与样品EC50的比值，反映待测样品抗PD-L1端的生物学活性。
实施例1.制备和纯化PD-L1/LAG-3双抗
根据PCT申请号PCT/CN2020/073964所述，获得同时结合PD-L1和LAG-3的双特异性抗体IGN-LP。PCT申请号PCT/CN2020/073964在此整体并入本文作为参考。
简言之，抗体在HEK293细胞中重组表达并通过过滤、层析、病毒灭活、过滤等工艺进行纯化。
实施例2.处方筛选试验
2.1实验步骤
共设计了4个处方，详细处方信息见表1。配制各个处方的缓冲液，将实施例1的 经纯化的本发明双特异性抗体蛋白超滤置换至各自的处方溶液中。置换完成后，将各处方蛋白浓度稀释至约20mg/ml，并加入聚山梨酯80。过滤分装至西林瓶中，加塞、轧盖，进行稳定性考察。检测项目为外观、可见异物、蛋白含量(UV法)、浊度(OD350nm法)、聚山梨酯80含量(HPLC-FLD法)、纯度(SEC-HPLC法和非还原型CE-SDS法)、电荷变异体(iCIEF法)和相对结合活性(直接ELISA法)。
表1.处方信息
Figure PCTCN2021107878-appb-000003
详细实验条件及取样计划见表2.
表2.实验条件及取样表
实验名称 处方 实验方案及取样点
40℃稳定性考察 处方1～处方4 40℃±2℃条件下放置，于0天、1周、2周和1个月取样
25℃稳定性考察 处方1～处方4 25℃±2℃条件下放置，于1个月和2个月取样
2.2判断标准
处方筛选实验采用表3所示标准进行质量判断
表3.质量判断标准
检测项目 质量标准
外观(观察法) 澄明至微乳光，无色至微黄色液体，无异物
可见异物(可见异物检查法) 符合《中华人民共和国药典》(2015年版，四部)0904[1]
蛋白含量(UV法) 变化率≤10％
聚山梨酯80含量(HPLC-FLD法) 应为0.3～0.7mg/ml
浊度(OD 350nm法) 变化值≤0.02
纯度(SEC-HPLC法) 主峰纯度变化值≤1％
纯度(非还原型CE-SDS法) 主峰变化值≤2％
相对结合活性(直接ELISA法) 应为70％～130％
2.3实验结果
处方筛选实验40℃稳定性考察结果详见表4，处方筛选实验纯度(SEC-HPLC法)变化趋势见图2、浊度(OD350nm法)变化趋势见图3、电荷变异体(iCIEF法)变化趋势见图4-5。在40℃±2℃条件下放置1个月，各处方外观、可见异物均合格；蛋白含量(UV法)、聚山梨酯80含量(HPLC-FLD法)、纯度(非还原型CE-SDS法)和相对结合活性(直接ELISA法)均未发生明显变化；处方4纯度(SEC-HPLC法)下降，主要表现为聚合体和片段增多，其余处方纯度(SEC-HPLC法)未发生明显变化；各处方浊度(OD350nm法)均有上升，但处方1上升幅度相对较小；各处方电荷变异体-酸性组分(iCIEF法)上升，主成分下降，碱性组分均未发生明显变化，其中处方1电荷变异体-酸性组分、主成分的变化幅度相对较小。
表4 40℃稳定性考察结果(40℃±2℃)
Figure PCTCN2021107878-appb-000004
Figure PCTCN2021107878-appb-000005
处方筛选实验25℃稳定性考察结果详见表5。在25℃±2℃条件下放置2个月，各处 方外观、可见异物均合格；蛋白含量(UV法)、浊度(OD350nm法)、聚山梨酯80含量(HPLC-FLD法)、纯度和相对结合活性(直接ELISA法)均未发生明显变化；各处方电荷变异体-酸性组分(iCIEF法)上升，主成分下降，碱性组分均未发生明显变化，其中处方4电荷变异体-酸性组分、主成分的变化幅度相对较大。
表5 25℃稳定性考察结果(25℃±2℃)
Figure PCTCN2021107878-appb-000006
Figure PCTCN2021107878-appb-000007
实施例3.长期稳定性考察试验
综合实施例2实验结果，选取处方1进行中试长期稳定性考察。在中试规模下按照处方1(20.0mg/ml重组抗淋巴细胞活化基因-3(LAG-3)和抗程序性死亡配体1(PD-L1)双特异性抗体、0.85mg/ml组氨酸、0.97mg/ml盐酸组氨酸、35.11mg/ml盐酸精氨酸、0.01mg/ml依地酸二钠、0.50mg/ml聚山梨酯80，pH 5.8-6.4)生产3批制剂成品，在5℃±3℃下进行长期稳定性考察，分别在0、3、6月进行取样检测。结果见表6。结果表明，该处方中蛋白稳定性良好，各批次间无显著差异，符合实际生产工艺质量标准。
表6 长期稳定性考察结果(5℃±3℃)
Figure PCTCN2021107878-appb-000008
Figure PCTCN2021107878-appb-000009
Figure PCTCN2021107878-appb-000010
实施例4.本发明抗LAG-3/PD-L1双特异性抗体的抗肿瘤活性
采用接种有MC38-huPD-L1 KI(MJ-1)结肠癌细胞的PD-L1转基因小鼠(MC38-huPD-L1 KI荷瘤小鼠模型，简称MC38/PD-L1模型)测定本发明的抗LAG-3/PD-L1双特异性抗体IGNLP(即如实施例1所述制备的IGN-LP)的抗肿瘤作用。
采用皮下接种的方式建立MC38-huPD-L1 KI荷瘤小鼠模型，成瘤后分组，给予不同抗体的治疗，监测给药期间各组小鼠肿瘤体积和体重变化，给药频率为2次/周，给药2周，共给药5次。监测频率均为2次/周，连续监测4周，给药剂量和方式如下所述。给药结束后计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝100％*(h-IgG对照组肿瘤体积–治疗组肿瘤体积)/(h-IgG对照组肿瘤体积–h-IgG对照组给药前肿瘤体积)，其中h-IgG对照组给药前肿瘤体积平均值为80mm 3。
小鼠：PD-L1转基因小鼠，雌性，7-8周(肿瘤细胞接种时的小鼠周龄)，体重18-20g，购自上海南方模式生物科技股份有限公司。小鼠在到达后驯化7天，随后开始研究。
细胞：小鼠的结肠癌细胞MC38(上海和元生物，HYC0116)购自上海和元生物，并敲入人PD-L1基因(南京银河生物医药有限公司)，获得包含人PD-L1的MC38细胞，用RPMI 1640培养基(Gibco，22400-071)培养，严格按照MC38培养要求进行常规传代培养用于后续体内实验。离心(400g/分，5分钟)收集细胞，在无菌RPMI 1640基础培养基中重悬细胞并调整细胞密度为5×10 6个/ml。PD-L1转基因小鼠右侧背部剃毛，皮下注射MC38-huPD-L1 KI细胞，0.2ml/只。肿瘤细胞接种5天后检测各只小鼠瘤体积，挑选出瘤平均体积在76-80mm 3范围内的小鼠按瘤体积随机分组。按如下给药方式，检测抗LAG-3/PD-L1抗体单独使用的抗肿瘤活性。
抗PD-L1抗体(人源化抗PD-L1抗体Nb-Fc)按照专利ZL201710657665.3中方法获得。
抗LAG-3抗体(ADI-31853)按照专利申请WO2019/129137中方法获得。
给药：将小鼠分为11组(每组6只小鼠)，每组分别皮下注射如下剂量的抗体：
(1)人IgG(equitech-Bio)，15mg/kg；
(2)抗PD-L1抗体(人源化抗PD-L1抗体Nb-Fc),1.25mg/kg；
(3)抗PD-L1抗体(人源化抗PD-L1抗体Nb-Fc),2.5mg/kg；
(4)抗PD-L1抗体(人源化抗PD-L1抗体Nb-Fc),5mg/kg；
(5)LAG-3(ADI-31853),10mg/kg；
(6)LAG-3(ADI-31853),10mg/kg+抗PD-L1抗体(人源化抗PD-L1抗体Nb-Fc),1.25mg/kg；
(7)LAG-3(ADI-31853),10mg/kg+抗PD-L1抗体(人源化抗PD-L1抗体Nb-Fc),2.5mg/kg；
(8)LAG-3(ADI-31853),10mg/kg+抗PD-L1抗体(人源化抗PD-L1抗体Nb-Fc),5mg/kg；
(9)抗LAG-3/PD-L1抗体(IGNLP),3mg/kg；
(10)抗LAG-3/PD-L1抗体(IGNLP),6mg/kg；
(11)抗LAG-3/PD-L1抗体(IGNLP),12mg/kg。
人IgG为获自Equitech-Bio的人IgG制备物。
在肿瘤细胞接种后的第5天、第9天、第12天、第15天和第18天，分别用如上抗体为每组小鼠按如上剂量给药。
分析：在整个研究期间每周测量两次肿瘤和体重，当肿瘤达到端点时(肿瘤体积>3000mm 3)或当小鼠具有>20％体重减轻时，使小鼠安乐死。采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L*W2/2。将来自每组的小鼠的肿瘤尺寸与时间作图。使用方差分析(ANOVA)来确定统计显著性。＜0.05的P值被视为在所有分析中具有统计显著性。
实验结果见下表，可见本申请的抗LAG-3/PD-L1抗体IGNLP单独使用时与IgG对照(equitech-Bio)或抗LAG-3或抗PD-L1抗体单独或组合使用相比，能显著抑制肿瘤的生长。
第29天肿瘤抑制率
组别 肿瘤体积 肿瘤抑制率(％) 肿瘤完全消失数目
人IgG 2411 - 0/6
Nb-Fc,1.25mg/kg 829 68 0/6
Nb-Fc,2.5mg/kg 1065 58 0/6
Nb-Fc,5mg/kg 902 65 0/6
ADI-31853 2942 - 0/6
ADI-31853+Nb-Fc,1.25mg/kg 705 73 0/6
ADI-31853+Nb-Fc,2.5mg/kg 1248 50 0/6
ADI-31853+Nb-Fc,5mg/kg 1198 52 0/6
IGNLP,3mg/kg 151 97 2/6
IGNLP,6mg/kg 367 88 0/6
IGNLP,12mg/kg 500 82 1/6
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (21)

  1. 一种液体抗体制剂，包含
    (i)PD-L1/LAG-3双抗蛋白；
    (ii)缓冲剂，
    (iii)稳定剂，和
    (iv)表面活性剂
    其中所述PD-L1/LAG-3双抗蛋白包含以下部分或由其组成：
    (a)式(I)的多肽链：
    VH-CH1-Fc-X-VHH；和
    (b)式(II)的多肽链：
    VL-CL；
    其中：
    VH表示重链可变区；
    CH表示重链恒定区；
    Fc包含CH2、CH3，以及任选的CH4；
    CH1、CH2、CH3和CH4分别表示重链恒定区的结构域1、2、3和4；
    X可以不存在，或者在存在时表示接头；
    VHH表示单结构域抗原结合位点；
    VL表示轻链可变区；
    CL表示轻链恒定区；
    任选地，CH1和Fc之间存在铰链区；
    其中所述VHH包含SEQ ID NO:6中所含的三个互补决定区域(VHH CDR)；和/或，所述VH包含如SEQ ID NO:2所示的重链可变区VH的3个互补决定区HCDR；和/或，所述VL包含如SEQ ID NO:8所示的重链可变区VL的3个互补决定区LCDR；
    优选地，所述液体抗体制剂还包含(v)螯合剂；
    优选地，所述液体抗体制剂的pH约为5.8-6.4，例如，pH为6.0±0.2或6.2±0.2，优选地pH约为6.0。
  2. 根据权利要求1所述的液体抗体制剂，特征在于所述液体抗体制剂中的PD-L1/LAG-3双抗蛋白的浓度为约10-200mg/ml，优选地为约20-100mg/ml，例如为约20、25、30、35、40、45、50、55、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，
    所述液体抗体制剂包含选自组氨酸-盐酸组氨酸缓冲体系；
    优选地，所述缓冲剂的浓度为约5-50mM，优选地为约5-30mM，例如，约5、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、甘露醇及其组合)、糖类(例如，蔗糖、海藻糖、麦芽糖及其组合)、氨基酸(例如精氨酸、盐酸精氨酸甲硫氨酸甘氨酸脯氨酸及组合)、和它们的任意组合，
    例如，所述稳定剂包含选自以下之一或多项：
    -选自山梨醇、甘露醇、或其组合的多元醇；
    -选自蔗糖、海藻糖、麦芽糖、或其组合的糖类；
    -选自盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸及组合的氨基酸。
  5. 根据权利要求1-4中任何一项所述的液体抗体制剂，特征在于所述稳定剂包含：
    (i)约为120-200mM的精氨酸，优选约150-180mM，例如150、155、160、165、170、175、180mM，或
    (ii)山梨醇和精氨酸的组合，所述精氨酸约为60-100mM，优选约70-90mM，例如70、75、80、85、90mM；所述山梨醇约为10-30mg/ml，优选约15-25mg/ml，例如15、16、17、18、19、20、21、22、23、24、25mg/ml，或
    (iii)约为60-100mg/ml的蔗糖，优选约70-90mg/ml，例如70,75,80,85,90mg/ml，或
    (iv)蔗糖和精氨酸的组合，所述精氨酸约为60-100mM，优选约70-90mM，例如70、75、80、85、90mM；所述蔗糖约为20-60mg/ml，优选约35-45mg/ml，例如35、36、37、38、39、40、41、42、43、44、45mg/ml，
    优选地，所述精氨酸为盐酸精氨酸。
  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、泊洛沙姆、聚乙二醇或其组合，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1-7中任何一项所述的液体抗体制剂，特征在于所述螯合剂是羧酸型螯合剂，优选为依地酸二钠。
  9. 根据权利要求1-8中任何一项所述的液体抗体制剂，特征在于所述螯合剂浓度为约0.008-0.018mg/ml，例如约0.008、0.009、0.010、0.012、0.014、0.018mg/ml。
  10. 根据权利要求1-9中任何一项所述的液体抗体制剂，特征在于所述VHH包含互补决定区域(CDR)VHH CDR1、VHH CDR2和VHH CDR3；其中所述VHH CDR1包含SEQ ID NO:10的氨基酸序列，或由所述氨基酸序列组成；所述VHH CDR2包含SEQ ID NO:11的氨基酸序列，或由所述氨基酸序列组成；所述VHH CDR3包含SEQ ID NO:12的氨基酸序列或由所述氨基酸序列组成；
    和/或，所述VH包含互补决定区域(CDR)HCDR1、HCDR2和HCDR3，其中HCDR1包含SEQ ID NO:13的氨基酸序列，或由所述氨基酸序列组成；HCDR2包含SEQ ID NO:14的氨基酸序列，或由所述氨基酸序列组成；HCDR3包含SEQ ID NO:15的氨基酸序列，或由所述氨基酸序列组成；
    和/或，所述VL包含互补决定区域(CDR)LCDR1、LCDR2和LCDR3，其中LCDR1包含SEQ ID NO:16的氨基酸序列，或由所述氨基酸序列组成；LCDR2包含SEQ ID NO:17的氨基酸序列，或由所述氨基酸序列组成；LCDR3包含SEQ ID NO:18的氨基酸序列，或由所述氨基酸序列组成。
  11. 根据权利要求1-10中任何一项所述的液体抗体制剂，特征在于所述VHH包含如SEQ ID NO:6所示的序列或由其组成；和/或，所述VH包含如SEQ ID NO:2所示的氨基酸序列或由其组成；和/或，所述VL包含如SEQ ID NO:8所示的氨基酸序列或由其组成。
  12. 根据权利要求1-11中任何一项所述的液体抗体制剂，特征在于所述式(I)中的VH-CH1-Fc包含SEQ ID NO:19的氨基酸序列或由其组成；和/或，所述的式(II)中的VL-CL包含SEQ ID NO:7的氨基酸序列或由其组成；
    较佳地：
    所述的式(I)的多肽链包含SEQ ID NO:1所示的序列或由其组成；和/或，所述的式(II)的多肽链包含SEQ ID NO:7所示的序列或由其组成。
  13. 根据权利要求1-12中任何一项所述的液体抗体制剂，特征在于所述PD-L1/LAG-3双抗在HEK293细胞或CHO细胞中重组表达。
  14. 根据权利要求1-13中任何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。
  15. 根据权利要求1-14中任一项所述的液体抗体制剂，其包含：
    (i)约20-100mg/ml的PD-L1/LAG-3双抗蛋白；
    (ii)约5-30mM的组氨酸缓冲剂；
    (iii)约150-180mM的精氨酸；或
    山梨醇和精氨酸的组合，所述精氨酸约为60-100mM，所述山梨醇约为10-30mg/ml；或
    约60-100mg/ml的蔗糖；或
    蔗糖和精氨酸的组合，所述精氨酸约为60-100mM，所述蔗糖约为20-60mg/ml；
    (iv)约0.2-0.8mg/ml聚山梨酯80；和
    (v)约0.008-0.018mg/ml依地酸二钠，
    其中所述液体制剂的pH为约5.8-6.4；
    或者，所述液体抗体制剂包含
    (i)约20-60mg/ml的PD-L1/LAG-3双抗蛋白；
    (ii)约5-15mM的组氨酸缓冲剂；
    (iii)约160-170mM的精氨酸；或
    山梨醇和精氨酸的组合，所述精氨酸约为70-90mM，所述山梨醇约为15-25mg/ml；或
    约70-90mg/ml的蔗糖；或
    蔗糖和精氨酸的组合，所述精氨酸约为70-90mM，所述蔗糖约为35-45mg/ml；
    (iv)约0.3-0.6mg/ml聚山梨酯80；和
    (v)约0.008-0.018mg/ml依地酸二钠，
    其中所述液体制剂的pH为约5.8-6.4；
    或者，所述液体抗体制剂包含
    (i)约20mg/ml的PD-L1/LAG-3双抗蛋白，约10mM组氨酸缓冲剂，约165mM盐酸精氨酸，约0.50mg/ml聚山梨酯80，约0.01mg/ml依地酸二钠，pH约6.0；或
    (ii)约20mg/ml的PD-L1/LAG-3双抗蛋白，约10mM组氨酸缓冲剂，约80mM盐酸精氨酸，约23.66mg/ml山梨醇，约0.50mg/ml聚山梨酯80，约0.01mg/ml依地酸二钠，pH约6.0；或
    (iii)约20mg/ml的PD-L1/LAG-3双抗蛋白，约10mM组氨酸缓冲剂，约80.00mg/ml蔗糖，约0.50mg/ml聚山梨酯80，约0.01mg/ml依地酸二钠，pH约6.0；或
    (iv)约20mg/ml的PD-L1/LAG-3双抗蛋白，约10mM组氨酸缓冲剂，约80mM盐酸精氨酸，约42.00mg/ml蔗糖，约0.50mg/ml聚山梨酯80，约0.01mg/ml依地酸二钠，pH约6.0。
  16. 根据权利要求1-15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25℃储存至少2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，主峰变化值小于1％，和/或制剂具有大于96％的纯度，优选大于97％、98％的纯度；
    (ii)通过非还原型CE-SDS法测量，主峰变化值小于2％，和/或制剂具有大于95％的纯度，优选大于96％、97％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PD-L1/LAG-3双抗蛋白的各组分(主成分、酸性组分和碱性组分)的变化值总和不超过40％和/或主成分的变化值不超过20％，例如在40℃±2℃储存1个月后变化值总和不超过约40％(例如不超过30％)或主成分变化值不超过约20％(例如不超过15％)，或在25℃储存2个月后变化值总和不超过约20％(例如约15％)或主成分变化值不超过约15％(例如不超过约10％)；
    (iv)通过ELISA法测量，相对于储存第0天的初始值，制剂中PD-L1/LAG-3双抗蛋白的相对结合活性为70％-130％，例如，90％-110％；
    或者，在5℃±3℃储存至少6个月后，是稳定的，优选地具有如下特征之一或多项：
    (i)纯度，通过SEC-HPLC法测量，主峰应≥95.0％；
    (ii)纯度，通过非还原型CE-SDS法测量，主峰应≥90.0％；
    (iii)电荷变异体，通过iCIEF法测量，主成分应≥58.0％；
    (iv)制剂中PD-L1/LAG-3双抗蛋白的相对结合活性，通过荧光素酶报告基因细胞测定法测定，抗体结合活性应为70-130％；
    (v)pH值，应为5.8～6.4。
  17. 一种固体抗体制剂，其通过固化权利要求1-16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。
  18. 递送装置，其包含权利要求1-16中任何一项的液体抗体制剂或权利要求17的固体抗体制剂。
  19. 预填装注射器，其包含权利要求1-16中任何一项的液体抗体制剂或权利要求17的固体抗体制剂，用于静脉内注射或者肌内注射。
  20. 根据权利要求1-16中任何一项的液体抗体制剂或权利要求17的固体抗体制剂的用途，用于制备在受试者中阻断LAG-3和/或PD-L1通路以降低或消除免疫抑制作用的递送装置或预填装注射器或药物。
  21. 根据权利要求1-16中任何一项的液体抗体制剂或权利要求17的固体抗体制剂 的用途，用于制备在受试者中治疗或预防肿瘤的递送装置或预填装注射器或药物，例如，所述肿瘤是胃肠道癌症。
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