WO2020259593A1 - 包含抗lag-3抗体的制剂、其制备方法及其用途 - Google Patents

包含抗lag-3抗体的制剂、其制备方法及其用途 Download PDF

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WO2020259593A1
WO2020259593A1 PCT/CN2020/098140 CN2020098140W WO2020259593A1 WO 2020259593 A1 WO2020259593 A1 WO 2020259593A1 CN 2020098140 W CN2020098140 W CN 2020098140W WO 2020259593 A1 WO2020259593 A1 WO 2020259593A1
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antibody
lag
liquid
preparation
antibody preparation
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PCT/CN2020/098140
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English (en)
French (fr)
Inventor
姚天怡
马一冬
汪音爵
周凯松
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信达生物制药(苏州)有限公司
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Priority to EP20833655.2A priority Critical patent/EP3991747A4/en
Priority to JP2021576991A priority patent/JP2022539088A/ja
Priority to CA3144116A priority patent/CA3144116A1/en
Priority to KR1020227002182A priority patent/KR20220036371A/ko
Priority to CN202080046468.8A priority patent/CN114007648B/zh
Priority to US17/621,867 priority patent/US20220251188A1/en
Priority to AU2020304108A priority patent/AU2020304108A1/en
Priority to BR112021026409A priority patent/BR112021026409A2/pt
Publication of WO2020259593A1 publication Critical patent/WO2020259593A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • 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
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to the field of antibody preparations. More specifically, the present invention relates to a pharmaceutical preparation comprising an antibody that specifically binds to LAG-3 (hereinafter also referred to as "anti-LAG-3 antibody”), a method for preparing the pharmaceutical preparation, and the pharmaceutical preparation Therapeutic and/or preventive use of
  • Lymphocyte activation gene 3 (LAG-3), also known as CD223, is a type I transmembrane protein encoded by the LAG-3 gene in the human body.
  • LAG-3 is a CD4-like protein, expressed on the surface of T cells (especially activated T cells), natural killer cells, B cells and plasmacytoid dendritic cells. LAG-3 has been shown to be an inhibitory receptor.
  • TIL tumor infiltrating lymphocytes
  • An anti-LAG-3 antibody that specifically binds to LAG-3 as a LAG-3 blocker is described in, for example, Chinese Patent Application No. 201811561512.X.
  • anti-LAG-3 antibody preparations that can be used to treat, prevent or delay various cancers, immune-related diseases, and T cell dysfunction diseases.
  • the antibody preparation In addition to preparing the antibody in a manner suitable for administration to a subject, the antibody preparation also needs to be formulated in a manner that maintains its stability during storage and subsequent use. For example, if the antibody is not properly formulated in the liquid, the antibody in the liquid solution tends to decompose, aggregate, or undergo undesirable chemical modification.
  • the stability of the antibody in the antibody preparation depends on the buffer, stabilizer and surfactant used in the preparation.
  • anti-LAG-3 antibodies Although some anti-LAG-3 antibodies are known, there is still a need in the art for novel pharmaceutical formulations containing anti-LAG-3 antibodies that are sufficiently stable and suitable for administration to a subject. Therefore, suitable anti-LAG-3 antibody preparations are needed to treat or prevent diseases.
  • the present invention meets the above-mentioned needs by providing pharmaceutical preparations containing antibodies that specifically bind to LAG-3.
  • the present invention provides a liquid antibody preparation comprising (i) an anti-LAG-3 antibody; (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant.
  • the anti-LAG-3 antibody may be any antibody that binds to LAG-3 molecules (such as human LAG-3 molecules) and blocks LAG-3 signal transduction, such as polyclonal antibodies, monoclonal antibodies, or a combination of both.
  • the anti-LAG-3 antibody is a monoclonal antibody.
  • the anti-LAG-3 antibody is a recombinant fully human anti-lymphocyte activation gene 3 (LAG-3) monoclonal antibody disclosed in Chinese application CN201811561512.X (filed on December 19, 2018) .
  • LAG-3 fully human anti-lymphocyte activation gene 3
  • the anti-LAG-3 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 7 or a sequence having at least 90% identity therewith, and
  • the light chain variable region includes the sequence of SEQ ID NO: 8 or a sequence with at least 90% identity:
  • the anti-LAG-3 antibody comprises:
  • the anti-LAG-3 antibody is an IgG4 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the sequence of SEQ ID NO: 9 or a sequence having at least 90% identity therewith, and wherein The light chain includes the sequence of SEQ ID NO: 10 or a sequence that is at least 90% identical to it:
  • the anti-LAG-3 antibody is the anti-LAG-3 monoclonal antibody ADI-31853 disclosed in Chinese application CN201811561512.X (filed on December 19, 2018), which is composed of SEQ ID NO: 9 The chain sequence and the light chain sequence of SEQ ID NO: 10.
  • the anti-LAG-3 antibody is an anti-LAG-3 antibody recombinantly expressed in 293 cells or CHO cells.
  • the concentration of the anti-LAG-3 antibody in the liquid antibody preparation of the present invention is about 1-100 mg/ml. In another embodiment, the concentration of the anti-LAG-3 antibody in the liquid antibody preparation of the present invention is about 5-50 mg/ml. In other embodiments, the concentration of the anti-LAG-3 antibody in the liquid antibody preparation of the present invention is about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg/ml.
  • the concentration of the buffer in the liquid antibody formulation of the present invention is about 1-10 mg/ml. In one embodiment, the concentration of the buffer in the liquid antibody formulation of the present invention is about 4-8 mg/ml, for example, about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 mg/ml.
  • the buffer is selected from citrate, citrate solvate or a combination thereof, more preferably citrate, citrate hydrate, for example, sodium citrate, citric acid dihydrate sodium.
  • the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 10-150 mg/ml. In one embodiment, the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 15-100 mg/ml, such as about 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/ml.
  • the stabilizer is selected from sorbitol, sucrose, trehalose, arginine, arginine hydrochloride or any combination thereof, more preferably sucrose, arginine and/or arginine hydrochloride.
  • the arginine and/or arginine hydrochloride is in an amount of about 10-40 mg/ml, preferably about 15-25 mg/ml (for example, about 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25 mg/ml).
  • the sucrose is present in an amount of about 20-100 mg/ml, preferably about 40-90 mg/ml (e.g., an amount of about 40, 50, 60, 70, 80, 90 mg/ml).
  • the concentration of the surfactant in the liquid antibody preparation of the present invention is about 0.1-1 mg/ml. In one embodiment, the concentration of the surfactant in the liquid antibody preparation of the present invention is about 0.2-0.8 mg/ml, for example about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml.
  • the surfactant is a nonionic surfactant. In one embodiment, the surfactant is selected from polysorbate surfactants. In a specific embodiment, the surfactant in the liquid antibody formulation of the present invention is polysorbate-80.
  • the liquid formulation may or may not include edetate (eg, disodium edetate) and/or methionine.
  • edetate eg, disodium edetate
  • methionine e.g, methionine
  • the pH of the liquid formulation is about 5.5-7.5. In some embodiments, the pH of the liquid formulation is any value from about 5.5 to 7.5, such as about 5.5, 6.0, 6.5, 7.0, 7.5.
  • the liquid preparation is a pharmaceutical preparation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection. In one embodiment, the liquid formulation is an intravenous infusion.
  • liquid antibody formulation of the invention comprises
  • the liquid formulation does not contain edetate (for example, disodium edetate) and methionine;
  • the pH of the liquid preparation is about 5.5-7.5, preferably about 6.0-7.0, more preferably 6.0 ⁇ 0.3, such as 6.0, and preferably, anhydrous citric acid is used to adjust to the pH value.
  • liquid antibody preparation of the invention comprises
  • the liquid formulation does not contain edetate (for example, disodium edetate) and methionine;
  • the pH of the liquid preparation is about 5.5-7.5, preferably about 6.0-7.0, more preferably 6.0 ⁇ 0.3, such as 6.0, and preferably, anhydrous citric acid is used to adjust to the pH value.
  • liquid antibody preparation of the invention comprises
  • the pH of the liquid preparation is about 5.5-7.5, preferably about 6.0-7.0, more preferably 6.0 ⁇ 0.3, such as 6.0, and preferably, anhydrous citric acid is used to adjust to the pH value.
  • liquid antibody preparation of the invention comprises
  • the pH of the liquid preparation is about 5.5-7.5, preferably about 6.0-7.0, more preferably 6.0 ⁇ 0.3, such as 6.0, and preferably, anhydrous citric acid is used to adjust to the pH value.
  • liquid antibody preparation of the invention comprises
  • the pH of the liquid preparation is about 5.5-7.5, preferably about 6.0-7.0, more preferably 6.0 ⁇ 0.3, such as 6.0, and preferably, anhydrous citric acid is used to adjust to the pH value.
  • liquid antibody preparation of the invention comprises
  • the pH of the liquid preparation is about 5.5-7.5, preferably about 6.0-7.0, more preferably 6.0 ⁇ 0.3, such as 6.0, and preferably, anhydrous citric acid is used to adjust to the pH value.
  • liquid antibody preparation of the invention comprises
  • the pH of the liquid preparation is about 5.5-7.5, preferably about 6.0-7.0, more preferably 6.0 ⁇ 0.3, such as 6.0, and preferably, anhydrous citric acid is used to adjust to the pH value.
  • the present invention provides a solid antibody preparation, which is obtained by subjecting the liquid antibody preparation of the present invention to curing treatment.
  • the curing treatment is performed by, for example, a crystallization method, a spray drying method, or a freeze drying method.
  • the solid antibody preparation is, for example, in the form of a lyophilized powder injection.
  • the solid antibody preparation can be reconstituted in a suitable solvent before use to form the reconstituted preparation of the present invention.
  • the reconstituted preparation is also a liquid antibody preparation of the present invention.
  • the appropriate solvent is selected from water for injection, organic solvent for injection, including but not limited to oil for injection, ethanol, propylene glycol, etc., or a combination thereof.
  • the liquid preparation of the present invention can be stored stably for a long time, for example, at least 24 months or more.
  • the liquid formulation of the present invention can be heated at about -80°C to about 45°C, for example -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about Store at 35°C, about 38°C, about 40°C, about 42°C or about 45°C for at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months , At least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months , At least 36 months, or longer, is stable.
  • the liquid formulation of the present invention can be stored stably for at least 24 months. In yet another embodiment, the liquid formulation of the invention is stable at at least 40°C. In yet another embodiment, the liquid formulation of the present invention is stable at about 2°C-8°C for at least 12 months, preferably at least 24 months. In one embodiment, the liquid formulation of the present invention is stable at room temperature or, for example, about 25°C for at least 3 months, preferably at least 6 months. In yet another embodiment, the liquid formulation of the invention remains stable at about 40°C for at least 1 month. In still another embodiment, the liquid formulation of the present invention can be stable at a temperature of about 5°C to 40°C, such as at a temperature of 25°C, for at least 1 day, such as 3 days or 5 days, under shaking.
  • the stability of the preparation after storage can be indicated by detecting changes in the appearance, visible foreign matter, protein content, purity, and/or charge variants of the preparation. In one embodiment, it can be in a high temperature stress test, for example after storage at 40°C ⁇ 2°C for at least 1 week, 2 weeks or preferably 1 month, or at 25°C ⁇ 2°C for at least 1 month or 2 Months later, the stability of the liquid preparation of the present invention was tested. In one embodiment, the oscillation stability of the liquid formulation of the present invention is tested by an oscillation test.
  • the stability of the liquid formulation of the present invention is visually inspected, wherein the liquid formulation of the present invention remains clear to slightly opalescent in appearance, a colorless to pale yellow liquid, and no foreign matter. In one embodiment, by visual inspection under a clarity detector, no visible foreign matter exists in the preparation. In one embodiment, after storage, the stability of the liquid formulation of the present invention is checked by measuring the change in protein content, for example, by ultraviolet spectrophotometry (UV) method, relative to the initial value on day 0 of storage, the rate of change in protein content is Not more than 20%, preferably not more than 10%, for example 7-8%, preferably not more than 5%.
  • UV ultraviolet spectrophotometry
  • the stability of the liquid preparation of the present invention is checked by measuring the change in turbidity of the liquid preparation of the present invention, for example, by the OD 350mm method, the change value is relative to the initial value on the 0th day of storage It does not exceed 0.04, more preferably does not exceed 0.03, and more preferably does not exceed 0.02.
  • the stability of the liquid preparation of the present invention is checked by measuring the change in purity of the liquid preparation of the present invention, wherein size exclusion high performance liquid chromatography (SEC-HPLC) is used to check the stability of the liquid preparation of the present invention.
  • SEC-HPLC size exclusion high performance liquid chromatography
  • the change value of monomer purity does not exceed 10%, for example, does not exceed 5%, 4%, 3%, for example 1-2%, preferably does not exceed 1%.
  • the stability of the liquid preparation of the invention is checked by measuring the purity change of the liquid preparation of the invention, wherein the stability of the liquid preparation of the invention is checked by non-reduced and/or reduced sodium lauryl sulfate capillary electrophoresis (CE- In the SDS) method, the change value of the monomer purity is reduced by no more than 10%, for example, no more than 5%, 4%, 3%, preferably no more than 2%, 1%, 0.5% or 0.1%.
  • CE- In the SDS non-reduced and/or reduced sodium lauryl sulfate capillary electrophoresis
  • the stability of the liquid preparation of the present invention is tested by imaging capillary isoelectric focusing electrophoresis (iCIEF), wherein relative to the initial value on day 0 of storage, the charge variants (principal components, The change value of acidic component or alkaline component) does not exceed 2%.
  • iCIEF capillary isoelectric focusing electrophoresis
  • the formulation is stable after storage, such as after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 3 months, or after storage at 40°C ⁇ 2°C for 1 month ,
  • the formulation is stable after storage, such as after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 3 months, or after storage at 40°C ⁇ 2°C for 1 month ,
  • the preparation anti-LAG-3 antibody has a purity greater than 90%, preferably greater than 95%, 96%, 97%, 98%, 99% purity;
  • the preparation has a purity greater than 90%, preferably greater than 95%, 96%, 97%, 98%, 99% purity;
  • the relative binding activity of the anti-LAG-3 antibody in the preparation is 70% to 130%, for example, 70%, 80%, 90%, 100% , 110%, 120%, 130%.
  • the present invention provides a delivery device comprising the liquid antibody preparation or solid antibody preparation of the present invention.
  • the delivery device of the present invention is provided in the form of a pre-filled syringe containing the liquid antibody preparation or solid antibody preparation of the present invention, for example for intravenous, subcutaneous, intradermal or intramuscular injection, intravenous infusion .
  • the present invention provides a method for delivering an anti-LAG-3 antibody to a subject, such as a mammal, comprising the step of administering the liquid antibody preparation or solid antibody preparation of the present invention to the subject, and the delivery is, for example, Implemented by using a delivery device with a pre-filled syringe.
  • the present invention provides the use of the liquid antibody preparation or solid antibody preparation of the present invention for preparing the treatment, prevention or delay of LAG-3 expressing cancer (especially metastatic cancer), immune-related Delivery devices (eg, pre-filled syringes) or drugs for diseases and T cell dysfunction diseases, especially for the treatment, prevention or delay of LAG-3 expressing cancers (especially metastatic cancers), such as various hematological tumors , Solid tumors, such as colon cancer.
  • LAG-3 expressing cancer especially metastatic cancer
  • immune-related Delivery devices eg, pre-filled syringes
  • drugs for diseases and T cell dysfunction diseases especially for the treatment, prevention or delay of LAG-3 expressing cancers (especially metastatic cancers), such as various hematological tumors , Solid tumors, such as colon cancer.
  • Figure 1 Shows the effect of pH on the purity of anti-LAG-3 antibody protein detected by SEC-HPLC.
  • Figure 2 shows the effect of pH detected by the iCIEF method on each charge variant of the anti-LAG-3 antibody protein.
  • Figure 3 A graph showing the change trend of the charge variant-acid component (iCIEF method) of anti-LAG-3 antibody formulations 1-7 in the high temperature stress test for 2 weeks.
  • Figure 4 A graph showing the change trend of the charge variant-principal component (iCIEF method) of anti-LAG-3 antibody prescriptions 1-7 in the high temperature stress test for 2 weeks.
  • Figure 5 A graph showing the change trend of the charge variant-alkaline component (iCIEF method) of anti-LAG-3 antibody formulations 1-7 in the high temperature stress test for 2 weeks.
  • Figure 6 Shows the turbidity (OD 350nm method) trend graph of anti-LAG-3 antibody formulations 8, 9, and 10 in the high temperature stress test.
  • Figure 7 shows the trend graph of the purity (SEC-HPLC method) of anti-LAG-3 antibody formulations 8, 9, and 10 in the high temperature stress test.
  • Figure 8 Shows the trend graph of the purity of anti-LAG-3 antibody formulations 8, 9, and 10 in the high temperature stress test (reduced CE-SDS method).
  • Figure 9 shows the change trend graph of the charge variant-acidic component (iCIEF method) of anti-LAG-3 antibody prescriptions 8, 9, and 10 in the high temperature stress test for 1 month.
  • Figure 10 A graph showing the change trend of the charge variant-principal component (iCIEF method) of anti-LAG-3 antibody formulations 8, 9, and 10 in the high temperature stress test for 1 month.
  • Figure 11 A graph showing the change trend of the charge variant-alkaline component (iCIEF method) of anti-LAG-3 antibody formulations 8, 9, and 10 in the high temperature stress test for 1 month.
  • Figure 12 shows the purity (reduced CE-SDS method) profile of anti-LAG-3 antibody formulation 9 at the beginning of the high temperature stress test and after 1 month of storage.
  • Figure 13 shows the mass spectrum (LC-MS method) of prescription 9 of anti-LAG-3 antibody placed in the high temperature stress test for 1 month.
  • Figure 14 shows the purity (SEC-HPLC method) profile of anti-LAG-3 antibody formulation 10 at the beginning of the high temperature stress test and after 1 month of storage.
  • the term “comprising” or “including” means to include the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprises” or “includes” when used, unless otherwise specified, it also covers the situation consisting of the stated elements, integers or steps.
  • an antibody variable region that "comprises” a specific sequence when referring to an antibody variable region that "comprises” a specific sequence, it is also intended to encompass the antibody variable region composed of the specific sequence.
  • the term "antibody” refers to a polypeptide comprising light chain and heavy chain immunoglobulin variable regions, which specifically recognize and bind to an antigen.
  • the antibody of the present invention is a full-length antibody, consisting of two heavy chains and two light chains, wherein each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region, each Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody of the present invention can also refer to an antigen-binding fragment of an antibody.
  • antibody preparation refers to a preparation in a form that allows the biological activity of the antibody as an active ingredient to be effectively exerted, and does not contain unacceptable toxicity to the subject to which the preparation is to be administered. Other components. Such antibody preparations are usually sterile. Generally, pharmaceutically acceptable excipients are included in antibody preparations.
  • a "pharmaceutically acceptable" excipient is an agent that can be reasonably administered to the mammal under test so that an effective dose of the active ingredient used in the formulation can be delivered to the subject. The concentration of the excipient is adapted to the mode of administration, for example, it may be acceptable for injection.
  • anti-LAG-3 antibody preparation is also abbreviated as "the antibody preparation of the present invention” herein, and means a preparation containing an anti-LAG-3 antibody as an active ingredient and a pharmaceutically acceptable excipient. After the anti-LAG-3 antibody is combined with a pharmaceutically acceptable excipient, the anti-LAG-3 antibody as an active ingredient is suitable for therapeutic or preventive administration to humans or non-human animals.
  • the antibody preparation of the present invention can be prepared, for example, as an aqueous liquid preparation, for example, a ready-to-use pre-filled syringe, or prepared as a lyophilized preparation, which is carried out by dissolving and/or suspending in a physiologically acceptable solution immediately before use Reconstitution (ie, reconstitution).
  • the anti-LAG-3 antibody formulation is in the form of a liquid formulation.
  • a “stable” antibody preparation is when the antibody in the preparation retains an acceptable degree of physical and/or chemical stability after storage under specific conditions. Although the antibody contained in the antibody preparation may not maintain 100% of its chemical structure after storage for a specific time, it usually maintains about 90%, about 95%, about 96%, about 97%, about 98% after storage for a specific time. Or about 99% of the structure or function of the antibody, the antibody preparation is considered “stable.”
  • the anti-LAG-3 antibody formulation of the present invention exhibits low to undetectable antibody aggregation or degradation or chemical modification during the manufacturing, preparation, transportation and long-term storage, so that there is little or even There is no loss of biological activity of anti-LAG-3 antibodies, showing high stability.
  • the anti-LAG-3 antibody preparation of the present invention substantially retains its physical and chemical stability after storage.
  • the liquid formulation of the present invention can be stable at room temperature or at 40°C for at least 1 month, and/or at 2-8°C for at least 24 months.
  • the stability can be measured at a selected temperature and selected storage time. For example, the storage time can be selected based on the expected shelf life of the formulation. Or an accelerated stability test can be used. In some embodiments, stability testing is performed by performing various stress tests on antibody preparations.
  • the formulated anti-LAG-3 antibody preparation can be filled into a glass bottle of suitable capacity for shaking stress to detect the shaking/shear stability of the antibody; or the formulated anti-LAG-3 antibody preparation can be filled into In a glass vial to test the antibody stability under high temperature stress.
  • the preparation After a period of storage, the preparation does not show aggregation, precipitation, turbidity and/or denaturation; or shows very little aggregation, precipitation, turbidity and/or denaturation, it can be considered that the antibody "maintains its physical stability" in the preparation.
  • the accumulation of antibodies in the preparation can potentially lead to an increased immune response in the patient, leading to safety issues. Therefore, there is a need to minimize or prevent aggregation of antibodies in the formulation.
  • Light scattering methods can be used to determine visible aggregates in the formulation.
  • SEC can be used to determine soluble aggregates in the formulation.
  • the stability of the preparation can be indicated by visually inspecting the appearance, color, and/or clarity of the preparation, or detecting the turbidity of the preparation by the OD 350nm method, or measuring the purity of the preparation by the non-reducing CE-SDS method.
  • the stability of the formulation is measured by determining the percentage of antibody monomers in the formulation after storage at a specific temperature for a specific time, wherein the higher the percentage of antibody monomers in the formulation, the higher the stability of the formulation .
  • an "acceptable degree" of physical stability can mean that at least about 92% of the anti-LAG-3 antibody monomer is detected in the formulation after storage at a specific temperature for a specific time.
  • storage at a specific temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months 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 an acceptable degree of physical stability indicates At least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-LAG-3 antibody monomer.
  • the specific temperature at which the pharmaceutical preparation is stored can be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C, about -20°C, about 0°C, About 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C.
  • the pharmaceutical preparation is considered stable.
  • the pharmaceutical preparation Considered to be stable. If after 9 months of storage at about 5°C, at least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-LAG-3 antibody monomer is detected, the pharmaceutical preparation Considered to be stable.
  • the antibody in the preparation After a period of storage, if the antibody in the preparation does not show significant chemical changes, it can be considered that the antibody "maintains its chemical stability" in the preparation.
  • Most chemical instabilities result from the formation of covalently modified forms of antibodies (for example, charge variants of antibodies).
  • charge variants of antibodies for example, by aspartic acid isomerization, N and C terminal modification, basic variants can be formed; by deamidation, sialylation and saccharification, acidic variants can be generated.
  • Chemical stability can be assessed by detecting and/or quantifying the chemically modified form of the antibody.
  • the charge variant of the antibody in the preparation can be detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing electrophoresis (iCIEF).
  • CEX cation exchange chromatography
  • iCIEF imaging capillary isoelectric focusing electrophoresis
  • the stability of the formulation is measured by determining the percentage change in the charge variant of the antibody in
  • the "acceptable degree" of chemical stability can mean that the percentage change of the charge variant (such as the main component or acidic component or alkaline component) in the preparation after storage at a certain temperature for a certain period of time does not exceed 30%, such as 20% .
  • the temperature for storing the pharmaceutical preparation can be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, or about 45°C.
  • the percentage change value of the acid component charge variant is less than about 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% Or 0.1%, the pharmaceutical preparation can be considered stable.
  • the pharmaceutical preparation can also be regarded as stable.
  • the pharmaceutical preparation can also be considered stable.
  • lyophilized preparation refers to a composition obtained or obtainable by freeze-drying a liquid preparation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.
  • reconstituted preparation refers to a liquid preparation obtained by dissolving and/or suspending a solid preparation (for example, a lyophilized preparation) in a physiologically acceptable solution.
  • room temperature refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, more preferably 25°C.
  • Stress conditions refer to environments that are chemically and/or physically unfavorable to the antibody protein, which can lead to unacceptable instability of the antibody protein.
  • High temperature stress refers to storing the antibody preparation at room temperature or even higher temperature (for example, 40°C ⁇ 2°C) for a period of time. Through the accelerated test of high temperature stress, the stability of the antibody preparation can be checked.
  • parenteral administration means administration methods other than enteral and local administration, usually by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, and intrathecal , Intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspine, epidural and intrasternal injections and infusions .
  • the stable anti-LAG-3 antibody formulations of the invention are administered to the subject parenterally.
  • the anti-LAG-3 antibody formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular or intravenous injection.
  • the present invention provides a stable liquid antibody preparation comprising (i) a recombinant fully human anti-LAG-3 monoclonal antibody; (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, the antibody preparation
  • the pH is about 5.5-7.5.
  • the liquid antibody preparation of the present invention is in the form of an injection preparation.
  • an “anti-LAG-3 antibody” refers to an antibody that can bind to LAG-3 molecules with sufficient affinity so that the antibody can be used as a therapeutic and/or preventive agent that targets LAG-3 molecules.
  • the antibody of the present invention is a recombinant fully human antibody.
  • the terms "fully human antibody” or “human antibody” are used interchangeably herein and refer to the antibody including variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences, and if the antibody Contains constant regions, which are also derived from human germline immunoglobulin sequences.
  • the human antibodies of the present invention may include amino acids that are not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or point-specific mutagenesis in vitro or somatic mutations in vivo).
  • human antibody is not intended to include antibodies in which the CDR sequences are derived from the germline of other mammalian species (eg, mice) and grafted into human framework sequences.
  • recombinant human antibody includes all human antibodies prepared, expressed, produced or isolated by recombinant means. These recombinant human antibodies have framework and CDR regions derived from variable regions of human germline immunoglobulin sequences.
  • recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when using human Ig sequence transgenic animals), thereby obtaining the amino acid sequences of the VH and VL regions of the recombinant antibody, although
  • the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, but do not naturally exist in the human antibody germline repertoire.
  • the anti-LAG-3 antibody can have a high affinity, such as 10 -7 M or less, preferably 10 -9 M to 10 -10 M.
  • K D specifically binds to human LAG-3, and thereby mediates a highly effective blocking effect on LAG-3 and its ligand binding.
  • the antibody LAG-3 antibody of the present invention comprises: SEQ ID NO: 7 or a heavy chain variable region (VH) having at least 90% identity; and SEQ ID NO: 8 or having at least 90% identical light chain variable region (VL).
  • a "variable region” or “variable domain” is a domain in the heavy or light chain of an antibody that participates in the binding of the antibody to its antigen.
  • the heavy chain variable region (VH) and light chain variable region (VL) can be further divided into hypervariable regions (HVR, also known as complementarity determining regions (CDR)), with more conservative regions (ie , Framework area (FR)).
  • HVR hypervariable regions
  • CDR complementarity determining regions
  • FR Framework area
  • Each VH and VL consists of three CDRs and 4 FRs, arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the anti-LAG-3 antibody of the present invention comprises the VH CDR1, 2 and 3 sequences of the heavy chain variable region of SEQ ID NO: 7 and the VL CDR1 of the light chain variable region of SEQ ID NO: 8 2 and 3 sequence.
  • “Complementarity determining region” or “CDR region” or “CDR” (herein used interchangeably with hypervariable region “HVR”) is an amino acid region in the variable region of an antibody that is mainly responsible for binding to an epitope.
  • the CDRs of the heavy and light chains are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • the CDRs located in the variable domain of the antibody heavy chain are called VH CDR1, VH CDR2, and VH CDR3, and the CDRs located in the variable domain of the antibody light chain are called VL CDR1, VL CDR2, and VL CDR3.
  • Various schemes are known in the art for determining the CDR sequence of a given VH or VL amino acid sequence.
  • the Kabat complementarity determining region is determined based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. 1991)).
  • Chothia refers to the position of structural loops (Chothia and Lesk, J.
  • AbM HVR is a compromise between Kabat HVR and Chothia structural loops, and is used by Oxford Molecular's AbM antibody modeling software. "Contact” HVR is based on the analysis of available complex crystal structures. HVR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., an exemplary CDR disclosed herein).
  • the anti-LAG-3 antibody of the present invention has the VH CDR1 of SEQ ID NO: 1, the VH CDR2 of SEQ ID NO: 2, the VH CDR3 of SEQ ID NO: 3; and the VL CDR1 of SEQ ID NO: 4 , VL CDR2 of SEQ ID NO: 5 and VL CDR3 of SEQ ID NO: 6.
  • the anti-LAG-3 antibody of the present invention may comprise a heavy chain variable region (VH) having at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 7; And/or a light chain variable region (VL) having at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 8.
  • VH heavy chain variable region
  • VL light chain variable region
  • sequence identity refers to the degree of sequence identity on a nucleotide-by-nucleotide or amino acid-by-amino-acid basis in the comparison window.
  • the "percentage of sequence identity” can be calculated in the following way: the two best aligned sequences are compared in the comparison window, and it is determined that the same nucleic acid base (for example, A, T, C, G, I) exists in the two sequences. ) Or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) The number of positions to get the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window (ie, the window size), and the result is multiplied by 100 to produce the sequence identity percentage.
  • the optimal alignment to determine the percent sequence identity can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithm needed to achieve the maximum alignment within the full-length sequence being compared or within the target sequence region.
  • the VH sequence of the antibody of the present invention has no more than 10, preferably no more than 5, 4 or 3 different residues compared to SEQ ID NO: 7, and preferably the different residues are Conservative amino acid substitutions.
  • the VL sequence of the antibody of the present invention has no more than 10, preferably no more than 5, 4 or 3 different residues compared to SEQ ID NO: 8, and preferably the different residues are Conservative amino acid substitutions.
  • Constant substitution refers to an amino acid change that results in the substitution of a certain amino acid with a chemically similar amino acid. It is well known in the art to provide conservative substitution tables of functionally similar amino acids. In any embodiment of the present invention, in a preferred aspect, the conservatively substituted residues are from conservative substitution table A below, preferably the preferred substituted residues shown in Table A.
  • the antibodies of the invention are antibodies in the form of IgG4.
  • Antibody in the form of IgG refers to the form of IgG to which the constant region of the heavy chain of the antibody belongs.
  • the heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different.
  • an antibody in the form of IgG4 means that the Ig domain of its heavy chain constant region is the Ig domain of IgG4.
  • the anti-LAG-3 antibody of the present invention is the anti-LAG-3 monoclonal antibody ADI-31853 disclosed in Chinese application CN201811561512.X (December 19, 2018), which has SEQ ID NO: 9 heavy chain and SEQ ID NO: 10 light chain.
  • the anti-LAG-3 antibody is a purified IgG4-type antibody produced by recombinant expression in 293 cells or CHO cells.
  • the antibody in the liquid formulation of the present invention exhibits significant anti-tumor activity.
  • a mouse tumor model vaccinated with mouse colon cancer cell CT26 (ATCC#CRL-2638) or human skin cancer cell A375 (ATCC# CRL-1619)
  • administration of the antibody preparation of the present invention can lead to tumor growth inhibition
  • the rate reaches about 50% or higher, such as 100%; and/or the tumor disappearance rate reaches above 60%.
  • the amount of the antibody or antigen-binding fragment thereof contained in the antibody preparation of the present invention may vary according to the specific purpose characteristics of the preparation, the specific environment, and the specific purpose for which the preparation is used.
  • the antibody preparation is a liquid preparation, which may contain about 1-100 mg/ml, preferably about 5-50 mg/ml, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50mg/ml anti-LAG-3 antibody.
  • the buffer is an agent that can maintain the pH of the solution within an acceptable range.
  • the buffering agent used in the formulation of the present invention can control the pH of the formulation of the present invention in a pH range of about 5.5-7.5, such as a pH of about 6.0-7.0, preferably a pH of 6.0 ⁇ 0.3.
  • the antibody formulation of the invention has a pH of about 5.5, 6.0, 6.5, 7.0 or 7.5, preferably a pH of about 6.0.
  • the buffering agent used in the formulation of the present invention is selected from citrate, citrate solvate (e.g., citrate hydrate), and combinations thereof, e.g., sodium citrate, lemon dihydrate Sodium and their combinations; preferably, the concentration of the buffer is about 1-10 mg/ml, preferably about 4-8 mg/ml, for example, about 4, 4.5, 5, 5.5, 6, 6.5, 7 , 7.5, 8mg/ml.
  • the buffer used in the formulation of the present invention is about 1-10 mg/ml sodium citrate or sodium citrate dihydrate, for example, about 2-8 mg/ml sodium citrate or sodium citrate dihydrate .
  • Suitable stabilizers for use in the present invention may be selected from sugars, polyols and amino acids and combinations thereof.
  • Sugars used as stabilizers include, but are not limited to, sucrose and trehalose.
  • the polyol used as a stabilizer includes but is not limited to sorbitol.
  • the amino acids used as stabilizers include but are not limited to arginine and arginine hydrochloride.
  • the stabilizer is in the liquid formulation of the present invention at about 10-150 mg/ml, more preferably about 15-100 mg/ml, for example, about 15, 20, 30, 40, 50, 60, Concentrations of 70, 80, 90, 100 mg/ml exist.
  • the liquid formulation of the present invention contains sucrose as a stabilizer.
  • the amount of sucrose in the liquid formulation of the present invention may be about 20-100 mg/ml, preferably about 40-90 mg/ml (for example, about 40, 50, 60, 70, 80, 90 mg/ml).
  • the liquid formulation of the present invention contains arginine and/or arginine hydrochloride as a stabilizer.
  • the amount of arginine and/or arginine hydrochloride in the liquid formulation of the present invention may be about 10-40 mg/ml, preferably about 15-25 mg/ml (for example, about 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25mg/ml).
  • the liquid formulation of the present invention contains a combination of sucrose, arginine and/or arginine hydrochloride as a stabilizer.
  • sucrose may be present in an amount of about 20-100 mg/ml, preferably about 40-90 mg/ml (e.g., about 40, 50, 60, 70, 80, 90 mg/ml).
  • arginine and/or arginine hydrochloride may be at about 10-40 mg/ml, preferably about 15-25 mg/ml (for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mg/ml).
  • surfactant refers to an organic substance having an amphiphilic structure; that is, they are composed of groups with opposite solubility tendencies, usually oil-soluble hydrocarbon chains and water-soluble ionic groups. group.
  • the surfactant in the liquid formulation of the present invention is a nonionic surfactant, for example, alkyl poly(ethylene oxide).
  • specific nonionic surfactants that can be included in the formulation of the present invention include, for example, polysorbates such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; Nick waits.
  • the liquid formulation of the present invention contains polysorbate-80 as a surfactant.
  • the amount of the surfactant contained in the antibody preparation of the present invention can be changed according to the specific purpose characteristics of the preparation, the specific environment, and the specific purpose of using the preparation.
  • the formulation may contain about 0.1-1 mg/ml, preferably about 0.2-0.8 mg/ml, for example about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml of polysorbate Class surfactants (for example, polysorbate-80).
  • the antibody liquid preparation of the present invention may or may not contain other excipients.
  • the antibody liquid formulation of the present invention may or may not contain edetate (eg, disodium edetate) and/or methionine.
  • the antibody liquid formulation of the present invention may or may not contain edetate (eg, disodium edetate).
  • edetate eg, disodium edetate
  • the antibody liquid formulation of the present invention may or may not contain methionine.
  • the antibody liquid formulation of the present invention does not contain edetate (e.g., edetate disodium) and/or methionine, which is combined with edetate (e.g., edetate disodium). Compared with the corresponding formulations of) and/or methionine, they have similar stability.
  • edetate e.g., edetate disodium
  • methionine methionine
  • excipients can also be used in the formulation of the present invention.
  • the excipients include, for example, flavoring agents, antimicrobial agents, sweeteners, antistatic agents, antioxidants, gelatin and the like.
  • These and other known pharmaceutical excipients and/or additives suitable for the formulation of the present invention are well known in the art, for example, listed in "The Handbook of Pharmaceutical Excipients, 4th edition, edited by Rowe et al., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippincott Williams & Wilkins (2005)".
  • the present invention provides stable formulations containing antibodies.
  • the antibodies used in the formulations of the present invention can be prepared using techniques known in the art for antibody production. For example, antibodies can be produced recombinantly. In a preferred embodiment, the antibody of the present invention is recombinantly produced in 293 cells or CHO cells.
  • recombinantly produced monoclonal antibodies can be purified by conventional purification methods to provide pharmaceutical substances with sufficient reproducibility and moderate purity for the preparation of antibody preparations.
  • a commercially available protein concentration filter such as Amicon's ultrafiltration device can be used to concentrate the supernatant from the expression system.
  • the antibody can be purified using methods such as chromatography, dialysis, and affinity purification.
  • Protein A is suitable as an affinity ligand for the purification of IgG1, IgG2 and IgG4 type antibodies.
  • Other antibody purification methods such as ion exchange chromatography, can also be used. After obtaining antibodies of sufficient purity, preparations containing antibodies can be prepared according to methods known in the art.
  • the following steps can be used for preparation: (1) After fermentation, the fermentation broth is centrifuged to clarify impurities such as cells to obtain the supernatant; (2) affinity chromatography (for example, specific for IgG1, IgG2, and IgG4 antibodies) Affinity protein A column) capture antibody; (3) virus inactivation; (4) purification and purification (usually CEX cation exchange chromatography can be used) to remove impurities in the protein; (4) virus filtration (to make the virus titer Reduce, for example, 4 log10 or more); (5) Ultrafiltration/diafiltration (which can be used to replace the protein in a formulation buffer that is conducive to its stability and concentrate it to a suitable concentration for injection). See, for example, B. Minow, P. Rogge, K. Thompson, BioProcess International, Vol. 10, No. 6, 2012, pp. 48-57.
  • affinity chromatography for example, specific for IgG1, IgG2, and IgG4 antibodies
  • Affinity protein A column capture antibody
  • antibodies may undergo aggregation, degradation or chemical modification, resulting in antibody heterogeneity (including size heterogeneity and charge heterogeneity), aggregates and fragments, etc., thereby affecting the quality of antibody preparations. Therefore, it is necessary to monitor the stability of antibody preparations.
  • Various methods are known in the art that can be used to test the stability of antibody preparations.
  • methods such as non-reduced CE-SDS and SEC-HPLC can be used to analyze the purity of antibody preparations and evaluate the aggregation level of antibodies; Capillary isoelectric focusing (cIEF), imaging capillary isoelectric focusing (iCIEF) and Ion exchange chromatography (IEX), etc., analyze charge variants in antibody preparations.
  • the stability of the preparation can be quickly judged by visually inspecting the appearance of the preparation.
  • the OD 350nm method can also be used to detect changes in the turbidity of the preparation, which can give information about the amount of soluble and insoluble aggregates.
  • ultraviolet spectrophotometry UV method
  • UV method ultraviolet spectrophotometry
  • the non-reduced CE-SDS method is a method for measuring the purity of monoclonal antibodies using capillary as the separation channel.
  • CE-SDS protein migration is driven by the surface charge caused by SDS binding, and the surface charge is proportional to the molecular weight of the protein. Since all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on molecular size or hydrodynamic radius can be achieved in the molecular sieve gel matrix of the capillary. This method has been widely used to monitor the purity of denatured intact antibodies.
  • the test sample is mixed with SDS sample buffer and iodoacetamide.
  • the mixture can be incubated at 68-72°C for about 10-15 minutes, and the supernatant centrifuged after cooling to room temperature for analysis.
  • a UV detector is used to detect the migration of the protein and obtain an electrophoretic spectrum.
  • the purity of the antibody preparation can be calculated as the percentage of the peak area of the main IgG peak to the sum of all peak areas.
  • Size exclusion high performance liquid chromatography is another important method for monoclonal antibody standards and quality control. This method is mainly based on the molecular size or hydrodynamic radius difference to separate molecules.
  • SEC-HPLC antibodies can be separated into three main forms: high molecular weight form (HMMS), main peak (mainly antibody monomer), and low molecular weight form (LMMS).
  • HMMS high molecular weight form
  • LMMS low molecular weight form
  • Antibody purity can be calculated as the percentage of the main peak area on the chromatogram of the sum of all peak areas.
  • the SEC-HPLC method the percentage of antibody monomers in the preparation product can be measured, and the content information of soluble aggregates and shears can be given.
  • Imaging capillary isoelectric focusing electrophoresis can be used to analyze the charge heterogeneity of monoclonal antibodies. This method can provide quantitative distribution of charge variants.
  • iCIEF achieves molecular separation based on the charge difference (apparent pI value) of molecules in the pH gradient.
  • the separation column is usually a short capillary (for example, a silica capillary with a length of 5 cm and an inner diameter of 100 ⁇ m).
  • the protein is focused in the capillary column under high voltage, and the focusing is performed by a whole column imaging detection system operating at 280 nM. Real-time online monitoring.
  • One advantage of this technology is that the whole column detection system can simultaneously record various charge variants of antibody samples.
  • icIEF the sample is mixed with urea and icIEF buffer, where the buffer contains methylcellulose, pi molecular weight standards and ampholytes.
  • iCIEF column such as an iCIEF column assembled by ProtionSimple on an iCIEF analyzer such as iCE280 Analyzer (Protein Simple, Santa Clara, CA).
  • iCE280 Analyzer Protein Simple, Santa Clara, CA.
  • the protein-related peak eluted before the main peak ie, the main component
  • the protein-related peak eluted after the main peak is classified as the basic component.
  • the relative amounts of the main component, acidic component and basic component can be expressed as a percentage of the total peak area.
  • the charge variant of the antibody in the antibody preparation can also be determined by cation exchange high performance liquid chromatography (CEX-HPLC).
  • CEX-HPLC cation exchange high performance liquid chromatography
  • Accelerated stability studies can be used to check the stability properties of products, which is conducive to the screening of stable pharmaceutical formulations.
  • the formulation sample can be placed at an elevated temperature, such as about 40°C ⁇ 2°C, 25°C ⁇ 2°C, for accelerated stability studies.
  • Detection indicators can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (iCIEF method).
  • a shaking test can be performed to investigate the shaking/shear stability of the formulation.
  • the preparation samples are dispensed into vials, stoppered and capped, set out, and subjected to a shaking test, such as shaking at 650 r/min for 3-5 days, and then inspect the appearance, protein content, turbidity and purity of the preparation.
  • the efficacy or biological activity of the antibody can be tested.
  • the ability of the antibody in the preparation to bind to its antigen can be tested.
  • Those skilled in the art know that a variety of methods can be used to quantify the specific binding of antibodies to antigens, such as immunoassay tests, ELISA, and the like.
  • the anti-LAG-3 antibody formulation of the present invention is stable. In one embodiment, after storage at about 25°C, 37°C, 40°C, or 45°C for at least 1 month or 2 months, for example, after storage at 40°C ⁇ 2°C for 1 month, the antibody preparation of the present invention
  • the purity of the anti-LAG-3 antibody in is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, such as by size exclusion chromatography or Measured by non-reduced CE-SDS.
  • the antibody preparation of the present invention At least 50%, preferably at least 55% of the middle anti-LAG-3 antibody is in non-basic and non-acidic form (ie, main peak or main charge form), as determined by imaging capillary focus electrophoresis.
  • the antibody preparation of the present invention comprising an anti-LAG-3 antibody of the present invention can be used to treat, ameliorate or prevent various diseases or disorders related to LAG-3.
  • LAG-3 related disease or disorder herein refers to a disease or disorder that can be treated (e.g., improved) or prevented with the anti-LAG-3 antibody preparation of the present invention. Any disease or condition that can benefit from the treatment of the antibody preparation of the present invention is applicable to the present invention.
  • the formulations of the invention comprising anti-LAG-3 antibodies can be used to modulate the immune response in a subject, especially for restoring, enhancing, stimulating or increasing the immune response in the subject.
  • the formulations of the invention containing anti-LAG-3 antibodies can be used to restore, enhance or stimulate antigen-specific T cell responses in a subject, for example, interleukin-2 (IL-2) or interferon- ⁇ (IFN- ⁇ ) is produced.
  • IL-2 interleukin-2
  • IFN- ⁇ interferon- ⁇
  • the preparation of the present invention containing the anti-LAG-3 antibody can be used to prevent or treat tumors in a subject, such as cancers including but not limited to solid tumors, hematological cancers (e.g., leukemia, lymphoma, myeloma) And its metastatic lesions.
  • the cancer is a solid tumor.
  • solid tumors include malignant tumors, such as sarcomas and cancers of multiple organ systems (e.g., adenocarcinoma), such as invasion of the lung, breast, lymph, gastrointestinal or colorectal tract, genitals, and genitourinary tract (e.g., kidney cells).
  • CNS for example, brain cells, nerve cells or glial cells
  • skin for example, melanoma
  • head and neck for example, head and neck squamous cell carcinoma (HNCC)
  • HNCC head and neck squamous cell carcinoma
  • melanoma colon cancer, gastric cancer, rectal cancer, renal cell carcinoma, breast cancer (e.g., breast cancer that does not express one, two, or all estrogen receptors, progesterone receptors, or Her2/neu, such as , Triple negative breast cancer), liver cancer, lung cancer (for example, non-small cell lung cancer (NSCLC) (for example, NSCLC with squamous and/or non-squamous structure) or small cell liver cancer), prostate cancer, head or neck Cancer (eg, HPV+ squamous cell carcinoma), small intestine cancer, and esophageal cancer.
  • NSCLC non-small cell lung cancer
  • head or neck Cancer eg, HPV+ squamous cell carcinoma
  • small intestine cancer small intestine cancer
  • esophageal cancer e.
  • hematological cancers include, but are not limited to, leukemia (e.g., myeloid leukemia, lymphoid leukemia, or chronic lymphocytic leukemia (CLL)), lymphoma (e.g., Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma) (NHL), diffuse large B-cell lymphoma (DLBCL), T-cell lymphoma or mantle cell lymphoma (MCL)) and myeloma, for example, multiple myeloma.
  • the cancer can be early, middle or late or metastatic cancer.
  • the tumor is a gastrointestinal tumor, such as colon cancer and the like.
  • the preparation of the present invention containing an anti-LAG-3 antibody can be used to prevent or treat infectious diseases in a subject.
  • infectious disease is a chronic infection, for example, the chronic infection is caused by a pathogen selected from bacteria, viruses, fungi, and protozoa.
  • formulations of the present invention containing anti-LAG-3 antibodies can be used to cause antibody-dependent cell-mediated cytotoxicity in a subject.
  • the present invention also provides the use of the preparation of the present invention in the preparation of a medicament, wherein the medicament is used to deliver anti-LAG-3 antibodies to mammals, or to treat, prevent or ameliorate one or more of the aforementioned diseases and disorders .
  • the mammal is a human.
  • the antibody formulation of the present invention can be administered to a subject or patient in various ways.
  • administration can be by infusion or by syringe.
  • the present invention provides a delivery device (e.g., a syringe) comprising the antibody formulation of the present invention (e.g., a pre-filled syringe).
  • the patient will receive an effective amount of anti-LAG-3 antibody as the main active ingredient, that is, an amount sufficient to treat, ameliorate or prevent the target disease or condition.
  • the therapeutic effect may include reducing physical symptoms.
  • the optimal effective amount and concentration of antibodies for any particular subject will depend on many factors, including the patient’s age, weight, health and/or gender, the nature and extent of the disease, the activity of the particular antibody, and the body’s Its clearance rate, and also includes any possible other treatments administered in combination with the antibody formulation.
  • the effective amount delivered can be determined within the judgment of the clinician.
  • the effective dose may be about 0.005 mg/kg body weight to about 50 mg/kg body weight, or about 0.1 mg/kg body weight to about 20 mg/kg body weight.
  • the application of known antibody-based drugs can provide some guidance.
  • the dosage can be a single dose schedule or a multiple dose schedule.
  • CE-SDS Sodium Lauryl Sulfate Capillary Gel Electrophoresis
  • iCIEF Imaging Capillary Isoelectric Focusing Electrophoresis
  • N/A means "Not applicable”.
  • the mobile phase is phosphate buffer (weigh 3.12g sodium dihydrogen phosphate dihydrate, 8.77g sodium chloride and 34.84g arginine, after dissolving in ultrapure water, adjust the pH to 6.8 with hydrochloric acid And dilute to 1000ml), the column protection solution is 0.05% (w/v) NaN 3 , the injection volume is 50 ⁇ l, the flow rate is 0.5ml/min, the collection time is 30 minutes, the column temperature is 25°C, and the detection wavelength is 280nm. Dilute the sample to be tested with ultrapure water to 2mg/ml as the test solution. Take the preparation buffer solution and dilute it with the same treatment method as the blank solution. Take 50 ⁇ l each of the blank solution and the test solution into the liquid chromatograph to start the test.
  • phosphate buffer weigh 3.12g sodium dihydrogen phosphate dihydrate, 8.77g sodium chloride and 34.84g arginine, after dissolving in ultrapure water, adjust the pH to 6.8 with hydro
  • the capillary is an uncoated capillary with an inner diameter of 50 ⁇ m, a total length of 30.2cm, and an effective length of 20.2cm. Wash the capillary column with 0.1mol/L sodium hydroxide, 0.1mol/L hydrochloric acid, ultrapure water, and 70psi electrophoresis gel before electrophoresis.
  • Sample injection conditions -5kV for 20 seconds; separation voltage: -15kV for 35 minutes.
  • the capillary column temperature is controlled at 25°C, and the detection wavelength is 220nm.
  • the capillary is an uncoated capillary with an inner diameter of 50 ⁇ m, a total length of 30.2cm, and an effective length of 20.2cm. Wash the capillary column with 0.1mol/L sodium hydroxide, 0.1mol/L hydrochloric acid, ultrapure water, and 70psi electrophoresis gel before electrophoresis.
  • Sample injection conditions -5kV for 20 seconds; separation voltage: -15kV for 35 minutes.
  • the capillary column temperature is controlled at 25°C, and the detection wavelength is 220nm.
  • iCIEF method imaging capillary isoelectric focusing electrophoresis (iCIEF method) detection.
  • the inner diameter of the capillary is 100 ⁇ m and the total length is 5cm.
  • MC solution 0.5% methylcellulose solution
  • ultrapure water should be used to rinse the capillary column.
  • the vacuum injection method is used to inject for 55 seconds, the pre-focusing voltage and time are 1.5kV and 1 minute, the focusing voltage and time are 3kV and 8 minutes, and the sampling time is 55 seconds.
  • the wavelength is 280nm.
  • Cathodic Stabilizer is 500mmol/L arginine solution
  • Anodic Stabilizer is 200mmol/L iminooxalic acid
  • 3mol/L urea improves protein solubility
  • 0.5% MC solution reduces protein and capillary The adhesion between. Dilute the test product to 0.5 mg/ml with water, take 20 ⁇ l of the diluted test product solution, add 83 ⁇ l of the premix solution to it and mix well to prepare the test sample solution. Use the preparation buffer to operate in the same way to prepare a blank solution.
  • Dilute streptavidin (Thermo, catalog number: 21125) with 1 ⁇ PBS to 1 ⁇ g/ml, 100 ⁇ l/well, and coat on 96-well microtiter plate at 37°C for 2 hours. After washing the plate, add blocking solution (5% FBS, 300 ⁇ l/well) at 37°C for 2h.
  • Dilute the biotinylated LAG-3 (Sino biological, catalog number: 16498-HNAH-B) with 1 ⁇ PBS to 0.5 ⁇ g/ml, 100 ⁇ l/well, and coat it on a 96-well microtiter plate at 37° C. for 0.5 h.
  • the anti-LAG-3 antibody was diluted with 2% FBS to 40 ⁇ g/ml in 100 ⁇ l/well, and the concentration was diluted 4-fold to the 12th concentration (0.01 ⁇ 10000ng/ml).
  • the EC 50 is calculated using Prism four-parameter fitting to reflect the binding activity of the anti-LAG-3 antibody and LAG-3.
  • the antibody is composed of the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10 , Is an IgG4 type antibody.
  • the anti-LAG-3 antibody ADI-31853 was recombinantly expressed and purified in 293 cells or CHO cells. For the samples used in the prescription screening test, they were purified by CEX (Cation Exchange Chromatography), and the samples had a protein content of about 11.5 mg/ml to about 25.0 mg/ml.
  • This example examines the stability of a formulation containing anti-LAG-3 antibodies at pH 5.0 to 8.0.
  • the results of the test on the effect of pH on the stability of the preparation are shown in Table 4.
  • the change trend diagram of the purity of the anti-LAG-3 antibody protein detected by SEC-HPLC is shown in Figure 1.
  • the charge variants of the anti-LAG-3 antibody protein detected by the iCIEF method The change trend chart is shown in Figure 2. It can be seen from Table 4, Figure 1 and Figure 2 that after each prescription is placed at 40°C ⁇ 2°C for one month, the anti-LAG-3 antibody is more effective in pH 5.5, pH 6.0, pH 6.5, pH 7.0, and pH 7.5 liquids. Stable, that is, the formulation can be implemented in the pH range of 5.5-7.5. Therefore, in subsequent experiments, pH 6.0 was used to design the formulation of LAG 3 antibody preparations.
  • a total of 7 prescriptions were designed, and the detailed prescription information is shown in Table 5.
  • Replace the antibody protein by ultrafiltration into the respective prescription solution.
  • the protein concentration of each prescription was diluted to about 20 mg/ml, and polysorbate 80 was added. Filter and dispense into vials, stopper and cap, and then conduct stability inspection. Stability investigation uses test methods including high temperature stress test, shaking test, freeze-thaw test and light test.
  • the detection indicators are appearance, visible foreign matter, protein content (UV method), turbidity (OD 350nm method), purity (SEC-HPLC method and non-reduced CE-SDS method), charge variant (iCIEF method) and relative binding activity (Direct ELISA method).
  • Prescriptions 1, 2, 4, 5, 6, and 7 are qualified in terms of appearance and visible foreign bodies; protein content (UV method) and purity (SEC-HPLC method and non-reducing CE-SDS method) are unchanged; charge variation The body-acid component (iCIEF method) increased; the charge variant-basic component (iCIEF method) decreased, and the charge variant-acid component of prescription 5, prescription 6, and prescription 7 increased relative to prescription 1, prescription 2.
  • Prescription 4 is relatively small; the charge variant-principal component (iCIEF method) of prescription 5 and prescription 7 is unchanged according to the judgment standard in Table 3 above.
  • N/A means no survey was designed.
  • N/A means no survey was designed.
  • N/A means no survey was designed.
  • a total of 3 prescriptions were designed, and the detailed prescription information is shown in Table 11.
  • the detection indicators are appearance, visible foreign matter, protein content (UV method), turbidity (OD 350nm method), purity (SEC-HPLC method, non-reduced CE-SDS method and reduced CE-SDS method), charge variant ( iCIEF method) and relative binding activity (direct ELISA method).
  • N/A means no survey was designed.
  • the freeze-thaw test results of the second prescription screening are shown in Table 15. After repeated freezing and thawing 6 times, the appearance and visible foreign matter of prescription 8, prescription 9, prescription 10 are all qualified; protein content (UV method), turbidity (OD 350nm method), purity (non-reduced CE-SDS method, reduced type) CE-SDS method and SEC-HPLC method), charge variants (iCIEF method) and relative binding activity (direct ELISA method) did not change.
  • N/A means no survey was designed.
  • the most preferred formulation is: about 20 mg/ml recombinant fully human anti-lymphocyte activation gene 3 (LAG-3) monoclonal antibody, about 5.88 mg/ml sodium citrate (dihydrate), about 21.07 mg /ml arginine hydrochloride, about 0.70mg/ml polysorbate 80, pH 6.0.
  • LAG-3 fully human anti-lymphocyte activation gene 3

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Abstract

本发明涉及包含抗LAG-3抗体的制剂，尤其涉及包含特异性结合LAG-3分子的抗体、缓冲剂、稳定剂和表面活性剂的药物制剂。此外，本发明还涉及这些制剂的疾病治疗或预防用途。

Description

包含抗LAG-3抗体的制剂、其制备方法及其用途 技术领域
本发明涉及抗体制剂领域。更具体而言，本发明涉及包含特异性结合LAG-3的抗体(下文中也称为“抗LAG-3抗体”)的药物制剂，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
淋巴细胞活化基因3(LAG-3)，也称为CD223，是由LAG-3基因在人体中编码的I型跨膜蛋白。LAG-3的分子性质和生物学功能已经有充分的表征和描述，参见例如Sierro等人，The CD4-like molecule LAG-3,biology and therapeutic applications，Expert Opin Ther Targets，2011，15(1)：91-101。LAG-3是一种CD4样蛋白，在T细胞(特别是活化的T细胞)、自然杀伤细胞、B细胞和浆细胞样树突状细胞表面表达。已显示LAG-3是一种抑制性受体。
已经在黑素瘤、结肠癌、胰腺癌、乳腺癌、肺癌、造血癌、头颈癌患者中报道了存在LAG-3 +肿瘤浸润淋巴细胞(TIL)(Demeure C.E.等人,T Lymphocytes infiltrating various tumour types express the MHC class II ligand lymphocyte activation gene-3(LAG-3):role of LAG-3/MHC class II interactions in cell-cell contacts,Eur.J.Cancer,2001,37(13):1709-1718)。在多种癌症小鼠模型中，通过使用抗LAG-3抗体来阻断LAG-3，由此恢复了CD8 +效应T细胞并减少了Treg细胞群体。
特异性结合LAG-3的作为LAG-3阻断剂的抗LAG-3抗体描述于例如中国专利申请号201811561512.X中。本领域中需要能够用来治疗、预防或延缓各种癌症、免疫相关疾病和T细胞功能障碍性疾病的抗LAG-3抗体制剂。
抗体制剂除了需要使抗体以适于施用给受试者的方式调配之外，还需要以在储存以及后续使用期间维持其稳定性的方式来调配。例如，如果抗体没有适当地在液体中得以调配，则液体溶液中的该抗体倾向于分解、聚集或发生不希望的化学修饰等。抗体在抗体制剂中的稳定性取决于制剂中所使用的缓冲剂、稳定剂和表面活性剂等。
尽管已知一些抗LAG-3抗体，但在本领域中对于含有足够稳定且适于施用给受试者的抗LAG-3抗体的新颖药物制剂仍存在需要。因此，需要合适的抗LAG-3抗体制剂，以用来治疗或预防疾病。
发明概述
本发明通过提供含有特异结合至LAG-3的抗体的药物制剂来满足上述需求。
在一个方面，本发明提供了一种液体抗体制剂，其包含(i)抗LAG-3抗体；(ii)缓冲剂，(iii)稳定剂，和(iv)表面活性剂。
所述抗LAG-3抗体可以为任何结合LAG-3分子(例如人LAG-3分子)并阻断LAG-3信号传导的抗体，例如多克隆抗体、单克隆抗体或者两者的组合。优选地，在一个实施方案中，所述抗LAG-3抗体为单克隆抗体。在一个实施方案中，所述抗LAG-3抗体为在中国申请CN201811561512.X(2018年12月19日递交)中公开的重组全人源抗淋巴细胞活化基因3(LAG-3)单克隆抗体。为了本申请的目的，该中国申请的全部内容特此并入本文作为参考。在一个实施方案中，抗LAG-3抗体包含重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:7的序列或与其具有至少90％同一性的序列，且轻链可变区包含SEQ ID NO:8的序列或与其具有至少90％同一性的序列：
Figure PCTCN2020098140-appb-000001
Figure PCTCN2020098140-appb-000002
在一个实施方案中，所述抗LAG-3抗体包含：
-GSIYSESYYWG(SEQ ID NO:1)的重链VH CDR1；
-SIVYSGYTYYNPSLKS(SEQ ID NO:2)的重链VH CDR2；
-ARVRTWDAAFDI(SEQ ID NO:3)的重链VH CDR3；
-QASQDISNYLN(SEQ ID NO:4)的轻链VL CDR1；
-DASNLET(SEQ ID NO:5)的轻链VL CDR2；和
-QQVLELPPWT(SEQ ID NO:6)的轻链VL CDR3。
在一个实施方案中，所述抗LAG-3抗体是包含重链和轻链的IgG4抗体，其中所述重链包含SEQ ID NO:9的序列或与其具有至少90％同一性的序列，且其中所述轻链包含SEQ ID NO:10的序列或与其具有至少90％同一性的序列：
Figure PCTCN2020098140-appb-000003
优选地，所述抗LAG-3抗体是中国申请CN201811561512.X(于2018年12月19日递交)中公开的抗LAG-3单克隆抗体ADI-31853，该抗体由SEQ ID NO：9的重链序列和SEQ ID NO:10的轻链序列组成。
在一个实施方案中，所述抗LAG-3抗体是在293细胞或CHO细胞中重组表达的抗LAG-3抗体。
在一个实施方案中，本发明的液体抗体制剂中的抗LAG-3抗体的浓度为约1-100mg/ml。在另一个实施方案中，本发明的液体抗体制剂中的抗LAG-3抗体的浓度为约5-50mg/ml。在其他实施方案中，本发明的液体抗体制剂中的抗LAG-3抗体的浓度为约5、10、15、20、25、30、35、40、45或50mg/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1-10mg/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4-8mg/ml，例如，约4、4.5、5、5.5、6、6.5、7、7.5、8mg/ml。
在一个实施方案中，所述缓冲剂选自柠檬酸盐、柠檬酸盐溶剂合物或它们的组合，更优选为柠檬酸盐、柠檬酸盐水合物，例如，柠檬酸钠、二水柠檬酸钠。
在一个实施方案中，本发明的液体抗体制剂中的稳定剂的浓度为约 10-150mg/ml。在一个实施方案中，本发明的液体抗体制剂中的稳定剂的浓度为约15-100mg/ml，例如约15、20、30、40、50、60、70、80、90、100mg/ml。
在一个实施方案中，所述稳定剂选自山梨醇、蔗糖、海藻糖、精氨酸、盐酸精氨酸或它们的任意组合，更优选为蔗糖、精氨酸和/或盐酸精氨酸。又在一个实施方案中，所述精氨酸和/或盐酸精氨酸以约10-40mg/ml，优选地约15-25mg/ml的量(例如，约15、16、17、18、19、20、21、22、23、24、25mg/ml的量)存在。又在一个实施方案中，所述蔗糖以约20-100mg/ml，优选地约40-90mg/ml的量(例如，约40、50、60、70、80、90mg/ml的量)存在。
在一个实施方案中，本发明的液体抗体制剂中的表面活性剂的浓度为约0.1-1mg/ml。在一个实施方案中，本发明的液体抗体制剂中的表面活性剂的浓度为约0.2-0.8mg/ml，例如约0.2、0.3、0.4、0.5、0.6、0.7、0.8mg/ml。
在一个实施方案中，所述表面活性剂是非离子型表面活性剂。在一个实施方案中，所述表面活性剂选自聚山梨酯类表面活性剂。在一个具体实施方案中，本发明的液体抗体制剂中的表面活性剂是聚山梨酯-80。
在一些实施方案中，所述液体制剂可以包含或可以不包含依地酸盐(例如，依地酸二钠)和/或甲硫氨酸。
在一个实施方案中，所述液体制剂的pH值为约5.5-7.5。在一些实施方案中，所述液体制剂的pH值为约5.5-7.5中的任意值，例如约5.5、6.0、6.5、7.0、7.5。
在一个实施方案中，所述液体制剂为药物制剂，优选为注射剂，更优选为皮下注射剂或静脉内注射剂。在一个实施方案中，所述液体制剂为静脉输注剂。
在一个实施方案中，本发明的液体抗体制剂包含
(i)约1-100mg/ml的抗LAG-3抗体；
(ii)约1-10mg/ml的柠檬酸钠或二水柠檬酸钠；
(iii)约10-150mg/ml蔗糖、精氨酸和/或盐酸精氨酸，和
(iv)约0.1-1mg/ml聚山梨醇酯80；
任选地，所述液体制剂不包含依地酸盐(例如，依地酸二钠)和甲硫氨酸；
其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值。
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约10-30mg/ml的抗LAG-3抗体；
(ii)约2-8mg/ml柠檬酸钠或二水柠檬酸钠；
(iii)约10-40mg/ml精氨酸和/或盐酸精氨酸和/或40-90mg/ml蔗糖，和
(iv)约0.2-0.8mg/ml聚山梨醇酯80；
任选地，所述液体制剂不包含依地酸盐(例如，依地酸二钠)和甲硫氨酸；
其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值。
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约20mg/ml的抗LAG-3抗体；
(ii)约5.88mg/ml二水柠檬酸钠；
(iii)约80mg/ml蔗糖，和
(iv)约0.3mg/ml聚山梨醇酯80；
其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值。
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约20mg/ml的抗LAG-3抗体；
(ii)约5.88mg/ml二水柠檬酸钠；
(iii)约17.42mg/ml精氨酸，和
(iv)约0.3mg/ml聚山梨醇酯80；
其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值。
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约20mg/ml的抗LAG-3抗体；
(ii)约5.88mg/ml二水柠檬酸钠；
(iii)约21.07mg/ml盐酸精氨酸，和
(iv)约0.7mg/ml聚山梨醇酯80；
其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值。
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约20mg/ml的抗LAG-3抗体；
(ii)约5.88mg/ml二水柠檬酸钠；
(iii)约50mg/ml蔗糖和约21.07mg/ml盐酸精氨酸，和
(iv)约0.7mg/ml聚山梨醇酯80；
其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值。
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约20mg/ml的抗LAG-3抗体；
(ii)约5.88mg/ml二水柠檬酸钠；
(iii)约50mg/ml蔗糖，和
(iv)约0.7mg/ml聚山梨醇酯80；
其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值。
另一方面，本发明提供了ー种固体抗体制剂，其是通过将本发明的液体抗体制剂经固化处理而获得的。所述固化处理是通过例如结晶法、喷雾干燥法、冷冻干燥法实施的。在一个优选的实施方案中，所述固体抗体制剂例如是冻干粉针剂形式。固体抗体制剂可在使用前，通过重构于适当的溶媒中，形成本发明的重构制剂。所述重构制剂也是一种本发明的液体抗体制剂。在一个实施方案中，所述适当的溶媒选自注射用水、注射用有机溶剂，包括但不限于注射用油、乙醇、丙二醇等，或其组合。
本发明的液体制剂可以长期稳定储存，例如至少24个月或更长时间。在一个实施方案中，本发明的液体制剂可以在约-80℃至约45℃，例如-80℃、约-30℃、约-20℃、约0℃、约5℃、约25℃、约35℃、约38℃、约40℃、约42℃或约45℃的条件下，储存至少10天、至少20天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，至少36个月，或更长时间，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12个月，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3个月，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1个月。在再一实施方案中，本发明的液体制剂在约5℃至40℃的温度，例如在25℃的温度，在振荡下可以保持稳定至少1天，例如3天或5天。
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、纯度、和/或电荷变异体的变化，来指示储存后制剂的稳定性。在一个实施方案中，可以在高温胁迫试验中，例如在40℃±2℃储存至少1周、2周或优选地1个月后，或在25℃±2℃储存至少1个月或2个月后，检测本发明液体制剂的稳定性。在一个实施方案中，通过振荡试验检测本发明液体制剂的振荡稳定性。
在一个实施方案中，在储存后，通过目视检查本发明液体制剂的稳定性，其中本发明液体制剂在外观上保持为澄明至微乳光，为无色至淡黄色液体，且无异物。在一个实施方案中，在澄明度检测仪下目视检查，制剂中无可见异物存在。在一个实施方案中，在储存后，通过测定蛋白含量变化，检查本发明液体制剂的稳定性，其中例如通过紫外分光光度(UV)法，相对于储存第0天的初始值，蛋白含量变化率不超过20％，优选不超过10％，例如7-8％，优选不超过5％。在一个实施方案中，在储存后，通过测定本发明液体制剂的浊度变化，检查本发明液体制剂的稳定性，其中例如通过OD 350mm法检测，相对于储存第0天的初始值，变化值不超过0.04，更优选地不超过0.03，更优选地不超过0.02。在一个实施方案中，在储存后，通过测定本发明液体制剂的纯度变化，检查本发明液体制剂的稳定性，其中通过尺寸排阻高效液相色谱法(SEC-HPLC)，相对于储存第0天的初始值，单体纯度的变化值不超过10％，例如不超过5％、4％、3％、例如1-2％，优选不超过1％。在一个实施方案中，在储存后，通过测定本发明液体制剂的纯度变化，检查本发明液体制剂的稳定性，其中通过非还原型和/或还原型十二烷基硫酸钠毛细管电泳(CE-SDS)法，单体纯度的变化值下降不超过10％，例如不超过5％、4％、3％、优选不超过2％、1％、0.5％或0.1％。在一个实施方案中，在储存后，通过成像毛细管等电聚焦电泳(iCIEF)检测本发明液体制剂的稳定性，其中相对于储存第0天的初始值，抗体的各电荷变异体(主成分、酸性组分或碱性组分)的变化值不超过2％。
在一个实施方案中，制剂在储存后，例如在2-8℃储存至少24个月后，或在室温储存至少3个月后，或在40℃±2℃储存1个月后，是稳定的，优选地具有如下特征之一或多项：
(i)通过SEC-HPLC法测量，制剂抗LAG-3抗体具有大于90％的纯度，优选大于95％、96％、97％、98％、99％的纯度；
(ii)通过还原型和非还原型CE-SDS法测量，制剂具有大于90％的纯度，优选大于95％、96％、97％、98％、99％的纯度；
(iii)通过iCIEF法测量，相对于储存第0天的初始值，制剂中抗LAG-3抗体的各组分(即主成分、酸性组分及碱性组分)变化值≤2％；
(iv)通过ELISA法测量，相对于储存第0天的初始值，制剂中抗LAG-3抗体的相对结合活性为70％～130％，例如，为70％、80％、90％、100％、110％、120％、130％。
在一个方面，本发明提供了一种递送装置，其包含本发明的液体抗体制剂或固体抗体制剂。在一个实施方案中，本发明的递送装置以包含本发明的液体抗体制剂或固体抗体制剂的预填装注射器形式提供，例如用于静脉内、皮下、皮内或者肌内注射、静脉内输注。
在又一方面，本发明提供向受试者，例如哺乳动物递送抗LAG-3抗体的方法，包括给予所述受试者本发明的液体抗体制剂或固体抗体制剂的步骤，所述递送是例如通过使用预填装注射器的递送装置实施的。
在又一方面，本发明提供本发明的液体抗体制剂或固体抗体制剂的用途，用于制备在受试者中治疗、预防或延缓表达LAG-3的癌症(特别是转移性癌症)、免疫相关疾病和T细胞功能障碍性疾病的递送装置(如，预填装注射器)或药物，特别地用于治疗、预防或延缓表达LAG-3的癌症(特别是转移性癌症)，例如各种血液肿瘤、实体瘤，如结肠癌。
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.显示了通过SEC-HPLC法检测的pH对抗LAG-3抗体蛋白纯度的影响。
图2.显示了通过iCIEF法检测的pH对抗LAG-3抗体蛋白各电荷变异体的影响。
图3.显示了抗LAG-3抗体的处方1-7在高温胁迫试验中放置2周的电荷变异体-酸性组分(iCIEF法)的变化趋势图。
图4.显示了抗LAG-3抗体的处方1-7在高温胁迫试验中放置2周的电荷变异体-主成分(iCIEF法)的变化趋势图。
图5.显示了抗LAG-3抗体的处方1-7在高温胁迫试验中放置2周的电荷变异体-碱性组分(iCIEF法)的变化趋势图。
图6.显示了抗LAG-3抗体的处方8、9、10在高温胁迫试验中浊度(OD 350nm法)的变化趋势图。
图7.显示了抗LAG-3抗体的处方8、9、10在高温胁迫试验中纯度(SEC-HPLC法)的变化趋势图。
图8.显示了抗LAG-3抗体的处方8、9、10在高温胁迫试验中纯度(还原型CE-SDS法)的变化趋势图。
图9.显示了抗LAG-3抗体的处方8、9、10在高温胁迫试验中放置1个月的电荷变异体-酸性组分(iCIEF法)的变化趋势图。
图10.显示了抗LAG-3抗体的处方8、9、10在高温胁迫试验中放置1个月的电荷变异体-主成分(iCIEF法)的变化趋势图。
图11.显示了抗LAG-3抗体的处方8、9、10在高温胁迫试验中放置1个月的电荷变异体-碱性组分(iCIEF法)的变化趋势图。
图12.显示了抗LAG-3抗体的处方9在高温胁迫试验中开始时和放置1个月的纯度(还原型CE-SDS法)图谱。
图13.显示了抗LAG-3抗体的处方9在高温胁迫试验中放置1个月的质谱(LC-MS法)图。
图14.显示了抗LAG-3抗体的处方10在高温胁迫试验中开始时和放置1个月的纯度(SEC-HPLC法)图谱。
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”是指包含轻链和重链免疫球蛋白可变区的多肽，所述免疫球蛋白可变区特异性识别并结合抗原。优选地，本发明的抗体是全长抗体，由两条重链和两条轻链组成，其中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，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。在一些实施方案中，本发明的抗体也可以指抗体的抗原结合片段。
术语“抗体制剂”指一种制备物，所述制备物处于允许作为活性成分的抗体的生物活性可以有效发挥的形式，并且不含有对于待施用该制剂的受试者而言具有不可接受毒性的其它组分。这类抗体制剂通常是无菌的。通常，抗体制剂中包含可药用赋形剂。“可药用”赋形剂是可以合理地施用至受试哺乳动物以便制剂中所用活性成分的有效剂量可以递送至受试者的试剂。赋形剂的浓度与施用模式相适应，例如可以是注射可接受的。
术语“抗LAG-3抗体制剂”在本文中也简称为“本发明的抗体制剂”，意指包含抗LAG-3抗体作为活性成分并包含可药用赋形剂的制备物。将抗LAG-3抗体与可药用赋形剂经所述组合后，作为活性成分的抗LAG-3抗体适于治疗性或预防性施与人类或非人类动物。本发明的抗体制剂可以例如制备成水性形式的液体制剂，例如，即用式预填装注射器，或者制备成冻干制剂，在即将使用前通过溶解和/或悬浮于生理可接受的溶液中进行重构(即，复溶)。在一些实施方案中，抗LAG-3抗体制剂是液体制剂形式。
“稳定的”抗体制剂是制剂中的抗体在储存于特定条件下之后保有可接受程度的物理稳定性和/或化学稳定性。尽管抗体制剂中所含的抗体在储存特定时间之后可能不会100％维持其化学结构，但通常在储存特定时间之后维持约90％、约95％、约96％、约97％、约98％或约99％的抗体结构或功能，则认为抗体制剂是“稳定的”。在一些具体的实施方案中，本发明的抗LAG-3抗体制剂在制造、制备、运输和长期储存过程中表现出低至检测不到的抗体聚集或降解或化学修饰，从而极少或甚至是没有抗LAG-3抗体的生物活性损失，表现出高度稳定性。在一些实施方案中，本发明的抗LAG-3抗体制剂在储存后，基本上保留其物理和化学稳定性。优选地，本发明液体制剂可以在室温或在40℃稳定至少1个月，和/或在2-8℃稳定至少24个月。
本领域已知多种分析技术可以用于测定蛋白质的稳定性，参见例如Peptide and Protein Drug Delivery,247-301,Vincent Lee Ed.,Marcel Dekker,Inc.,New York,N.Y.,Pubs(1991)and Jones,A.Adv.Drug Delivery Rev.10:29-90(1993)。可以在选定的温度和选定的储存时间测量稳定性。例如，可以基于预期的制剂货架期来选 择储存时间。或者可以使用加速稳定性试验。在一些实施方案中，通过对抗体制剂进行各种胁迫测试来进行稳定性测试。这些测试可以代表调配的抗体制剂在制造、储存或运输期间可能遭遇到的极端条件，也可以代表在非制造、储存或运输期间可能使抗体制剂中的抗体的不稳定性加速的条件。例如，可以将经调配的抗LAG-3抗体制剂充填至合适容量的玻璃瓶中用于振荡胁迫以检测抗体的振荡/剪切稳定性；或者可以将经调配的抗LAG-3抗体制剂充填至玻璃小瓶中以检验在高温胁迫下的抗体稳定性。
经一段储存时间后，制剂不显示聚集、沉淀、混浊和/或变性；或显示非常少的聚集、沉淀、混浊和/或变性，则可以认为抗体在制剂中“保持其物理稳定性”。由于制剂中抗体的聚集可以潜在地导致患者增加的免疫反应，从而导致安全性问题。因此，需要使在制剂中的抗体聚集最小化或防止聚集。光散射法可以用于测定制剂中的可见聚集物。SEC可以用于测定制剂中的可溶性聚集物。此外，可以通过目视检查制剂的外观、颜色和/或澄清度、或者通过OD 350nm法检测制剂的浊度、或者通过非还原型CE-SDS法测定制剂的纯度，来指示制剂的稳定性。在一个实施方案中，通过测定在特定温度下储存特定时间之后制剂中的抗体单体的百分比来测量制剂的稳定性，其中制剂中的抗体单体的百分比越高，则制剂的稳定性越高。
“可接受程度的”物理稳定性可以表示于特定温度下储存特定时间之后，在制剂中检测到至少约92％的抗LAG-3抗体单体。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92％、93％、94％、95％、96％、97％、98％、99％的抗LAG-3抗体单体。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92％、93％、94％、95％、96％、97％、98％、99％的抗LAG-3抗体单体，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92％、93％、94％、95％、96％、97％、98％、99％的抗LAG-3抗体单体，则药物制剂视为是稳定的。若储存于约5℃9个月之后，检测到至少约92％、93％、94％、95％、96％、97％、98％、99％的抗LAG-3抗体单体，则药物制剂视为是稳定的。
经一段储存时间后，如果制剂中的抗体不显示显著的化学改变，则可以认为抗体在制剂中“保持其化学稳定性”。大多数化学不稳定性源自于形成了抗体的共价修饰形式(例如，抗体的电荷变异体)。例如由天冬氨酸异构化、N和C末端修饰，可以形成碱性变异体；由脱酰胺化、唾液酸化和糖化，可以产生酸性变异体。化学稳定性可以通过检测和/或定量抗体的化学改变形式来评估。例如，可以通过阳离子交换色谱(CEX)或成像毛细管等电聚焦电泳(iCIEF)检测制剂中抗体的电荷变异体。在一个实施方案中，通过测定在特定温度下储存特定时间之后制剂中抗体的电荷变异体百分比变化值来测量制剂的稳定性，其中该变化值越小，则制剂的稳定性越高。
“可接受程度”的化学稳定性可以表示于特定温度下储存特定时间之后制剂中电荷变异体(例如主成分或酸性组分或碱性组分)的百分比变化值不超过30％，例如20％。在一些实施方案中，在特定温度储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个 月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月或更久后，可接受程度的化学稳定性可以表现为酸性组分电荷变异体的百分比变化值不超过约25％、20％、15％、10％、5％、4％、3％、2％、或1％。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，酸性组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，酸性组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，酸性组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％，则药物制剂亦可被视为是稳定的。
术语“冻干制剂”是指通过液体制剂的冷冻干燥处理得到或能够得到的组合物。优选地，其为具有少于5％、优选少于3％水含量的固体组合物。
术语“重构制剂”是指将固体制剂(例如冻干制剂)溶解和/或悬浮于生理可接受的溶液中得到的液体制剂。
文中使用的术语“室温”是指15℃至30℃、优选20℃至27℃、更优选25℃的温度。
“胁迫条件”是指在化学和/或物理上不利于抗体蛋白的环境，所述环境可以导致不可接受的抗体蛋白失稳定。“高温胁迫”是指，将抗体制剂置于室温或甚至于更高温度(例如40℃±2℃)储存一段时间。通过高温胁迫加速试验，可以检查抗体制剂的稳定性。
如本文所使用，术语“肠胃外施用”意指肠内和局部给药以外的给药方式，通常通过注射或输注方式，并且包括但不限于，静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下(subcuticular)、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射以及输注。在一些实施方案中，本发明的稳定抗LAG-3抗体制剂肠胃外施用于受试者。在一个实施方案中，本发明的抗LAG-3抗体制剂以皮下、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)重组全人源抗LAG-3单克隆抗体；(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5.5-7.5。在一个优选方案中，本发明的液体抗体制剂是注射制剂形式。
(i)抗LAG-3抗体
“抗LAG-3抗体”是指这样的抗体，所述抗体能够以足够的亲和力结合LAG-3分子，以致所述抗体可以用作靶向LAG-3分子的治疗剂和/或预防剂。
本发明的抗体是重组全人源抗体。术语“全人源抗体”或“人抗体”在本文中可以互换使用，指该抗体包括其中构架区和CDR区二者均源自人种系免疫球蛋白序列的可变区，而且如果抗体含有恒定区，恒定区也源自人种系免疫球蛋白序列。本发明的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸(例如，通过体外随机或点特异诱变或体内体细胞突变引入的突变)。然而，如本文所使用的，术语“人抗体”不意欲包括其中的CDR序列衍生自其他哺乳动物物种(如，小鼠)的 种系而移植入人构架序列的抗体。如本文所用，术语“重组人抗体”包括所有通过重组方式制备、表达、产生或分离的人抗体。这些重组人抗体具有构架区和CDR区源自人种系免疫球蛋白序列的可变区。然而，在某些实施方案中，可以对重组人抗体进行体外诱变(或使用人Ig序列转基因动物时为体内体细胞诱变)，由此得到重组抗体的VH和VL区的氨基酸序列，尽管所述重组抗体的VH和VL区的氨基酸序列源自人种系VH和VL序列并与之相关、但是并不天然存在于体内的人抗体种系库中。
在一些实施方案中，如通过生物光干涉法测量，所述抗LAG-3抗体能够以高的亲和力，例如以10 -7M或更小、优选地以10 -9M至10 -10M的K D特异性结合人LAG-3，并由此介导对LAG-3及其配体结合的高效阻断作用。
在一些实施方案中，本发明抗体LAG-3抗体包含：SEQ ID NO:7或与之具有至少90％同一性的重链可变区(VH)；和SEQ ID NO:8或与之具有至少90％同一性的轻链可变区(VL)。“可变区”或“可变结构域”是抗体的重链或轻链中参与抗体与其抗原的结合的结构域。一般，重链可变区(VH)和轻链可变区(VL)可以进一步再划分为高变区(HVR，又称作互补决定区(CDR))，其间插有较保守的区域(即，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。
在一些实施方案中，本发明的抗LAG-3抗体包含SEQ ID NO:7的重链可变区的VH CDR1、2和3序列和SEQ ID NO：8的轻链可变区的VL CDR1、2和3序列。“互补决定区”或“CDR区”或“CDR”(在本文中与超变区“HVR”可以互换使用)，是抗体可变区中主要负责与抗原表位结合的氨基酸区域。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作VH CDR1、VH CDR2和VH CDR3，而位于抗体轻链可变结构域内的CDR被称作VL CDR1、VL CDR2和VL CDR3。本领域公知多种用于在一个给定的VH或VL氨基酸序列中确定其CDR序列的方案。例如，Kabat互补决定区(CDR)是基于序列变异性确定的并且是最常用的(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))。而Chothia指的是结构环的位置(Chothia和Lesk，J.Mol.Biol.196:901-917(1987))。AbM HVR是Kabat HVR和Chothia结构环之间的折中，并且由Oxford Molecular的AbM抗体建模软件使用。“接触性”(Contact)HVR基于对可获得的复杂晶体结构的分析。HVR也可以基于与参考CDR序列(例如本文公开的示例性CDR)具有相同的Kabat编号位置而确定。在一个实施方案中，本发明抗LAG-3抗体具有SEQ ID NO：1的VH CDR1，SEQ ID NO:2的VH CDR2，SEQ ID NO:3的VH CDR3；和SEQ ID NO:4的VL CDR1，SEQ ID NO:5的VL CDR2和SEQ ID NO:6的VL CDR3。
在一些实施方案中，本发明的抗LAG-3抗体可以包含与SEQ ID NO:7具有至少90％,95％,98％或99％或更高同一性的重链可变区(VH)；和/或与SEQ ID NO:8具有至少90％,95％,98％或99％或更高同一性的轻链可变区(VL)。在本文中，“序列同一性”是指在比较窗中以逐个核苷酸或逐个氨基酸为基础的序列相同的程度。可以通过以下方式计算“序列同一性百分比”：将两条最佳比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100， 以产生序列同一性百分比。为了确定序列同一性百分数而进行的最佳比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。
在一些实施方案中，本发明抗体的VH序列与SEQ ID NO：7相比具有不超过10个，优选地不超过5个、4个或3个不同残基，优选地所述不同残基为保守氨基酸替代。在一些实施方案中，本发明抗体的VL序列与SEQ ID NO:8相比具有不超过10个，优选地不超过5个、4个或3个不同残基，优选地所述不同残基为保守氨基酸替代。“保守性取代”是指导致某个氨基酸置换为化学上相似的氨基酸的氨基酸改变。提供功能上相似氨基酸的保守性置换表是本领域熟知的。在本发明任一实施方案中，在一个优选的方面，保守取代残基来自以下的保守替代表A，优选地为表A中所示优选置换残基。
表A
原始残基 示例性取代 优选的保守氨基酸取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp；Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；正亮氨酸 Leu
Leu(L) 正亮氨酸；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸 Leu
在一些实施方案中，本发明的抗体是IgG4形式的抗体。“IgG形式的抗体”是指抗体的重链恒定区所属于的IgG形式。所有同一型的抗体的重链恒定区都是相同的，不同型的抗体之间的重链恒定区不同。例如，IgG4形式的抗体是指其重链恒定区Ig结构域为IgG4的Ig结构域。
在一个优选的实施方案中，本发明的抗LAG-3抗体是中国申请CN201811561512.X(2018年12月19日)中公开的抗LAG-3单克隆抗体ADI-31853，其具有SEQ ID NO:9的重链和SEQ ID NO:10的轻链。在一个实施方案中，该抗LAG-3抗体是由293细胞或CHO细胞重组表达产生并经纯化的IgG4型抗体。优选地，在本发明液体制剂中所述抗体表现出显著的抗肿瘤活性。例如，在接种了小鼠结肠癌细胞CT26(ATCC#CRL-2638)或人的皮肤癌细胞A375(ATCC# CRL-1619)的小鼠肿瘤模型中，施用本发明的抗体制剂可以导致肿瘤生长抑制率达到约50％或更高，例如100％；和/或肿瘤消失率达到60％以上。
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1-100mg/ml，优选地为约5-50mg/ml，例如约5、10、15、20、25、30、35、40、45或50mg/ml抗LAG-3抗体。
(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5.5-7.5的pH范围，例如约6.0-7.0的pH，优选地6.0±0.3的pH。在一个具体的实施方案中，本发明的抗体制剂具有约5.5、6.0、6.5、7.0或7.5的pH，优选地约6.0的pH。
在一些实施方案中，用于本发明制剂中的缓冲剂选自柠檬酸盐、柠檬酸盐溶剂合物(例如，柠檬酸盐水合物)和它们的组合，例如，柠檬酸钠、二水柠檬酸钠和它们的组合；优选地，所述缓冲剂的浓度为约1-10mg/ml，优选地为约4-8mg/ml，例如，约4、4.5、5、5.5、6、6.5、7、7.5、8mg/ml。
在一个实施方案中，用于本发明制剂中的缓冲剂是约1-10mg/ml的柠檬酸钠或二水柠檬酸钠，例如，约2-8mg/ml柠檬酸钠或二水柠檬酸钠。
(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和海藻糖。对于作为稳定剂的多元醇包括但不限于山梨醇。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。在一些实施方案中，所述稳定剂在本发明的液体制剂中以约10-150mg/ml，更优选地约15-100mg/ml，例如，约15、20、30、40、50、60、70、80、90、100mg/ml的浓度存在。
在一个实施方案中，本发明液体制剂包含蔗糖作为稳定剂。蔗糖在本发明液体制剂中的量可以是约20-100mg/ml，优选地约40-90mg/ml(例如，约40、50、60、70、80、90mg/ml)。
在一个实施方案中，本发明液体制剂包含精氨酸和/或盐酸精氨酸作为稳定剂。精氨酸和/或盐酸精氨酸在本发明液体制剂中的量可以是约10-40mg/ml，优选地约15-25mg/ml(例如，约15、16、17、18、19、20、21、22、23、24、25mg/ml)。
在一个实施方案中，本发明液体制剂包含蔗糖、精氨酸和/或盐酸精氨酸的组合作为稳定剂。该组合中，蔗糖可以以约20-100mg/ml，优选地约40-90mg/ml(例如，约40、50、60、70、80、90mg/ml)的量存在。在该组合中，精氨酸和/或盐酸精氨酸可以以约10-40mg/ml，优选地约15-25mg/ml(例如，约15、16、17、18、19、20、21、22、23、24、25mg/ml)的量存在。
(iv)表面活性剂
如本文所使用的，术语“表面活性剂”是指具有两亲结构的有机物质；即，它们由相反的溶解性倾向的基团所组成，通常是油溶性的烃链和水溶性的离子基团。
在一个实施方案中，本发明的液体制剂中的表面活性剂是非离子型表面活性剂，例如，烷基聚(环氧乙烯)。可包括在本发明制剂中的特定非离子型表面活性剂包括，例如聚山梨酯，诸如聚山梨酯-20、聚山梨酯-80、聚山梨酯-60、或聚山梨酯-40；普洛尼克等。在一个优选实施方案中，本发明的液体制剂中包含聚山梨酯-80作为表面活性剂。
本发明抗体制剂中所含的表面活性剂的量可随制剂的特定目的特性、特定环 境、和使用制剂的特定目的而改变。在优选的一些实施方案中，制剂可含有约0.1-1mg/ml，优选地约0.2-0.8mg/ml，例如约0.2、0.3、0.4、0.5、0.6、0.7、0.8mg/ml的聚山梨酯类表面活性剂(例如，聚山梨酯-80)。
(v)其它赋形剂
本发明的抗体液体制剂中可以包含或不包含其它赋形剂。
在一些实施方案中，本发明的抗体液体制剂可以包含或可以不包含依地酸盐(例如，依地酸二钠)和/或甲硫氨酸。
在一个实施方案中，本发明的抗体液体制剂可以包含或不包含依地酸盐(例如，依地酸二钠)。
在一个实施方案中，本发明的抗体液体制剂可以包含或不包含甲硫氨酸。
在一个实施方案中，本发明的抗体液体制剂不包含依地酸盐(例如，依地酸二钠)和/或甲硫氨酸，其与添加依地酸盐(例如，依地酸二钠)和/或甲硫氨酸的相应制剂相比较，具有类似的稳定性。
出于其他考虑，也可在本发明制剂中使用其它的赋形剂。所述赋形剂包括，例如，调味剂、抗微生物剂、甜味剂、抗静电剂、抗氧化剂、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)”。
II.制剂的制备
本发明提供包含抗体的稳定制剂。在本发明制剂中使用的抗体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；(4)病毒过滤(使病毒滴度降低例如4log10以上)；(5)超滤/渗滤(可以用于将蛋白置换于利于其稳定的制剂缓冲液中并浓缩至合适的浓度供注射用)。参见例如，B.Minow,P.Rogge,K.Thompson,BioProcess International,Vol.10,No.6,2012,pp.48–57。
III.制剂的分析方法
在抗体制剂的储存过程中，抗体可能会发生聚集、降解或化学修饰，导致抗体异质性(包括大小异质性和电荷异质性)以及聚集物和片段等，从而影响抗体制剂的质量。因此，有必要进行抗体制剂稳定性的监测。
在本领域中已知多种方法可以用于检测抗体制剂的稳定性。例如，可以通过非还原型CE-SDS和SEC-HPLC等方法，分析抗体制剂的纯度和评估抗体的聚集水平；可以通过毛细管等电聚焦电泳(cIEF)、成像毛细管等电聚焦电泳(iCIEF)和离子交换色谱(IEX)等，分析抗体制剂中的电荷变异体。此外，可以通过目视检测制剂外观，快速地判断制剂的稳定性。也可以使用OD 350nm法检测制剂的浊度改变，该方法可以给出有关可溶性和不溶性聚集物量的信息。此外，可以使用紫外分光光度法(UV法)检测制剂中的蛋白质含量变化。
非还原型CE-SDS法是一种以毛细管为分离通道进行的单克隆抗体纯度测定方法。在CE-SDS中，蛋白迁移由SDS结合引起的表面电荷来驱动，而该表面电荷与蛋白质的分子量成正比。由于所有的SDS-蛋白质复合物都具有相似的质量-电荷比，故可以在毛细管的分子筛凝胶基质中，实现基于分子的大小或流体动力学半径的电泳分离。该方法已经被广泛地用于监测变性的完整抗体的纯度。一般，在非还原CE-SDS法中，供试样品与SDS样品缓冲液和碘乙酰胺混合。之后，混合物可以于68-72℃孵育约10-15分钟，冷却至室温后离心的上清液用于分析。采用紫外检测器检测蛋白的迁移，获得电泳谱图。抗体制剂纯度可以计算为IgG主峰的峰面积占所有峰面积之和的百分比。关于CE-SDS法的进一步描述，可以参见例如Richard R.等,Application of CE SDS gel in development of biopharmaceutical antibody-based products,Electrophoresis,2008,29,3612-3620。
尺寸排阻高效液相色谱法，即SEC-HPLC法，是用于单克隆抗体标准和质控的另一重要方法。该方法主要依据分子的尺寸大小或流体动力学半径差异来进行分子的分离。通过SEC-HPLC，抗体可以分离出三种主要形式：高分子量形式(HMMS)、主峰(主要是抗体单体)、和低分子量形式(LMMS)。抗体纯度可以计算为色谱图上主峰面积占所有峰面积之和的百分比。通过SEC-HPLC法，可以测量制剂产品中抗体单体的百分数，给出可溶性聚集物和剪切物的含量信息。关于SEC-HPLC法的进一步描述，可以参见例如，J.Pharm.Scien.,83:1645-1650,(1994)；Pharm.Res.,11:485(1994)；J.Pharm.Bio.Anal.,15:1928(1997)；J.Pharm.Bio.Anal.,14:1133-1140(1986)。此外，也可以参见例如，R.Yang等，High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography(SE-UHPLC),Journal of Pharmaceutical and Biomedical Analysis(2015),http://dx.doi.org/10.1016/j.jpba.2015.02.032；和Alexandre Goyon等,Protocols for the analytical characterization of therapeutic monoclonal antibodies.I－Non-denaturing chromatographic techniques,Journal of Chromatography，http://dx.doi.org/10.1016/j.jchromb.2017.05.010。
成像毛细管等电聚焦电泳(iCIEF)可以用于分析单克隆抗体的电荷异质性。该方法可以提供电荷变异体的定量分布情况。iCIEF基于分子在pH梯度中的电荷差异(表观pI值)来实现分子分离的目的。在iCIEF中，分离柱通常是短毛细管(例如,5cm长,100μm内径的二氧化硅毛细管)，蛋白质在高电压下在毛细管柱中聚焦，并通过在280nM操作的全柱成像检测系统对聚焦进行实时在线监测。该技术的一个优点是，可以通过该全柱检测系统同时记录抗体样品的各种电荷变异体。一般而言，在icIEF中，将样品与尿素和icIEF缓冲液混合，其中所述缓冲液含有甲基纤维素、pI分子量标准和ampholytes。然后，可以在iCIEF分析仪例如iCE280分析仪(Protein  Simple,Santa Clara,CA)上，使用iCIEF柱例如ProtionSimple组装的iCIEF柱，在样品聚焦一定时间后，测定280nm的吸光度，获得聚焦mAb电荷变异体的谱图。在iCEIF谱图中，在主峰(即主成分)之前洗脱的蛋白相关峰被分类为酸性组分；相对地，在主峰之后洗脱的蛋白相关峰被分类为碱性组分。主成分、酸性组分和碱性组分的相对量可以表示为占总峰面积的百分数。关于iCIEF的进一步描述，可以参见例如，Salas-Solano O等,Robustness of iCIEF methodology for the analysis of monoclonal antibodies:an interlaboratory study,J Sep Sci.2012 Nov；35(22):3124-9.doi:10.1002/jssc.201200633.Epub 2012 Oct 15；和Dada OO等,Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation,Electrophoresis.2015Nov；36(21-22):2695-2702.doi:10.1002/elps.201500219.Epub 2015 Sep 18.
也可以通过阳离子交换高效液相色谱法(CEX-HPLC)测定抗体制剂中抗体的电荷变异体。在该测定法中，以比主峰的保留时间更早从CEX-HPLC柱洗脱出的峰被标记为“酸性峰”，而那些以比主峰的保留时间更晚从CEX-HPLC柱洗脱出的峰被标记为“碱性峰”。
加速稳定性研究可以用于检查产品的稳定性性质，有利于筛选稳定药物制剂形式。例如，可以将制剂样品放置于升高的温度，例如约40℃±2℃、25℃±2℃条件下进行加速稳定性研究。检测指标可以包括外观、可见异物、蛋白含量、浊度、纯度(SEC-HPLC法、非还原型CE-SDS法)和电荷变异体(iCIEF法)。
可以进行振荡试验，考察制剂的振荡/剪切稳定性。例如，将制剂样品分装至西林瓶，加塞轧盖后放样，进行振荡试验，例如650r/min振荡3-5天，之后检测制剂的外观、蛋白含量、浊度和纯度。
此外，可以检测抗体的功效或生物活性。例如，可以检测制剂中抗体与其抗原的结合能力。本领域技术人员已知多种方法可以用于定量抗体与抗原的特异性结合，例如免疫测定试验，ELISA等。
本发明的抗LAG-3抗体制剂是稳定的。在一个实施方案中，于约25℃、37℃、40℃、或45℃储存至少1个月或2个月后，例如，在40℃±2℃储存1个月后，本发明的抗体制剂中的抗LAG-3抗体纯度是至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％以上，如通过尺寸排阻色谱法或通过非还原型CE-SDS所测定。在一个实施方案中，于约25℃、37℃、40℃、或45℃储存至少1个月或2个月后，例如，在40℃±2℃储存1个月后，本发明的抗体制剂中抗LAG-3抗体的至少50％，优选至少55％是非碱性及非酸性形式(亦即，主峰或主要电荷形式)，如通过成像毛细管聚焦电泳法所测定。
IV.制剂的用途
本发明的包含抗LAG-3抗体的本发明的抗体制剂可以用于治疗、改善或预防LAG-3相关的多种疾病或病症。“LAG-3相关的疾病或病症”在本文中指可以用本发明抗LAG-3抗体制剂进行治疗(例如改善)或预防的疾病或病症。任何可以得益于本发明抗体制剂治疗的疾病或病症都适用于本发明。
在一个方面，包含抗LAG-3抗体的本发明制剂能够用于调节受试者中的免疫反应，尤其是用于恢复、增强、刺激或增加受试者中的免疫反应。在一些实施方案中，包含抗LAG-3抗体的本发明制剂能够用于恢复、增强或刺激受试者中的抗原特异性T细胞反应，例如，抗原特异性T细胞反应中的白介素-2(IL-2)或干扰素-γ(IFN-γ)产生。
在另一方面，包含抗LAG-3抗体的本发明制剂能够用于预防或治疗受试者的肿瘤，例如癌症包括但不限于实体瘤、血液学癌(例如，白血病、淋巴瘤、骨髓瘤) 及其转移性病灶。在一个实施方案中，癌症是实体瘤。实体瘤的例子包括恶性肿瘤，例如，多个器官系统的肉瘤和癌(例如，腺癌)，如侵袭肺、乳腺、淋巴、胃肠道或结直肠、生殖器和生殖泌尿道(例如，肾细胞、膀胱细胞、膀胱细胞)、咽、CNS(例如，脑细胞、神经细胞或神经胶质细胞)、皮肤(例如，黑素瘤)、头部和颈部(例如，头颈鳞状细胞癌(HNCC))和胰的那些。例如，黑素瘤、结肠癌、胃癌、直肠癌、肾细胞癌、乳腺癌(例如，不表达一种、两种或全部雌激素受体、孕酮受体或Her2/neu的乳腺癌，例如，三阴性乳腺癌)、肝癌、肺癌(例如，非小细胞肺癌(NSCLC)(例如，具有鳞状和/或非鳞状结构的NSCLC)或小细胞肝癌)、前列腺癌、头部或颈部癌(例如，HPV+鳞状细胞癌)、小肠癌和食道癌。血液学癌的例子包括但不限于白血病(例如，髓样白血病、淋巴样白血病或慢性淋巴细胞白血病(CLL))、淋巴瘤(例如，霍奇金淋巴瘤(HL)、非霍奇金淋巴瘤(NHL)、弥漫性大B细胞淋巴瘤(DLBCL)、T细胞淋巴瘤或套细胞淋巴瘤(MCL))和骨髓瘤，例如，多发性骨髓瘤。癌症可以处于早期、中期或晚期或是转移性癌。在一个实施方案中，肿瘤是胃肠道肿瘤，例如结肠癌等。
在另一方面，包含抗LAG-3抗体的本发明制剂能够用于预防或治疗受试者的感染性疾病。在一些实施方案中，感染性疾病是慢性感染，例如，所述慢性感染是由于选自细菌、病毒、真菌和原生动物的病原体导致的。
在另一方面，包含抗LAG-3抗体的本发明制剂能够用于在受试者中引起抗体依赖性细胞介导的细胞毒性。
本发明也提供本发明的制剂在制备药物中的用途，其中所述药物用于向哺乳动物递送抗LAG-3抗体，或用于治疗、预防或改善上述疾病和病症中的一种或多种。优选地，哺乳动物是人。
可以以多种途径将本发明的抗体制剂施用于受试者或患者。例如，施用可以通过输注或通过注射器进行。因此，在一个方面，本发明提供了一种递送装置(例如注射器)，其包含本发明的抗体制剂(例如，预填装注射器)。患者将接受有效量的抗LAG-3抗体作为主要活性成分，即足以治疗、改善或预防目的疾病或病症的量。
治疗效果可包括减少生理症状。用于任何特定受试者的抗体的最佳有效量和浓度将取决于多种因素，包括患者的年龄、体重、健康状况和/或性别、疾病的性质和程度、特定抗体的活性，身体对其清除率，并且也包括与所述抗体制剂组合施用的任何可能的其它治疗。对于具体的情况，所递送的有效量可以在临床医师的判断范围内来确定。取决于待治疗的适应症，有效剂量可为约0.005mg/kg体重至约50mg/kg体重，或约0.1mg/kg体重至约20mg/kg体重。在这方面，已知的基于抗体的药物的应用可以提供一定的指导。剂量可以是单剂量方案或多剂量方案。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
缩略词描述
CE-SDS：十二烷基硫酸钠毛细管凝胶电泳
ELISA：酶联免疫吸附测定法
FLD：荧光检测器
HPLC：高效液相色谱法
iCIEF：成像毛细管等电聚焦电泳
SEC-HPLC：尺寸排阻高效液相色谱法
实施例
为了开发出重组全人源抗淋巴细胞活化基因3(LAG-3)单克隆抗体注射液长期稳定储存的制剂处方，确保产品在有效期内(至少24个月)的质量可控，设计了处方筛选试验，考察了不同辅料对LAG-3抗体制剂稳定性的影响。试验所用材料和方法如下：
材料和方法
1.1.本发明的制剂研究中使用的材料
Figure PCTCN2020098140-appb-000004
注：N/A表示“不适用”(Not applicable)。
1.2.本发明的制剂研究中使用的仪器设备
Figure PCTCN2020098140-appb-000005
1.3.制剂稳定性的检测项目和检测方法
对抗体制剂检测了以下项目：(1)检测外观以及是否存在可见异物；(2) 通过紫外法(UV法)测定制剂中的蛋白质含量；(3)通过OD 350nm法检测制剂的浊度；(4)通过尺寸排阻色谱法，例如，尺寸排阻高效液相色谱法(size-exclusion chromatography-HPLC；SEC-HPLC)测定抗体制剂的纯度，表示为单体的面积占所有峰面积之和的百分数；(5)通过还原型十二烷基硫酸钠毛细管电泳(还原型CE-SDS)和/或非还原型十二烷基硫酸钠毛细管电泳(非还原型CE-SDS)测定抗体制剂的纯度，表示为单体的面积占所有峰面积之和的百分数；(6)通过成像毛细管等电聚焦电泳法(iCIEF法)测定抗体制剂中电荷变异体，表示为主成分、酸性组分和碱性组分的百分数；(7)通过免疫测定法，例如，直接ELISA法测定抗体制剂中抗LAG-3抗体的相对结合活性。
可见异物检测
按照国家药典委员会，中华人民共和国药典(2015年版，四部通则0904“可见异物检查法”).北京:中国医药科技出版社.2015中所记载的方法，采用澄明度检测仪(天津天大天发生产，型号YB-2)，检查样品中的可见异物。
蛋白含量测定
使用紫外分光光度计(日本岛津生产，型号UV-1800)测定样品中的蛋白质含量。
浊度测定
使用紫外分光光度计(日本岛津生产，型号UV-1800)，测定样品在350nm的吸光度，确定样品浊度。
纯度(SEC-HPLC法)
使用体积排阻色谱柱分离，流动相为磷酸盐缓冲液(称取3.12g二水合磷酸二氢钠，8.77g氯化钠和34.84g精氨酸，超纯水溶解后用盐酸调节pH至6.8并定容至1000ml)，色谱柱保护液为0.05％(w/v)NaN 3，进样量50μl，流速0.5ml/分钟，采集时间30分钟，柱温25℃，检测波长280nm。取待测样品用超纯水稀释至2mg/ml，作为供试品溶液。取制剂缓冲液用上述相同处理方式稀释后做为空白溶液。取空白溶液、供试品溶液各50μl注入液相色谱仪，开始检测。
纯度(还原型CE-SDS法)
采用毛细管凝胶电泳法检测。毛细管为无涂层毛细管，内径50μm，总长30.2cm，有效长度20.2cm。电泳前分别使用0.1mol/L氢氧化钠、0.1mol/L盐酸、超纯水、电泳胶70psi冲洗毛细管柱。将待测样品用适量超纯水稀释至2.0mg/ml，取以上稀释后的样品50μl于1.5ml离心管中，分别向其中加入45μl pH 6.5的样品缓冲液(称取一水柠檬酸0.32g，十二水合磷酸氢二钠2.45g，溶于45ml超纯水中，定容至50ml，制得柠檬酸-磷酸盐缓冲液，精密量取该缓冲液200μl，加10％(w/v)十二烷基硫酸钠溶液80μl，加水至1ml，混匀，即得)、1μl内标(10kDa蛋白质，5mg/mL)(Beckman Coulter，货号：390953)和5μl β-巯基乙醇，充分混匀后70±2℃加热10±2分钟，冷却至室温后转移至样品瓶作为供试品溶液。取与供试品相同体积的制剂缓冲液，按上述方法同样操作，制得空白溶液。样品进样条件：-5kV 20秒；分离电压：-15kV 35分钟。毛细管柱温控制在25℃，检测波长为220nm。
纯度(非还原型CE-SDS法)
采用毛细管凝胶电泳法检测。毛细管为无涂层毛细管，内径50μm，总长30.2cm，有效长度20.2cm。电泳前分别使用0.1mol/L氢氧化钠、0.1mol/L盐酸、超纯水、电泳胶70psi冲洗毛细管柱。将待测样品用适量超纯水稀释至2.0mg/ml，取以上稀释后的样品50μl于1.5ml离心管中，分别向其中加入45μl pH 6.5的样 品缓冲液(称取一水柠檬酸0.32g，十二水合磷酸氢二钠2.45g，溶于45ml超纯水中，定容至50ml，制得柠檬酸-磷酸盐缓冲液，精密量取该缓冲液200μl，加10％(w/v)十二烷基硫酸钠溶液80μl，加水至1ml，混匀，即得)、1μl内标(10kDa蛋白质，5mg/mL)(Beckman Coulter，货号：390953)和5μl 250mmol/L NEM溶液(称取N-乙基顺丁稀二酰亚胺62mg，溶于2ml超纯水中)，充分混匀后70±2℃加热10±2分钟，冷却至室温后转移至样品瓶作为供试品溶液。取与供试品相同体积的制剂缓冲液，按上述方法同样操作，制得空白溶液。样品进样条件：-5kV20秒；分离电压：-15kV 35分钟。毛细管柱温控制在25℃，检测波长为220nm。
电荷变异体(iCIEF法)
采用成像毛细管等电聚焦电泳(iCIEF法)检测。毛细管内径100μm，总长5cm。样品电泳前需分别使用0.5％甲基纤维素溶液(下文中也缩写为MC溶液)、超纯水冲洗毛细管柱。采用真空进样方法进样55秒，预聚焦电压及时间为1.5kV 1分钟，聚焦电压及时间为3kV 8分钟，进样时间55秒，样品盘温度为10℃，毛细管柱温为室温，检测波长为280nm。阴极稳定剂(Cathodic Stabilizer)为500mmol/L精氨酸溶液，阳极稳定剂(Anodic Stabilizer)为200mmol/L亚氨基乙二酸，3mol/L尿素提高蛋白溶解性，0.5％MC溶液降低蛋白与毛细管之间的粘附。将供试品用水稀释至0.5mg/ml，取上述稀释后的供试品溶液20μl，向其中加入83μl预混液充分混匀制得待测样品溶液。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)
用1×PBS稀释链亲和素(Thermo，货号：21125)至1μg/ml，100μl/孔，37℃2h包被于96孔酶标板上。洗板后加封闭液(5％FBS，300μl/孔)37℃封闭2h。用1×PBS稀释生物素化的LAG-3(Sino biological，货号：16498-HNAH-B)至0.5μg/ml，100μl/孔，37℃0.5h包被于96孔酶标板上。100μl/孔以2％FBS稀释抗LAG-3抗体至40μg/ml，4倍梯度稀释至第12个浓度(0.01～10000ng/ml)。将梯度稀释的供试品以100μl/孔加入到洗过的酶标板中，37℃恒温培养箱中孵育30min。洗板后加入以2％FBS稀释的HRP缀合山羊抗人IgG-Fc片段(美国BETHYL，货号A80-104P)作为二抗(30000倍稀释，100μl/孔)37℃反应20min。洗板后加入100μl TMB显色液，显色10min后，每孔加入100μl的1mol/L H 2SO 4终止反应。以620nm为参比波长，测450nm处的OD值。以各浓度梯度样品的浓度值作为横坐标，各梯度样品的OD 450nm-OD 620nm值为纵坐标，应用Prism四参数拟合计算EC 50反映抗LAG-3抗体与LAG-3的结合活性。
实施例1.制备和纯化抗LAG-3抗体
根据CN201811561512.X所述制备和纯化了特异性结合LAG-3的新型抗LAG-3抗体ADI-31853，该抗体由SEQ ID NO：9的重链序列和SEQ ID NO：10的轻链序列组成，为IgG4型抗体。简言之，抗LAG-3抗体ADI-31853在293细胞或CHO细胞中重组表达并纯化。对于用于处方筛选试验的样品，通过CEX(阳离子交换色谱)纯化，样品分别具有约11.5mg/ml至约25.0mg/ml的蛋白含量。
实施例2.pH对制剂的稳定性影响试验
本实施例考察了包含抗LAG-3抗体的制剂在pH 5.0至8.0的稳定性。
2.1 pH影响试验步骤：
共设计了7个pH值，详细信息见表1。配制各个处方的缓冲液，将抗LAG-3抗体蛋白超滤置换至各处方溶液中。置换后，将各处方蛋白含量调整至约20mg/ml， 并加入聚山梨酯80。过滤分装至西林瓶中，加塞、轧盖，进行高温胁迫稳定性考察，通过SEC-HPLC法检测抗LAG-3抗体蛋白纯度，并通过iCIEF法检测电荷变异体。
表1处方信息表
序号 处方信息
处方A 20mM组氨酸，5％(w/v)山梨醇，0.20mg/ml聚山梨酯80，pH 5.0
处方B 20mM组氨酸，5％(w/v)山梨醇，0.20mg/ml聚山梨酯80，pH 5.5
处方C 20mM组氨酸，5％(w/v)山梨醇，0.20mg/ml聚山梨酯80，pH 6.0
处方D 20mM组氨酸，5％(w/v)山梨醇，0.20mg/ml聚山梨酯80，pH 6.5
处方E 20mM组氨酸，5％(w/v)山梨醇，0.20mg/ml聚山梨酯80，pH 7.0
处方F 20mM组氨酸，5％(w/v)山梨醇，0.20mg/ml聚山梨酯80，pH 7.5
处方G 20mM组氨酸，5％(w/v)山梨醇，0.20mg/ml聚山梨酯80，pH 8.0
详细试验条件及取样计划见表2。
表2.试验条件及取样表
Figure PCTCN2020098140-appb-000006
2.2 判断标准
根据对产品的认识以及仪器和方法的精密度，设定了样品检测指标数值与初始值相比质量未发生变化的判定标准，用以判断样品是否发生了变化，具体见表3。
表3.质量未发生变化的判断标准
Figure PCTCN2020098140-appb-000007
2.3 pH影响试验的结果
pH对制剂的稳定性影响试验结果见表4，通过SEC-HPLC法检测的抗LAG-3抗体蛋白纯度的变化趋势图见图1，通过iCIEF法检测的抗LAG-3抗体蛋白各电荷变异体的变化趋势图见图2。由表4、图1和图2可见，各处方在40℃±2℃条件下放置一个月后，抗LAG-3抗体在pH 5.5、pH 6.0、pH 6.5、pH 7.0、pH 7.5的液体中较为稳定，即该制剂可在pH 5.5-7.5范围内实施。因此，在后续实验中，采用pH 6.0进行LAG 3抗体制剂的处方设计。
Figure PCTCN2020098140-appb-000008
实施例3.处方筛选试验之一
3.1 处方筛选试验之一的步骤
共设计了7个处方，详细处方信息见表5。按照表5配制各个处方的缓冲液.。将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，然后进行稳定性考察。稳定性考察使用了包括高温胁迫试验、振荡试验、冻融试验和光照试验在内的试验方法。检测指标为外观、可见异物、蛋白含量(UV法)、浊度(OD 350nm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5.筛选试验之一中使用的处方信息表
Figure PCTCN2020098140-appb-000009
详细试验条件及取样计划见表6。
表6.试验条件及取样表
Figure PCTCN2020098140-appb-000010
3.2 判断标准
见上文2.2节，表3。
3.3 处方筛选试验之一的结果
(1)高温胁迫试验
通过高温胁迫试验筛选处方的结果详见表7、以及图3－图5。
在40℃±2℃条件下放置2周，仅处方3外观出现白色沉淀。处方1、2、4、5、6、7在外观、可见异物方面均合格；蛋白含量(UV法)和纯度(SEC-HPLC法和非还原型CE-SDS法)均未发生变化；电荷变异体-酸性组分(iCIEF法)增加；电荷变异体-碱性组分(iCIEF法)减少，其中处方5、处方6和处方7的电荷变异体-酸性组分上升幅度相对处方1、处方2、处方4而言较小；处方5和处方7的电荷变异体-主成分(iCIEF法)根据上表3的判定标准为未发生变化。
Figure PCTCN2020098140-appb-000011
(2)振荡试验
通过振荡试验筛选处方的结果详见表8。结果表明，在650转/分钟条件下振荡5天，处方1、处方2和处方4这3组处方外观、可见异物均合格；蛋白含量(UV法)、浊度(OD 350nm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)均未发生变化。
表8.处方筛选振荡试验结果
Figure PCTCN2020098140-appb-000012
注：N/A表示未设计考察。
(3)冻融试验
通过冻融试验筛选处方的结果详见表9。反复冻融6次后，处方1、处方2和 处方4这3组处方外观、可见异物均合格；蛋白含量(UV法)、浊度(OD 350nm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)均未发生变化。
表9.处方筛选冻融试验结果
Figure PCTCN2020098140-appb-000013
注：N/A表示未设计考察。
(4)光照试验
通过光照试验筛选处方的结果详见表10。在600Lux±50Lux照度下照射5天，处方1、处方2和处方4这3组处方外观、可见异物均合格；蛋白含量(UV法)、浊度(OD 350nm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)均未发生变化。
表10.处方筛选光照试验结果
Figure PCTCN2020098140-appb-000014
Figure PCTCN2020098140-appb-000015
注：N/A表示未设计考察。
结论
对处方1-处方7进行高温胁迫试验的结果显示，辅料蔗糖、精氨酸、海藻糖和山梨醇可用作抗LAG-3抗体的稳定剂，其中辅料蔗糖和精氨酸更优于海藻糖和山梨醇，能够更有效抑制蛋白电荷变异体-酸性组分的上升和主成分的下降。
使用处方1、处方2和处方4进行振荡试验、冻融试验和光照试验的结果显示，这3个处方均有良好的稳定性，且处方间无差异，因此，添加依地酸二钠或甲硫氨酸对产品稳定性影响不大。从处方简单化和安全性角度出发，处方中不添加依地酸二钠或甲硫氨酸。
实施例4.处方筛选试验之二
4.1 处方筛选试验之二的步骤
共设计了3个处方，详细处方信息见表11。配制各个处方的缓冲液，将抗LAG-3抗体ADI-31853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，包括高温胁迫试验、振荡试验和冻融试验。检测指标为外观、可见异物、蛋白含量(UV法)、浊度(OD 350nm法)、纯度(SEC-HPLC法、非还原型CE-SDS法和还原型CE-SDS法)，电荷变异体(iCIEF法)和相对结合活性(直接ELISA法)。
表11.处方筛选试验之二的处方信息表
Figure PCTCN2020098140-appb-000016
详细试验条件及取样计划见表12。
表12.试验条件及取样表
Figure PCTCN2020098140-appb-000017
4.2 判断标准
见上文2.2节，表3。
4.3 处方筛选试验之二结果
(1)高温胁迫试验
高温胁迫试验的结果详见表13和图6-图14。在40℃±2℃条件下放置1周，处方8、处方9、处方10的电荷变异体-酸性组分(iCIEF法)上升，碱性组分下降；在40℃±2℃条件下放置2周，处方9和处方10纯度(还原型CE-SDS法)下降，结合质谱检测结果表明主要是发生了糖化反应；处方10电荷变异体-主成分下降；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)、纯度(非还原型CE-SDS法)、相对结合活性(直接ELISA法)均合格；处方8纯度(还原型CE-SDS法和SEC-HPLC法)和处方9纯度(SEC-HPLC法)均未发生变化；处方8、处方9、处方10的浊度上升；处方8、处方9、处方10的电荷变异体-主成分下降，其中处方8下降幅度相对较小；处方10的纯度(SEC-HPLC法)下降，主要表现为聚合体增多。
表13.处方筛选之二的高温胁迫试验结果(40℃±2℃)
Figure PCTCN2020098140-appb-000018
Figure PCTCN2020098140-appb-000019
(2)振荡试验
处方筛选之二的振荡试验结果详见表14。以650转/分钟条件下振荡3天，处方8、处方9、处方10的外观、可见异物均合格；蛋白含量(UV法)、浊度(OD 350nm法)、纯度(非还原型CE-SDS法、还原型CE-SDS法和SEC-HPLC法)、电荷变异体(iCIEF法)和相对结合活性(直接ELISA法)均未发生变化。
表14.处方筛选之二的振荡试验结果
Figure PCTCN2020098140-appb-000020
Figure PCTCN2020098140-appb-000021
注：N/A表示未设计考察。
(3)冻融试验
处方筛选之二的冻融试验结果详见表15。反复冻融6次后，处方8、处方9、处方10的外观、可见异物均合格；蛋白含量(UV法)、浊度(OD 350nm法)、纯度(非还原型CE-SDS法、还原型CE-SDS法和SEC-HPLC法)、电荷变异体(iCIEF法)和相对结合活性(直接ELISA法)均未发生变化。
表15.处方筛选之二的冻融试验结果
Figure PCTCN2020098140-appb-000022
Figure PCTCN2020098140-appb-000023
注：N/A表示未设计考察。
结论
高温胁迫试验结果显示，从电荷变异体(iCIEF法)的检测结果看，处方8电荷变异体-酸性组分的上升与主成分的下降变化幅度相对较小；从纯度(SEC-HPLC法)的检测结果看，处方8和处方9更有优势，能够更有效抑制蛋白在40℃高温下聚合体的增多，从纯度(还原型CE-SDS法)的检测结果看，处方8更有优势，未在40℃高温下发生糖化反应。由此，确定最优选的制剂方案为：约20mg/ml重组全人源抗淋巴细胞活化基因3(LAG-3)单克隆抗体、约5.88mg/ml柠檬酸钠(二水)、约21.07mg/ml盐酸精氨酸、约0.70mg/ml聚山梨酯80，pH 6.0。
以上描述了本发明的示例性实施方案，本领域技术人员应当理解的是，这些公开内容仅是示例性的，在本发明的范围内可以进行各种其它替换、适应和修改。因此，本发明不限于文中列举的具体实施方案。

Claims (18)

  1. 一种具有pH约为5.5-7.5的液体抗体制剂，例如，pH约为6.0的液体抗体制剂，包含
    (i)抗LAG-3抗体；
    (ii)缓冲剂，
    (iii)稳定剂，和
    (iv)表面活性剂，
    其中，所述抗LAG-3抗体包含：
    -GSIYSESYYWG(SEQ ID NO:1)的重链VH CDR1；
    -SIVYSGYTYYNPSLKS(SEQ ID NO:2)的重链VH CDR2；
    -ARVRTWDAAFDI(SEQ ID NO:3)的重链VH CDR3；
    -QASQDISNYLN(SEQ ID NO:4)的轻链VL CDR1；
    -DASNLET(SEQ ID NO:5)的轻链VL CDR2；和
    -QQVLELPPWT(SEQ ID NO:6)的轻链VL CDR3。
  2. 权利要求1的液体抗体制剂，特征在于所述液体抗体制剂中的抗LAG-3抗体的浓度为约1-100mg/ml，优选地为约5-50mg/ml，例如约5、10、15、20、25、30、35、40、45或50mg/ml。
  3. 根据权利要求1或2所述的液体抗体制剂，特征在于所述液体抗体制剂中的缓冲剂选自柠檬酸盐、柠檬酸盐溶剂合物(例如，柠檬酸盐水合物)或它们的组合，例如，柠檬酸钠、二水柠檬酸钠或它们的组合；优选地，所述缓冲剂的浓度为约1-10mg/ml，优选地为约4-8mg/ml，例如，约4、4.5、5、5.5、6、6.5、7、7.5、8mg/ml。
  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山梨醇、蔗糖、海藻糖、精氨酸、盐酸精氨酸或它们的任意组合，更优选为蔗糖、精氨酸和/或盐酸精氨酸；优选地，所述稳定剂的浓度为约10-150mg/ml，更优选地为约15-100mg/ml，例如，约15、20、30、40、50、60、70、80、90、100mg/ml。
  5. 根据权利要求1-4中任何一项所述的液体抗体制剂，特征在于所述液体抗体制剂包含精氨酸和/或盐酸精氨酸作为稳定剂，优选地精氨酸和/或盐酸精氨酸以约10-40mg/ml，优选地约15-25mg/ml的量(例如，约15、16、17、18、19、20、21、22、23、24、25mg/ml的量)存在；和/或包含蔗糖作为稳定剂，优选地蔗糖以约20-100mg/ml，优选地约40-90mg/ml的量(例如，约40、50、60、70、80、90mg/ml的量)存在。
  6. 根据权利要求1-5中任何一项所述的液体抗体制剂，特征在于所述液体抗体制剂中的表面活性剂选自聚山梨酯类表面活性剂，优选为聚山梨酯-80。
  7. 根据权利要求1-6中任何一项所述的液体抗体制剂，特征在于所述表面活性剂的浓度为约0.1-1mg/ml，优选地为约0.2-0.8mg/ml，例如约0.2、0.3、0.4、0.5、0.6、0.7、0.8mg/ml。
  8. 根据权利要求1-7中任何一项所述的液体抗体制剂，特征在于所述液体制剂可以包含或可以不包含依地酸盐(例如，依地酸二钠)和/或甲硫氨酸。
  9. 根据权利要求1-8中任何一项所述的液体抗体制剂，特征在于所述抗LAG-3抗体包含重链可变区VH和轻链可变区VL，其中重链可变区包含SEQ ID NO:7的序列或与其具有至少90％,95％,98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:8的序列或与其具有至少90％,95％,98％或99％同一性的序列。
  10. 根据权利要求1-9中任何一项所述的液体抗体制剂，特征在于所述抗LAG-3抗体是IgG4型抗体，优选地包含SEQ ID NO:9或与之具有至少90％,95％,98％或99％同一性的重链序列以及SEQ ID NO：10或与之具有至少90％,95％,98％或99％同一性的轻链序列。
  11. 根据权利要求1-10中任何一项所述的液体抗体制剂，特征在于所述抗LAG-3抗体在293细胞或CHO细胞中重组表达。
  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。
  13. 根据权利要求1所述的液体抗体制剂，其包含：
    (i)约1-100mg/ml的抗LAG-3抗体；
    (ii)约1-10mg/ml的柠檬酸钠或二水柠檬酸钠；
    (iii)约10-150mg/ml蔗糖、精氨酸和/或盐酸精氨酸，和
    (iv)约0.1-1mg/ml聚山梨醇酯80；
    任选地，所述液体制剂不包含依地酸盐(例如，依地酸二钠)和甲硫氨酸；
    其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值；
    例如，所述液体抗体制剂包含
    (i)约10-30mg/ml的抗LAG-3抗体；
    (ii)约2-8mg/ml柠檬酸钠或二水柠檬酸钠；
    (iii)约10-40mg/ml精氨酸和/或盐酸精氨酸和/或40-90mg/ml蔗糖，和
    (iv)约0.2-0.8mg/ml聚山梨醇酯80；
    任选地，所述液体制剂不包含依地酸盐(例如，依地酸二钠)和甲硫氨酸；
    其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值；
    或者，所述液体抗体制剂包含
    (i)约20mg/ml的抗LAG-3抗体；
    (ii)约5.88mg/ml二水柠檬酸钠；
    (iii)约80mg/ml蔗糖，和
    (iv)约0.3mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值；
    或者，所述液体抗体制剂包含
    (i)约20mg/ml的抗LAG-3抗体；
    (ii)约5.88mg/ml二水柠檬酸钠；
    (iii)约17.42mg/ml精氨酸，和
    (iv)约0.3mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值；
    或者，所述液体抗体制剂包含
    (i)约20mg/ml的抗LAG-3抗体；
    (ii)约5.88mg/ml二水柠檬酸钠；
    (iii)约21.07mg/ml盐酸精氨酸，和
    (iv)约0.7mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值；
    或者，所述液体抗体制剂包含
    (i)约20mg/ml的抗LAG-3抗体；
    (ii)约5.88mg/ml二水柠檬酸钠；
    (iii)约50mg/ml蔗糖和约21.07mg/ml盐酸精氨酸，和
    (iv)约0.7mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值；
    或者，所述液体抗体制剂包含
    (i)约20mg/ml的抗LAG-3抗体；
    (ii)约5.88mg/ml二水柠檬酸钠；
    (iii)约50mg/ml蔗糖，和
    (iv)约0.7mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH约为5.5-7.5，优选地约为6.0-7.0，更优选地6.0±0.3，例如6.0，优选地，使用无水柠檬酸调节至所述pH值。
  14. 根据权利要求1-13中任何一项所述的液体抗体制剂，其特征在于，该制剂在储存后，例如在2-8℃储存至少24个月后，或在室温储存至少3个月后，或在40℃±2℃储存1个月后，是稳定的，优选地具有如下特征之一或多项：
    (i)通过SEC-HPLC法测量，制剂具有大于90％的纯度，优选大于95％、96％、97％、98％、99％的纯度；；
    (ii)通过还原型和非还原型CE-SDS法测量，制剂具有大于90％的纯度，优选大于95％、96％、97％、98％、99％的纯度；
    (iii)通过iCIEF法测量，相对于储存第0天的初始值，制剂中抗LAG-3抗体的各组分(即主成分、酸性组分及碱性组分)变化值≤2％；
    (iv)通过ELISA法测量，相对于储存第0天的初始值，制剂中抗LAG-3抗体的相对结合活性为70％-130％，例如，为70％、80％、90％、100％、110％、120％、130％。
  15. ー种固体抗体制剂，其通过固化权利要求1-14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。
  16. 递送装置，其包含权利要求1-14中任何一项的液体抗体制剂或权利要求15的固体抗体制剂。
  17. 预填装注射器，其包含权利要求1-14中任何一项的液体抗体制剂或权利要求15的固体抗体制剂，用于静脉内注射或者肌内注射。
  18. 根据权利要求1-14中任何一项的液体抗体制剂或权利要求15的固体抗体制剂的用途，用于制备治疗、预防或延缓表达LAG-3的癌症(特别是转移性癌症)、免疫相关疾病和T细胞功能障碍性疾病的药物。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018204374A1 (en) * 2017-05-02 2018-11-08 Merck Sharp & Dohme Corp. Formulations of anti-lag3 antibodies and co-formulations of anti-lag3 antibodies and anti-pd-1 antibodies
WO2018222722A2 (en) * 2017-05-30 2018-12-06 Bristol-Myers Squibb Company Compositions comprising an anti-lag-3 antibody or an anti-lag-3 antibody and an anti-pd-1 or anti-pd-l1 antibody
CN109970857A (zh) * 2017-12-27 2019-07-05 信达生物制药(苏州)有限公司 抗pd-l1抗体及其用途
CN109970860A (zh) * 2017-12-27 2019-07-05 信达生物制药(苏州)有限公司 三链抗体、其制备方法及其用途
CN109970856A (zh) * 2017-12-27 2019-07-05 信达生物制药(苏州)有限公司 抗lag-3抗体及其用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
UY34887A (es) * 2012-07-02 2013-12-31 Bristol Myers Squibb Company Una Corporacion Del Estado De Delaware Optimización de anticuerpos que se fijan al gen de activación de linfocitos 3 (lag-3) y sus usos
TWI756187B (zh) * 2015-10-09 2022-03-01 美商再生元醫藥公司 抗ｌａｇ３抗體及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018204374A1 (en) * 2017-05-02 2018-11-08 Merck Sharp & Dohme Corp. Formulations of anti-lag3 antibodies and co-formulations of anti-lag3 antibodies and anti-pd-1 antibodies
WO2018222722A2 (en) * 2017-05-30 2018-12-06 Bristol-Myers Squibb Company Compositions comprising an anti-lag-3 antibody or an anti-lag-3 antibody and an anti-pd-1 or anti-pd-l1 antibody
CN109970857A (zh) * 2017-12-27 2019-07-05 信达生物制药(苏州)有限公司 抗pd-l1抗体及其用途
CN109970860A (zh) * 2017-12-27 2019-07-05 信达生物制药(苏州)有限公司 三链抗体、其制备方法及其用途
CN109970856A (zh) * 2017-12-27 2019-07-05 信达生物制药(苏州)有限公司 抗lag-3抗体及其用途

Non-Patent Citations (21)

* Cited by examiner, † Cited by third party
Title
"Remington: the Science and Practice of Pharmacy", 2005, LIPPINCOTT WILLIAMS & WILKINS
"The Handbook of Pharmaceutical Excipients", 2003, AMERICAN PHARMACEUTICALS ASSOCIATION
ALEXANDRE GOYON ET AL.: "Protocols for the analytical characterization of therapeutic monoclonal antibodies, I-Non-denaturing chromatographic techniques", JOURNAL OF CHROMATOGRAPHY, Retrieved from the Internet <URL:http://dx.doi.org/10.1016/j.jchromb.2017.05.010>
B. MINOWP. ROGGEK. THOMPSON, BIOPROCESS INTERNATIONAL, vol. 10, no. 6, 2012, pages 48 - 57
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
DADA OO: "Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation", ELECTROPHORESIS, vol. 36, no. 21-22, 18 September 2015 (2015-09-18), pages 2695 - 2702
DEMEURE C.E. ET AL.: "T Lymphocytes infiltrating various tumour types express the MHC class II ligand lymphocyte activation gene-3 (LAG-3): role of LAG-3/MHC class II interactions in cell-cell contacts", EUR. J. CANCER, vol. 37, no. 13, 2001, pages 1709 - 1718, XP004301143, DOI: 10.1016/S0959-8049(01)00184-8
J. PHARM. BIO. ANAL., vol. 14, 1986, pages 1133 - 1140
J. PHARM. BIO. ANAL., vol. 15, 1997, pages 1928
J. PHARM. SCIEN., vol. 83, 1994, pages 1645 - 1650
JONES, A., ADV. DRUG DELIVERY REV., vol. 10, 1993, pages 29 - 90
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH, pages: 247 - 301
KELLEY ET AL.: "Weak partitioning chromatography for anion exchange purification of monoclonal antibodies", BIOTECHNOLOGY AND BIOENGINEERING, vol. 101, 2008, pages 553 - 566
NATIONAL PHARMACOPOEIA COMMITTEE: "Pharmacopoeia of the People's Republic of China", vol. IV, 2015, CHINA MEDICAL SCIENCE PRESS, article "Test for Visible Particles"
PHARM. RES., vol. 11, 1994, pages 485
R. YANG ET AL.: "High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography (SE-UHPLC", JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, 2015, Retrieved from the Internet <URL:http://dx.doi.org/10.1016/jjpba.2015.02.032>
RICHARD R. ET AL.: "Application of CE SDS gel in development of biopharmaceutical antibody-based products", ELECTROPHORESIS, vol. 29, 2008, pages 3612 - 3620
SALAS-SOLANO O ET AL.: "Robustness of iCIEF methodology for the analysis of monoclonal antibodies: an interlaboratory study", J SEP SCI, vol. 35, no. 22, 15 October 2012 (2012-10-15), pages 3124 - 9
See also references of EP3991747A4
SIERRO ET AL.: "The CD4-like molecule LAG-3, biology and therapeutic applications", EXPERT OPIN THER TARGETS, vol. 15, no. 1, 2011, pages 91 - 101, XP009178261, DOI: 10.1517/14712598.2011.540563
TUGCU ET AL.: "Maximizing productivity of chromatography steps for purification of monoclonal antibodies", BIOTECHNOLOGY AND BIOENGINEERING, vol. 99, 2008, pages 599 - 613, XP002556043, DOI: 10.1002/bit.21604

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