WO2021147854A1 - 重组全人源抗tigit单克隆抗体制剂及其制备方法和用途 - Google Patents

重组全人源抗tigit单克隆抗体制剂及其制备方法和用途 Download PDF

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WO2021147854A1
WO2021147854A1 PCT/CN2021/072690 CN2021072690W WO2021147854A1 WO 2021147854 A1 WO2021147854 A1 WO 2021147854A1 CN 2021072690 W CN2021072690 W CN 2021072690W WO 2021147854 A1 WO2021147854 A1 WO 2021147854A1
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antibody
preparation
liquid
antibody preparation
tigit
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PCT/CN2021/072690
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English (en)
French (fr)
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张海桃
马丽强
汪音爵
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信达生物制药(苏州)有限公司
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Priority to CN202180010322.2A priority Critical patent/CN115052622A/zh
Priority to AU2021211799A priority patent/AU2021211799A1/en
Priority to US17/793,503 priority patent/US20230080706A1/en
Priority to CA3168600A priority patent/CA3168600A1/en
Priority to EP21744016.3A priority patent/EP4094777A4/en
Priority to JP2022544137A priority patent/JP2023511356A/ja
Publication of WO2021147854A1 publication Critical patent/WO2021147854A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • A61K47/16—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
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of antibody preparations. More specifically, the present invention relates to an antibody (also called anti-TIGIT antibody) comprising a recombinant fully human anti-T cell immune receptor containing an immunoglobulin motif and an immunoreceptor tyrosine inhibitory motif domain
  • an antibody also called anti-TIGIT antibody
  • Drug stability is one of the important indicators to ensure the effectiveness and safety of drugs. Obtaining a good formulation prescription is a key condition to ensure that the drug maintains its effectiveness and safety during the shelf life.
  • TIGIT T cell immune receptor containing immunoglobulin motif and immunoreceptor tyrosine inhibitory motif domain, also known as WUCAM, Vstm3 or Vsig9
  • WUCAM immunoglobulin motif and immunoreceptor tyrosine inhibitory motif domain
  • Vsig9 T cell immune receptor containing immunoglobulin motif and immunoreceptor tyrosine inhibitory motif domain
  • the present invention meets the above-mentioned needs by providing a pharmaceutical preparation containing an antibody that specifically binds to TIGIT.
  • the antibody preparation of the present invention exhibits excellent stability against various stability influencing factors (such as temperature, repeated freezing and thawing, and shaking).
  • the present invention provides a liquid antibody preparation comprising (i) an anti-TIGIT antibody protein; (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant.
  • the anti-TIGIT antibody comprises a heavy chain variable region VH and a light chain variable region VL, 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 that is at least 90% identical to it:
  • the anti-TIGIT antibody comprises:
  • the anti-TIGIT antibody is an IgG4 type antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the sequence of SEQ ID NO: 9 or a sequence that is at least 90% identical to it, 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-TIGIT antibody is the anti-TIGIT monoclonal antibody ADI-30278 disclosed in PCT Application No. PCT/CN2019/097665 (International Filing Date: July 25, 2019), which is composed of SEQ ID NO: 9
  • the heavy chain sequence and the light chain sequence of SEQ ID NO: 10 are composed.
  • the anti-TIGIT antibody is an anti-TIGIT antibody recombinantly expressed in 293 cells or CHO cells.
  • the concentration of the anti-TIGIT antibody in the liquid antibody preparation of the present invention is about 1-150 mg/ml. In another embodiment, the concentration of the anti-TIGIT antibody in the liquid antibody preparation of the present invention is about 10-100 mg/ml, preferably 20-60 mg/ml, especially about 50 mg/ml. In other embodiments, the concentration of the anti-TIGIT antibody in the liquid antibody formulation of the present invention is about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 mg/ml.
  • the concentration of the buffer in the liquid antibody formulation of the present invention is about 5-50 mM. In one embodiment, the concentration of the buffer in the liquid antibody formulation of the present invention is about 5-30 mM, for example, about 5, 10, 15, 20, 25, 30 mM. In one embodiment, the buffer is a histidine buffer, and preferably, the buffer consists of histidine and histidine hydrochloride. In a preferred embodiment, the buffer is about 5-30 mM histidine buffer, for example 10-20 mM, such as about 10 mM histidine.
  • the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 50-500 mM. In one embodiment, the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 100-400 mM, for example about 100, 150, 200, 250, 300, 350, 400 mM.
  • the stabilizer is selected from polyols (e.g., sorbitol), sugars (e.g., sucrose), amino acids (e.g., arginine or arginine hydrochloride), and any combination thereof.
  • the stabilizer contains about 20-80 mg/ml sorbitol, such as 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80 mg/ml sorbitol.
  • the stabilizer contains about 20-60 mg/ml sucrose, for example, 20, 30, 40, 50, 60 mg/ml sucrose.
  • the stabilizer further comprises arginine, such as about 25mM-200mM, such as 50-150mM, or 50mM-120mM, preferably about 60-100mM, for example, about 60, 65, 70, 75, 80, 85, 90mM arginine.
  • arginine as a stabilizer is provided by arginine hydrochloride.
  • the stabilizer comprises a combination of sorbitol and arginine; or a combination of sucrose and arginine.
  • a combination of about 20-40 mg/ml sorbitol and about 50-100 mM arginine, or about 30-60 mg/ml sucrose and about 50-100 mM arginine may be used.
  • the liquid antibody formulation of the present invention contains about 30-60 mg/ml (such as about 50 mg/ml) sorbitol, or about 20-30 mg/ml (such as about 25 mg/ml) sorbitol and 80-90 mM (Approximately 85mM) Arginine.
  • 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 pH of the liquid formulation is about 5.0-6.0. In some embodiments, the pH of the liquid formulation is about any value from 5.0 to 6.0, for example, about 5.0, 5.2, 5.4, 5.6, 5.8, 6.0. Preferably, the pH of the formulation is 5.2 ⁇ 0.2 or 5.5 ⁇ 0.2, preferably the pH is 5.2.
  • the liquid antibody preparation of the invention comprises:
  • Histidine buffer of about 5-50 mM, such as 5-30 mM, such as 5, 10, 15, 20, 25, 30 mM;
  • polysorbate 80 for example, 0.1, 0.2.0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg/ml;
  • the pH of the liquid formulation is about 5.0-5.5, for example, about 5.2.
  • the liquid antibody preparation may contain
  • anti-TIGIT antibody protein such as 20, 30, 40, 50, 55, 60 mg/ml;
  • the pH of the liquid formulation is about 5.0-5.5, for example, about 5.2.
  • the liquid antibody preparation comprises:
  • the liquid formulation of the present invention can be stored stably for a long period of time, for example, at least 24 months or more.
  • the liquid formulation of the present invention can be heated at about -80°C to about 45°C, for example -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about Store at 35°C, about 38°C, about 40°C, about 42°C or about 45°C for at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months , At least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months , At least 36 months, or longer, and stable.
  • the liquid formulation of the present invention can be stored stably for at least 24 months.
  • the liquid formulation of the present invention is stable at at least 40°C.
  • the liquid formulation of the present invention is stable at about 2°C-8°C for at least 3 months, preferably at least 12 months, and more preferably at least 24 months.
  • the liquid formulation of the present invention is stable at room temperature or, for example, about 25°C for at least 2 months, preferably at least 3 months, and more preferably at least 6 months.
  • the liquid formulation of the present invention remains stable at about 40°C for at least 2 weeks, preferably at least 1 month.
  • the stability of the preparation can be indicated by detecting changes in the appearance, visible foreign matter, protein content, turbidity, purity, and/or charge variants of the preparation. In one embodiment, it can be in a forced experiment of high temperature stress, for example after storage at 40°C ⁇ 2°C for at least 1 week, 2 weeks or preferably 1 month, or in an accelerated experiment, such as at 25°C ⁇ 2°C After storage for at least 1 month or 2 months, or in long-term experiments, such as storage at 5°C ⁇ 3°C for at least 2 months or 3 months, or in shaking experiments (for example, at room temperature, protected from light, 650r/min Under the conditions of shaking for 5 days), and/or in a freeze-thaw experiment (for example, repeated freeze-thaw at -30°C/room temperature 6 times), the stability of the liquid preparation of the present invention is tested. In one embodiment, the stability of the liquid formulation of the present invention is tested relative to the initial value, for example, the initial value on day 0 of storage, or the
  • the stability of the liquid formulation of the present invention is visually checked, wherein the liquid formulation of the present invention remains clear to slightly opalescent in appearance , It is a colorless to light yellow liquid with no foreign matter. In one embodiment, under a visual inspection under a clarity detector, no visible foreign matter is present in the preparation.
  • 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), relative From the initial value, the protein content change rate is not more than 20%, preferably not more than 10%, such as 7-8%, more preferably not more than 5%, 2% or 1%.
  • the stability of the liquid formulation of the present invention is checked by measuring the turbidity change of the liquid formulation of the present invention, for example, by the OD 350mm method.
  • the change value does not exceed 0.06, preferably does not exceed 0.05, more preferably does not exceed 0.04, and does not exceed 0.02.
  • the stability of the liquid formulation of the present invention is checked by measuring the change in purity of the liquid formulation of the present invention, wherein the stability of the liquid formulation of the present invention is checked by the size exclusion Chromatography (SEC-HPLC), relative to the initial value, the change value of monomer purity (or main peak change value) does not exceed 10%, such as not more than 5%, 4%, 3%, for example, the change value does not exceed 2%, Preferably it 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 dodecane Based on sodium sulfate capillary electrophoresis (CE-SDS) method, relative to the initial value, the change value of the monomer purity (or the main peak change value) is reduced by no more than 10%, for example, no more than 5%, 4%, 3%, 2% or 1%.
  • CE-SDS sodium sulfate capillary electrophoresis
  • the stability of the liquid preparation of the present invention is tested by cation exchange high performance liquid chromatography (CEX-HPLC), which is relative to The initial value, the total change value of the antibody's charge variants (principal component, acidic component and basic component) does not exceed 50%, for example, does not exceed 40%, 30%, 20%, 10%, 5%, and/ Or the change value of the main component does not exceed 20%, 15%, 10%, 8%, 5%.
  • CEX-HPLC cation exchange high performance liquid chromatography
  • the stability of the liquid preparation of the present invention is tested by a direct ELISA method, wherein the relative binding activity of the antibody is 70 relative to the initial value. -130%, for example, 70, 80, 90, 93, 95, 98, 100, 103, 105, 108, 110, 115, 120, 125, 130%, preferably 90-110%.
  • the liquid formulation of the present invention is stable after storage, for example, after storage at 25°C for at least 2 months, or after storage at 40°C ⁇ 2°C for 1 month, and preferably has one of the following characteristics or Multiple items: relative to the initial value on day 0 of storage,
  • the main peak change value is less than 1%, and/or the preparation has a purity of greater than 96%, preferably greater than 97%, 98%;
  • the main peak change value is less than 2%, and/or the preparation has a purity of greater than 96%, preferably greater than 97%, 98%;
  • the total change value after storage at 40°C ⁇ 2°C for 1 month does not exceed about 40% (for example, not more than 35%, 30%, 25%, 20%, 15%, 10%) or the main component change value does not exceed 20% (for example, no more than 15%, 12%, 10%, 8%), or
  • the total change value does not exceed about 20% (for example, not more than 15%, 14%, 13%, 12%) or the main component change value does not exceed about 15% (for example, not more than 10 %, 8%, 7%, 6%, 5%);
  • the relative binding activity of the anti-TIGIT antibody protein in the preparation is 70%-130%, for example, 90,93,95,98,100,103,105,108,110,115,120%, for example, 90%-110%;
  • the liquid formulation of the present invention is stable under shaking and/or repeated freezing and thawing.
  • the preparation is stable under shaking or repeated freezing and thawing, for example, after shaking for 5 days under the condition of 650r/min at room temperature and avoiding light, or after repeated freezing and thawing at -30°C/room temperature for 6 times, it has the following characteristics:
  • the main peak change value is less than 1%, and/or the preparation has a purity greater than 96%, preferably greater than 97%, 98%, 99%;
  • the main peak change value is less than 1%, and/or the preparation has a purity of greater than 96%, preferably greater than 97%, 98%;
  • the relative binding activity of the anti-TIGIT antibody protein in the preparation is 70%-130%, for example, 90%-110%;
  • the liquid preparation of the present invention is a pharmaceutical preparation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection.
  • the liquid formulation is an intravenous infusion agent.
  • the present invention provides a solid antibody preparation, which is obtained by subjecting the liquid antibody preparation of the present invention to a 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 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-TIGIT antibody protein to a subject, such as a mammal, comprising the step of administering the liquid antibody preparation or solid antibody preparation of the present invention to the subject, and the delivery is, for example, by Implemented 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 to prepare a delivery device or a preventive device that blocks the binding of TIGIT to CD155 in a subject to reduce or eliminate the immunosuppressive effect of TIGIT.
  • a delivery device or a preventive device that blocks the binding of TIGIT to CD155 in a subject to reduce or eliminate the immunosuppressive effect of TIGIT.
  • the tumors being, for example, cancers, including but not limited to the gastrointestinal tract Cancer, such as colon cancer.
  • the present invention also provides a liquid antibody preparation or solid antibody preparation of the present invention or a delivery device (such as a pre-filled syringe) or drug containing the liquid antibody preparation or solid antibody preparation of the present invention by administering the liquid antibody preparation or solid antibody preparation of the present invention to a subject.
  • a delivery device such as a pre-filled syringe
  • drug containing the liquid antibody preparation or solid antibody preparation of the present invention by administering the liquid antibody preparation or solid antibody preparation of the present invention to a subject.
  • the method of blocking the combination of TIGIT and CD155 to reduce or eliminate the immunosuppressive effect of TIGIT are examples of blocking the combination of TIGIT and CD155 to reduce or eliminate the immunosuppressive effect of TIGIT.
  • the present invention also provides a method for treating a subject by administering the liquid antibody preparation or solid antibody preparation of the present invention or a delivery device (such as a pre-filled syringe) or drug containing the liquid antibody preparation or solid antibody preparation to a subject Patients’ diseases, such as the above-mentioned tumor or pathogen infection methods.
  • a delivery device such as a pre-filled syringe
  • drug containing the liquid antibody preparation or solid antibody preparation to a subject Patients’ diseases, such as the above-mentioned tumor or pathogen infection methods.
  • Figure 1 shows that in the pH screening experiment of Example 1, the change trend graph of the antibody charge variant-principal component under different pH conditions detected by the CEX-HPLC method.
  • FIG. 1 Shows that in the pH screening experiment of Example 1, the CEX-HPLC method was used to detect the change trend graph of the antibody charge variant-acidic component under different pH conditions (CEX-HPLC method).
  • Fig. 3 shows a graph showing the change trend of charge variants detected by the CEX-HPLC method in the 40°C stability confirmation experiment in Example 2.
  • Fig. 4 shows a graph showing the change trend of charge variants detected by the CEX-HPLC method in the 25°C stability confirmation experiment of Example 2.
  • the term “comprising” or “including” means including 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 encompasses 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.
  • TIGIT refers to "T cell immune receptor containing Ig and ITIM domains".
  • the expression also includes variants, isotypes, homologs, and species homologs of TIGIT.
  • TIGIT is also called VSIG9, VSTM3, WUCAM.
  • the amino acid and nucleic acid sequences of human and murine GITR can be found in GenBank accession numbers NP_776160 (human amino acid sequence) and NP_001139797 (murine amino acid sequence).
  • the TIGIT protein may also include a fragment of TIGIT, such as a fragment containing an extracellular domain, such as a fragment that retains the ability to bind to any antibody of the present invention.
  • antibody is used in the broadest sense and refers to a protein containing an antigen binding site, encompassing natural antibodies and artificial antibodies of various structures, including but not limited to intact antibodies and antigen-binding fragments of antibodies.
  • the terms “whole antibody”, “full-length antibody”, “full antibody” and “whole antibody” are used interchangeably herein to refer to at least two heavy chains (H) and two Light chain (L) glycoprotein.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region.
  • the heavy chain constant region is composed of three structural domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • the VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDR)), with more conservative regions (framework regions (FR)) interposed between them.
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four
  • the FR composition is arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant region does not directly participate in the binding of the antibody to the antigen, but displays a variety of effector functions.
  • human antibody or “fully human antibody” are used interchangeably herein and refer to antibodies that include variable regions in which both framework regions and CDR regions are derived from human germline immunoglobulin sequences. Moreover, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • the human antibodies of the present invention may include amino acid sequences not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or point-specific mutagenesis in vitro or somatic mutations in vivo), such as in CDRs, especially in CDR3.
  • the term "human antibody” does not include antibodies in which CDR sequences are derived from the germline of other mammalian species (eg, mice) and transplanted into human framework sequences.
  • antibody preparation refers to a preparation that is in a form that allows the biological activity of the antibody as an active ingredient to be effectively exerted, and does not contain unacceptable toxicity to the subject to which the preparation is to be administered. Other components. Such antibody preparations are usually sterile. Generally, pharmaceutically acceptable excipients are included in antibody preparations.
  • a "pharmaceutically acceptable" excipient is an agent that can be reasonably administered to a tested mammal so that an effective dose of the active ingredient used in the formulation can be delivered to the subject. The concentration of the excipient is adapted to the mode of administration, for example, it may be acceptable for injection.
  • anti-TIGIT antibody preparation is also abbreviated as "antibody preparation of the present invention” herein, and means a preparation containing an anti-TIGIT antibody protein as an active ingredient and a pharmaceutically acceptable excipient. After the anti-TIGIT antibody protein is combined with a pharmaceutically acceptable excipient, the anti-TIGIT antibody protein as the active ingredient is suitable for therapeutic or preventive administration to humans or non-human animals.
  • the antibody preparations of the present invention can be prepared, for example, as liquid preparations in aqueous form, for example, ready-to-use prefilled syringes, or prepared as lyophilized preparations by dissolving and/or suspending in a physiologically acceptable solution immediately before use. Reconstitution (ie, reconstitution).
  • the anti-TIGIT antibody protein preparation is in the form of a liquid preparation.
  • a “stable” antibody preparation means that the antibody in the preparation retains an acceptable degree of physical stability and/or chemical stability after being stored under specific conditions, or after shaking, or after repeated freezing and thawing. Although the antibody contained in the antibody preparation may not maintain 100% of its chemical structure after storage, shaking or repeated freezing and thawing, it is usually maintained at about 90%, about 95%, about 96%, about 97%, about 98%.
  • the antibody preparation is considered "stable.”
  • the anti-TIGIT antibody protein 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 no The biological activity of the anti-TIGIT antibody protein is lost, showing a high degree of stability.
  • the anti-TIGIT antibody protein preparation of the present invention substantially retains its physical and chemical stability after storage, shaking, and/or repeated freezing and thawing.
  • the liquid preparation of the present invention can be stable at room temperature or at 40°C for at least 2 weeks, and/or at 25°C for at least 2 months, and/or at 2-8°C for at least 24 months.
  • the liquid preparation of the present invention can be stable after shaking at 650 r/min at room temperature for 5 days and/or after repeated freezing and thawing at -30°C/room temperature for 1-6 times.
  • the stability can be measured at a selected temperature and selected storage time. For example, the storage time can be selected based on the expected shelf life of the formulation. Alternatively, an accelerated stability test can be used. In some embodiments, the stability test is performed by performing various stress tests on the antibody preparation.
  • the formulated anti-TIGIT antibody protein preparation can be filled into a glass vial to test the stability of the antibody under high temperature stress.
  • the formulated anti-TIGIT antibody protein preparation can be filled into a glass vial and shaken at 650 r/min at room temperature and protected from light for 5 days, and then the stability of the antibody can be checked.
  • the stability of the antibody is checked.
  • freezing and storing below -30°C for 1 day and then thawing at room temperature is a freeze-thaw cycle.
  • the preparation After a period of storage, or after shaking for a period of time, or after repeated freezing and thawing many times, the preparation does not show aggregation, precipitation, turbidity and/or denaturation; or shows very little aggregation, precipitation, turbidity and/or denaturation, then It can be said that the antibody "maintains its physical stability" in the formulation.
  • the accumulation of antibodies in the preparation can potentially lead to an increased immune response in patients, leading to safety issues. Therefore, there is a need to minimize or prevent aggregation of the antibody in the formulation.
  • the light scattering method can be used to determine the visible aggregates in the formulation.
  • SEC-HPLC 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 or after shaking or after repeated freezing and thawing, wherein the percentage of antibody monomers in the formulation The higher, the higher the stability of the formulation.
  • “Acceptable degree” of physical stability can mean that at least about 90% of the anti-TIGIT antibody protein monomer is detected in the preparation after storage at a specific temperature for a specific time, after shaking or after repeated freezing and thawing.
  • an acceptable degree of physical stability indicates At least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-TIGIT antibody protein monomer.
  • the specific temperature at which the pharmaceutical preparation is stored may 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 is considered stable. If stored at about 25°C for 2 months, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-TIGIT antibody protein list is detected Body, the pharmaceutical preparation is considered stable. If stored at about 5°C for 9 months, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-TIGIT antibody protein list is detected. Body, the pharmaceutical preparation is considered stable.
  • the antibody in the preparation After a period of storage, or after shaking for a period of time, or after repeated freezing and thawing many times, 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, charge variants of antibodies.
  • chemical stability can be assessed by detecting and/or quantifying the chemically altered 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 formulation after being stored at a specific temperature for a specific time or after shaking or repeated freezing and thawing several times, wherein the smaller the change is , The higher the stability of the formulation.
  • the "acceptable degree" of chemical stability can mean that the charge variants (such as the main component or acidic component or alkali) in the preparation after storage at a specific temperature for a specific period of time, or after shaking for a period of time, or after repeated freezing and thawing many times.
  • the percentage change value of the sexual component does not exceed 40%, such as not more than 30%, not more than 20%; or the sum of the percentage change values of the charge variants (main component, acidic component and alkaline component) does not exceed 60% , For example, no more than 50%, no more than 30%.
  • an acceptable degree of chemical stability can be The percentage change value of the main component charge variant does not exceed about 50%, 40%, 30%, 20%, or 15%; or the sum of the percentage change value of the charge variant does not exceed about 60%, 50%, or 30 %.
  • the storage temperature of the pharmaceutical preparation can be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, or about 45°C.
  • the pharmaceutical preparation can be considered stable.
  • the pharmaceutical preparation can also be considered stable. If the percentage change value of the principal component charge variant is less than about 50%, 40%, 30%, 20%, 16%, 15%, 14%, 13%, 12%, 10%, 5%, or 4%, 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, for example, high temperature, shaking, freezing and thawing.
  • High temperature stress refers to storing the antibody preparation at room temperature or even at a higher temperature (for example, 40°C ⁇ 2°C) for a period of time. Through the accelerated test of high temperature stress, the stability of the antibody preparation can be checked.
  • parenteral administration means administration methods other than enteral and local administration, usually by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, and intrathecal , Intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspine, epidural and intrasternal injections and infusions .
  • the stabilized anti-TIGIT antibody protein formulation of the present invention is administered to the subject parenterally.
  • the anti-TIGIT antibody protein formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular, or intravenous injection.
  • the present invention provides a stable liquid antibody preparation comprising (i) an anti-TIGIT antibody, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, and the pH of the antibody preparation is about 5.0-6.0.
  • the liquid antibody preparation of the present invention is in the form of an injection preparation.
  • an "anti-TIGIT antibody” refers to an antibody that can bind to a TIGIT molecule with sufficient affinity so that the antibody can be used as a therapeutic and/or preventive agent that targets the TIGIT molecule.
  • the anti-TIGIT antibody in the antibody preparation of the present invention can have a high affinity, for example, with a K of 10 -7 M or less, preferably 10-20 ⁇ 10 -10 M.
  • D specifically binds to human TIGIT, and thereby mediates efficient blocking of the binding of TIGIT and its ligand CD155, reducing or eliminating the inhibitory signal transduction caused by TIGIT binding to its ligand.
  • the anti-TIGIT antibody in the antibody preparation of the present invention inhibits the growth of tumors (such as gastrointestinal tumors, preferably colorectal cancer) containing infiltrating lymphocytes expressing human TIGIT, and is preferably used in combination with an anti-PD1 antibody ,
  • tumors such as gastrointestinal tumors, preferably colorectal cancer
  • an anti-PD1 antibody preferably used in combination with an anti-PD1 antibody .
  • the anti-tumor effect is significantly better than that when a single antibody is administered.
  • the anti-TIGIT antibody in the antibody preparation of the present invention comprises: SEQ ID NO: 7 or a heavy chain variable region (VH) with at least 90% identity; and SEQ ID NO: 8 or a heavy chain variable region (VH) A light chain variable region (VL) with at least 90% identity.
  • 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, CDR
  • the anti-TIGIT antibody in the antibody preparation of the present invention comprises the VH CDR1, 2 and 3 sequences in the heavy chain variable region shown in SEQ ID NO: 7 and the light chain variable region shown in SEQ ID NO: 8
  • the "complementarity determining region” or “CDR region” or “CDR” (herein can be used interchangeably with the hypervariable region “HVR”) is the amino acid region in the variable region of an antibody that is mainly responsible for binding to an epitope.
  • the CDRs of the heavy chain and light chain are usually referred to as 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 for determining the CDR sequence of a given VH or VL amino acid sequence are known in the art.
  • the Kabat complementarity determining region (CDR) is determined based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. 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-TIGIT antibody of the present invention has VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, VH CDR3 of SEQ ID NO: 3; and VL CDR1 of SEQ ID NO: 4, SEQ VL CDR2 of ID NO: 5 and VL CDR3 of SEQ ID NO: 6.
  • the anti-TIGIT antibody in the antibody preparation of the present invention may comprise a heavy chain variable region (VH) having at least 90%, 95%, 98% or 99% or more identity with SEQ ID NO: 7 ; And/or a light chain variable region (VL) with 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 "percent sequence identity” can be calculated in the following way: the two best aligned sequences are compared in the comparison window, and the same nucleic acid bases (for example, A, T, C, G, I, etc.) are present 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 algorithms required 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, 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 derived from the conservative substitution table A below, preferably the preferred substituted residues shown in Table A.
  • the anti-TIGIT antibody in the antibody preparation of the invention is an antibody in the form of IgG.
  • 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 IgG form are the same, and the heavy chain constant regions of antibodies of different IgG forms are different.
  • an antibody in the form of IgG1 means that the Ig domain of its heavy chain constant region is the Ig domain of IgG1.
  • the anti-TIGIT antibody in the antibody preparation of the present invention is the anti-TIGIT monoclonal antibody ADI-30278 disclosed in PCT application number PCT/CN2019/097665 (International filing date: July 25, 2019), It has a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10.
  • the anti-TIGIT antibody is a purified IgG4 type antibody produced by recombinant expression in CHO cells.
  • the antibody in the liquid formulation of the present invention exhibits significant anti-tumor activity. For example, in a mouse tumor model inoculated with mouse MC38 cells, administration of the antibody preparation of the present invention can lead to a significant tumor suppressing effect, especially when combined with an anti-PD-1 antibody.
  • 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 1-150 mg/ml, preferably about 10-100 mg/mL, such as about 10, 15, 20, 25, 30, 35, 40, 50, 60. , 70, 80, 90 or 100mg/ml anti-TIGIT 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 5.0-6.0, for example, a pH of about 5.0-5.5.
  • the antibody formulations of the invention have a pH of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, or 5.8.
  • the antibody preparation of the present invention has a pH of 5.2 ⁇ 0.2 or 5.5 ⁇ 0.2, preferably a pH of 5.2.
  • the formulation of the present invention comprises a buffer system selected from the group consisting of histidine-histidine hydrochloride buffer system, citrate-sodium citrate buffer system, acetic acid-sodium acetate buffer system, phosphate buffer system , Preferably histidine-histidine hydrochloride buffer system.
  • the buffer used in the formulation of the present invention is a histidine buffer, especially a buffer system composed of histidine and histidine hydrochloride.
  • the concentration of histidine in the histidine buffer of the present invention is about 5-50 mM, especially about 5-30 mM, for example, about 5, 10, 15, 20, 25, 30 mM.
  • the formulation of the invention contains about 10 mM histidine.
  • the histidine buffer used in the formulation of the present invention consists of, for example, about 0.21 mg/ml histidine and about 1.81 mg/ml histidine hydrochloride.
  • 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, trehalose, maltose and combinations.
  • the polyol used as a stabilizer includes, but is not limited to, sorbitol, mannitol, or a combination thereof.
  • the amino acids used as stabilizers include, but are not limited to, arginine, arginine hydrochloride, methionine, glycine, proline, and combinations.
  • the stabilizer comprises one or more selected from the following:
  • -A polyol selected from sorbitol, mannitol, or a combination thereof, about 10-100 mg/ml, preferably 20-40 mg/ml, such as 25 mg/ml; or 40-60 mg/ml, such as 50 mg/ml;
  • -A sugar selected from sucrose, trehalose, maltose, or a combination thereof, for example, about 10-100 mg/ml, preferably 30-60 mg/ml, such as 40 mg/ml;
  • -Amino acids selected from arginine hydrochloride, methionine, glycine, proline and combinations thereof, for example, 20-200 mM, such as about 50-110 mM, for example, 70-100 mM, especially about 80-90 mM.
  • the liquid formulation of the present invention contains sucrose as a stabilizer.
  • the amount of sucrose in the liquid formulation of the present invention may be about 10-100 mg/ml, preferably 30-60 mg/ml, for example 40 mg/ml.
  • the liquid formulation of the present invention contains sorbitol as a stabilizer.
  • the amount of sorbitol in the liquid formulation of the present invention may be about 10-100 mg/ml, preferably 20-40 mg/ml, such as 25 mg/ml; or 40-60 mg/ml, such as 50 mg/ml.
  • the liquid formulation of the present invention contains arginine as a stabilizer.
  • the amount of arginine in the liquid preparation of the present invention may be about 50-110 mM, for example, 70-100 mM, especially about 80-90 mM, for example, about 17.91 mg/ml arginine hydrochloride.
  • the liquid formulation of the present invention contains sorbitol as a single stabilizer.
  • the amount of sorbitol in the liquid formulation of the present invention may be about 30-70 mg/ml, for example 40-60 mg/ml.
  • sorbitol may be present in an amount of about 30, 35, 40, 45, 50, 55, 60, 65 or 70 mg/ml, preferably in an amount of about 50 mg/ml.
  • the liquid formulation of the present invention contains a combination of sorbitol and arginine as a stabilizer.
  • sorbitol may be present in an amount of about 10-60 mg/ml, preferably 15-40 mg/ml, such as 20-35 mg/ml, for example, about 20, 21, 22, 23, 24, 25, 26 ,27,28,29,30mg/ml.
  • arginine may be present in an amount of about 70-100 mM, especially about 85 mM.
  • the liquid preparation of the present invention contains about 20-30 mg/ml sorbitol and about 15-20 mg/ml arginine hydrochloride. More preferably, the liquid formulation of the present invention contains about 25 mg/ml of sorbitol and about 17.91 mg/ml of arginine hydrochloride.
  • the liquid formulation of the present invention contains a combination of sucrose and arginine as a stabilizer.
  • sucrose may be present in an amount of about 10-60 mg/ml, preferably 20-50 mg/ml, such as 30-40 mg/ml, for example, it may be about 30, 32, 34, 36, 38, 40, 42. 44,46,48,50mg/ml.
  • arginine may be present in an amount of about 70-100 mM, especially about 85 mM.
  • the liquid preparation of the present invention contains about 30-50 mg/ml sucrose and about 15-20 mg/ml arginine hydrochloride. More preferably, the liquid preparation of the present invention contains about 40 mg/ml of sucrose and about 17.91 mg/ml of arginine hydrochloride.
  • surfactant refers to an organic substance with 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).
  • 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; Sam et al.
  • the liquid formulation of the present invention contains polysorbate-80 as a surfactant.
  • the surfactants that can be used in the liquid formulations of the present invention include, but are not limited to, polysorbate surfactants (e.g., polysorbate 80, polysorbate 20), poloxamers, and polyethylene. Glycol.
  • 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 for which the preparation is used.
  • the formulation may contain about 0.01-5 mg/ml, preferably about 0.1-1 mg/ml, for example about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg/ml Surfactant, especially polysorbate-80, preferably about 0.5mg/ml polysorbate-80.
  • the antibody liquid formulation of the present invention optionally contains other excipients.
  • the other excipients include, for example, antimicrobial agents, antistatic agents, antioxidants, chelating agents, 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 anti-TIGIT antibody protein.
  • the anti-TIGIT antibody protein used in the formulation 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.
  • a preparation containing the antibody can be prepared according to methods known in the art.
  • the following steps can be used for preparation: (1) After the 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; (5) virus filtration (to make the virus titer) Reduce, for example, 4 log10 or more); (6) Ultrafiltration/diafiltration (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.
  • the stability research of biological products generally includes real-time stability research under actual storage conditions (long-term stability research), accelerated stability research and forced condition test research.
  • Stability research should explore and optimize the research conditions according to the research purpose and the product's own characteristics; for various influencing factors (such as temperature, repeated freezing and thawing, vibration, etc.), develop stability studies such as long-term, accelerated and/or forced condition tests Program. Accelerated and compulsory condition tests are helpful to understand the stability of the product under short-term deviation from storage conditions and extreme conditions, and provide supporting data for the determination of the expiration date and storage conditions.
  • antibodies may undergo aggregation, degradation, or chemical modification, resulting in antibody heterogeneity (including size heterogeneity and charge heterogeneity), aggregates and fragments, etc. Affect the quality of antibody preparations. Therefore, it is necessary to monitor the stability of antibody preparations.
  • Various methods are known in the art that can be used to test the stability of antibody preparations.
  • methods such as reduced CE-SDS, non-reduced CE-SDS, and SEC-HPLC can be used to analyze the purity of antibody preparations and evaluate the aggregation level of antibodies; capillary isoelectric focusing (cIEF), imaging capillary, etc.
  • Focused electrophoresis (iCIEF) and ion exchange chromatography (IEX) are used to analyze charge variants in antibody preparations.
  • the stability of the preparation can be quickly judged by visually inspecting the appearance of the preparation.
  • the OD 350nm method can also be used to detect changes in the turbidity of the preparation, which can give information about the amount of soluble and insoluble aggregates.
  • ultraviolet spectrophotometry UV method
  • UV method ultraviolet spectrophotometry
  • the non-reduced CE-SDS method is a method of antibody purity determination using capillary as a separation channel.
  • CE-SDS protein migration is driven by the surface charge caused by SDS binding, and the surface charge is proportional to the molecular weight of the protein. Since all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on molecular size or hydrodynamic radius can be achieved in the molecular sieve gel matrix of the capillary. This method has been widely used to monitor the purity of denatured intact antibodies.
  • the test sample is mixed with 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 is used for analysis.
  • a UV detector is used to detect the migration of the protein and obtain an electrophoresis spectrum.
  • the purity of the antibody preparation can be calculated as the percentage of the peak area of the main IgG peak to the sum of all peak areas.
  • Size exclusion high performance liquid chromatography is another important method for antibody standards and quality control. This method is mainly based on the size of the molecule or the difference in hydrodynamic radius to separate the 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 to 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.
  • the charge variant of the antibody in the antibody preparation can be determined by cation exchange high performance liquid chromatography (CEX-HPLC).
  • CEX-HPLC cation exchange high performance liquid chromatography
  • the peaks eluted from the CEX-HPLC column earlier than the retention time of the main peak (or main component) are marked as “acidic peaks” (or acidic components), and those with a retention time greater than that of the main peak
  • the peaks eluted from the CEX-HPLC column later are labeled as "basic peaks” (or basic components).
  • Accelerated stability studies can be used to check the stability properties of products, which is conducive to the screening of stable pharmaceutical formulations.
  • a sample of the formulation can be placed at an elevated temperature, such as about 40°C ⁇ 2°C, 25°C ⁇ 2°C, for accelerated stability studies.
  • shaking experiments or repeated freezing and thawing experiments can be performed to test the stability properties of the product. For example, oscillate at 650r/min at room temperature and avoid light for 1-5 days to perform an oscillation experiment.
  • a product frozen at -30°C for one day and then thawing at room temperature can be used as a freeze-thaw cycle, and repeated freeze-thaw experiments can be performed, in which repeated freeze-thaw cycles can be implemented 1-6 times.
  • the detection indicators of product stability can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (iCIEF method, CEX-HPLC method).
  • the efficacy or biological activity of the antibody can be tested.
  • the binding ability of the antibody and its antigen molecule (TIGIT) in the preparation can be tested.
  • TIGIT antigen molecule
  • the antibody preparation of the present invention containing the anti-TIGIT antibody protein of the present invention has reduced immunosuppression, and can be used to treat or prevent tumors, pathogen infections, and the like.
  • the formulation of the present invention can be used to block the binding of TIGIT to CD155 in a subject to reduce or eliminate the immunosuppressive effect of TIGIT.
  • the formulations of the present invention can be used to treat or prevent tumors and pathogen infections in subjects.
  • the tumor is, for example, a gastrointestinal cancer, such as colon cancer.
  • the formulation of the invention may be administered in combination with a second therapeutic agent, such as an anti-PD-1 antibody.
  • the present invention also provides the use of the preparation of the present invention in the preparation of medicines, wherein the medicines are used to deliver anti-TIGIT antibody proteins to mammals.
  • the present invention also provides a method for the preparation of the present invention to treat or prevent one or more of the above-mentioned diseases and diseases.
  • the mammal is a human.
  • the antibody formulation of the present invention can be administered to a subject or patient in a variety of ways.
  • administration can be by infusion or by syringe.
  • the present invention provides a delivery device (e.g., a syringe) comprising the antibody preparation of the present invention (e.g., a pre-filled syringe).
  • the patient will receive an effective amount of anti-TIGIT antibody protein as the main active ingredient, that is, an amount sufficient to treat, ameliorate or prevent the target disease or condition.
  • the therapeutic effect may include reducing physical symptoms.
  • the optimal effective amount and concentration of antibodies for any particular subject will depend on many factors, including the patient’s age, weight, health and/or gender, the nature and extent of the disease, the activity of the particular antibody, and the body’s Its clearance rate, and also includes any possible other treatments administered in combination with the antibody formulation. For specific situations, the effective amount delivered can be determined within the judgment of the clinician.
  • Recombinant fully human anti-TIGIT monoclonal antibody ADI-30278 is an antibody independently developed by Cinda Biopharmaceutical (Suzhou) Co., Ltd. and is disclosed in PCT application number PCT/CN2019/097665.
  • the antibody can effectively block the binding of TIGIT to its ligand CD155, relieve the inhibitory effect of CD155 on the downstream IL2 signaling pathway, and inhibit tumor growth when administered in vivo, especially when combined with anti-PD-1 antibody, the tumor suppressor effect Especially remarkable.
  • N/A means not applicable.
  • the inspection items during the whole research process mainly include: (1) detection of appearance and presence of visible foreign matter; (2) determination of protein content in preparations by ultraviolet method (UV method); (3) detection of turbidity of preparations by OD350nm method; 4) Determine the purity of the antibody preparation by size exclusion high performance liquid chromatography (SEC-HPLC), expressed as the percentage of the area of the main peak to the sum of all peak areas; (5) Pass the non-reduced sodium lauryl sulfate capillary Electrophoresis (non-reduced CE-SDS) was used to determine the purity of the antibody preparation, expressed as the percentage of the area of the main peak to the sum of all peak areas; (6) The charge variant in the antibody preparation was measured by CEX-HPLC method, expressed as the main component, The percentage of acidic and alkaline components; (7) The relative binding activity of the anti-TIGIT antibody to the TIGIT antigen in the antibody preparation is determined by direct ELISA.
  • SEC-HPLC size exclusion high performance liquid chromatography
  • 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. Take the sample to be tested and dilute it to 2mg/ml with ultrapure water as the test solution. Take the preparation buffer solution and dilute it with the same treatment method as above and use it as a 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
  • 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.
  • mobile phase A is 10mmol/L phosphate buffer (weigh NaH2PO4 ⁇ 2H2O 0.51g, Na2HPO4 ⁇ 12H2O 2.40g dissolved in 800ml ultrapure water, dilute to 1000ml, Use ⁇ 0.22 ⁇ m membrane filter), mobile phase B is 10mmol/L phosphate + 200mmol/L sodium chloride buffer (weigh NaH2PO4 ⁇ 2H2O 0.51g, Na2HPO4 ⁇ 12H2O 2.40g, NaCl 11.69g dissolved in 800ml ultrapure In water, dilute to 1000ml, filter with ⁇ 0.22 ⁇ m membrane).
  • a 96-well microtiter plate was coated with human TIGIT antigen (purchased from Acrobiosystems, TIT-H52H3) at 0.5 ⁇ g/ml, 100 ⁇ l/well, 4°C overnight. After washing the plate, add blocking solution (2%BSA-PBST, 300 ⁇ l/well) at 37°C for 2h. Add 100 ⁇ l/well of the test substance in gradient dilution to the ELISA plate where the blocking solution is discarded. Set the negative control to add 100 ⁇ l diluent (2%BSA-PBST) to each well, and incubate in a constant temperature incubator at 37°C for 60min.
  • human TIGIT antigen purchased from Acrobiosystems, TIT-H52H3
  • HRP-conjugated goat anti-human IgG-Fc fragment (BETHYL, USA, catalog number A80-104P) diluted with 2% BSA-PBST was added as a secondary antibody (100000 times dilution, 100 ⁇ l/well), and reacted at 37°C for 30 min.
  • After washing the plate add 100 ⁇ l of TMB color developing solution to each well.
  • After 10 minutes of color development add 100 ⁇ l of 1mol/L H 2 SO 4 to each well to stop the reaction. Measure the OD value at 450nm with 620nm as the reference wavelength.
  • a concentration value of each sample as the abscissa the concentration gradient, OD450nm-OD620nm value for the vertical gradient of each sample, four parameter fit calculation application Prism EC 50 reflects the binding activity of the antibody with the antigen.
  • Opposing anti-TIGIT antibody binding activity (%) (test sample for the EC 50 / EC reference article 50) * 100%, wherein the reference product is not stable any stress processing.
  • the anti-TIGIT antibody ADI-30278 that specifically binds to TIGIT was obtained.
  • the antibody has the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10, and is a fully human antibody.
  • PCT application number PCT/CN2019/097665 is hereby incorporated by reference in its entirety.
  • antibodies are recombinantly expressed in CHO cells and purified through processes such as filtration, chromatography, virus inactivation, and filtration.
  • the sample used for the following pH screening test is a product purified by affinity chromatography with a protein content of 17.0 mg/ml.
  • the sample used for the following prescription determination test is a product purified by nanofiltration membrane filtration and has a protein content of 11.5 mg/ml.
  • the judgment standard of the quality of the sample detection index value compared with the initial value is set to judge whether the sample has changed.
  • the judgment standard is shown in Table 2.
  • Test items Judgment criteria unchanged Appearance (observation method) Clear to slightly opalescent, colorless to light yellow liquid, no foreign matter Visible foreign body (Visible foreign body inspection method) Comply with "The Pharmacopoeia of the People's Republic of China" (2015 edition, four), 0904 Protein content (UV method) Change rate ⁇ 10% Turbidity (OD 350nm method) Change value ⁇ 0.02 Purity (SEC-HPLC method) Change of main peak ⁇ 1% Purity (non-reduced CE-SDS method) Change of main peak ⁇ 2% Charge variant (CEX-HPLC method) Change value of main component and each component of acid and alkali ⁇ 2% Relative binding activity (direct ELISA method) Should be 70% ⁇ 130%
  • the test results of protein content are shown in Table 3. The results showed that the protein content of the samples at pH 5.0, pH 5.5, and pH 6.0 did not change significantly after being placed at 40°C ⁇ 2°C for 2 weeks.
  • N/A means that the test is not set.
  • N/A means that the test is not set.
  • formulation 2 was finally selected as the antibody formulation formulation.
  • Its composition is: 50.0mg/ml recombinant fully human anti-TIGIT antibody, 0.21mg/ml histidine, 1.81mg/ml histidine hydrochloride, 25.00mg/ml sorbitol, 17.91mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.2.

Abstract

本发明提供了包含抗TIGIT抗体的制剂，其包含抗TIGIT抗体、缓冲剂、稳定剂和表面活性剂。此外，本发明还提供了这些制剂在疾病治疗或预防中的用途。

Description

重组全人源抗TIGIT单克隆抗体制剂及其制备方法和用途 技术领域
本发明涉及抗体制剂领域。更具体而言，本发明涉及包含重组的全人源抗含免疫球蛋白基序和免疫受体酪氨酸抑制基序结构域的T细胞免疫受体的抗体(也称为抗TIGIT抗体)的药物制剂，尤其是稳定的液体制剂，以及用于制备所述药物制剂的方法，以及所述药物制剂的治疗和/或预防用途。
背景技术
药品稳定性是保证药品有效性和安全性的重要指标之一。获得良好的制剂处方是保证药品在货架期内保持其有效性和安全性的关键条件。然而，由于抗体本身及其降解途径的复杂性，目前尚不可能对优化抗体稳定性所需的制剂条件作出预测。尤其是考虑到，不同抗体通常具有十分不同的CDR序列，而这些序列差异会导致不同抗体在溶液中具有不同的稳定性性质。因此，基于对人用抗体在安全性和有效性上的严格要求，有必要针对每个抗体单独进行最佳制剂配方的优化。
TIGIT(含免疫球蛋白基序和免疫受体酪氨酸抑制基序结构域的T细胞免疫受体，也称为WUCAM、Vstm3或Vsig9)最初是利用生物信息学比对的方法作为CD28家族成员之一被发现。已经证实，结合人TIGIT的抗体可用于治疗癌症。参见例如WO 2006/124667。在小鼠模型中，PD-L1和TIGIT二者的抗体阻断可以导致协同提高CD8+T细胞介导的肿瘤排斥。Grogan et al.(2014)J.Immunol.192(1)Suppl.203.15；Johnston et al.(2014)Cancer Cell 26:1-15。在黑色素瘤的动物模型中得到类似结果。Inozume et al.(2014)J.Invest.Dermatol.134:S121-Abstract 693。与TIGIT高特异性结合的抗TIGIT抗体已经描述在例如PCT申请号PCT/CN2019/097665中。
尽管一些抗TIGIT抗体制剂已经被提出，但在本领域中对于含有足够稳定且适于施用给人受试者的抗TIGIT抗体的新药物制剂仍存在需要。此外，对于这样的抗体制剂，寻找制剂处方的简单和易用性，也是有利的。
发明概述
本发明通过提供含有特异结合至TIGIT的抗体的药物制剂来满足上述需求。本发明的抗体制剂针对多种稳定性影响因素(诸如温度、反复冻融、振荡)，均表现出了优良的稳定性。
在一个方面，因此，本发明提供了一种液体抗体制剂，其包含(i)抗TIGIT抗体蛋白；(ii)缓冲剂，(iii)稳定剂，和(iv)表面活性剂。
在一个实施方案中，抗TIGIT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％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 PCTCN2021072690-appb-000001
在一个实施方案中，所述抗TIGIT抗体包含：
-YTFTEYYMH(SEQ ID NO:1)的重链VH CDR1；
-IISPSAGSTKYAQKFQG(SEQ ID NO:2)的重链VH CDR2；
-ARDHDIRLAGRLADY(SEQ ID NO:3)的重链VH CDR3；
-RASQGISSWLA(SEQ ID NO:4)的轻链VL CDR1；
-AASSLQS(SEQ ID NO:5)的轻链VL CDR2；和
-QQAVILPIT(SEQ ID NO:6)的轻链VL CDR3。
在一个实施方案中，所述抗TIGIT抗体是包含重链和轻链的IgG4型抗体，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 PCTCN2021072690-appb-000002
优选地，所述抗TIGIT抗体是PCT申请号PCT/CN2019/097665(国际申请日：2019年7月25日)中公开的抗TIGIT单克隆抗体ADI-30278，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TIGIT抗体是在293细胞或CHO细胞中重组表达的抗TIGIT抗体。
在一个实施方案中，本发明的液体抗体制剂中的抗TIGIT抗体的浓度为约1-150mg/ml。在另一个实施方案中，本发明的液体抗体制剂中的抗TIGIT抗体的浓度为约10-100mg/ml，优选20-60mg/ml，尤其是约50mg/ml。在其他实施方案中，本发明的液体抗体制剂中的抗TIGIT抗体的浓度为约10、15、20、25、30、35、40、50、60、70、80、90或100mg/ml。
在一个实施方案中，本发明的液体抗体制剂中的缓冲剂的浓度为约5-50mM。在一个实施方案中，本发明的液体抗体制剂中的缓冲剂的浓度为约5-30mM，例如，约5、10、15、20、25、30mM。在一个实施方案中，所述缓冲剂是组氨酸缓冲剂，优选地，所述缓冲剂由组氨酸和盐酸组氨酸组成。在一个优选的实施方案中，所述缓冲剂是大约5-30mM组氨酸缓 冲剂，例如10-20mM，如大约10mM组氨酸。
在一个实施方案中，本发明的液体抗体制剂中的稳定剂的浓度为约50-500mM。在一个实施方案中，本发明的液体抗体制剂中的稳定剂的浓度为约100-400mM，例如约100、150、200、250、300、350、400mM。
在一个实施方案中，所述稳定剂选自多元醇(例如，山梨醇)、糖类(例如，蔗糖)、氨基酸(例如精氨酸或盐酸精氨酸)、和它们的任意组合。在一个实施方案中，所述稳定剂包含约20-80mg/ml的山梨醇，例如20、25、30、35、40、45、50、55、60、70、80mg/ml山梨醇。又在一个实施方案中，所述稳定剂包含约20-60mg/ml的蔗糖，例如，20、30、40、50、60mg/ml蔗糖。在一个优选实施方案中，所述稳定剂还包含精氨酸，例如约25mM-200mM,例如50-150mM，或50mM-120mM，优选地为约60-100mM，例如，约60、65、70、75、80、85、90mM精氨酸。优选地，由盐酸精氨酸提供作为稳定剂的精氨酸。
在一个优选实施方案中，所述稳定剂包含山梨醇和精氨酸的组合；或蔗糖和精氨酸的组合。例如，可以使用约20-40mg/ml的山梨醇和约50－100mM的精氨酸的组合，或约30-60mg/ml的蔗糖和约50－100mM的精氨酸。
在优选的实施方案中，本发明的液体抗体制剂包含大约30-60mg/ml(如大约50mg/ml)山梨醇、或大约20-30mg/ml(如大约25mg/ml)山梨醇与80-90mM(大约85mM)精氨酸。
在一个实施方案中，本发明的液体抗体制剂中的表面活性剂的浓度为约0.1-1mg/ml。在一个实施方案中，本发明的液体抗体制剂中的表面活性剂的浓度为约0.2-0.8mg/ml，例如约0.2、0.3、0.4、0.5、0.6、0.7、0.8mg/ml。
在一个实施方案中，所述表面活性剂是非离子型表面活性剂。在一个实施方案中，所述表面活性剂选自聚山梨酯类表面活性剂。在一个具体实施方案中，本发明的液体抗体制剂中的表面活性剂是聚山梨酯-80。
在一个实施方案中，所述液体制剂的pH值为约5.0-6.0。在一些实施方案中，所述液体制剂的pH值为约5.0-6.0中的任意值，例如约5.0、5.2、5.4、5.6、5.8、6.0。优选地，所述制剂的pH为5.2±0.2或5.5±0.2，优选地pH为5.2。
在一个实施方案中，本发明的液体抗体制剂包含：
(i)约10-100mg/ml的抗TIGIT抗体蛋白，例如10-80mg/ml，例如10,20,30,40,50,60,70或80mg/ml抗体蛋白；
(ii)约5-50mM的组氨酸缓冲剂,例如5-30mM，例如5,10,15,20,25,30mM；
(iii)约50-300mM的山梨醇、蔗糖、或它们的任意组合，例如，50,80,100,120,140,160,180,200,250,300mM；
(iv)约0.1-1mg/ml聚山梨酯80,例如,0.1,0.2.0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0mg/ml；和
(v)任选地，约50-120mM的精氨酸，
其中所述液体制剂的pH为约5.0-5.5，例如，约5.2。
例如，所述液体抗体制剂可以包含
(i)约10-60mg/ml抗TIGIT抗体蛋白，例如20,30,40,50,55,60mg/ml；
(ii)约10mM的组氨酸缓冲剂；
(iii)约10-50mg/ml山梨醇或蔗糖，例如10,15,20,25,30,35,40,45，50mg/ml山梨醇，或例如10，15,20,25,30,35,40,45，50mg/ml蔗糖；
(iv)约0.2-0.8mg/ml，例如0.2,0.3,0.4,0.5,0.6,0.7,0.8mg/ml，例如0.3-0.6mg/ml聚山梨酯80；和
(v)约60-100mM，例如60,65,70,75,80,85,90,95,100mM,尤其是约85mM的精氨酸，
其中所述液体制剂的pH为约5.0-5.5，例如，约5.2。
在一个优选方案中，所述液体抗体制剂包含：
(i)约50mg/ml的抗TIGIT抗体蛋白，约0.21mg/ml组氨酸，约1.81mg/ml盐酸组氨酸，约25.00mg/ml山梨醇，约17.91mg/ml盐酸精氨酸，约0.50mg/ml聚山梨酯80，pH约5.2；或
(ii)约50mg/ml的抗TIGIT抗体蛋白，约0.21mg/ml组氨酸，约1.81mg/ml盐酸组氨酸，约25.00mg/ml山梨醇，约17.91mg/ml盐酸精氨酸，约0.20mg/ml聚山梨酯80，pH约5.2；或
(iii)约0.21mg/ml组氨酸，约1.81mg/ml盐酸组氨酸，约40.00mg/ml蔗糖，约17.91mg/ml盐酸精氨酸，约0.20mg/ml聚山梨酯80，pH 5.2。
本发明的液体制剂可以长期稳定储存，例如至少24个月或更长时间。在一个实施方案中，本发明的液体制剂可以在约-80℃至约45℃，例如-80℃、约-30℃、约-20℃、约0℃、约5℃、约25℃、约35℃、约38℃、约40℃、约42℃或约45℃的条件下，储存至少10天、至少20天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，至少36个月，或更长时间，且是稳定的。
在一个实施方案中，本发明的液体制剂可以稳定储存至少24个月。在再一实施方案中，本发明的液体制剂在至少40℃是稳定的。在再一实施方案中，本发明的液体制剂在约2℃-8℃保持稳定至少3个月，优选至少12个月，更优选至少24个月。在一个实施方案中，本发明的液体制剂在室温或例如约25℃保持稳定至少2个月，优选至少3个月，更优选至少6个月。在再一实施方案中，本发明的液体制剂在约40℃保持稳定至少2周、优选至少1个月。
在一个实施方案中，可以通过检测制剂的外观、可见异物、蛋白含量、浊度、纯度、和/或电荷变异体的变化，来指示制剂的稳定性。在一个实施方案中，可以在高温胁迫的强制实验中，例如在40℃±2℃储存至少1周、2周或优选地1个月后，或在加速实验中，例如在25℃±2℃储存至少1个月或2个月后，或在长期实验中，例如在5℃±3℃储存至少2个月或3个月后，或在振荡实验中(例如在室温、避光650r/min条件下振荡5天)，和/或在冻融实验中(例如，在-30℃/室温反复冻融6次)，检测本发明液体制剂的稳定性。在一个实施方案中，相对于初始值，例如储存第0天的初始值、或振荡或冻融实验前的初始值，检测本发明液体制剂的稳定性。
在一个实施方案中，在储存后，或在振荡实验后，或在冻融实验后，通过目视检查本发明液体制剂的稳定性，其中本发明液体制剂在外观上保持为澄明至微乳光，为无色至淡黄色液体，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，或在振荡实验后，或在冻融实验后，通过测定蛋白含量变化，检查本发明液体制剂的稳定性，其中例如通过紫外分光光度(UV)法，相对于初始值，蛋白含量变化率不超过20％，优选不超过10％，例如7-8％，更优选不超过5％，2％或1％。在一个实施方案中，在储存后，或在振荡实验后，或在冻融实验后，通过测定本发明液体制剂的浊度变化，检查本发明液体制剂的稳定性，其中例如通过OD 350mm法检测，相对于初始值，变化值不超过0.06，优选地不超过0.05，更优选地不超过0.04，不超过0.02。在一个实施方案中，在储存后，或在振荡实验后，或在冻融实验后，通过测定本发明液体制剂的纯度变化，检查本发明液体制剂的稳定性，其中通过体积排阻高效液相色谱法(SEC-HPLC)，相对于初始值，单体纯度的变化值(或主峰变化值)不超过10％，例如不超过5％、4％、3％、例如变化值不超过2％，优选不超过1％。在一个实施方案中，在储存后，或在振荡实验后，或在冻融实验后，通过测定本发明液体制剂的纯度变化，检查本发明液体制剂的稳定性，其中通过非还原型十二烷基硫酸钠毛细管电泳(CE-SDS)法，相对于初始值，单体纯度的变化值(或主峰变化值)下降不超过10％，例如不超过5％、4％、3％,2％或1％。在一个实施方案中，在储存后，或在在振荡实验后，或在冻融实验后，通过阳离子交换高效液相色谱法(CEX-HPLC)，检测本发明液体制剂的稳定性，其中相对于初始值，抗体的电荷变异体(主成分、酸性组分和碱性组分)的变化值总和不超过50％，例如不超过40％、30％、20％、10％、5％，和/或主成分的变化值不超过20％,15％,10％,8％,5％。在一个实施方案中，在储存后，或在振荡实验后，或在冻融实验后，通过直接ELISA法，检测本发明液体制剂的稳定性，其中相对于初始值，抗体的相对结合活性为70-130％，例如，70,80,90,93,95,98,100,103,105,108,110,115,120,125,130％,优选90-110％。
在一个实施方案中，本发明液体制剂在储存后，例如在25℃储存至少2个月后，或在40℃±2℃储存1个月后，是稳定的，优选地具有如下特征之一或多项：相对于储存第0天的初始值，
(i)通过SEC-HPLC法测量，主峰变化值小于1％，和/或制剂具有大于96％的纯度，优选大于97％、98％的纯度；
(ii)通过非还原型CE-SDS法测量，主峰变化值小于2％，和/或制剂具有大于96％的纯度，优选大于97％、98％的纯度；
(iii)通过CEX-HPLC法测量，制剂中抗TIGIT抗体蛋白的各组分(主成分、酸性组分和碱性组分)的变化值总和不超过40％和/或主成分的变化值不超过20％，
例如在40℃±2℃储存1个月后变化值总和不超过约40％(例如，不超过35％，30％，25％,20％,15％,10％)或主成分变化值不超过20％(例如，不超过15％,12％,10％,8％)，或
例如在25℃储存2个月后变化值总和不超过约20％(例如，不超过15％，14％,13％,12％)或主成分变化值不超过约15％(例如，不超过10％，8％,7％,6％,5％)；
(iv)通过ELISA法测量，制剂中抗TIGIT抗体蛋白的相对结合活性为70％-130％，例如，90,93,95,98,100,103,105,108,110,115,120％，例如90％-110％；
在优选的实施方案中，本发明液体制剂在振荡和/或反复冻融下是稳定的。
优选地，该制剂在振荡下或在反复冻融下是稳定的，例如，在室温避光650r/min条件下振荡5天或在-30℃/室温反复冻融6次后，具有如下特征之一或多项：
(i)通过SEC-HPLC法测量，主峰变化值小于1％，和/或制剂具有大于96％的纯度，优选大于97％、98％、99％的纯度；
(ii)通过非还原型CE-SDS法测量，主峰变化值小于1％，和/或制剂具有大于96％的纯度，优选大于97％、98％的纯度；
(iii)通过CEX-HPLC法测量，制剂中抗TIGIT抗体蛋白的各组分(主成分、酸性组分和碱性组分)的变化值总和不超过2％；
(iv)通过ELISA法测量，制剂中抗TIGIT抗体蛋白的相对结合活性为70％-130％，例如，90％-110％；
在一个方面中，本发明的液体制剂为药物制剂，优选为注射剂，更优选为皮下注射剂或静脉内注射剂。在一个实施方案中，所述液体制剂为静脉输注剂。
另一方面，本发明提供了ー种固体抗体制剂，其是通过将本发明的液体抗体制剂经固化处理而获得的。所述固化处理是通过例如结晶法、喷雾干燥法或冷冻干燥法实施的。在一个优选的实施方案中，所述固体抗体制剂例如是冻干粉针剂形式。固体抗体制剂可在使用前，通过重构于适当的溶媒中，形成本发明的重构制剂。所述重构制剂也是一种本发明的液体抗体制剂。在一个实施方案中，所述适当的溶媒选自注射用水、注射用有机溶剂，包括但不限于注射用油、乙醇、丙二醇等，或其组合。
在一个方面，本发明提供了一种递送装置，其包含本发明的液体抗体制剂或固体抗体制剂。在一个实施方案中，本发明的递送装置以包含本发明的液体抗体制剂或固体抗体制剂的预填装注射器形式提供，例如用于静脉内、皮下、皮内或者肌内注射、静脉内输注。
在又一方面，本发明提供向受试者，例如哺乳动物递送抗TIGIT抗体蛋白的方法，包括给予所述受试者本发明的液体抗体制剂或固体抗体制剂的步骤，所述递送是例如通过使用预填装注射器的递送装置实施的。
在又一方面，本发明提供本发明的液体抗体制剂或固体抗体制剂的用途，用于制备在受试者中阻断TIGIT与CD155的结合以降低或消除TIGIT的免疫抑制作用的递送装置或预填装注射器或药物，或用于制备在受试者中治疗或预防肿瘤、病原体感染的递送装置(如，预填装注射器)或药物，所述肿瘤为例如癌症，包括但不限于胃肠道癌症，例如结肠癌。
本发明还提供了一种通过向受试者施用本发明的液体抗体制剂或固体抗体制剂或包含该液体抗体制剂或固体抗体制剂的递送装置(如，预填装注射器)或药物，在受试者中阻断TIGIT与CD155的结合以降低或消除TIGIT的免疫抑制作用的方法。
本发明还提供了一种通过向受试者施用本发明的液体抗体制剂或固体抗体制剂或包含该液体抗体制剂或固体抗体制剂的递送装置(如，预填装注射器)或药物,治疗受试者的疾病， 例如上述肿瘤或病原体感染的方法。
本发明的其它实施方案将通过参阅此后的详细说明而清楚明了。
附图简述
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1显示，在实施例1的pH筛选实验中，通过CEX-HPLC法检测，在不同pH条件下，抗体电荷变异体-主成分的变化趋势图。
图2.显示，在实施例1的pH筛选实验中，通过CEX-HPLC法检测，在不同pH条件下，抗体电荷变异体-酸性组分的变化趋势图(CEX-HPLC法)。
图3.显示，在实施例2的40℃稳定性确认实验中，通过CEX-HPLC法检测的电荷变异体变化趋势图。
图4.显示，在实施例2的25℃稳定性确认实验中，通过CEX-HPLC法检测的电荷变异体变化趋势图。
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“TIGIT”是指"含Ig和ITIM结构域的T细胞免疫受体"。该表述也包括TIGIT的变体、同种型、同源物、和物种同源物。TIGIT也称作VSIG9,VSTM3,WUCAM。人和鼠形式的GITR的氨基酸和核酸序列可以参见GenBank登记号NP_776160(人氨基酸序列)和NP_001139797(鼠氨基酸序列)。TIGIT蛋白也可包括TIGIT的片段，诸如包含胞外结 构域的片段，例如保持与本发明任何抗体结合能力的片段。
在本文中，术语“抗体”以最广意义使用，指包含抗原结合位点的蛋白质，涵盖各种结构的天然抗体和人工抗体，包括但不限于完整抗体和抗体的抗原结合片段。
术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换地用来指包含由二硫键相互连接的至少两条重链(H)和两条轻链(L)的糖蛋白。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH区和VL区可以进一步再划分为超变区(为互补决定区(CDR)，其间插有较保守的区域(为构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。
术语“人抗体”或“全人源抗体”在本文中可以互换使用，指包括其中构架区和CDR区二者均源自人种系免疫球蛋白序列的可变区的抗体。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序列(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)，例如在CDR——尤其在CDR3中。然而，如本文所使用的，术语“人抗体”不包括其中的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。
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TIGIT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TIGIT抗体蛋白作为活性成分并包含可药用赋形剂的制备物。将抗TIGIT抗体蛋白与可药用赋形剂组合后，作为活性成分的抗TIGIT抗体蛋白适于治疗性或预防性施与人类或非人类动物。本发明的抗体制剂可以例如制备成水性形式的液体制剂，例如，即用预填装式注射器，或者制备成冻干制剂，在即将使用前通过溶解和/或悬浮于生理可接受的溶液中进行重构(即，复溶)。在一些实施方案中，抗TIGIT抗体蛋白制剂是液体制剂形式。
“稳定的”抗体制剂是指，制剂中的抗体在储存于特定条件下之后、或在振荡后、或在反复冻融后保有可接受程度的物理稳定性和/或化学稳定性。尽管抗体制剂中所含的抗体在储存、振荡或反复冻融之后可能不会100％维持其化学结构，但通常如果维持约90％、约95％、约96％、约97％、约98％或约99％的抗体结构或功能，则认为抗体制剂是“稳定的”。在一些具体的实施方案中，本发明的抗TIGIT抗体蛋白制剂在制造、制备、运输和长期储存过程中表现出低至检测不到的抗体聚集或降解或化学修饰，从而极少或甚至是没有抗TIGIT抗体蛋白的生物活性损失，表现出高度稳定性。在一些实施方案中，本发明的抗TIGIT抗体蛋白制剂在储存、振荡和/或反复冻融后，基本上保留其物理和化学稳定性。优选地，本发明液体制 剂可以在室温或在40℃稳定至少2周，和/或在25℃稳定至少2个月，和/或在2-8℃稳定至少24个月。优选地，本发明液体制剂可以在室温避光650r/min振荡5天后和/或在-30℃/室温反复冻融1-6次后是稳定的。
本领域已知多种分析技术可以用于测定蛋白质的稳定性，参见例如Peptide and Protein Drug Delivery,247-301,Vincent Lee Ed.,Marcel Dekker,Inc.,New York,N.Y.,Pubs(1991)and Jones,A.Adv.Drug Delivery Rev.10:29-90(1993)。可以在选定的温度和选定的储存时间测量稳定性。例如，可以基于预期的制剂货架期来选择储存时间。备选地，可以使用加速稳定性试验。在一些实施方案中，通过对抗体制剂进行各种胁迫测试来进行稳定性测试。这些测试可以代表调配的抗体制剂在制造、储存或运输期间可能遭遇到的极端条件，也可以代表在非制造、储存或运输期间可能使抗体制剂中的抗体的不稳定性加速的条件。例如，可以将经调配的抗TIGIT抗体蛋白制剂充填至玻璃小瓶中以检验在高温胁迫下的抗体稳定性。再例如，可以将经调配的抗TIGIT抗体蛋白制剂充填至玻璃小瓶中并在室温避光650r/min条件下振荡5天后，检查抗体的稳定性。再例如，将将经调配的抗TIGIT抗体蛋白制剂充填至玻璃小瓶中并在-30℃/室温反复冻融1-6次后，检查抗体的稳定性。在一个实施方案中，在-30℃以下冻存1天后室温解冻，为一个冻融循环。
经一段储存时间后、或经振荡一段时间后或经反复冻融多次后，制剂不显示聚集、沉淀、混浊和/或变性；或显示非常少的聚集、沉淀、混浊和/或变性，则可以认为抗体在制剂中“保持其物理稳定性”。由于制剂中抗体的聚集可以潜在地导致患者增加的免疫反应，从而导致安全性问题。因此，需要使在制剂中的抗体聚集最小化或防止聚集。光散射法可以用于测定制剂中的可见聚集物。SEC-HPLC可以用于测定制剂中的可溶性聚集物。此外，可以通过目视检查制剂的外观、颜色和/或澄清度、或者通过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或在振荡后或在反复冻融后制剂中的抗体单体的百分比来测量制剂的稳定性，其中制剂中的抗体单体的百分比越高，则制剂的稳定性越高。
“可接受程度的”物理稳定性可以表示，于特定温度下储存特定时间之后，振荡后或反复冻融后，在制剂中检测到至少约90％的抗TIGIT抗体蛋白单体。在一些实施方案中，在特定温度储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月或更久后，可接受程度的物理稳定性表示至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗TIGIT抗体蛋白单体。当评估物理稳定性时，药物制剂储存的特定温度可为约-80℃至约45℃的任一温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约4℃-8℃、约5℃、约25℃、约35℃、约37℃、约40℃、约42℃或约45℃。例如，若储存于约40℃±2℃1个月或4周之后，检测到至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗TIGIT抗体蛋白单体，则药物制剂视为是稳定的。若储存于约25℃2个月之后，检测到至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗TIGIT抗体蛋白单体，则药物制剂视为是 稳定的。若储存于约5℃9个月之后，检测到至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗TIGIT抗体蛋白单体，则药物制剂视为是稳定的。
经一段储存时间后、或经振荡一段时间后或经反复冻融多次后，如果制剂中的抗体不显示显著的化学改变，则可以认为抗体在制剂中“保持其化学稳定性”。大多数化学不稳定性源自于形成了抗体的共价修饰形式(例如，抗体的电荷变异体)。例如由天冬氨酸异构化、N和C末端修饰，可以形成碱性变异体；由脱酰胺化、唾液酸化和糖化，可以产生酸性变异体。化学稳定性可以通过检测和/或定量抗体的化学改变形式来评估。例如，可以通过阳离子交换色谱(CEX)或成像毛细管等电聚焦电泳(iCIEF)检测制剂中抗体的电荷变异体。在一个实施方案中，通过测定在特定温度下储存特定时间之后或经振荡后或反复冻融多次后制剂中抗体的电荷变异体百分比变化值来测量制剂的稳定性，其中该变化值越小，则制剂的稳定性越高。
“可接受程度”的化学稳定性可以表示于特定温度下储存特定时间之后、或经振荡一段时间后或经反复冻融多次后，制剂中电荷变异体(例如主成分或酸性组分或碱性组分)的百分比变化值不超过40％，例如不超过30％、不超过20％；或电荷变异体(主成分、酸性组分和碱性组分)的百分比变化值总和不超过60％，例如不超过50％、不超过30％。在一些实施方案中，在特定温度储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月或更久后，可接受程度的化学稳定性可以表现为主成分电荷变异体的百分比变化值不超过约50％、40％、30％、20％、或15％；或电荷变异体的百分比变化值总和不超过约60％、50％、或30％。当评估化学稳定性时，储存药物制剂的温度可为约-80℃至约45℃的任一温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约4℃-8℃、约5℃、约25℃或约45℃。例如，若在储存于5℃24个月之后，主成分电荷变异体的百分比变化值少于约25％、24％、23％、22％、21％、20％、19％、18％、17％、16％、15％、14％、13％、12％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、0.5％或0.1％，则药物制剂可被视为是稳定的。若在储存于25℃2个月后，主成分电荷变异体的百分比变化值少于约20％、19％、18％、17％、16％、15％、14％、13％、12％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、0.5％或0.1％，则药物制剂亦可被视为是稳定的。若在储存于40℃1个月之后，主成分电荷变异体的百分比变化值少于约50％、40％、30％、20％、16％、15％、14％、13％、12％、10％、5％或4％，则药物制剂亦可被视为是稳定的。
术语“冻干制剂”是指通过液体制剂的冷冻干燥处理得到或能够得到的组合物。优选地，其为具有少于5％、优选少于3％水含量的固体组合物。
术语“重构制剂”是指将固体制剂(例如冻干制剂)溶解和/或悬浮于生理可接受的溶液中得到的液体制剂。
文中使用的术语“室温”是指15℃至30℃、优选20℃至27℃、更优选25℃的温度。
“胁迫条件”是指在化学和/或物理上不利于抗体蛋白的环境，所述环境可以导致不可接受的抗体蛋白失稳定，例如，高温、振荡、冻融。“高温胁迫”是指，将抗体制剂置于室温或甚至于更高温度(例如40℃±2℃)储存一段时间。通过高温胁迫加速试验，可以检查抗体制剂的 稳定性。
如本文所使用，术语“肠胃外施用”意指肠内和局部给药以外的给药方式，通常通过注射或输注方式，并且包括但不限于，静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下(subcuticular)、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射以及输注。在一些实施方案中，本发明的稳定抗TIGIT抗体蛋白制剂肠胃外施用于受试者。在一个实施方案中，本发明的抗TIGIT抗体蛋白制剂以皮下、皮内、肌内或静脉内注射方式施用于受试者。
I.抗体制剂
本发明提供稳定的液体抗体制剂，其包含(i)抗TIGIT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5.0-6.0。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TIGIT抗体
“抗TIGIT抗体”是指这样的抗体，所述抗体能够以足够的亲和力结合TIGIT分子，以致所述抗体可以用作靶向TIGIT分子的治疗剂和/或预防剂。
在一些实施方案中，如通过生物光干涉法测量，本发明抗体制剂中的抗TIGIT抗体能够以高亲和力，例如以10 -7M或更小、优选地以10-20x 10 -10M的K D特异性结合人TIGIT，并由此介导对TIGIT及其配体CD155结合的高效阻断作用，减少或消除由TIGIT结合其配体引起的抑制性信号传导。在一些实施方案中，本发明抗体制剂中的抗TIGIT抗体抑制含有表达人TIGIT的浸润淋巴细胞的肿瘤(例如胃肠道肿瘤，优选结肠直肠癌)的生长，且优选地与抗PD1抗体联用时，获得显著好于单独一种抗体施用时的抗肿瘤效果。
在一些实施方案中，本发明抗体制剂中的抗TIGIT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，本发明抗体制剂中的抗TIGIT抗体包含SEQ ID NO:7所示重链可变区中的VH CDR1、2和3序列和SEQ ID NO：8所示轻链可变区中的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TIGIT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，本发明抗体制剂中的抗TIGIT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，本发明抗体制剂中的抗TIGIT抗体是IgG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。相同IgG形式的所有抗体的重链恒定区都是相同的，不同IgG形式的抗体之间的重链恒定区不同。例如，IgG1形式的抗体是指其重链恒定区Ig结构域为IgG1的Ig结构域。
在一个优选的实施方案中，本发明抗体制剂中的抗TIGIT抗体是PCT申请号PCT/CN2019/097665(国际申请日：2019年7月25日)中公开的抗TIGIT单克隆抗体ADI-30278，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TIGIT抗体是由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的所述抗体表现出显著的抗肿瘤活性。例如，在接种了小鼠MC38细胞的小鼠肿瘤模型中，施用本发明的抗体制剂可以导致显著的肿瘤抑制效应，尤其是与抗PD-1抗体联用时。
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1-150mg/ml，优选地约10-100mg/mL，例如约10、15、20、25、30、35、40、50、60、70、80、90或100mg/ml抗TIGIT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5.0-6.0的pH范围，例如约5.0-5.5的pH。在一些具体的实施方案中，本发明的抗体制剂具有约5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、或5.8的pH。例如，本发明的抗体制剂具有pH为5.2±0.2或5.5±0.2，优选地pH为5.2。
在一些实施方案中，本发明制剂包含选自以下的缓冲体系：组氨酸-盐酸组氨酸缓冲体系，枸橼酸-枸橼酸钠缓冲体系，醋酸-醋酸钠缓冲体系，磷酸盐缓冲体系，优选组氨酸-盐酸组氨酸缓冲体系。
在一些实施方案中，用于本发明制剂中的缓冲剂为组氨酸缓冲剂，尤其是由组氨酸和盐酸组氨酸组成的缓冲体系。在一些实施方案中，本发明的组氨酸缓冲剂中组氨酸的浓度为约5-50mM，尤其是约5-30mM，例如，约5、10、15、20、25、30mM。在一个实施方案中，本发明制剂包含约10mM组氨酸。在另一个实施方案中，用于本发明制剂的组氨酸缓冲剂由 例如约0.21mg/ml的组氨酸和约1.81mg/ml的盐酸组氨酸组成。
(iii).稳定剂
用于本发明的合适的稳定剂可以选自糖、多元醇和氨基酸及其组合。对于作为稳定剂的糖包括但不限于蔗糖，海藻糖、麦芽糖及组合。对于作为稳定剂的多元醇包括但不限于山梨醇、甘露醇、或其组合。对于作为稳定剂的氨基酸包括但不限于精氨酸、盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸及组合。
例如，在一些实施方案中，稳定剂包含选自以下之一或多项：
-选自山梨醇、甘露醇、或其组合的多元醇，约10-100mg/ml，优选地20-40mg/ml，例如25mg/ml；或40-60mg/ml，例如50mg/ml；
-选自蔗糖、海藻糖、麦芽糖、或其组合的糖类，例如约10-100mg/ml，优选地30-60mg/ml，例如40mg/ml；
-选自盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸及组合的氨基酸，例如，20-200mM,如约50-110mM，例如，70-100mM，尤其是约80-90mM。
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10-100mg/ml，优选地30-60mg/ml，例如40mg/ml。
在一个实施方案中，本发明液体制剂包含山梨醇作为稳定剂。山梨醇在本发明液体制剂中的量可以是约10-100mg/ml，优选地20-40mg/ml，例如25mg/ml；或40-60mg/ml，例如50mg/ml。
在一个实施方案中，本发明液体制剂包含精氨酸作为稳定剂。精氨酸在本发明液体制剂中的量可以是约50-110mM，例如，70-100mM，尤其是约80-90mM，例如约17.91mg/ml盐酸精氨酸。
在一个实施方案中，本发明液体制剂包含山梨醇作为单一稳定剂。在此实施方案中，山梨醇在本发明液体制剂中的量可以是约30-70mg/ml,例如40-60mg/ml。例如，山梨醇可以以约30,35,40,45,50,55,60,65或70mg/ml的量存在，优选地以约50mg/ml的量存在。
在一个实施方案中，本发明液体制剂包含山梨醇和精氨酸的组合作为稳定剂。该组合中，山梨醇可以以约10-60mg/ml，优选地15-40mg/ml,例如20-35mg/ml的量存在，例如可以为大约20,21,22,23,24,25,26,27,28,29,30mg/ml。在该组合中，精氨酸可以以约70-100mM，尤其是约85mM的量存在。优选地，本发明液体制剂包含约20-30mg/ml的山梨醇和约15-20mg/ml的盐酸精氨酸。更优选地，本发明液体制剂包含约25mg/ml的山梨醇和约17.91mg/ml的盐酸精氨酸。
在一个实施方案中，本发明液体制剂包含蔗糖和精氨酸的组合作为稳定剂。该组合中，蔗糖可以以约10-60mg/ml，优选地20-50mg/ml,例如30-40mg/ml的量存在，例如可以为大约30,32,34,36,38,40,42,44,46,48,50mg/ml。在该组合中，精氨酸可以以约70-100mM，尤其是约85mM的量存在。优选地，本发明液体制剂包含约30-50mg/ml的蔗糖和约15-20mg/ml的盐酸精氨酸。更优选地，本发明液体制剂包含约40mg/ml的蔗糖和约17.91mg/ml的盐酸精氨酸。
(iv).表面活性剂
如本文所使用的，术语“表面活性剂”是指具有两亲结构的有机物质；即，它们由相反的溶解性倾向的基团所组成，通常是油溶性的烃链和水溶性的离子基团。
在一个实施方案中，本发明的液体制剂中的表面活性剂是非离子型表面活性剂，例如，烷基聚(环氧乙烯)。可包括在本发明制剂中的特定非离子型表面活性剂包括，例如聚山梨酯，诸如聚山梨酯-20、聚山梨酯-80、聚山梨酯-60、或聚山梨酯-40；泊洛沙姆等。在一个优选实施方案中，本发明的液体制剂中包含聚山梨酯-80作为表面活性剂。
在一些实施方案中，可以用于本发明液体制剂中的表面活性剂包括但不限于，聚山梨酯类表面活性剂(例如聚山梨酯80，聚山梨酯20)、泊洛沙姆和聚乙二醇。
本发明抗体制剂中所含的表面活性剂的量可随制剂的特定目的特性、特定环境、和使用制剂的特定目的而改变。在优选的一些实施方案中，制剂可含有约0.01-5mg/ml，优选地为约0.1-1mg/ml，例如约0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0mg/ml的表面活性剂，尤其是聚山梨酯-80，优选地约0.5mg/ml的聚山梨酯-80。
(v)其它赋形剂
本发明的抗体液体制剂中任选地包含其它赋形剂。所述其它赋形剂包括，例如，抗微生物剂、抗静电剂、抗氧化剂、螯合剂、明胶等等。这些和另外已知的药物赋形剂和/或适用于本发明制剂的添加剂是本领域公知的，例如，列出于“The Handbook of Pharmaceutical Excipients，第4版，Rowe等人编，American Pharmaceuticals Association(2003)；和Remington:the Science and Practice of Pharmacy，第21版，Gennaro编，Lippincott Williams&Wilkins(2005)”。
II.制剂的制备
本发明提供了包含抗TIGIT抗体蛋白的稳定制剂。在本发明制剂中使用的抗TIGIT抗体蛋白可以使用本领域已知的用于生产抗体的技术进行制备。例如，可以重组制备抗体。在一个优选的实施方案中，本发明的抗体在293细胞或CHO细胞中重组制备。
抗体作为药物的活性成分的应用现在已经很广泛。用于将治疗性抗体纯化至药用级的技术是本领域公知的。例如，Tugcu等(Maximizing productivity of chromatography steps for purification of monoclonal antibodies,Biotechnology and Bioengineering 99(2008)599–613.)描述在蛋白A捕获步骤后使用离子交换色谱(阴离子IEX和/或阳离子CEX色谱)的单克隆抗体三柱纯化方法。Kelley等(Weak partitioning chromatography for anion exchange purification of monoclonal antibodies,Biotechnology and Bioengineering 101(2008)553–566)描述了两柱纯化法，其中在蛋白A亲和色谱后使用弱分配阴离子交换树脂。
一般，重组产生的单克隆抗体可以利用常规的纯化方法纯化，以提供具有足够的可重复性和适度纯度的药物物质用于抗体制剂的配制。例如，在抗体从重组表达细胞分泌至培养基中后，可以使用商业可得的蛋白浓缩过滤器例如Amicon的超滤装置，浓缩来自该表达系统的上清液。之后，可以使用例如色谱、透析和亲和纯化等方式进行抗体的纯化。蛋白A适应 于作为亲和配体用于纯化IgG1、IgG2和IgG4型抗体。也可以使用其它抗体纯化方法，例如离子交换色谱。在获得足够纯度的抗体后，可以按照本领域已知的方法，制备包含抗体的制剂。
例如，可以采用如下步骤进行制备：(1)在发酵结束后将发酵液离心澄清去除细胞等杂质以获得上清；(2)使用亲和层析(例如对IgG1、IgG2和IgG4型抗体具有特异亲和力的蛋白A柱)捕获抗体；(3)进行病毒灭活；(4)精制纯化(一般可以采用CEX阳离子交换层析)，以去除蛋白中的杂质；(5)病毒过滤(使病毒滴度降低例如4log10以上)；(6)超滤/渗滤(可以用于将蛋白置换于利于其稳定的制剂缓冲液中并浓缩至合适的浓度供注射用)。参见例如，B.Minow,P.Rogge,K.Thompson,BioProcess International,Vol.10,No.6,2012,pp.48–57。
III.制剂的分析方法
生物制品稳定性研究一般包括实际贮存条件下的实时稳定性研究(长期稳定性研究)、加速稳定性研究和强制条件试验研究。稳定性研究应根据研究目的和产品自身特性对研究条件进行摸索和优化；针对各种影响因素(如温度、反复冻融、振动等)，制定长期、加速和/或强制条件试验等稳定性研究方案。加速和强制条件试验有利于了解产品在短期偏离保存条件和极端情况下产品的稳定性情况，并为有效期和保存条件的确定提供支持性数据。
在抗体制剂的储存、振荡或反复冻融过程中，抗体可能会发生聚集、降解或化学修饰，导致抗体异质性(包括大小异质性和电荷异质性)以及聚集物和片段等，从而影响抗体制剂的质量。因此，有必要进行抗体制剂稳定性的监测。
在本领域中已知多种方法可以用于检测抗体制剂的稳定性。例如，可以通过还原型CE-SDS、非还原型CE-SDS和SEC-HPLC等方法，分析抗体制剂的纯度和评估抗体的聚集水平；可以通过毛细管等电聚焦电泳(cIEF)、成像毛细管等电聚焦电泳(iCIEF)和离子交换色谱(IEX)等，分析抗体制剂中的电荷变异体。此外，可以通过目视检测制剂外观，快速地判断制剂的稳定性。也可以使用OD 350nm法检测制剂的浊度改变，该方法可以给出有关可溶性和不溶性聚集物量的信息。此外，可以使用紫外分光光度法(UV法)检测制剂中的蛋白质含量变化。
非还原型CE-SDS法是一种以毛细管为分离通道进行的抗体纯度测定方法。在CE-SDS中，蛋白迁移由SDS结合引起的表面电荷来驱动，而该表面电荷与蛋白质的分子量成正比。由于所有的SDS-蛋白质复合物都具有相似的质量-电荷比，故可以在毛细管的分子筛凝胶基质中，实现基于分子的大小或流体动力学半径的电泳分离。该方法已经被广泛地用于监测变性的完整抗体的纯度。一般，在非还原型CE-SDS法中，供试样品与SDS样品缓冲液和碘乙酰胺混合。之后，混合物可以于68-72℃孵育约10-15分钟，冷却至室温后离心的上清液用于分析。采用紫外检测器检测蛋白的迁移，获得电泳谱图。抗体制剂纯度可以计算为IgG主峰的峰面积占所有峰面积之和的百分比。关于CE-SDS法的进一步描述，可以参见例如Richard R.等,Application of CE SDS gel in development of biopharmaceutical antibody-based products,Electrophoresis,2008,29,3612-3620。
体积排阻高效液相色谱法，即SEC-HPLC法，是用于抗体标准和质控的另一重要方法。该方法主要依据分子的尺寸大小或流体动力学半径差异来进行分子的分离。通过SEC-HPLC，抗体可以分离出三种主要形式：高分子量形式(HMMS)、主峰(主要是抗体单体)、和低分子量形式(LMMS)。抗体纯度可以计算为色谱图上主峰面积占所有峰面积之和的百分比。通过SEC-HPLC法，可以测量制剂产品中抗体单体的百分数，给出可溶性聚集物和剪切物的含量信息。关于SEC-HPLC法的进一步描述，可以参见例如，J.Pharm.Scien.,83:1645-1650,(1994)；Pharm.Res.,11:485(1994)；J.Pharm.Bio.Anal.,15:1928(1997)；J.Pharm.Bio.Anal.,14:1133-1140(1986)。此外，也可以参见例如，R.Yang等，High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography(SE-UHPLC),Journal of Pharmaceutical and Biomedical Analysis(2015),http://dx.doi.org/10.1016/j.jpba.2015.02.032；和Alexandre Goyon等,Protocols for the analytical characterization of therapeutic monoclonal antibodies.I–Non-denaturing chromatographic techniques,Journal of Chromatography，http://dx.doi.org/10.1016/j.jchromb.2017.05.010。
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(或主成分)的保留时间更早从CEX-HPLC柱洗脱出的峰被标记为“酸性峰”(或酸性组分)，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”(或碱性组分)。
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。此外，可以进行振荡实验或反复冻融实验来检测产品的稳定性性质。例如，在室温避光650r/min振荡1-5天，进行振荡实验。例如，可以将在-30℃以下冻存产品一天后室温融化作为一个冻融循环，进行反复冻融实验，其中可以实施1-6次反复冻融循环。产品稳定性的检测指标可以包括外观、可见异物、蛋白含量、浊度、纯度(SEC-HPLC法、非还原型CE-SDS法)和电荷变异体(iCIEF法、CEX-HPLC法)。此外，可以检测抗体的功效或生物活性。例如，可以检测制剂中抗体与其抗原分子(TIGIT)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。
IV.制剂的用途
本发明的包含抗TIGIT抗体蛋白的本发明抗体制剂具有减少免疫抑制，并能够用于治疗或预防肿瘤、病原体感染等。
在一个实施方案中，本发明制剂可以用于在受试者中阻断TIGIT与CD155的结合以降低或消除TIGIT的免疫抑制作用。在另一实施方案中，本发明制剂可以用于在受试者中治疗或预防肿瘤、病原体感染。所述肿瘤例如是胃肠道癌症，例如结肠癌。
在一个实施方案中，本发明制剂可以与第二治疗剂，例如抗PD-1抗体组合施用。
本发明也提供了本发明的制剂在制备药物中的用途，其中所述药物用于向哺乳动物递送抗TIGIT抗体蛋白。本发明还提供了本发明的制剂用于治疗或预防一种或多种上述疾病和病 症的方法。优选地，哺乳动物是人。
可以以多种途径将本发明的抗体制剂施用于受试者或患者。例如，施用可以通过输注或通过注射器进行。因此，在一个方面，本发明提供了一种递送装置(例如注射器)，其包含本发明的抗体制剂(例如，预填装注射器)。患者将接受有效量的抗TIGIT抗体蛋白作为主要活性成分，即足以治疗、改善或预防目的疾病或病症的量。
治疗效果可包括减少生理症状。用于任何特定受试者的抗体的最佳有效量和浓度将取决于多种因素，包括患者的年龄、体重、健康状况和/或性别、疾病的性质和程度、特定抗体的活性，身体对其清除率，并且也包括与所述抗体制剂组合施用的任何可能的其它治疗。对于具体的情况，所递送的有效量可以在临床医师的判断范围内来确定。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
缩略词描述
缩略词 全称
CE-SDS 十二烷基硫酸钠毛细管凝胶电泳
CEX-HPLC 阳离子交换高效液相色谱
ELISA 酶联免疫吸附测定
SEC-HPLC 体积排阻高效液相色谱
实施例
重组全人源抗TIGIT单克隆抗体ADI-30278，是信达生物制药(苏州)有限公司自主研发的抗体，在PCT申请号PCT/CN2019/097665中公开。该抗体能够有效地阻断TIGIT与其配体CD155的结合，解除CD155对下游IL2信号通路的抑制作用，并且在体内施用时抑制肿瘤生长，尤其是在与抗PD-1抗体联用时，抑瘤效果尤为显著。
为了开发出适用于该全人源抗体的长期稳定储存且简单易用的注射剂制剂处方，通过40℃强制和25℃加速稳定性实验，考察了不同pH值、不同稳定剂及表面活性剂的含量对于该抗体蛋白质量的影响，最终筛选出有利于其稳定的制剂处方。整个研究过程中所用的材料和方法如下：
材料和方法
1.1.本发明的制剂研究中使用的材料
Figure PCTCN2021072690-appb-000003
Figure PCTCN2021072690-appb-000004
注：N/A表示不适用。
1.2.本发明的制剂研究中使用的仪器设备
名称 产地及品牌 型号 编号
电子天平 德国Sartorius BSA3202S PD-A1-186
电子天平 瑞士梅特勒 XPE3003S PD-A1-247
恒温恒湿箱 德国BINDER KBF P 720 PD-A1-070
生化培养箱 上海精宏 SHP-150 PD-A1-200
医用冷藏箱 青岛海尔 HYC-360 PD-A1-166
医用冷藏箱 青岛海尔 HYC-360 PD-A1-165
超低温冰箱 美国Thermo 907 PD-A1-175
澄明度检测仪 天津天大天发 YB-2 PD-A1-033
紫外可见分光光度计 日本岛津 UV-1800 AS-A1-037
pH计 瑞士梅特勒 FE20 PD-A1-161
多通道微量分光光度计 美国Thermo Nanodrop 8000 PD-A1-052
台式冷冻离心机 美国Thermo SL16R PD-A1-082
洁净工作台 苏州Airtech SW-CJ-2FD QC-A1-011
中流量手动蠕动泵 英国Watson Marlow 520S/R2 PD-A1-235
灌装机 丹麦Watson Marlow FP50 PD-C14-115
不溶性微粒检测仪 天津天大天发 GWJ-8 QC-A1-094
1.3.制剂稳定性的检测项目和检测方法
整个研究过程中检测项目主要包括：(1)检测外观以及是否存在可见异物；(2)通过 紫外法(UV法)测定制剂中的蛋白质含量；(3)通过OD350nm法检测制剂的浊度；(4)通过体积排阻高效液相色谱法(SEC-HPLC)测定抗体制剂的纯度，表示为主峰的面积占所有峰面积之和的百分数；(5)通过非还原型十二烷基硫酸钠毛细管电泳(非还原型CE-SDS)测定抗体制剂的纯度，表示为主峰的面积占所有峰面积之和的百分数；(6)通过CEX-HPLC法测定抗体制剂中电荷变异体，表示为主成分、酸性组分和碱性组分的百分数；(7)通过直接ELISA法测定抗体制剂中抗TIGIT抗体对TIGIT抗原的相对结合活性。
可见异物检测
按照国家药典委员会，中华人民共和国药典(2015年版，四部通则0904“可见异物检查法”，北京：中国医药科技出版社.2015)中所记载的方法，采用澄明度检测仪(天津天大天发生产，型号YB-2)和不溶性微粒检测仪(天津天大天发生产，型号GWJ-8)，检查样品中的可见异物。
蛋白含量测定
使用紫外分光光度计(日本岛津生产，型号UV-1800)或多通道微量分光光度计(美国Thermo生产，型号Nanodrop 8000)，测定样品中的蛋白质含量。
浊度测定
使用紫外分光光度计(日本岛津生产，型号UV-1800)，测定样品在350nm的吸光度，确定样品浊度。
纯度(SEC-HPLC法)
使用体积排阻色谱柱分离，流动相为磷酸盐缓冲液(称取3.12g二水合磷酸二氢钠，8.77g氯化钠和34.84g精氨酸，超纯水溶解后用盐酸调节pH至6.8并定容至1000ml)，色谱柱保护液为0.05％(w/v)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(非还原型CE-SDS法)
采用毛细管凝胶电泳法检测。毛细管为无涂层毛细管，内径50μm，总长30.2cm，有效长度20.2cm。电泳前分别使用0.1mol/L氢氧化钠、0.1mol/L盐酸、超纯水、电泳胶70psi冲洗毛细管柱。将待测样品用适量超纯水稀释至2.0mg/ml，取以上稀释后的样品50μl于1.5ml离心管中，分别向其中加入45μl pH 6.5的样品缓冲液(称取一水柠檬酸0.32g，十二水合磷酸氢二钠2.45g，溶于45ml超纯水中，定容至50ml，制得柠檬酸-磷酸盐缓冲液，精密量取该缓冲液200μl，加10％(w/v)十二烷基硫酸钠溶液80μl，加水至1ml，混匀，即得)、1μl内标(10kDa蛋白质，5mg/mL)(Beckman Coulter，货号：390953)和5μl 250mmol/L NEM溶液(称取N-乙基顺丁稀二酰亚胺62mg，溶于2ml超纯水中)，充分混匀后70±2℃加热10±2分钟，冷却至室温后转移至样品瓶作为供试品溶液。取与供试品相同体积的制剂缓冲液，按上述方法同样操作，制得空白溶液。样品进样条件：-5kV 20秒；分离电压：-15kV 35 分钟。毛细管柱温控制在25℃，检测波长为220nm。
电荷变异体(CEX-HPLC法)
采用阳离子交换色谱法(CEX-HPLC法)检测。使用MabPac SCX-10强阳离子交换色谱柱分离，流动相A为10mmol/L磷酸盐缓冲液(称取NaH2PO4·2H2O 0.51g，Na2HPO4·12H2O 2.40g溶解于800ml超纯水中，定容到1000ml，使用Φ0.22μm滤膜过滤)，流动相B为10mmol/L磷酸盐+200mmol/L氯化钠缓冲液(称取NaH2PO4·2H2O 0.51g，Na2HPO4·12H2O 2.40g，NaCl 11.69g溶解于800ml超纯水中，定容到1000ml，使用Φ0.22μm滤膜过滤)。用超纯水将样品稀释至2.0mg/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作为空白溶液。取空白溶液、供试品溶液各50μl注入液相色谱仪，流动相流速1.0ml/min，采集时间35分钟，柱温35℃，检测波长280nm，样品盘温度10℃。进样分析，根据面积归一化法，计算主成分、酸性组分及碱性组分含量。
相对结合活性(直接ELISA法)
用人TIGIT抗原(购自Acrobiosystems，TIT-H52H3)以0.5μg/ml，100μl/孔，4℃过夜包被96孔酶标板。洗板后加封闭液(2％BSA-PBST，300μl/孔)37℃封闭2h。将梯度稀释的供试品以100μl/孔加入到弃去封闭液的酶标板中，设置阴性对照每孔只加100μl稀释液(2％BSA-PBST)，37℃恒温培养箱中孵育60min。洗板后加入以2％BSA-PBST稀释的HRP缀合山羊抗人IgG-Fc片段(美国BETHYL，货号A80-104P)作为二抗(100000倍稀释，100μl/孔)，37℃反应30min。洗板后每孔加入100μl TMB显色液，显色10min后，每孔加入100μl的1mol/L H 2SO 4终止反应。以620nm为参比波长，测450nm处的OD值。以各浓度梯度样品的浓度值作为横坐标，各梯度样品的OD450nm-OD620nm值为纵坐标，应用Prism四参数拟合计算EC 50反映抗体与各抗原的结合活性。相对结合活性(％)＝(供试品的EC 50/参比品的EC 50)＊100％，其中所述参比品为未经任何胁迫处理的稳定的抗TIGIT抗体。
实施例1.制备和纯化抗TIGIT抗体
根据PCT申请号PCT/CN2019/097665所述，获得特异性结合TIGIT的抗TIGIT抗体ADI-30278。该抗体具有SEQ ID NO：9的重链序列和SEQ ID NO：10的轻链序列，为全人源抗体。PCT申请号PCT/CN2019/097665在此整体并入本文作为参考。
简言之，抗体在CHO细胞中重组表达并通过过滤、层析、病毒灭活、过滤等工艺进行纯化。用于以下pH筛选试验的样品为经亲和层析纯化的产品，具有17.0mg/ml的蛋白含量。用于以下处方确定试验的样品为经纳滤膜过滤纯化的产品，具有11.5mg/ml的蛋白含量。
实施例2.pH筛选试验
2.1实验步骤
本实施例考察pH 5.0、5.5、6.0、6.5和7.0的组氨酸缓冲体系对实施例1的经纯化的抗TIGIT抗体稳定性的影响，以获得较优的pH值范围。
配制10mM组氨酸，5％(w/v)山梨醇缓冲液，用盐酸将pH分别调节为5.0、5.5、6.0、 6.5和7.0，将实施例1的经纯化的抗TIGIT抗体超滤置换到上述不同pH值的缓冲液中，调节蛋白含量至约20mg/ml；加入聚山梨酯80至0.20mg/ml；过滤分装至西林瓶，加塞，轧盖。将上述样品于40℃±2℃条件下进行稳定性考察，具体实验方案见表1。
表1.实验方案
Figure PCTCN2021072690-appb-000005
注：(1)X表示该点取样。(2)上述时间点取样后均先放入超低温冰箱中冻存待检。
2.2判断标准
根据对产品的认识以及仪器和方法的精密度，设定了样品检测指标数值与初始值相比质量未发生变化的判断标准，用以判断样品是否发生了变化，判断标准见表2。
表2.质量未发生变化的判断标准
检测项目 未发生变化的判断标准
外观(观察法) 澄明至微乳光，无色至淡黄色液体，无异物
可见异物(可见异物检查法) 符合《中华人民共和国药典》(2015年版，四部)则0904
蛋白含量(UV法) 变化率≤10％
浊度(OD 350nm法) 变化值≤0.02
纯度(SEC-HPLC法) 主峰变化值≤1％
纯度(非还原型CE-SDS法) 主峰变化值≤2％
电荷变异体(CEX-HPLC法) 主成分及酸碱各组分变化值≤2％
相对结合活性(直接ELISA法) 应为70％～130％
2.3实验结果
(1)外观和可见异物
pH 6.5和pH 7.0样品在浓缩换液结束后，样品外观出现浑浊、有沉淀，故不进行加速稳定性考察。pH 5.0、pH 5.5和pH 6.0样品在40℃±2℃条件下放置2周，外观、可见异物均合格。
(2)蛋白含量
蛋白含量检测结果见表3。结果表明，在40℃±2℃条件下放置2周，pH 5.0、pH 5.5和pH 6.0样品蛋白含量均未发生显著变化。
表3.pH筛选蛋白含量结果(UV法，mg/ml)
Figure PCTCN2021072690-appb-000006
(3)浊度
浊度结果见表4。结果表明，在40℃±2℃条件下放置2周，样品pH 5.0和pH 5.5浊度未发生变化；样品pH 6.0的浊度有一定的上升趋势。
表4.pH筛选浊度结果(OD 350nm法)
Figure PCTCN2021072690-appb-000007
(4)纯度
纯度(SEC-HPLC法和非还原型CE-SDS法)结果见表5。结果表明，在40℃±2℃条件下放置2周，pH 5.0、pH 5.5和pH 6.0样品纯度均未发生明显变化。
表5.pH筛选纯度结果
Figure PCTCN2021072690-appb-000008
(5)电荷变异体
电荷变异体(CEX-HPLC法)结果见表6，其变化趋势见图1和图2。结果表明，在40℃±2℃条件下放置2周，pH 5.0、pH 5.5和pH 6.0样品主成分和酸性组分均发生明显变化。与0天比较，pH 5.0、pH 5.5和pH 6.0样品主成分分别下降4.8％、4.4％和7.2％，酸性组分分别上升3.6％、4.3％和7.4％，综上所述，从电荷变异体检测结果分析，抗体蛋白在pH 5.0和pH 5.5条件下更为稳定。
表6.pH筛选电荷变异体结果(CEX-HPLC法，％)
Figure PCTCN2021072690-appb-000009
Figure PCTCN2021072690-appb-000010
综上所述，pH筛选实验结果表明，抗体制剂处方组氨酸缓冲体系的pH在5.0～5.5之间较为合适。选定pH 5.2，进行下一轮处方确定实验。
实施例3.处方确定实验
3.1实验步骤
根据上述pH筛选实验结果并结合制剂处方开发平台经验，考察不同稳定剂(山梨醇、蔗糖和盐酸精氨酸)以及聚山梨酯80含量对抗体蛋白稳定性的影响，共设计了3个处方，详细处方信息见表7。
表7.处方信息表
Figure PCTCN2021072690-appb-000011
按照表7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，调节各处方蛋白含量至约50mg/ml；加入聚山梨酯80。过滤分装至西林瓶中，加塞，轧盖。将上述样品进行振荡、冻融和不同温度条件下的稳定性考察。具体方案见表8。
表8.实验条件及取样计划
Figure PCTCN2021072690-appb-000012
注：“*”表示设置该点考察，但未检测。
3.2判断标准
判断标准具体参见实施例2中的表2。
3.3实验结果
(1)振荡实验
振荡实验结果详见表9。结果表明，在室温、避光650r/min条件下振荡5天，3组处方样品外观、可见异物均合格；蛋白含量、纯度、电荷变异体和相对结合活性均未发生明显变化。
表9.振荡实验结果
Figure PCTCN2021072690-appb-000013
注：N/A表示未设置该项检测。
(2)冻融实验
冻融实验结果详见表10。反复冻融6次后，3组处方样品外观、可见异物均合格；蛋白含量、纯度、电荷变异体和相对结合活性均未发生明显变化。
表10.冻融实验结果
Figure PCTCN2021072690-appb-000014
上述实验结果表明，在振荡和冻融实验中处方1、2和3稳定性无明显差异。
考虑到抗体给药方式采用静脉输注，样品由生理盐水稀释后，聚山梨酯80含量过低会影响蛋白稳定性。另外，鉴于山梨醇和蔗糖对蛋白的保护作用无差异，选择非还原型的山梨醇，可降低在长期存放过程中辅料之间发生糖化反应的风险，因此选择处方2在40℃和25℃条件开展稳定性确认实验。
(3)40℃稳定性确认实验
40℃稳定性确认实验结果详见表11。在40℃条件下放置1个月，样品外观、可见异物均合格。蛋白含量、纯度和相对结合活性均未发生明显变化；电荷变异体变化较明显，主成分下降13.5％，酸性组分上升8.4％，碱性组分上升5.1％，其变化趋势见图3。结合制剂处方开发平台经验，此电荷变异体变化在可接受范围之内。
表11. 40℃稳定性确认实验结果
Figure PCTCN2021072690-appb-000015
注：N/A表示未设置该项检测。
(4)25℃稳定性确认实验
25℃稳定性确认实验结果详见表12。在25℃条件下放置2个月，样品外观、可见异物均合格。蛋白含量和纯度均未发生明显变化；电荷变异体变化较明显，主成分下降6.4％，酸性组分上升3.6％，碱性组分上升2.9％，其变化趋势见图5。结合制剂处方开发平台经验，此电荷变异体变化在可接受范围之内。
表12. 25℃稳定性确认实验结果
Figure PCTCN2021072690-appb-000016
根据上述实验结果及制剂处方开发平台经验，最终选定处方2为抗体制剂处方。其组成 为：50.0mg/ml重组全人源抗TIGIT抗体、0.21mg/ml组氨酸、1.81mg/ml盐酸组氨酸、25.00mg/ml山梨醇、17.91mg/ml盐酸精氨酸、0.50mg/ml聚山梨酯80，pH 5.2。
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 (19)

  1. 一种液体抗体制剂，包含
    (i)抗TIGIT抗体蛋白；
    (ii)缓冲剂，
    (iii)稳定剂，和
    (iv)表面活性剂，
    其中所述抗TIGIT抗体蛋白包含
    -YTFTEYYMH(SEQ ID NO:1)的重链VH CDR1；
    -IISPSAGSTKYAQKFQG(SEQ ID NO:2)的重链VH CDR2；
    -ARDHDIRLAGRLADY(SEQ ID NO:3)的重链VH CDR3；
    -RASQGISSWLA(SEQ ID NO:4)的轻链VL CDR1；
    -AASSLQS(SEQ ID NO:5)的轻链VL CDR2；和
    -QQAVILPIT(SEQ ID NO:6)的轻链VL CDR3，
    优选地，所述液体抗体制剂的pH约为5.0-6.0，例如，pH为5.2±0.2或5.5±0.2，优选地pH为5.2。
  2. 根据权利要求1的液体抗体制剂，特征在于所述液体抗体制剂中的抗TIGIT抗体蛋白的浓度为约1-100mg/ml，优选地为约10-70mg/mL，例如约10、15、20、25、30、35、40、50、55、60、70mg/ml。
  3. 根据权利要求1或2所述的液体抗体制剂，特征在于，
    所述液体抗体制剂包含选自组氨酸-盐酸组氨酸缓冲体系，枸橼酸-枸橼酸钠缓冲体系，醋酸-醋酸钠缓冲体系，和磷酸盐缓冲体系的缓冲剂，
    优选地，所述液体抗体制剂中的缓冲剂选自组氨酸、盐酸组氨酸和它们的组合；
    优选地，所述缓冲剂的浓度为约5-50mM，优选地为约5-30mM，例如，约5、10、15、20、25、30mM。
  4. 根据权利要求1-3中任何一项所述的液体抗体制剂，特征在于所述稳定剂选自多元醇(例如，山梨醇、甘露醇及其组合)、糖类(例如，蔗糖、海藻糖、麦芽糖及其组合)、氨基酸(例如精氨酸、盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸及组合)、和它们的任意组合，
    例如，所述稳定剂包含选自以下之一或多项：
    -选自山梨醇、甘露醇、或其组合的多元醇；
    -选自蔗糖、海藻糖、麦芽糖、或其组合的糖类；
    -选自盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸及组合的氨基酸。
  5. 根据权利要求1-4中任何一项所述的液体抗体制剂，特征在于所述稳定剂选自：
    (i)约20-40mg/ml的山梨醇和约50－100mM的精氨酸的组合，或
    (ii)约30-60mg/ml的蔗糖和约50－100mM的精氨酸的组合。
  6. 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中的表 面活性剂选自聚山梨酯类表面活性剂、泊洛沙姆、聚乙二醇或其组合，优选为聚山梨酯-80。
  7. 根据权利要求1-5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，特征在于所述抗TIGIT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。
  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TIGIT抗体是IgG型抗体(例如，IgG4亚型抗体)，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。
  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TIGIT抗体在293细胞或CHO细胞中重组表达。
  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。
  12. 根据权利要求1-11中任一项所述的液体抗体制剂，其包含：
    (i)约10-100mg/ml的抗TIGIT抗体蛋白；
    (ii)约5-50mM的组氨酸缓冲剂；
    (iii)约50-300mM的山梨醇或蔗糖；
    (iv)约0.1-1mg/ml聚山梨酯80；和
    (v)任选地，约50-120mM的精氨酸，
    其中所述液体制剂的pH为约5.0-5.5，例如，约5.2；
    例如，所述液体抗体制剂包含
    (i)约10-60mg/ml，例如20-50mg/ml的抗TIGIT抗体蛋白；
    (ii)约10mM的组氨酸缓冲剂；
    (iii)约10-50mg/ml山梨醇或蔗糖，优选20-30mg/ml山梨醇，或30-50mg/ml蔗糖；
    (iv)约0.2-0.8mg/ml，例如0.3-0.6mg/ml聚山梨酯80；和
    (v)约60-100mM，例如约85mM的精氨酸，
    其中所述液体制剂的pH为约5.0-5.5，例如，约5.2；
    或者，所述液体抗体制剂包含
    (i)约50mg/ml的抗TIGIT抗体蛋白，约0.21mg/ml组氨酸，约1.81mg/ml盐酸组氨酸，约25.00mg/ml山梨醇，约17.91mg/ml盐酸精氨酸，约0.50mg/ml聚山梨酯80，pH约5.2；或
    (ii)约50mg/ml的抗TIGIT抗体蛋白，约0.21mg/ml组氨酸，约1.81mg/ml盐酸组氨酸，约25.00mg/ml山梨醇，约17.91mg/ml盐酸精氨酸，约0.20mg/ml聚山梨酯80，pH约5.2；或
    (iii)0.21mg/ml组氨酸，1.81mg/ml盐酸组氨酸，40.00mg/ml蔗糖，17.91mg/ml盐酸精氨酸，0.20mg/ml聚山梨酯80，pH 5.2。
  13. 根据权利要求1-12中任何一项所述的液体抗体制剂，其特征在于，该制剂在储存后，例如在25℃储存至少2个月后，或在40℃±2℃储存1个月后，是稳定的，优选地具有如下特征之一或多项：
    (i)通过SEC-HPLC法测量，主峰变化值小于1％，和/或制剂具有大于96％的纯度，优选大于97％、98％的纯度；
    (ii)通过非还原型CE-SDS法测量，主峰变化值小于2％，和/或制剂具有大于96％的纯度，优选大于97％、98％的纯度；
    (iii)通过CEX-HPLC法测量，相对于储存第0天的初始值，制剂中抗TIGIT抗体蛋白的各组分(主成分、酸性组分和碱性组分)的变化值总和不超过40％和/或主成分的变化值不超过20％，例如在40℃±2℃储存1个月后变化值总和不超过约40％(例如不超过30％)或主成分变化值不超过约20％(例如不超过15％)，或在25℃储存2个月后变化值总和不超过约20％(例如约15％)或主成分变化值不超过约15％(例如不超过约10％)；
    (iv)通过ELISA法测量，相对于储存第0天的初始值，制剂中抗TIGIT抗体蛋白的相对结合活性为70％-130％，例如，90％-110％；
    更优选地，该制剂在振荡和/或反复冻融下是稳定的。
  14. 一种固体抗体制剂，其通过固化权利要求1-13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。
  15. 递送装置，其包含权利要求1-13中任何一项的液体抗体制剂或权利要求14的固体抗体制剂。
  16. 预填装注射器，其包含权利要求1-13中任何一项的液体抗体制剂或权利要求14的固体抗体制剂，用于静脉内注射或者肌内注射。
  17. 根据权利要求1-13中任何一项的液体抗体制剂或权利要求14的固体抗体制剂的用途，用于制备在受试者中阻断TIGIT与CD155的结合以降低或消除TIGIT的免疫抑制作用的递送装置或预填装注射器或药物。
  18. 根据权利要求1-13中任何一项的液体抗体制剂或权利要求14的固体抗体制剂的用途，用于制备在受试者中治疗或预防肿瘤、病原体感染的递送装置或预填装注射器或药物，例如，所述肿瘤是胃肠道癌症，例如结肠癌。
  19. 根据权利要求17或18的用途，其中所述抗TIGIT抗体与第二治疗剂，例如抗PD-1抗体组合施用。
PCT/CN2021/072690 2020-01-21 2021-01-19 重组全人源抗tigit单克隆抗体制剂及其制备方法和用途 WO2021147854A1 (zh)

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