WO2017054646A1 - 一种抗pd-1抗体制剂及其在医药上的应用 - Google Patents

一种抗pd-1抗体制剂及其在医药上的应用 Download PDF

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Publication number
WO2017054646A1
WO2017054646A1 PCT/CN2016/098982 CN2016098982W WO2017054646A1 WO 2017054646 A1 WO2017054646 A1 WO 2017054646A1 CN 2016098982 W CN2016098982 W CN 2016098982W WO 2017054646 A1 WO2017054646 A1 WO 2017054646A1
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cancer
antibody
pharmaceutical preparation
polysorbate
preparation according
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PCT/CN2016/098982
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English (en)
French (fr)
Inventor
李�杰
颜贞
王萍萍
方言
陶维康
张连山
孙飘扬
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to UAA201804310A priority Critical patent/UA124259C2/uk
Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to AU2016329960A priority patent/AU2016329960A1/en
Priority to CN201680003949.4A priority patent/CN106999591B/zh
Priority to JP2018515764A priority patent/JP6921062B2/ja
Priority to BR112018005349-0A priority patent/BR112018005349A2/zh
Priority to RU2018110333A priority patent/RU2731418C2/ru
Priority to KR1020187011041A priority patent/KR20180054791A/ko
Priority to US15/761,552 priority patent/US10786567B2/en
Priority to CA2999079A priority patent/CA2999079A1/en
Priority to EP16850270.6A priority patent/EP3357508A4/en
Priority to MX2018003306A priority patent/MX2018003306A/es
Publication of WO2017054646A1 publication Critical patent/WO2017054646A1/zh
Priority to US16/948,077 priority patent/US20210000954A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • 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
    • 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
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to a pharmaceutical preparation comprising an anti-PD-1 antibody and an antigen-binding fragment thereof, a process for the preparation thereof, and the use of the preparation.
  • tumor immunotherapy is to maximize the patient's own immune system response to the tumor. It not only activates the original immune system response in the body, but also maintains the duration of the immune system response and the intensity of the response. key.
  • PD-l Programmed death-l
  • CTL-4 cytotoxic T Iymphocyte antigen 4
  • PD-1 has two ligands, PD-L1 and PD-L2.
  • the new study found that high PD-L1 protein expression was detected in human tumor tissues such as breast cancer, lung cancer, gastric cancer, colon cancer, kidney cancer, and melanoma, and the expression level of PD-L1 was closely related to the clinical and prognosis of patients. . Since PD-L1 plays a role in suppressing T cell proliferation by the second signaling pathway, blocking the binding between PD-L1/PD-1 has become a very potential emerging target in the field of tumor immunotherapy.
  • WO 2015/085847 discloses a new class of anti-PD-1 antibodies which have the characteristics of high affinity and long half-life, and are expected to have a better therapeutic effect on the above diseases.
  • these new anti-PD-1 antibodies are extremely unstable and difficult to make into clinically available formulations, and PCT does not have any specific description of how they are formulated. Therefore, it is necessary to conduct intensive studies on these antibodies to obtain a stable and convenient clinical use preparation.
  • the stable pharmaceutical preparation of the present invention contains an anti-PD-1 antibody, an antigen-binding fragment thereof, and a buffer.
  • the pharmaceutical preparations may also contain at least one stabilizer, and optionally may also contain a surfactant.
  • the anti-PD-1 antibody and antigen-binding fragment thereof comprise any one or more CDR region sequences selected from the following or an amino acid sequence having at least 85% sequence homology thereto :
  • Antibody heavy chain variable region HCDR region sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3;
  • Antibody light chain variable region LCDR region sequence SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6.
  • amino acid sequences are shown in the following table:
  • sequence homology may be selected by conventional methods in order to improve the affinity or immunogenicity or stability of the antibody or other conventional physical and chemical properties and biological activities.
  • a further preferred anti-PD-1 antibody has the heavy chain of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8:
  • the concentration of the anti-PD-1 antibody may be 1-60 mg/ml, preferably 20-50 mg/ml, more preferably 35-45 mg/ml, and most preferably 40 mg/ml.
  • the buffering agent of the present invention is selected from one or more of acetate, citrate, succinate, and phosphate, and the phosphate may be selected from the group consisting of sodium dihydrogen phosphate and potassium dihydrogen phosphate;
  • a preferred buffer is acetate, and the amount thereof is not particularly limited, and in the embodiment of the present invention, for example, is 1 to 50 mM, preferably 2 to 20 mM, more preferably 5 to 15 mM, and most preferably 10 mM.
  • the pH of the pharmaceutical preparation of the invention may range from 4.5 to 6.0, preferably from 4.8 to 5.6, most preferably the pH is 5.2.
  • the at least one stabilizer of the present invention is selected from the group consisting of sugars or amino acids.
  • the sugar is preferably a disaccharide, selected From sucrose, lactic acid, trehalose, maltose, preferably trehalose, most preferably alpha, alpha-dihydrate trehalose.
  • the amount of the disaccharide to be used is not particularly limited, and in the embodiment of the present invention, for example, is 30 to 120 mg/ml, preferably 60 to 100 mg/ml, more preferably 85 to 95 mg/ml, and most preferably 90 mg/ml.
  • the surfactant of the present invention may be selected from the group consisting of polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, and the polyoxyethylene sorbitan fatty acid ester may be selected from polysorbates. 20, 40, 60 or 80.
  • the amount of the surfactant to be used is not particularly limited, and in the embodiment of the present invention, for example, is 0.01 to 1 mg/ml, preferably 0.05 to 0.5 mg/ml, more preferably 0.1 to 0.4 mg/ml, and most preferably 0.2 mg/ml. .
  • the stable pharmaceutical preparation of the present invention is an injectable pharmaceutical preparation.
  • the stable pharmaceutical formulation consists of an anti-PD-1 antibody, a buffer, a disaccharide, a surfactant, optionally including water.
  • the stable pharmaceutical formulation comprises:
  • An anti-PD-1 antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8;
  • the injectable pharmaceutical preparation may be in the form of an injection or may be further prepared in the form of a lyophilized powder.
  • the lyophilized powder can be prepared by conventional methods in the art.
  • the invention also provides an injection formed by reconstitution after lyophilization powder, which can be directly used for injection.
  • the pharmaceutical preparation of the invention can effectively inhibit the aggregation and deamidation of the antibody, thereby preventing the degradation of the antibody product therein, and obtaining a stable injection composition, which can be stably stored at 25 ° C for 6 months. Stable storage for 12 months at 2-8 °C. Further, the pharmaceutical composition of the present invention has a protective effect on oxidative degradation of proteins, and is also compatible with glass and stainless steel containers, and can be stably present in these containers.
  • the pharmaceutical preparation of the present invention for use in the prevention or treatment of a PD-1 mediated disease or condition, preferably a cancer; more preferably a cancer expressing PD-L1; said cancer being most preferably a mammary gland Cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma; most preferred are non-small cell lung cancer, melanoma and kidney cancer.
  • a pharmaceutical preparation according to the invention for the preparation of a medicament for the prevention or treatment of a PD-1 mediated disease or condition, said disease being preferably a cancer; more preferably a cancer expressing PD-L1;
  • the cancer is most preferably breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma; most preferably non-small cell lung cancer, melanoma and kidney cancer.
  • a method for preventing or treating a PD-1 mediated disease or condition preferably a cancer; more preferably a cancer expressing PD-L1; said cancer being most preferably breast cancer, lung cancer, Gastric cancer, intestinal cancer, renal cancer, melanoma; most preferably non-small cell lung cancer, melanoma and renal cancer, the method comprising administering a pharmaceutical formulation of the invention.
  • the present invention relates to a stable pharmaceutical liquid preparation comprising a high concentration of an antibody against PD-1.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the antibody of the present invention includes a murine antibody, a chimeric antibody, a humanized antibody, preferably a humanized antibody.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, PD-1). It has been shown that fragments of full length antibodies can be utilized for antigen binding function of antibodies.
  • the antigen binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact immunoglobulin.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding.
  • One of the most commonly used definitions of the six CDRs is provided by Kabat E. A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • Kabat definition of a CDR applies only to the LCDR1, LCDR2, and LCDR3 of the light chain variable domain, as well as the HCDR1, HCDR2, and HCDR3 of the heavy chain variable domain.
  • the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or a variant thereof.
  • the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the murine hybridoma cell, and the variable region gene of the human antibody is cloned as needed, and the murine variable region gene is cloned.
  • Human constant region gene After insertion into a chimeric gene, it is inserted into an expression vector, and finally a chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of murine CDR sequences into human antibody variable region frameworks, ie different types of human germline antibodies An antibody produced in a framework sequence. It is possible to overcome the heterologous reaction induced by the chimeric antibody by carrying a large amount of the mouse protein component.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • pharmaceutical formulation means an article that is in a form that allows for a clear and effective biological activity of the active ingredient, and which does not contain additional components that are toxic to the subject to which the formulation is to be administered.
  • liquid as used herein in connection with a formulation according to the invention means a formulation which is liquid at atmospheric pressure at a temperature of at least about 2 ° C to about 8 ° C.
  • stabilizer means a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and application.
  • Stabilizers include, but are not limited to, sugars, amino acids, polyols, surfactants, antioxidants, preservatives, cyclodextrins, polyethylene glycols such as PEG 3000, 3350, 4000, 6000, albumin, for example, as defined below Human serum albumin (HSA), bovine serum albumin (BSA), salts such as sodium chloride, magnesium chloride, calcium chloride, chelating agents such as EDTA, as defined hereinafter.
  • HSA Human serum albumin
  • BSA bovine serum albumin
  • the stabilizer specifically used in the present invention is selected from the group consisting of sugars. More specifically, the stabilizer is selected from the group consisting of sucrose, trehalose, sorbitol.
  • the stabilizer may be present in the formulation in an amount of from 30 mg/ml to 100 mg/ml, preferably from 60 mg/ml to 90 mg/ml. More specifically, sucrose or trehalose was used as a stabilizer in an amount of 90 mg/ml.
  • a “stable” formulation is one in which the protein (e.g., antibody) contained therein substantially retains its physical and chemical stability upon storage and thus retains its biological activity.
  • a “stable liquid drug antibody formulation” is a liquid antibody formulation that does not exhibit significant changes at refrigeration temperatures (2-8 ° C) for at least 12 months, particularly 2 years, and more particularly 3 years.
  • the criteria for stability are as follows: no more than 10%, especially 5%, of the antibody monomer is degraded when measured by size exclusion chromatography (SEC-HPLC). In addition, by visual analysis, the solution was colorless or clear to a slight milky white.
  • the protein concentration of the formulation has no more than a +/- 10% change. No more than 10%, especially 5%, of aggregation is formed. Stability is measured by methods known in the art such as UV spectroscopy, size exclusion chromatography (SEC-HPLC), ion exchange chromatography (IE-HPLC), turbidimetry and visual inspection.
  • programmed death 1 means programmed death 1
  • protein PD-1 protein PD-1
  • PD-1 protein PD-1
  • PD1 protein PD-1
  • PDCD1 protein PD-1
  • hPD-1 protein PD-1
  • hPD-I used interchangeably, including variants, isoforms, species homologs of human PD-1, and analogs having at least one co-epitope with PD-1.
  • NCBI Reference Sequence NM_005018.1.
  • anti-PD-1 antibody refers to the ability to bind PD-1 protein with sufficient affinity such that the antibody can target PD-1 Used as a diagnostic and/or therapeutic agent in proteins.
  • binding PD-1 protein refers to the binding of an antibody to PD-1 protein in a BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden) or in an ELISA, wherein the purified PD-1 protein is to be purified. Or PD-1 protein CHO transfectants were coated on microtiter plates.
  • the concentration of the antibody against the PD-1 protein contained in the pharmaceutical preparation is in the range of 1 mg/ml to 60 mg/ml, preferably in the range of 20 mg/ml to 50 mg/ml, and most preferably 40 mg/ml.
  • surfactant denotes a pharmaceutically acceptable excipient for protecting a protein preparation against physical stress such as agitation and shear.
  • pharmaceutically acceptable surfactants include: polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (such as those sold under the trademark BrijTM ) , and polyoxyethylene-poly Oxypropylene copolymer (Ploronic, Pluronic).
  • polyoxyethylene sorbitan-fatty acid esters are polysorbate 20 ( sold under the trademark Tween 20TM) and polysorbate 80 ( sold under the trademark Tween 80TM).
  • buffer denotes a pharmaceutically acceptable excipient that stabilizes the pH of the pharmaceutical formulation.
  • Suitable buffers are well known in the art and can be found in the literature.
  • Preferred pharmaceutically acceptable buffers include, but are not limited to, histidine buffer, citrate buffer, succinate buffer, acetate buffer, arginine buffer, phosphate buffer or mixture.
  • Buffers of particular interest include citrate buffer or acetate buffer which is pH adjusted using acids or bases known in the art.
  • the above buffer is usually used in an amount of from about 1 mM to 50 mM, preferably from about 10 mM to 30 mM, most preferably 10 mM.
  • the pH can be adjusted to a value in the range of 4.5-6.0, independently of the buffer used, using acids or bases known in the art (eg, hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, and citric acid, sodium hydroxide, and potassium hydroxide). In particular, it is adjusted to a value in the range of 4.8-5.6, most particularly to pH 5.2.
  • acids or bases known in the art (eg, hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, and citric acid, sodium hydroxide, and potassium hydroxide).
  • acids or bases known in the art eg, hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, and citric acid, sodium hydroxide, and potassium hydroxide.
  • it is adjusted to a value in the range of 4.8-5.6, most particularly to pH 5.2.
  • the stabilized anti-PD-1 antibody pharmaceutical formulation of the invention may further comprise an antioxidant as a second stabilizer.
  • Antioxidant is a pharmaceutically acceptable excipient that prevents oxidation of the active pharmaceutical ingredient.
  • Antioxidants include, but are not limited to, chelating agents such as EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, Cysteine, methionine, ethanol, benzyl alcohol and n-acetylcysteine.
  • chelating agents such as EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, Cysteine, methionine
  • sugar denotes a monosaccharide or an oligosaccharide.
  • Monosaccharides are monomeric carbohydrates that are not hydrolyzable by acids, including monosaccharides and their derivatives, such as amino sugars. Examples of monosaccharides include glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, and neuraminic acid.
  • Oligosaccharides are branched or linear carbohydrates composed of more than one monomeric sugar unit linked via a glycosidic linkage. The monomeric sugar units within the oligosaccharide may be the same or different.
  • oligosaccharides are disaccharides, trisaccharides, tetrasaccharides, Five sugars and the like. Unlike polysaccharides, monosaccharides and oligosaccharides are water soluble. Examples of oligosaccharides include sucrose, trehalose, lactose, maltose, and raffinose. Specifically, the sugar is selected from the group consisting of sucrose and trehalose.
  • amino acid denotes a pharmaceutically acceptable organic molecule having an amino moiety at the alpha position of the carboxyl group.
  • amino acids include arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, and tyrosine.
  • Amino acids are typically employed in amounts of from about 5 to 500 mM, particularly from about 5 to about 200 mM, more specifically from about 100 to about 150 mM.
  • cryoprotectant means a pharmaceutically acceptable excipient that protects an unstable active ingredient (eg, a protein) from destabilizing conditions during a freeze-drying process, subsequent storage, and reconstitution.
  • Cryoprotectants include, but are not limited to, sugars, polyols (eg, sugar alcohols), and amino acids.
  • the cryoprotectant may be selected from the group consisting of sugars such as sucrose, trehalose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, neuraminic acid, amino sugars such as glucosamine , galactosamine, N-methylglucamine ("meglumine”), polyols such as mannitol and sorbitol, and amino acids such as arginine and glycine or mixtures thereof.
  • the cryoprotectant is preferably a disaccharide.
  • the present inventors have surprisingly found that disaccharides are more stable to the formulation than monosaccharides, polyols, amino acids.
  • the pharmaceutical preparations may also contain a tonicity agent.
  • a tonicity agent denotes a pharmaceutically acceptable tonicity agent for adjusting the tonicity of a formulation.
  • the formulation may be hypotonic, isotonic or hypertonic. Isotonicity usually involves the relative osmotic pressure of the solution, often relative to the osmotic pressure of human serum. Formulations according to the invention may be hypotonic, isotonic or hypertonic, but are preferably isotonic.
  • An isotonic formulation is a liquid or a liquid that is reconstituted from a solid form (e.g., from a lyophilized form) and represents a solution having the same degree of tonicity as some other solution to which it is compared, such as physiological saline solutions and serum.
  • Suitable tonicity agents include, but are not limited to, sodium chloride, potassium chloride, glycerin, and any component selected from the group consisting of amino acids, sugars, and especially glucose.
  • the tonicity agent is typically used in an amount from about 5 mM to about 500 mM.
  • stabilizers and tonicity agents there is a group of compounds that can function in two ways, ie they can be both stabilizers and tonicity agents.
  • sugars examples thereof can be found in the following collections: sugars, amino acids, polyols, cyclodextrins, polyglycols, and salts.
  • An example of a sugar that can be both a stabilizer and a tonicity agent is trehalose.
  • the pharmaceutical preparations may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by sterilization operations and by the inclusion of different antibacterial and antifungal agents (for example, methylparaben, chlorobutanol, phenol, sorbic acid, etc.).
  • Preservatives are typically employed in amounts of from about 0.001 to about 2% (w/v).
  • Preservatives include, but are not limited to, ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl paraben or propyl paraben, benzalkonium chloride.
  • a stable pharmaceutical preparation of an antibody against a PD-1 protein according to the present invention can be used for preventing or treating a PD-1 mediated disease or condition, preferably a cancer; more preferably a cancer expressing PD-L1;
  • the cancer is most preferably breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma; most preferably non-small cell lung cancer, melanoma and kidney cancer.
  • the pharmaceutical preparation according to the present invention can be administered intravenously without the need for an in-line filter, and thus is farther than conventional preparations which require an in-line filter to be applied.
  • Inline filters must be used such as It is installed in the infusion line of intravenous drugs to prevent the application of any particles, air or microorganisms that may be present in the intravenous solution or line. Particles of 5-20 micron size and larger have the ability to block blood flow through the pulmonary capillaries, which can lead to complications such as pulmonary embolism. Foreign particles can also cause phlebitis at the injection site, and filters can help reduce the incidence of phlebitis.
  • Stable preparations to be used for in vivo administration must be sterile. This is easily achieved by filtration through a sterile filtration membrane.
  • Stable anti-PD-1 antibody pharmaceutical formulations according to the present invention can be prepared by methods known in the art, such as ultrafiltration-diafiltration, dialysis, addition and mixing, freeze drying, reconstitution, and combinations thereof. Examples of the preparation of the preparation according to the present invention can be found below.
  • the stabilized anti-PD-1 antibody pharmaceutical preparations according to the invention may also be in lyophilized form or in liquid form reconstituted from lyophilized form.
  • the "lyophilized form” is prepared by freeze drying methods known in the art.
  • the lyophilized powder often has a residual moisture content of from about 0.1% to about 5% (w/w) and is present as a powder or a physically stable cake.
  • the "reconstituted form” can be obtained from the lyophilized powder by rapid dissolution after addition of the reconstitution medium.
  • Suitable remodeling media include, but are not limited to, water for injection (WFI), water for inhibiting bacterial injection (BWFI), sodium chloride solution (eg, 0.9% (w/v) NaCl), glucose solution (eg, 5% (w/).
  • v) Glucose a surfactant-containing solution (eg, 0.01% (w/v) polysorbate 20 and a pH buffer solution (eg, acetate buffer solution).
  • FIG 1 shows the effect of buffer system on the thermal stability of anti-PD-1 antibodies
  • Figure 2 shows the effect of sugar on the thermal stability of anti-PD-1 antibodies.
  • the preparation process of the invention is as follows:
  • the first step anti-PD-1 antibody stock solution before the filtration of the central control sampling to detect antibody protein concentration. After passing the control, The stock solution was passed through a 0.22 ⁇ m PVDF filter and the filtrate was collected.
  • the PD-1 antibody has the heavy chain of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8, which is prepared according to the method disclosed in WO 2015/085847.
  • the second step adjust the loading to 5.3ml, fill the filtrate in a 20ml vial, and add half of the plug, respectively, at the beginning of filling, in the middle of filling, at the end of filling, sampling and control the difference in loading.
  • the third step the liquid medicine after filling and filling is filled into a freeze-drying box, and lyophilized according to the following freeze-drying process. After the lyophilization procedure is finished, the vacuum is stoppered.
  • the fourth step open the capping machine, add the aluminum cover, and carry out the capping.
  • Step 5 Visual inspection to confirm that the product has no defects such as collapse and inaccurate loading.
  • Print and paste the vial label print the tray label, fold the tray, box, and sticker box labels.
  • the absorption peak at 280 nm was detected by an ultraviolet spectrophotometer (manufacturer: Thermo, model Nanodrop 2000).
  • the 0.22 ⁇ m PVDF filter cartridge is Millipore Millipak-100.
  • the filling machine is Chutian Technology KGS8/2-X2 linear filling and stoppering machine.
  • the difference in the control volume is measured using an electronic balance (manufacturer: Sartorius, model BSA423S).
  • the lyophilized Lyo-B (SIP.CIP) vacuum freeze dryer was used for freeze drying.
  • the capping machine uses Shandong Penglai DZG-130 knife type automatic capping machine.
  • the protein heat denaturation temperature (Tm) was determined using a GE MicroCal VP-Capillary DSC differential scanning calorimeter.
  • the DLS (Dynamic Light Scattering) average particle size was measured using a Malvern Zetasizer Nano ZS nanoparticle size potentiometer.
  • the anti-PD-1 antibody was prepared as a preparation containing 10 mM acetic acid (sodium), 90 mg/mL ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/mL polysorbate 20 at a pH of 4.8–5.6, and the antibody protein concentration was 40 mg/mL. .
  • Each preparation was filtered and lyophilized in a 20 mL neutral borosilicate glass controlled injection bottle filled with 5 mL/bottle, and the glass bottle was sealed with a halogenated butyl rubber stopper with a freeze-dried sterile powder.
  • Store lyophilized product Stability analysis was performed at 25 ° C and 40 ° C. The results showed that the anti-PD-1 antibody was very stable at pH 4.8 - 5.6.
  • Buffer 1 10 mM acetic acid (sodium), pH 5.0;
  • Buffer 2 10 mM disodium hydrogen phosphate (citric acid), pH 5.0;
  • Buffer 3 10 mM succinic acid (sodium), pH 5.0;
  • Buffer 4 10 mM citric acid (sodium), pH 5.0.
  • the thermal stability of the anti-PD-1 antibody in each preparation was measured by differential scanning calorimetry (DSC).
  • DSC differential scanning calorimetry
  • Tm midpoint temperature analysis of the thermal denaturation of the drug showed that the stability of the anti-PD-1 antibody in the acetate buffer salt system was significantly better than that of the succinate, citrate, and disodium hydrogen phosphate, lemon, as shown in FIG. Acid buffer system.
  • the DSC technique was used to screen PD-1 antibody preparations with a protein concentration of 1 mg/mL, containing different sugars and concentrations:
  • buffer 1 10 mM acetic acid (sodium), 90 mg / mL sucrose, pH 5.2;
  • buffer 2 10 mM acetic acid (sodium), 30 mg / mL ⁇ , ⁇ -dihydrate trehalose, pH 5.2;
  • buffer 3 10 mM acetic acid (sodium), 60 mg / mL ⁇ , ⁇ -dihydrate trehalose, pH 5.2;
  • buffer 4 10 mM acetic acid (sodium), 90 mg / mL ⁇ , ⁇ -dihydrate trehalose, pH 5.2;
  • the Tm value indicates that the anti-PD-1 antibody has the best thermal stability when the ⁇ , ⁇ -dihydrate trehalose concentration reaches 90 mg/mL.
  • PD-1 antibody protein concentration 40 mg/mL, containing 10 mM acetic acid (sodium), 90 mg/mL ⁇ , ⁇ -dihydrate trehalose, pH 5.2 anti-PD-1 in a buffer containing the following surfactants at different concentrations
  • Antibody preparation 40 mg/mL, containing 10 mM acetic acid (sodium), 90 mg/mL ⁇ , ⁇ -dihydrate trehalose, pH 5.2 anti-PD-1 in a buffer containing the following surfactants at different concentrations Antibody preparation:
  • Each preparation was filled into a 20 mL vial filled with 5 mL/vial and sealed with a plastic plug.
  • the drug was placed on a 25 ° C constant temperature shaker and shaken at 500 rpm.
  • the stability results indicated that 0.1-0.4 mg/mL polysorbate 20 effectively prevented the aggregation of anti-PD-1 antibodies and the formation of agglomerated large particles.
  • the anti-PD-1 antibody was formulated at 40 mg/mL in 10 mM acetic acid (sodium), 90 mg/mL ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/mL polysorbate 20, pH 5.2.
  • the antibody was filled in a 20 mL vial at 5 mL/vial and lyophilized and sealed with a lyophilized plug.
  • the lyophilized samples were placed under 4500 ⁇ 500Lx intense light irradiation or 40 ⁇ 2°C for 10 days, or 4°C ⁇ 40°C for 3 times, or -20°C ⁇ 40°C for 3 times.
  • the stability of the drug was evaluated by protein content, purity and activity assay. The results showed that the anti-PD-1 antibody was stable in the prescription, and could still meet the requirements after strong light, high temperature or low temperature, and freeze-thaw cycle.
  • the anti-PD-1 antibody was formulated at 40 mg/mL in 10 mM acetic acid (sodium), 90 mg/mL ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/mL polysorbate 20, pH 5.2.
  • the formulations were separately filled in glass bottles and 316L stainless steel cans and allowed to stand at room temperature for 24 hours. Analysis of protein content and purity indicated that the anti-PD-1 antibody was stable within 24 hours.
  • the formulation is compatible with 316L stainless steel cans.
  • Anti-PD-1 antibody was prepared at 40 mg/mL in 10 mM acetic acid (sodium), 90 mg/mL ⁇ , ⁇ -dihydrate Trehalose, 0.2 mg/mL polysorbate 20, pH 5.2.
  • the antibody was filled in a 20 mL vial at 5 mL/vial, lyophilized at a drying temperature of -15 ° C, -10 ° C and -5 ° C, respectively, and sealed with a lyophilized rubber stopper.
  • the lyophilized product was stored at 25 ° C for stability analysis. The results show that -10 ° C ⁇ -5 ° C is the best drying temperature for the freeze-drying process.
  • the stable pharmaceutical preparation provided by the present invention comprises: an anti-PD-1 antibody (the heavy chain amino acid sequence of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8); and a combination of any of the following stable buffers :
  • the concentration of the anti-PD-1 antibody is in the range of 1 mg/ml to 60 mg/ml, preferably 20-50 mg/ml, more preferably 35-45 mg/ml, and most preferably 40 mg/ml.
  • the implementable scheme may be selected from, but not limited to, the following combinations:
  • anti-PD-1 antibody 40 mg/ml, 90 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/ml polysorbate 20, and pH 5.2 10 mM acetate buffer;
  • anti-PD-1 antibody 1 mg/ml, 30 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/ml polysorbate 20, and pH mM 10 mM acetate buffer;
  • anti-PD-1 antibody 20 mg/ml, 60 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/ml polysorbate 20, and pH 4.8 1 mM acetate buffer;
  • anti-PD-1 antibody 35 mg/ml, 85 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/ml polysorbate 20, and pH 5.6 2 mM acetate buffer;
  • anti-PD-1 antibody 45 mg/ml, 95 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 5 mM acetate buffer pH 6.0;
  • anti-PD-1 antibody 50 mg/ml, 100 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/ml polysorbate 20, and pH 5.2 15 mM acetate buffer;
  • anti-PD-1 antibody 60 mg/ml, 90 mg/ml sucrose, 0.2 mg/ml polysorbate 400, and 30 mM acetate buffer pH 5.2;
  • anti-PD-1 antibody 40 mg/ml, 90 mg/ml lactose, 0.2 mg/ml polysorbate 60, and 50 mM acetate buffer pH 4.5;
  • anti-PD-1 antibody 40 mg/ml, 90 mg/ml maltose, 0.2 mg/ml polyoxyethylene hydrogenated castor oil, and 10 mM acetate buffer pH 5.2;
  • anti-PD-1 antibody 40 mg/ml, 90 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.01 mg/ml, glycerin fatty acid ester, and 10 mM acetate buffer pH 5.2;
  • anti-PD-1 antibody 40 mg/ml, 90 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.05 mg/ml polysorbate 20, and pH 5.2 10 mM acetate buffer;
  • anti-PD-1 antibody 40 mg/ml, 90 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.4 mg/ml polysorbate 20, and pH 5.2 10 mM acetate buffer;
  • anti-PD-1 antibody 40 mg/ml, 90 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.5 mg/ml polysorbate 20, and pH 5.2 10 mM acetate buffer;

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Abstract

一种稳定的抗PD-1抗体药物制剂及其在医药上的应用。该抗PD-1抗体药物制剂含有抗PD-1抗体、缓冲剂，还可以含有至少一种稳定剂，任选的还可以含有表面活性剂。本发明的抗PD-1抗体药物制剂可以有效地抑制抗体的聚集和脱酰胺作用，从而阻止抗体产品的降解，得到稳定的注射药物制剂。

Description

一种抗PD-1抗体制剂及其在医药上的应用 技术领域
本发明涉及包含抗PD-1抗体及其抗原结合片段的药物制剂、其制备方法以及该制剂的用途。
背景技术
肿瘤的免疫逃逸机制与机体对肿瘤的免疫应答之间存在着极为复杂的关系。肿瘤免疫治疗早期肿瘤特异性的杀伤性T细胞是有其生物活性的，但随着肿瘤生长后期失去了杀伤的功能。所以肿瘤免疫治疗是为了最大限度的提高患者自身对肿瘤的免疫系统反应，它不但要在体内激活原有的免疫系统反应，更要维持免疫系统反应的持续时间和反应强度，才是免疫治疗肿瘤的关键。
程序性死亡分子1(programmed death-l,PD-l)是1992年发现的表达在T细胞表面的一个蛋白受体，参与到细胞的凋亡过程之中。PD-l属于CD28家族，与细胞毒性T淋巴细胞抗原4(cytotoxic T Iymphocyte antigen 4,CTLA-4)具有23％的氨基酸同源性，但其表达却与CTLA不同，主要表达在活化的T细胞、B细胞和髓系细胞上。PD-1有两个配体，分别为PD-L1和PD-L2。新的研究发现乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤等人类肿瘤组织中检测到高PD-L1蛋白的表达，且PD-L1的表达水平和患者的临床及预后紧密相关。由于PD-L1起到着第二信号通路抑制T细胞增殖的作用，所以阻断PD-L1/PD-1之间结合成为了肿瘤免疫治疗领域一个非常有潜力的新兴靶点。
WO2015/085847公开了一类新的抗PD-1抗体，所述的抗体具有亲和力高、半衰期长的特点，预期其对上述疾病具有更好的疗效。然而这些新的抗PD-1抗体极不稳定，难以制成临床可用的制剂，PCT对于它们如何制成制剂没有任何具体的描述。因此，有必要对这些抗体进行深入研究，以得到稳定、方便临床使用的制剂。
发明内容
本发明的目的在于提供一种稳定的抗PD-1抗体制剂。
本发明的稳定的药物制剂含有抗PD-1抗体及其抗原结合片段、缓冲剂。所述药物制剂还可以含有至少一种稳定剂，任选的还可以含有表面活性剂。
本发明所述的药物制剂中，所述的抗PD-1抗体及其抗原结合片段其包含任意1个或多个选自以下的CDR区序列或与其具有至少85％序列同源性的氨基酸序列：
抗体重链可变区HCDR区序列：SEQ ID NO:1，SEQ ID NO:2，SEQ ID NO:3；和
抗体轻链可变区LCDR区序列：SEQ ID NO:4，SEQ ID NO:5，SEQ ID NO:6。
所述氨基酸序列如下表所示：
Figure PCTCN2016098982-appb-000001
所述的序列同源性可以是为了改进抗体的亲和性或免疫源性或稳定性或其它常规的理化特性及生物活性，以常规方法筛选而得。
进一步优选的抗PD-1抗体具有SEQ ID NO:7的重链和SEQ ID NO:8的轻链氨基酸序列：
Figure PCTCN2016098982-appb-000002
所述的抗PD-1抗体的浓度可以是1-60mg/ml，优选20-50mg/ml，更优选35-45mg/ml，最优选40mg/ml。
本发明所述的缓冲剂选自醋酸盐、柠檬酸盐、琥珀酸盐、以及磷酸盐中的一种或几种，所述的磷酸盐可选自磷酸二氢钠、磷酸二氢钾；优选的缓冲剂为醋酸盐，其用量没有特别限制，在本发明的实施方案中，例如是1至50mM，优选2至20mM，更优选5-15mM，最优选10mM。
本发明所述药物制剂的pH值范围可以在4.5-6.0，优选4.8-5.6,最优选pH值是5.2。
本发明所述的至少一种稳定剂选自糖或氨基酸。其中所述的糖优选二糖，选 自蔗糖、乳酸、海藻糖、麦芽糖，优选海藻糖，最优选α,α-二水合海藻糖。所述二糖的用量无需特别限制，在本发明的实施方案中，例如是30至120mg/ml，优选60至100mg/ml，更优选85至95mg/ml，最优选90mg/ml。
本发明所述的表面活性剂可以选自聚氧乙烯氢化蓖麻油、甘油脂肪酸酯、聚氧乙烯山梨醇酐脂肪酸酯，所述聚氧乙烯山梨醇酐脂肪酸酯可以选自聚山梨酯20、40、60或80。所述表面活性剂的用量无需特别限制，在本发明的实施方案中，例如是0.01至1mg/ml，优选0.05至0.5mg/ml，更优选0.1至0.4mg/ml，最优选0.2mg/ml。
本发明所述的稳定的药物制剂是一种可注射的药物制剂。
在本发明的一个实施方案中，所述稳定的药物制剂由抗PD-1抗体、缓冲剂、二糖，表面活性剂组成，任选包括水。
在本发明的一个实施方案中，所述稳定的药物制剂包含：
抗PD-1抗体，所述人抗体包含SEQ ID NO:7的重链氨基酸序列和SEQ ID NO:8的轻链氨基酸序列；和
(i)90mg/mlα,α-二水合海藻糖，和pH5.2的10mM醋酸盐缓冲液；或者
(ii)90mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.2的10mM醋酸盐缓冲液；或者
(iii)90mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.4的20mM醋酸盐缓冲液；或者
(iv)60mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨酯20，和pH5.0的20mM醋酸盐缓冲液；或者
(v)60mg/mlα,α-二水合海藻糖，0.1mg/ml的聚山梨酯20，和pH5.2的20mM醋酸盐缓冲液；或者
(vi)60mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.2的10mM醋酸盐缓冲液；或者
(vii)30mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨酯20，和pH4.8的10mM醋酸盐缓冲液；或者
(viii)30mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.2的30mM醋酸盐缓冲液；或者
(ix)30mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨酯20，和pH5.6的10mM醋酸盐缓冲液。
所述的可注射的药物制剂可以是注射液或者进一步制备成冻干粉形式。所述的冻干粉可以由本领域的常规方法制备。
本发明还提供了一种经冻干粉复溶后重建形成的注射液，可直接用于注射。
本发明的药物制剂可以有效抑制抗体的聚集和脱酰胺作用，从而阻止其中抗体产品的降解，得到稳定性的注射组合物，在25℃条件下可以稳定性存放6个月， 2-8℃条件下可以稳定存放12个月。并且，本发明的药物组合物对于蛋白氧化降解具有保护作用，还能够与玻璃、不锈钢容器相容，在这些容器中也能够稳定存在。
本发明所述的药物制剂，其用于预防或治疗PD-1介导的疾病或病症，所述的疾病优选为癌症；更优选为表达PD-L1的癌症；所述的癌症最优选为乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤；最优选为非小细胞肺癌、黑素瘤和肾癌。
本发明所述的药物制剂用于制备药物的用途，所述药物用于预防或治疗PD-1介导的疾病或病症，所述的疾病优选为癌症；更优选为表达PD-L1的癌症；所述的癌症最优选为乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤；最优选为非小细胞肺癌、黑素瘤和肾癌。
一种方法，其用于预防或治疗PD-1介导的疾病或病症，所述的疾病优选为癌症；更优选为表达PD-L1的癌症；所述的癌症最优选为乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤；最优选为非小细胞肺癌、黑素瘤和肾癌，所述方法包括施用本发明所述的药物制剂。
术语
本发明涉及一种稳定的药物液体制剂，其包含高浓度的针对PD-1的抗体。
本发明所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。
本发明的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
术语抗体的“抗原结合片段”(或简称“抗体片段”)是指抗体的保持特异性结合抗原(例如，PD-1)的能力的一个或多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的LCDR1、LCDR2和LCDR3，以及重链可变结构域的HCDR1、HCDR2和HCDR3。
在本发明中，本发明所述的抗体轻链可变区可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本发明中，本发明所述的抗体重链可变区可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因 连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。
术语“药物制剂”表示这样的制品：其处于允许活性成分的生物活性明确有效的形式，且其不含有对所述制剂要施用的受试者有毒的额外组分。
本文中结合根据本发明的制剂使用的术语“液体”表示，在大气压下在至少约2℃至约8℃的温度为液体的制剂。
术语"稳定剂"表示药学可接受的赋形剂，其在制造，储存和应用过程中保护活性药物成分和/或制剂免受化学和/或物理降解。由Cleland,J.L.,M.F.Powell,等人(1993)."The development of stable protein fomulations:a close look at protein aggregation,deamidation,and oxidation."Crit Rev Ther Drug Carrier Syst 10(4):307-77,Wang,W.(1999).“Instability,stabilization,and formulation of liquid protein pharmaceuticals."Int J Pharm 185(2):129-88.,Wang,W.(2000)."Lyophilization and development of solid protein pharmaceuticals."Int J Ph arm203(1-2):1-60.和Chi,E.Y.,S.Krishnan,等人(2003)."Physical stability of proteins in aqueous solution:mechanism and driving forces in nonnative protein aggregation."Pharm Res 20(9):1325-36综述了蛋白质药物的化学和物理降解途径。稳定剂包括但不限于如以下定义的糖,氨基酸,多元醇,表面活性剂,抗氧化剂,防腐剂,环糊精,聚乙二醇,例如PEG 3000、3350、4000、6000，白蛋白，例如人血清白蛋白(HSA)，牛血清白蛋白(BSA),盐,例如氯化钠，氯化镁，氯化钙，螯合剂,例如EDTA，如在后面定义的。在本发明中具体地使用的稳定剂选自糖。更具体地，所述稳定剂选自蔗糖、海藻糖、山梨糖醇。稳定剂可以以下述量存在于制剂中：30mg/ml至100mg/ml的量，优选60mg/ml至90mg/ml的量。更具体地，以90mg/ml的量使用蔗糖或海藻糖作为稳定剂。
“稳定的”制剂是这样的制剂：其中所含的蛋白(例如抗体)在贮存后基本上保留它的物理和化学稳定性，且因而保留它的生物活性。
“稳定的液体药物抗体制剂”是在冷藏温度(2-8℃)至少12个月，特别是2年，并且更特别是3年观察不到显著改变的液体抗体制剂。稳定性的标准如下:当通过分子排阻色谱(SEC-HPLC)测量时，不多于10％,特别是5％的抗体单体被降解。此外,通过视觉分析，溶液无色或清澈到轻微的乳白色。制剂的蛋白质浓度具有不多于+/-10％的改变。形成不多于10％,特别是5％的聚集。通过本领域已知的方法比如UV光谱法,分子排阻色谱(SEC-HPLC),离子交换色谱(IE-HPLC),比浊法和肉眼检查测量稳定性。
术语“程序性死亡1”、“程序性细胞死亡1”、“蛋白PD-1”、“PD-1”、“PD1”、“PDCD1”、“hPD-1”和“hPD-I”和互换使用，包括人PD-1的变体、同种型(isoform)、物种同系物，及与PD-1具有至少一个共同表位的类似物。完整的PD-1序列可以根据NCBI Reference Sequence:NM_005018.1查到。
术语“抗PD-1抗体”、“针对PD-1的抗体”和“针对PD-1蛋白的抗体”指能够以足够的亲和力结合PD-1蛋白，使得所述抗体可在靶向PD-1蛋白中用作诊断剂和/或治疗剂。本文中使用的术语“结合PD-1蛋白”是指，在BIAcore测定(Pharmacia Biosensor AB，Uppsala，瑞典)中或在ELISA中抗体对PD-1蛋白的结合，其中将经纯化的PD-1蛋白或PD-1蛋白CHO转染子包被在微量滴定板上。
药物制剂所含的针对PD-1蛋白的抗体的浓度是在1mg/ml至60mg/ml的范围内，优选是在20mg/ml至50mg/ml的范围内，最优选40mg/ml。
本文中使用的术语“表面活性剂”表示，用于保护蛋白制剂抵抗物理应力(如搅拌和剪切)的药学上可接受的赋形剂。药学上可接受的表面活性剂的例子包括：聚氧乙烯脱水山梨糖醇脂肪酸酯(吐温)、聚氧乙烯烷基醚(例如在商标BrijTM下销售的那些)和聚氧乙烯-聚氧丙烯共聚物(泊洛沙姆，Pluronic)。聚氧乙烯脱水山梨糖醇-脂肪酸酯的例子是聚山梨酯20(在商标吐温20TM下销售)和聚山梨酯80(在商标吐温80TM下销售)。
本文中使用的术语“缓冲液”表示稳定药物制剂的pH的药学上可接受的赋形剂。合适的缓冲液是本领域众所周知的，且可以在文献中找到。优选的药学上可接受的缓冲液包括但不限于：组氨酸缓冲液、柠檬酸盐缓冲液、琥珀酸盐缓冲液、醋酸盐缓冲液、精氨酸缓冲液、磷酸盐缓冲液或其混合物。特别感兴趣的缓冲液包含柠檬酸盐缓冲液或醋酸盐缓冲液，其用本领域已知的酸或碱进行pH调节。上述的缓冲液通常以约1mM至50mM、优选约10mM至30mM、最优选10mM的量使用。独立于使用的缓冲液，用本领域已知的酸或碱(例如盐酸、乙酸、磷酸、硫酸和柠檬酸、氢氧化钠和氢氧化钾)可以将pH调至在4.5-6.0范围内的值，特别是调至在4.8-5.6范围内的值，最特别地是调至pH5.2。
在某些实施方案中，本发明的稳定的抗PD-1抗体药物制剂还可以包含抗氧化剂作为第二种稳定剂。“抗氧化剂”是阻止活性药物成分的氧化的药学上可接受的赋形剂。抗氧化剂包括、但不限于：螯合剂诸如EDTA、柠檬酸、抗坏血酸、丁羟甲苯(BHT)、丁羟茴香醚(BHA)、亚硫酸钠、对氨基苯甲酸、谷胱甘肽、没食子酸丙酯、半胱氨酸、甲硫氨酸、乙醇、苯甲醇和正乙酰基半胱氨酸。
本文中使用的术语“糖”表示单糖或寡糖。单糖是不可被酸水解的单体碳水化合物，包括单糖和它们的衍生物，例如氨基糖。单糖的例子包括葡萄糖、果糖、半乳糖、甘露糖、山梨糖、核糖、脱氧核糖、神经氨酸。寡糖是由超过一个经由糖苷键连接的单体糖单元组成的支链或直链碳水化合物。在寡糖内的单体糖单元可以是相同的或不同的。取决于单体糖单元的数目，寡糖是二糖、三糖、四糖、 五糖诸如此类。与多糖不同，单糖和寡糖是水溶性的。寡糖的例子包括蔗糖、海藻糖、乳糖、麦芽糖和棉子糖。具体地，糖选自蔗糖和海藻糖。
本文中使用的术语“氨基酸”表示，具有位于羧基的α-位的氨基部分的药学上可接受的有机分子。氨基酸的例子包括精氨酸、甘氨酸、鸟氨酸、赖氨酸、组氨酸、谷氨酸、天冬氨酸、异亮氨酸、亮氨酸、丙氨酸、苯丙氨酸、酪氨酸、色氨酸、甲硫氨酸、丝氨酸、脯氨酸。通常以下述量使用氨基酸：约5-500mM的量，特别是约5至约200mM的量，更特别是约100至约150mM的量。
术语“稳定剂”也包括冷冻保护剂。术语“冷冻保护剂”表示，在冷冻干燥过程、随后的贮存和重构中保护不稳定的活性成分(例如蛋白)抵抗失稳条件的药学上可接受的赋形剂。冷冻保护剂包括、但不限于糖、多元醇(例如糖醇)和氨基酸。具体地，冷冻保护剂可以选自：糖类诸如蔗糖、海藻糖、乳糖、葡萄糖、甘露糖、麦芽糖、半乳糖、果糖、山梨糖、棉子糖、神经氨酸，氨基糖类诸如葡糖胺、半乳糖胺、N-甲基葡糖胺(“葡甲胺”)，多元醇类诸如甘露醇和山梨糖醇，和氨基酸类诸如精氨酸和甘氨酸或其混合物。所述的冷冻保护剂优选二糖。本发明惊奇地发现，二糖对制剂的稳定性优于单糖，多元醇，氨基酸。
药物制剂还可以含有张度剂。本文中使用的术语“张度剂”表示用于调节制剂的张度的药学上可接受的张度剂。所述制剂可以是低渗的、等渗的或高渗的。等渗性通常涉及溶液的相对渗透压，经常是相对于人血清的渗透压。根据本发明的制剂可以是低渗的、等渗的或高渗的，但是优选地是等渗的。等渗制剂是液体或从固体形式(例如从低压冻干形式)重构的液体，且表示具有与它所对比的某种其它溶液(诸如生理学的盐溶液和血清)相同的张度的溶液。合适的张度剂包括、但不限于氯化钠、氯化钾、甘油和选自以下的任意组分：氨基酸，糖，尤其是葡萄糖。通常以约5mM至约500mM的量使用张度剂。在稳定剂和张度剂内，存在一组可以以两种方式起作用的化合物，即它们可以同时为稳定剂和张度剂。其例子可以在以下集合中找到：糖、氨基酸、多元醇、环糊精、聚二醇和盐。可以同时为稳定剂和张度剂的糖的一个例子是海藻糖。
所述药物制剂还可以含有佐剂诸如防腐剂、润湿剂、乳化剂和分散剂。通过灭菌操作和通过包含不同的抗细菌剂和抗真菌剂(例如，对羟基苯甲酸甲酯、三氯叔丁醇、苯酚、山梨酸等)，可以确保阻止微生物的存在。通常以约0.001至约2％(w/v)的量使用防腐剂。防腐剂包括、但不限于：乙醇、苯甲醇、苯酚、间甲酚、对氯-间甲酚、对羟基苯甲酸甲酯或对羟基苯甲酸丙酯、苯扎氯铵。
根据本发明的针对PD-1蛋白的抗体的稳定的药物制剂可以用于预防或治疗PD-1介导的疾病或病症，所述的疾病优选为癌症；更优选为表达PD-L1的癌症；所述的癌症最优选为乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤；最优选为非小细胞肺癌、黑素瘤和肾癌。
通过静脉内(i.v.)、皮下(s.c.)或任意其它胃肠外施用方式(如药学领域中已知 的那些方式)，可以施用根据本发明的稳定的抗PD-1抗体药物制剂。
考虑到它们的高稳定性，根据本发明的药物制剂可以在不需要线内过滤器(in-line filter)的情况下静脉内施用，且因而比需要用线内过滤器才能施用的常规制剂远远更方便地操作。必须将线内过滤器诸如
Figure PCTCN2016098982-appb-000003
安装在静脉内药物的输注线中，以防止可能存在于静脉内溶液或线中的任何颗粒、空气或微生物的施用。5-20微米尺寸和更大的颗粒具有阻塞穿过肺毛细血管的血流的能力，这会导致并发症诸如肺栓塞。外来颗粒还可以在注射部位处造成静脉炎，并且过滤器可以帮助降低静脉炎的发病率。
要用于体内施用的稳定制剂必须是无菌的。这通过穿过无菌过滤膜的过滤容易地实现。
通过本领域已知的方法，例如超滤-透析过滤、透析、添加和混合、冷冻干燥、重构及其组合，可以制备根据本发明的稳定的抗PD-1抗体药物制剂。可以在下文中找到根据本发明的制剂的制备例子。
根据本发明的稳定的抗PD-1抗体药物制剂还可以呈低压冻干形式或呈从低压冻干形式重构的液体形式。通过本领域已知的冷冻干燥方法，制备“低压冻干形式”。所述冻干粉剂经常具有约0.1-5％(w/w)的残余含水量，且作为粉末或物理上稳定的饼状物存在。通过在加入重构介质以后的快速溶解，可以从冻干粉剂得到“重构形式”。合适的重构介质包括、但不限于：注射用水(WFI)、抑制细菌注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％(w/v)葡萄糖)、含有表面活性剂的溶液(例如0.01％(w/v)聚山梨酯20和pH缓冲溶液(例如醋酸盐缓冲溶液)。
附图说明
图1显示缓冲体系对于抗PD-1抗体热稳定性的影响
图2显示糖对于抗PD-1抗体热稳定性的影响。
具体实施方式
通过以下实施例进一步详细说明本发明。这些实施例仅用于说明性目的，而并不用于限制本发明的范围。
本发明实施例中未注明具体条件的实验方法，通常按照常规条件；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例
本发明制备工艺如下：
第一步：抗PD-1抗体原液过滤前中控取样检测抗体蛋白浓度。中控合格后， 将原液过0.22μm PVDF滤芯，收集滤液。所述的PD-1抗体具有SEQ ID NO:7的重链和SEQ ID NO:8的轻链氨基酸序列，其按照WO2015/085847公开的方法制备。
第二步：调节装量至5.3ml，将滤液灌装于20ml西林瓶中，半加塞，分别于灌装开始、灌装中间、灌装结束时取样中控检测装量差异。
第三步：将灌装加塞后的药液装入冻干箱中，按以下冻干工艺冻干。冻干程序结束后，真空加塞。
Figure PCTCN2016098982-appb-000004
第四步：开启轧盖机，加铝盖，进行轧盖。
第五步：目检，确认产品无塌陷、装量不准等缺陷。打印、粘贴西林瓶标签；打印纸盒标签，折叠纸盒，装盒，贴纸盒标签。
中控蛋白浓度的测定用紫外分光光度计检测280nm处吸收峰(生产商：Thermo,型号Nanodrop 2000)。
0.22μm PVDF滤芯采用密理博Millipak-100。
灌装机为楚天科技KGS8/2-X2直线式灌装加塞机。
中控装量差异检测使用电子天平称量(生产商：Sartorius,型号BSA423S)。
冷冻干燥使用东富龙Lyo-B(SIP.CIP)真空冷冻干燥机。
轧盖机用山东蓬莱DZG-130型刀式自动轧盖机。
目检外观采用天津精拓YB-2A澄明度检测仪。
实施例中HPLC(SEC和IEC)的检测使用安捷伦1200DAD高压液相色谱仪(TSK gel SuperSW mAb HR 300×7.8mm色谱柱和ProPacTM WCX-10BioLC TM,250×4mm色谱柱)。
蛋白热变性温度(Tm)的测定使用GE公司MicroCal VP-Capillary DSC差示扫描热量计。
DLS(Dynamic Light Scattering)平均粒径的测定用马尔文Zetasizer Nano ZS纳米粒度电位仪。
实施例1
将抗PD-1抗体分别配制为pH 4.8–5.6的含10mM醋酸(钠)，90mg/mLα,α-二水合海藻糖，0.2mg/mL聚山梨酯20的制剂，抗体蛋白浓度为40mg/mL。过滤每种制剂并以5mL/瓶填充入20mL中性硼硅玻璃管制注射剂瓶中进行冻干，所述玻璃瓶用注射液用冷冻干燥无菌粉末用卤化丁基橡胶塞封口。将冻干品保存 于25℃和40℃进行稳定性分析。结果显示抗PD-1抗体在pH 4.8–5.6均非常稳定。
表1.pH对于抗PD-1抗体的降解的效应
Figure PCTCN2016098982-appb-000005
实施例2
在下列缓冲液中，配置蛋白浓度为1mg/mL的抗PD-1抗体制剂：
1)缓冲液1：10mM醋酸(钠)，pH 5.0；
2)缓冲液2：10mM磷酸氢二钠(柠檬酸)，pH 5.0；
3)缓冲液3：10mM琥珀酸(钠)，pH 5.0；
4)缓冲液4：10mM柠檬酸(钠)，pH 5.0。
用差示扫描量热法(differential scanning calorimetry,DSC)测量抗PD-1抗体在每种制剂中的热稳定性。药品的热变性中点温度(Tm)分析显示，如图1所示，抗PD-1抗体在醋酸缓冲盐体系中的稳定性明显优于琥珀酸盐、柠檬酸盐和磷酸氢二钠、柠檬酸盐缓冲体系。
实施例3
使用DSC技术筛选PD-1抗体制剂蛋白浓度为1mg/mL，含不同糖及浓度的处方：
1)缓冲液1：10mM醋酸(钠)，90mg/mL蔗糖，pH 5.2；
2)缓冲液2：10mM醋酸(钠)，30mg/mLα,α-二水合海藻糖，pH 5.2；
3)缓冲液3：10mM醋酸(钠)，60mg/mLα,α-二水合海藻糖，pH 5.2；
4)缓冲液4：10mM醋酸(钠)，90mg/mLα,α-二水合海藻糖，pH 5.2；
参照图2，Tm值表明，当α,α-二水合海藻糖浓度达到90mg/mL时抗PD-1抗体的热稳定性最好。
实施例4
在含下列不同浓度表面活性剂的缓冲液中，制备PD-1抗体蛋白浓度为40mg/mL，含10mM醋酸(钠)，90mg/mLα,α-二水合海藻糖，pH 5.2的抗PD-1抗体制剂：
1)不含表面活性剂；
2)0.1mg/mL聚山梨酯20；
3)0.2mg/mL聚山梨酯20；；
4)0.3mg/mL聚山梨酯20；
5)0.4mg/mL聚山梨酯20；
6)0.2mg/mL聚山梨酯80；
将每种制剂填充入以5mL/瓶填充入20mL西林瓶中，并用覆膜胶塞封口。药品置于25℃恒温摇床上，以500rpm的速度振摇。稳定性结果表明，0.1-0.4mg/mL聚山梨酯20，有效防止了抗PD-1抗体的聚集和团聚大颗粒物的形成。
表2.表面活性剂对于25℃，500rpm振摇的抗PD-1抗体聚集的影响
Figure PCTCN2016098982-appb-000006
实施例5
抗PD-1抗体以40mg/mL配制在10mM醋酸(钠)，90mg/mLα,α-二水合海藻糖，0.2mg/mL聚山梨酯20，pH 5.2中。将抗体以5mL/瓶填充入20mL西林瓶中冻干，并用冻干胶塞封口。冻干后的样品分别置于4500±500Lx强光照射或40±2℃高温放置10天，或4℃～40℃低温循环3次，或-20℃～40℃冻融循环3次。通过蛋白含量，纯度与活性检测评估药品稳定性，结果证明抗PD-1抗体在该处方中是比较稳定的，经强光、高温或低温、冻融循环后仍能够符要求。
表3.抗PD-1抗体制剂在强光、高温或低温、冻融循环时的稳定性
Figure PCTCN2016098982-appb-000007
实施例6
抗PD-1抗体以40mg/mL配制在10mM醋酸(钠)，90mg/mLα,α-二水合海藻糖，0.2mg/mL聚山梨酯20，pH 5.2中。将制剂分别填充在玻璃瓶和316L不锈钢罐中，室温放置24小时。蛋白含量和纯度分析表明，抗PD-1抗体在24小时内是稳定的。该制剂与316L不锈钢罐可以相容。
表4.在不锈钢罐中抗PD-1抗体的稳定性
Figure PCTCN2016098982-appb-000008
实施例7
抗PD-1抗体以40mg/mL配制在10mM醋酸(钠)，90mg/mLα,α-二水合 海藻糖，0.2mg/mL聚山梨酯20，pH 5.2中。将抗体以5mL/瓶填充入20mL西林瓶中，分别以-15℃、-10℃和-5℃的一次干燥温度进行冻干，并用冻干胶塞封口。将冻干品保存于25℃进行稳定性分析。结果表明，-10℃～-5℃为冻干工艺最佳的一次干燥温度。
表5.用不同一次干燥工艺制备的抗PD-1抗体制剂稳定性
Figure PCTCN2016098982-appb-000009
实施例8其它制剂配方
本发明提供的稳定的药物制剂包含：抗PD-1抗体(SEQ ID NO:7的重链氨基酸序列和SEQ ID NO:8的轻链氨基酸序列)；和任选自以下的稳定缓冲液的组合：
(i)90mg/mlα,α-二水合海藻糖，和pH5.2的10mM醋酸盐缓冲液；
(ii)90mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.2的10mM醋酸盐缓冲液；
(iii)90mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.4的20mM醋酸盐缓冲液；
(iv)60mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨酯20，和pH5.0的20mM醋酸盐缓冲液；
(v)60mg/mlα,α-二水合海藻糖，0.1mg/ml的聚山梨酯20，和pH5.2的20mM醋酸盐缓冲液；
(vi)60mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.2的10mM醋酸盐缓冲液；
(vii)30mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨酯20，和pH4.8的10mM醋酸盐缓冲液；
(viii)30mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.2的30mM醋酸盐缓冲液；或者
(ix)30mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨酯20，和pH5.6的10mM醋酸盐缓冲液。
以上实施例中，抗PD-1抗体的浓度在1mg/ml至60mg/ml的范围内，优选20-50mg/ml，更优选35-45mg/ml，最优选40mg/ml。可实施的方案可选自，但不限于以下组合：
(1)抗PD-1抗体40mg/ml，90mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.2的10mM醋酸盐缓冲液；
(2)抗PD-1抗体1mg/ml，30mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH4.5的10mM醋酸盐缓冲液；
(3)抗PD-1抗体20mg/ml，60mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH4.8的1mM醋酸盐缓冲液；
(4)抗PD-1抗体35mg/ml，85mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.6的2mM醋酸盐缓冲液；
(5)抗PD-1抗体45mg/ml，95mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH6.0的5mM醋酸盐缓冲液；
(6)抗PD-1抗体50mg/ml，100mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.2的15mM醋酸盐缓冲液；
(7)抗PD-1抗体60mg/ml，90mg/ml蔗糖，0.2mg/ml的聚山梨酯400，和pH5.2的30mM醋酸盐缓冲液；
(8)抗PD-1抗体40mg/ml，90mg/ml乳糖，0.2mg/ml的聚山梨酯60，和pH4.5的50mM醋酸盐缓冲液；
(9)抗PD-1抗体40mg/ml，90mg/ml海藻糖，0.2mg/ml的聚山梨酯80，和pH5.2的10mM醋酸盐缓冲液；
(10)抗PD-1抗体40mg/ml，90mg/ml麦芽糖，0.2mg/ml的聚氧乙烯氢化蓖麻油，和pH5.2的10mM醋酸盐缓冲液；
(11)抗PD-1抗体40mg/ml，90mg/mlα,α-二水合海藻糖，0.01mg/ml的、甘油脂肪酸酯，和pH5.2的10mM醋酸盐缓冲液；
(12)抗PD-1抗体40mg/ml，90mg/mlα,α-二水合海藻糖，0.05mg/ml的聚山梨酯20，和pH5.2的10mM醋酸盐缓冲液；
(13)抗PD-1抗体40mg/ml，90mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨酯20，和pH5.2的10mM醋酸盐缓冲液；
(14)抗PD-1抗体40mg/ml，90mg/mlα,α-二水合海藻糖，0.5mg/ml的聚山梨酯20，和pH5.2的10mM醋酸盐缓冲液；
(15)抗PD-1抗体40mg/ml，90mg/mlα,α-二水合海藻糖，1mg/ml的聚山梨酯20，和pH5.2的10mM醋酸盐缓冲液。

Claims (21)

  1. 一种稳定的抗PD-1抗体药物制剂，其包含抗PD-1抗体及其抗原结合片段和缓冲剂。
  2. 根据权利要求1所述的药物制剂，其中所述抗PD-1抗体及其抗原结合片段其包含任意1个或多个选自以下的CDR区序列或与其具有至少85％序列同一性的氨基酸序列：
    抗体重链可变区HCDR区序列：SEQ ID NO:1，SEQ ID NO:2，SEQ ID NO:3；和
    抗体轻链可变区LCDR区序列：SEQ ID NO:4，SEQ ID NO:5，SEQ ID NO:6。
  3. 根据权利要求1或2所述的药物制剂，其中所述抗PD-1抗体具有SEQ ID NO:7的重链和SEQ ID NO:8的轻链氨基酸序列。
  4. 根据权利要求1-3中的任一项所述的药物制剂，其中所述抗PD-1抗体的浓度在1mg/ml至60mg/ml的范围内，优选20-50mg/ml，更优选35-45mg/ml，最优选40mg/ml。
  5. 根据权利要求1-4中的任一项所述的药物制剂，其中所述缓冲剂选自醋酸盐、柠檬酸盐、琥珀酸盐、以及磷酸盐中的一种或几种，优选醋酸盐缓冲剂。
  6. 根据权利要求5所述的药物制剂，其中所述缓冲剂的浓度为1至50mM，优选2至30mM，更优选5-15mM，最优选10mM。
  7. 根据权利要求1-6中的任一项所述的药物制剂，其中所述制剂的pH值范围可以在4.5-6.0，优选4.8-5.6，最优选pH值是5.2。
  8. 根据权利要求1-7中的任一项所述的药物制剂，还含有至少一种稳定剂。
  9. 根据权利要求8所述的药物制剂，所述的至少一种稳定剂选自糖或氨基酸。
  10. 根据权利要求9所述的药物制剂，其中所述的糖为二糖，优选选自蔗糖、乳糖、海藻糖、麦芽糖，更优选海藻糖，最优选α,α-二水合海藻糖。
  11. 根据权利要求9或10所述的药物制剂，其中所述二糖的浓度为30至120mg/ml，优选60至100mg/ml，更优选85至95mg/ml，最优选90mg/ml。
  12. 根据权利要求8-11中的任一项所述的药物制剂，还包括表面活性剂，其中所述的表面活性剂选自聚氧乙烯氢化蓖麻油、甘油脂肪酸酯、聚氧乙烯山梨醇酐脂肪酸酯，优选所述聚氧乙烯山梨醇酐脂肪酸酯为聚山梨酯20、40、60或80，最优选聚山梨酯20。
  13. 根据权利要求12所述的药物制剂，其中所述表面活性剂浓度为0.01至1mg/ml，优选0.05至0.5mg/ml，更优选0.1至0.4mg/ml，最优选0.2mg/ml。
  14. 根据权利要求1-13中的任一项所述的药物制剂，其包含：抗PD-1抗体，所述抗体包含SEQ ID NO:7的重链氨基酸序列和SEQ ID NO:8的轻链氨基酸序列；和
    (i)90mg/mlα,α-二水合海藻糖，和pH5.2的10mM醋酸盐缓冲液；或者
    (ii)90mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.2的10mM醋酸盐缓冲液；或者
    (iii)90mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.4的20mM醋酸盐缓冲液；或者
    (iv)60mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨酯20，和pH5.0的20mM醋酸盐缓冲液；或者
    (v)60mg/mlα,α-二水合海藻糖，0.1mg/ml的聚山梨酯20，和pH5.2的20mM醋酸盐缓冲液；或者
    (vi)60mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.2的10mM醋酸盐缓冲液；或者
    (vii)30mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨酯20，和pH4.8的10mM醋酸盐缓冲液；或者
    (viii)30mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨酯20，和pH5.2的30mM醋酸盐缓冲液；或者
    (ix)30mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨酯20，和pH5.6的10mM醋酸盐缓冲液。
  15. 根据权利要求1-14中的任一项所述的稳定的抗PD-1抗体药物制剂，所述的药物制剂是一种可注射的药物制剂，其还含有注射用水。
  16. 由权利要求15所述的药物制剂制得的冻干粉。
  17. 由权利要求16所述的冻干粉，其冻干工艺最佳的干燥温度为-10℃～-5℃。
  18. 由权利要求15或16所述的冻干粉，其复溶后得到的注射液。
  19. 根据权利要求1-18中的任一项所述的稳定的抗PD-1抗体药物制剂，其用于预防或治疗PD-1介导的疾病或病症，所述的疾病优选为癌症；更优选为表达PD-L1的癌症；所述的癌症最优选为乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤；最优选为非小细胞肺癌、黑素瘤和肾癌。
  20. 根据权利要求1-18中的任一项所述的稳定的抗PD-1抗体药物制剂用于制备药物的用途，所述药物用于预防或治疗PD-1介导的疾病或病症，所述的疾病优选为癌症；更优选为表达PD-L1的癌症；所述的癌症最优选为乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤；最优选为非小细胞肺癌、黑素瘤和肾癌。
  21. 一种方法，其用于预防或治疗PD-1介导的疾病或病症，所述的疾病优选为癌症；更优选为表达PD-L1的癌症；所述的癌症最优选为乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤；最优选为非小细胞肺癌、黑素瘤和肾癌，所述方法包括施用根据权利要求1-18中的任一项所述的稳定的抗PD-1抗体药物制剂。
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WO2018204368A1 (en) 2017-05-02 2018-11-08 Merck Sharp & Dohme Corp. Stable formulations of programmed death receptor 1 (pd-1) antibodies and methods of use thereof
WO2019072220A1 (zh) * 2017-10-13 2019-04-18 江苏恒瑞医药股份有限公司 Pd-1抗体和表观遗传调节剂联合在制备治疗肿瘤的药物中的用途
WO2019076277A1 (zh) * 2017-10-17 2019-04-25 江苏恒瑞医药股份有限公司 抗pd-1抗体和抗lag-3抗体联合在制备治疗肿瘤的药物中的用途
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MX2018003306A (es) 2018-05-16
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US20180339045A1 (en) 2018-11-29
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