WO2018223958A1 - 一种含c-Met抗体药物偶联物的药物组合物及其用途 - Google Patents

一种含c-Met抗体药物偶联物的药物组合物及其用途 Download PDF

Info

Publication number
WO2018223958A1
WO2018223958A1 PCT/CN2018/089955 CN2018089955W WO2018223958A1 WO 2018223958 A1 WO2018223958 A1 WO 2018223958A1 CN 2018089955 W CN2018089955 W CN 2018089955W WO 2018223958 A1 WO2018223958 A1 WO 2018223958A1
Authority
WO
WIPO (PCT)
Prior art keywords
pharmaceutical composition
cancer
met
antibody
drug conjugate
Prior art date
Application number
PCT/CN2018/089955
Other languages
English (en)
French (fr)
Inventor
方晶晶
颜贞
童桂梅
刘洵
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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 CA3064470A priority Critical patent/CA3064470A1/en
Priority to KR1020197038495A priority patent/KR20200012937A/ko
Priority to CN201880011419.3A priority patent/CN110382008A/zh
Priority to AU2018280485A priority patent/AU2018280485A1/en
Priority to MX2019014666A priority patent/MX2019014666A/es
Priority to US16/618,974 priority patent/US20200129633A1/en
Priority to BR112019025591-5A priority patent/BR112019025591A2/pt
Publication of WO2018223958A1 publication Critical patent/WO2018223958A1/zh

Links

Classifications

    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • A61K47/26—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
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • 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
    • 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

Definitions

  • the invention belongs to the field of pharmaceutical preparations, and in particular relates to a pharmaceutical composition comprising a c-Met antibody drug conjugate, and the use thereof as a medicament.
  • the c-Met proto-oncogene is located on the long arm of human chromosome 7 (7q31) and is over 120 kb in size. It encodes a c-Met protein precursor with a molecular weight of approximately 150 kD and is locally glycosylated to generate a 170 kD glycoprotein. Shears into the alpha subunit (50 kDa) and the beta subunit (140 kDa), which are linked by disulfide bonds to form a mature c-Met protein receptor.
  • the heterodimer comprises two strands, the beta strand having an extracellular region, a transmembrane region (also referred to as a membrane stretch fragment), and an intracellular region (including an intracellular tyrosine kinase binding site).
  • the alpha chain has only the extracellular portion, but it is highly glycosylated and attached to the beta chain via a disulfide bond.
  • the extracellular region of the two subunits is the recognition site for the corresponding ligand, and the intracellular region has tyrosine kinase activity.
  • c-Met activation The mechanism of c-Met activation is divided into three types: one is the activation mechanism dependent on HGF, the other is independent of HGF activation mechanism, and the third is through other membrane pathways, such as CD44, adhesin, and receptors on the surface of hyaluronic acid membrane. RON signaling pathway and so on. The most common of these is the activation mechanism that relies on HGF.
  • the N-terminus of HGF binds to c-Met, promotes dimerization and autophosphorylation of Tyr1234 and Tyr1235 on the ⁇ chain, and phosphorylation of Tyr1349 and Tyr1356 near the C-terminus produces binding sites for multiple adaptor proteins.
  • c-Met has cross-talk with other membrane receptors, and it is now known that this cross-linking promotes tumor formation and metastasis, since c-Met is the intersection of many pathways leading to tumor formation and metastasis. With c-Met as a target, simultaneous interference with many pathways can be achieved relatively easily, and c-Met has become a promising target for anti-tumor production and metastasis treatment.
  • Antibody drug conjugates link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds, making full use of the specificity of antibodies for tumor cell specific or high expression of antigen binding. And the efficiency of cytotoxins to avoid toxic side effects on normal cells. This means that antibody drug conjugates bind precisely to tumor cells and reduce the effects on normal cells compared to traditional chemotherapeutic drugs.
  • the ADC drug consists of an antibody (targeting moiety), a linker and a toxin.
  • the good targeting part determines the specificity of the ADC drug, which includes not only specific targeted binding, but also effective endocytosis.
  • antibody drugs especially ADCs
  • stable formulation studies of antibody drugs are particularly important.
  • c-Met ADC the formulation is not an optimal formulation composition.
  • the present invention provides a pharmaceutical (formulation) composition comprising a c-Met ADC that is sufficiently stable and more suitable for administration.
  • the present invention provides a pharmaceutical composition comprising a c-Met antibody drug conjugate, and other excipients.
  • a pharmaceutical composition comprising a c-Met antibody drug conjugate, and a buffer, preferably a succinate or citrate buffer, more preferably a succinate buffer.
  • the pharmaceutical composition wherein the c-Met antibody drug conjugate concentration is from 1 mg/ml to 30 mg/ml, preferably from about 1 mg/ml to 20 mg/ml, further preferably from about 5 mg/ml to 20 mg/ml, most preferably 10-20 mg/ml, non-limiting examples include 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml , 9mg/ml, 10mg/ml, 11mg/ml, 12mg/ml, 13mg/ml, 14mg/ml, 15mg/ml, 16mg/ml, 17mg/ml, 18mg/ml, 19mg/ml, 20mg/ml and 30mg /ml.
  • the pharmaceutical compositions have a pH of from about 5.0 to 6.0, preferably from about 5.0 to 5.5, and most preferably from 5.0, 5.1, 5.2, 5.3, 5.4, 5.5.
  • the pharmaceutical composition wherein the buffer concentration is from about 5 mM to 30 mM, preferably from 5 mM to 20 mM, further preferably from about 10 mM to 20 mM, more preferably from about 10 mM to 15 mM, and most preferably 10 mM.
  • the pharmaceutical composition also includes its sugar.
  • "Sugar” comprises the conventional composition (CH2O) n and its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars and the like.
  • Preferred sugars are non-reducing disaccharides, more preferably trehalose or sucrose.
  • a pharmaceutical composition further comprising a disaccharide, preferably from trehalose or sucrose, most preferably trehalose.
  • the pharmaceutical composition wherein the concentration of the sugar is from about 40 mg/ml to about 80 mg/ml, preferably from 50 mg/ml to about 70 mg/ml, more preferably from 55 mg/ml to about 65 mg/ml, Non-limiting examples include 55 mg/ml, 57 mg/ml, 59 mg/ml, 60 mg/ml, 61 mg/ml, 63 mg/ml, 65 mg/ml.
  • the pharmaceutical composition further comprising a surfactant
  • a surfactant may be selected from the group consisting of polysorbate 20, polysorbate 80, polyhydroxyl, Triton, sodium dodecyl sulfate, lauryl sulfonate Sodium, sodium octyl sulphate, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine , myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauramide-propyl Base-betaine, cocaramidopropyl-betaine, linoleidopropyl-betaine, myristylpropyl-betaine, palmi
  • the concentration of the surfactant in the pharmaceutical composition is from about 0.05 mg/ml to 1.0 mg/ml, further preferably from 0.05 mg/ml to 0.4 mg/ml, more preferably from 0.1 mg/ml to 0.4 mg. /ml, most preferably from 0.1 mg/ml to 0.2 mg/ml, non-limiting examples include 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml.
  • composition comprising:
  • the pharmaceutical composition comprises: (a) 1-20 mg/ml of c-Met antibody drug conjugate; (b) 10-20 mM succinate buffer, pH 5.0-5.5; (c) 60 mg/ml of ⁇ , ⁇ -dihydrate trehalose; (d) 0.05-0.4 mg/ml of polysorbate 20.
  • the pharmaceutical composition comprises: (a) 5-20 mg/ml of c-Met antibody drug conjugate; (b) 10-20 mM succinate buffer, pH 5.0-5.5; (c) 50-70 mg/ml of ⁇ , ⁇ -dihydrate trehalose; (d) 0.1-0.2 mg/ml of polysorbate 20.
  • the pharmaceutical composition wherein the antibody in the c-Met antibody drug conjugate is Ab-10
  • the Ab-10 antibody heavy chain sequence is set forth in SEQ ID NO: 24 of WO2016/165580A1
  • Ab The -10 antibody light chain sequence is set forth in SEQ ID NO: 27 of WO2016/165580A1.
  • the pharmaceutical composition wherein the c-Met antibody drug conjugate is ADC-12, has the structure shown by:
  • y ranges from 1-8, preferably 2-5; y can be a decimal.
  • the pharmaceutical composition comprises: (a) 1-30 mg/ml of ADC-12; (b) 10-30 mM succinate buffer or citrate buffer, pH 5.0-5.5 (c) 40-80 mg/ml of ⁇ , ⁇ -dihydrate trehalose; (d) 0.05-0.4 mg/ml of polysorbate 20.
  • the pharmaceutical composition comprises: (a) 1-20 mg/ml of ADC-12; (b) 10-20 mM succinate buffer, pH 5.0-5.5; (c) 40- 80 mg/ml of ⁇ , ⁇ -dihydrate trehalose; (d) 0.05-0.4 mg/ml of polysorbate 20.
  • the pharmaceutical composition comprises: (a) 1-20 mg/ml of ADC-12; (b) 10-20 mM succinate buffer, pH 5.0-5.5; (c) 60 mg/ 1 g of ⁇ , ⁇ -dihydrate trehalose; (d) 0.05-0.4 mg/ml of polysorbate 20.
  • the pharmaceutical composition comprises: (a) 5-20 mg/ml of ADC-12; (b) 10-20 mM succinate buffer, pH 5.0-5.5; (c) 50- 70 mg/ml of ⁇ , ⁇ -dihydrate trehalose; (d) 0.1-0.2 mg/ml of polysorbate 20.
  • the pharmaceutical composition comprises: 1 mg/ml ADC-12, 20 mM succinate buffer, pH 5.5 or 6.0.
  • the pharmaceutical composition comprises: 1 mg/ml ADC-12, 20 mM citrate buffer, pH 5.0, 5.5 or 6.0.
  • the pharmaceutical composition comprises: 20 mg/ml ADC-12, 10 mM succinate buffer or citrate buffer, 60 mg/ml sucrose, 0.2 mg/ml polysorbate 20, pH 5.5.
  • the pharmaceutical composition comprises: 20 mg/ml ADC-12, 10 mM succinate buffer, 60 mg/ml alpha, alpha-dihydrate trehalose, 0.05-0.4 mg/ml polysorbate Ester 20, pH 5.0-5.5.
  • the pharmaceutical composition comprises: 20 mg/ml ADC-12, 10 mM succinate buffer, 60 mg/ml alpha, alpha-dihydrate trehalose, 0.2 mg/ml polysorbate 20 , pH 5.0-5.5.
  • the pharmaceutical composition comprises: 20 mg/ml ADC-12, 10 mM succinate buffer, 60 mg/ml alpha, alpha-dihydrate trehalose, 0.2 mg/ml polysorbate 20 , pH 5.3.
  • Also provided herein is a method of preparing a lyophilized formulation comprising a c-Met antibody drug conjugate comprising the step of lyophilizing the aforementioned pharmaceutical composition.
  • a method of preparing a lyophilized formulation comprising a c-Met antibody drug conjugate wherein the lyophilization comprises, in order, a step of pre-freezing, one-time drying, and secondary drying.
  • the purpose of pre-freezing is to freeze the product to obtain a crystalline solid.
  • the pre-freezing temperature and the pre-freezing speed are two important process parameters.
  • the pre-freezing temperature of the present invention is optimally set to -45 ° C, and the pre-freezing speed is set to 1 ° C / min (starting from -5 ° C).
  • One drying also known as main drying, is the main stage of sample freeze drying.
  • the goal is to remove the ice from the product while maintaining the shape of the product, minimizing damage to the product. If the temperature and vacuum of one drying are not properly selected, it will cause the product to collapse. Higher temperatures and vacuums will accelerate lyophilization efficiency, but at the same time increase the risk of product collapse.
  • the temperature of the primary drying of the present invention may be a temperature conventional in the art, for example, -27 to -20 ° C, preferably -20 ° C.
  • the temperature for the primary drying of the present invention is preferably 0.1 mbar.
  • Secondary drying also known as analytical drying, is the primary step in removing bound water from an article by pumping an ultimate vacuum (0.01 mbar) and elevated temperature (25-40 °C).
  • the secondary drying temperature is chosen to be at the low end of the temperature range, ie 25 °C.
  • the freeze-drying conditions may vary with the size and type of the formulation and sample holding container and the volume of the liquid during actual production.
  • a lyophilized formulation comprising a c-Met antibody drug conjugate prepared by the foregoing method.
  • a lyophilized formulation wherein the lyophilized formulation is reconstituted to form any of the pharmaceutical compositions described above, preferably reconstituted with water for injection.
  • a pharmaceutical composition or lyophilized formulation as described above for the manufacture of a medicament for the treatment of a disease or condition associated with c-Met, wherein said disease or condition is preferably cancer; more preferably expression of c-Met Cancer; more preferably gastric cancer, pancreatic cancer, lung cancer (eg, non-small cell lung cancer), glioblastoma, sarcoma, colorectal cancer, renal cancer, hepatocellular carcinoma, melanoma, and breast cancer expressing c-Met; Most preferred are gastric cancer, pancreatic cancer, non-small cell lung cancer, and kidney cancer.
  • Also provided herein is a method of treating and preventing a disease or condition associated with c-Met comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the foregoing or a lyophilized formulation; wherein the disease is preferably cancer; more preferably Cancer expressing c-Met; more preferably gastric cancer, pancreatic cancer, lung cancer (eg, non-small cell lung cancer), glioblastoma, sarcoma, colorectal cancer, renal cancer, hepatocellular carcinoma, melanin expressing c-Met Tumors and breast cancer; most preferred are gastric cancer, pancreatic cancer, non-small cell lung cancer, and kidney cancer.
  • the disease is preferably cancer; more preferably Cancer expressing c-Met; more preferably gastric cancer, pancreatic cancer, lung cancer (eg, non-small cell lung cancer), glioblastoma, sarcoma, colorectal cancer, renal cancer, hepatocellular carcinoma, melanin
  • an article of manufacture comprising a container containing the pharmaceutical composition or lyophilized formulation described above.
  • the container is preferably a glass bottle, a liquid storage bag or a 316L stainless steel can.
  • the antibody in the c-Met antibody drug conjugate in the pharmaceutical composition comprises a sequence identical to 85% or more of the amino acid sequence set forth in SEQ ID NO: 24 of WO 2016/165580 A1. Sex, if at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
  • the sequence identity heavy chain sequence preferably has more than 95% sequence identity; and the amino acid sequence set forth in SEQ ID NO: 27 of WO 2016/165580 A1 has greater than 85% sequence identity, such as having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of light chain sequences, Preferably, it has more than 95% sequence identity.
  • the formulation is stable at 2-8 ° C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the formulation is stable at 40 ° C for at least 7 days, at least 14 days or at least 28 days.
  • Buffer refers to a buffer that is resistant to changes in pH by the action of its acid-base conjugate component.
  • the buffer of the present invention has a pH of from about 4.5 to 6.0, preferably from about 5.0 to 6.0, more preferably from about 5.0 to 5.5, most preferably 5.3.
  • Examples of the buffer which controls the pH in this range include acetate buffer, succinate buffer, gluconate buffer, histidine buffer, oxalate buffer, lactate buffer, and phosphoric acid. Salt buffer, citrate buffer, tartrate buffer, fumarate buffer, glycylglycine and other organic acid buffers.
  • a preferred buffering agent of the invention is a succinate buffer or a citrate buffer, more preferably a succinate buffer.
  • succinate buffer is a buffer that includes succinate ions.
  • succinate buffers include sodium succinate-succinate, histidine succinate, potassium succinate-succinate, calcium succinate-succinate, and the like.
  • a preferred succinate buffer of the invention is sodium succinate-succinate.
  • citrate buffer is a buffer that includes citrate ions.
  • the citrate buffer include citric acid-sodium citrate, citrate histidine, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like.
  • a preferred citrate buffer of the invention is citric acid-sodium citrate.
  • “Pharmaceutical composition” means a mixture comprising one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiological/pharmaceutically acceptable Carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • “pharmaceutical composition” and “formulation” are not mutually exclusive.
  • the pharmaceutical composition of the present invention is capable of achieving a stable effect: a pharmaceutical composition in which the antibody substantially retains its physical stability and/or chemical stability and/or biological activity after storage, preferably, a drug
  • the composition retains substantially its physical and chemical stability and its biological activity upon storage.
  • the shelf life is generally selected based on the predetermined shelf life of the pharmaceutical composition.
  • Freeze-dried formulation means a formulation obtained by vacuum-drying a pharmaceutical composition or liquid or solution formulation in liquid or solution form.
  • a stable pharmaceutical preparation is one in which no significant change is observed in the following conditions: at a refrigerated temperature (2-8 ° C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 year.
  • stable liquid preparations include liquid preparations which exhibit desirable characteristics after storage at temperatures including 25 ° C and 40 ° C including periods of 1 month, 3 months, and 6 months.
  • Typical acceptable criteria for stability are as follows: typically no more than about 10%, preferably no more than about 5%, of antibody monomers are degraded by SEC-HPLC.
  • the pharmaceutical liquid formulation is colorless or clear to slightly milky white.
  • the concentration, pH and osmolality of the formulation have a variation of no more than ⁇ 10%.
  • a truncation of no more than about 10%, preferably no more than about 5% is generally observed.
  • Aggregation is generally formed no more than about 10%, preferably no more than about 5%.
  • the antibody did not show significant aggregation increase, precipitation and/or denaturation if visually examined for color and/or clarity, or by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS). Then the antibody "retains its physical stability" in the pharmaceutical preparation. Changes in protein conformation can be assessed by fluorescence spectroscopy, which determines the tertiary structure of the protein, and by FTIR spectroscopy, which determines the secondary structure of the protein.
  • the antibody does not show a significant chemical change, the antibody "retains its chemical stability" in the pharmaceutical formulation.
  • Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein.
  • Degradation processes that frequently alter the chemical structure of a protein include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (by peptide mapping such as mass spectrometry or MALDI/TOF/MS) Method to evaluate), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide spectroscopy, isoaspartic acid measurement, etc.) and isomerization (by measuring the content of isoaspartic acid, Peptide mapping, etc. to evaluate).
  • An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of biological activity exhibited in the preparation of the pharmaceutical formulation.
  • the biological activity of an antibody can be determined, for example, by antigen binding assays.
  • Antibody drug conjugate is the use of a monoclonal antibody or antibody fragment by a stable chemical linker compound with a biologically active cytotoxin or a small molecule drug with cell killing activity, making full use of the antibody.
  • the specificity of the tumor cells is specific or highly expressed, and the efficiency of cytotoxin is high, avoiding the toxic side effects on normal cells. This means that antibody drug conjugates bind precisely to tumor cells and reduce the effects on normal cells compared to traditional chemotherapeutic drugs.
  • c-Met antibody drug conjugate refers to an antibody drug conjugate (ADC) formed by the attachment of an antibody targeting c-Met to a cytotoxin or a small molecule drug via a chemical linker. This includes, but is not limited to, ADC-12 of the present invention.
  • ADC antibody drug conjugate
  • 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 light chain 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 of the present invention may further comprise a heavy chain constant region comprising IgG1, IgG2, IgG3, IgG4 or a variant thereof of human or murine origin.
  • variable region The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, being the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • CDR complementarity determining region
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment of the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDE2-3) in number and position, or conform to the numbering rules of kabat and chothia. (HCDR1).
  • the antibody of the present invention includes a murine antibody, a chimeric antibody, a humanized antibody, preferably a humanized antibody.
  • murine antibody is in the present invention a monoclonal antibody to an antigen prepared according to the knowledge and skill in the art.
  • the test subject is injected with an antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • 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 mouse hybridoma cell, and the mouse variable region gene is cloned as needed, and the mouse variable region is cloned.
  • the gene is ligated into a human vector after being ligated into a human chimeric gene, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the antibody light chain of the c-Met chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the c-Met chimeric antibody further comprises a heavy chain constant region of human IgGl, IgG2, IgG3, IgG4 or variants thereof.
  • the constant region of a human antibody may be selected from the heavy chain constant region of human IgGl, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising a human IgG2 or IgG4 heavy chain constant region, or without ADCC after amino acid mutation (antibody-dependent) Cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG4.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into human antibody variable region frameworks, ie different types of human germline An antibody produced in an antibody framework sequence. It is possible to overcome the strong antibody variable antibody response induced by chimeric antibodies by carrying a large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, etc.
  • humanized antibodies of the invention also include humanized antibodies that are further affinity matured by phage display.
  • identity also referred to as “consistency” or “similarity” refers to the proportion of the sequence of identical bases or amino acid residues between the detection sequence and the target sequence during sequence alignment.
  • the "antigen-binding fragment” as used in the present invention refers to a Fab fragment having antigen-binding activity, a Fab' fragment, a F(ab')2 fragment, and a ScFv fragment which binds to human c-Met, and other utilization energy.
  • a fragment of human C-Met formed by the VH and VL regions of a human c-Met-binding antibody; comprising one or more selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 2 of the antibody of the present invention CDR region.
  • the Fv fragment contains the antibody heavy chain variable region and the light chain variable region, but has no constant region and has the smallest antibody fragment of the entire antigen binding site.
  • Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the desired structure for antigen binding.
  • the two antibody variable regions can also be joined by a different linker into a single polypeptide chain, referred to as a single chain antibody or a single chain Fv (sFv).
  • binding to c-Met in the present invention means that it can interact with human c-Met.
  • antigen binding site refers to a three-dimensional spatial site recognized by a c-Met antibody or antigen-binding fragment, either continuously or discontinuously on the antigen.
  • c-Met antibody is an antibody that specifically binds to c-Met, including, but not limited to, the c-Met antibody disclosed in WO 2016/165580 A1.
  • Ab-10 of WO2016 / 165580A1 disclosed c-Met antibody Ab-10, which is a heavy chain amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFSLSNYGVHWVRQAPGKGLEWLAVIWSGGSTNYAAAFVSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARNHDNPYNYAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
  • ADC-12 is a c-Met antibody drug conjugate formed by the attachment of Ab-10 via a chemical linker to a small molecule toxin. Has the structure shown in ADC-12 below
  • y ranges from 1 to 8, preferably from 2 to 5; y can be a decimal.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region.
  • the human FR germline sequence can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN 014441351. obtain.
  • the engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human c-Met. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by a conventional technique.
  • purification is carried out using an A or G Sepharose FF column containing an adjusted buffer.
  • the non-specifically bound components are washed away.
  • the bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange.
  • the resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
  • Constantly modified refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • Amino acid sequence "identity” refers to sequence similarity between two proteins or polypeptides. When the positions in both comparison sequences are occupied by the same amino acid residue, for example if one position of both polypeptides is occupied by the same amino acid residue, then the molecules are identical at that position.
  • Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al, respectively. (1977) Nucleic Acids Res. 25: 3389-3402.
  • Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administeristering and “treating” also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, for example a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases to induce such symptoms to degenerate or to inhibit the progression of such symptoms to any degree of clinical right measurement.
  • the amount of therapeutic agent also referred to as "therapeutically effective amount" effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient.
  • Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition. While embodiments of the invention (e.g., methods of treatment or preparations) may be ineffective in ameliorating the symptoms of each target disease, any statistical test methods known in the art such as Student's t-test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • any statistical test methods known in the art such as Student's t-test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the methodological route and dosage of the administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Tm value refers to the heat denaturation temperature of the protein, that is, the temperature at which half of the protein is unfolded, and the spatial structure of the protein is destroyed at this time, so the higher the Tm value, the higher the thermal stability of the protein.
  • N,N-diisopropylethylamine (0.29 Ml, 1.67 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea Fluorophosphate (152.3 mg, 0.40 mmol).
  • the reaction system was stirred at room temperature for 1 hour under an argon atmosphere. After the reaction was completed, 10 ml of water was added and the mixture was evaporated and evaporated.
  • reaction mixture was concentrated under reduced pressure, and then diluted with 5 Ml of dichloromethane.
  • the system was layered and the aqueous layer was extracted with dichloromethane (5Ml ⁇ 3).
  • dichloromethane layers were washed with a saturated sodium chloride solution (10 mL) and dried over anhydrous sodium sulfate.
  • the preparation method of the Ab-10 mAb is the same as the preparation method of the Ab-10 mAb disclosed in Examples 1 to 3 and 5 to 6 in WO2016/165580A1.
  • the first step the ADC-12 stock solution is filtered and the central control sample is tested for sterility.
  • the stock solution was passed through a 0.22 ⁇ m PVDF filter and the filtrate was collected.
  • the ADC-12 is an anti-c-Met antibody ADC, wherein the c-MET antibody is Ab-10, having a heavy chain as shown in SEQ ID NO: 24 of WO 2016/165580 A1 and a light as shown in SEQ ID NO: chain.
  • the second step adjust the loading to 4.2ml, fill the filtrate in a 15ml vial, and add half of the plug, respectively, at the beginning of filling, middle of filling, and at the end of filling, the difference between sampling and control.
  • the third step the filling and filling of the liquid medicine into the freeze-drying box, and performing a freeze-drying process.
  • the freeze-drying step includes steps of pre-freezing, primary drying, and secondary drying. After the lyophilization procedure is finished, the vacuum is stoppered. Exemplary lyophilization parameters are as follows:
  • 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 experimental design of the buffer system, buffer concentration, pH value, sugar type and sugar concentration of ADC-12 preparation was carried out, and the Tm value of the sample was determined by DSC technique, and the prescription of the preparation was initially screened.
  • the buffer system, buffer concentration, pH value, sugar type and sugar concentration were used as the factors, and the Tm value was used as the response value to design the test.
  • the design table was generated, and the Tm value was determined according to the experimental group of the design table.
  • freeze-drying was carried out using a Toufong LYO-3 (SIP, CIP) vacuum freeze dryer.
  • SE-HPLC was tested using an Agilent 1200 DAD high pressure liquid chromatograph (Waters Xbridge Protein BEH SEC) Column).
  • the CE-SDS was tested using a Beckman PA800plus capillary electrophoresis apparatus (SDS-Gel MW Analysis Kit).
  • 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 thermal stability of ADC-12 in each preparation was measured by differential scanning calorimetry (DSC) (see Table 1 for the test results).
  • N/A means that this ingredient is not added.
  • the acetate buffer system may cause a pH shift due to volatilization during lyophilization.
  • the buffer range of the phosphate buffer (pH 6.0-8.0) is too overlapping with the isoelectric point range of ADC-12 and should not be used. Therefore, two buffer systems of succinate and citrate with relatively high Tm onset and Tm were selected.
  • ADC-12 preparation was prepared with 10 mM succinic acid (sodium succinate) or citric acid (sodium citrate) as buffer at pH 5.5, containing 60 mg/ml sucrose, 0.2 mg/ml polysorbate 20, and final concentration of ADC-12. It is 20 mg/ml. It was lyophilized in a 15 mL vial filled with 4 mL/vial and sealed with a lyophilized rubber stopper. The lyophilized product was placed at 25 ° C for investigation. The results show that the succinic acid (sodium succinate) system is slightly better than the citric acid (sodium citrate) system.
  • M0 indicates the 0th month
  • D15 indicates the 15th day
  • M1 indicates the first month
  • M3 indicates the third month
  • M6 indicates the sixth month.
  • the ADC-12 preparation was prepared with a buffer containing 10 mM succinic acid-succinate at pH 4.8-5.8, containing 60 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/ml polysorbate 20 preparation, ADC-12 The final concentration was 20 mg/ml.
  • Each formulation was filtered and lyophilized in a 15 mL neutral borosilicate glass controlled injection vial filled with 4 mL/bottle, which was sealed with a rubber stopper.
  • the lyophilized product was stored at 25 ° C for stability analysis.
  • the stability results of ADC-12 at 25 ° C and 0-6 months at different pHs are shown in Table 3. The results show that ADC-12 is very stable at pH 5.0–5.5.
  • M0 indicates the 0th month
  • M3 indicates the 3rd month
  • M6 indicates the 6th month.
  • ADC-12 preparation was prepared with 60 mg/ml ⁇ , ⁇ -dihydrate trehalose or sucrose as buffer at pH 5.5. The final concentration of ADC-12 was 20 mg/ml, containing 10 mM sodium succinate-succinate, 0.2 mg/ml. Polysorbate 20. Each formulation was filtered and filled into a 15 mL vial at 4 mL/vial, lyophilized and sealed with a lyophilized plug. The lyophilized product was stored at 25 ° C and 2-8 ° C for stability analysis. The results show that ADC-12 is more stable in the trehalose system.
  • M0 indicates the 0th month
  • M3 indicates the 3rd month
  • M6 indicates the 6th month.
  • M0 indicates the 0th month
  • M3 indicates the 3rd month
  • M6 indicates the 6th month.
  • the ADC-12 formulation was prepared in a buffer of pH 5.5 containing the following surfactants at different concentrations to prepare a final concentration of ADC-12 of 20 mg/ml, containing 10 mM sodium succinate-succinate, 60 mg/ml alpha, alpha-dihydrate seaweed Sugar, ADC-12 preparation:
  • the sample is placed in a -35 ° C refrigerator for 12 h, and then transferred to 2-8 ° C for 12 h, which is a freeze-thaw cycle. Repeat a total of 5 cycles.
  • the stability results showed that 0.05-0.4 mg/ml polysorbate 20 effectively prevented the aggregation of ADC-12 during freezing and thawing.
  • ADC-12 preparation was prepared with 10 mM succinic acid (sodium) as pH 5.3, containing 60 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/ml polysorbate 20, and the final concentration of ADC-12 was 20 mg/ml. .
  • the antibody was filled in a 15 mL vial at 4 mL/vial, lyophilized at a drying temperature of -27 ° C, -20 ° C and -15 ° C, respectively, and sealed with a lyophilized rubber stopper. The results show that -20 ° C is the best drying temperature for the freeze-drying process.
  • ADC-12 preparation was prepared with 10 mM succinic acid (sodium) as pH 5.3, containing 60 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/ml polysorbate 20, and the final concentration of ADC-12 was 20 mg/ml. .
  • the preparations were filled in glass bottles, liquid storage bags and 316L stainless steel tanks, respectively, and left at 2-8 ° C for 24 hours. Analysis of protein content and purity indicated (see Table 8) that ADC-12 was stable within 24 hours.
  • the formulation is compatible with 316L stainless steel cans, glass bottles and liquid storage bags.
  • the ADC-12 preparation was prepared with 10 mM succinic acid (sodium) as pH 5.3, containing 60 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/ml polysorbate 20, and the final concentration of ADC-12 was 10 mg/ml. , 1mg / ml, also has good stability.
  • N/A means not detected.
  • the stable pharmaceutical formulation provided by the present invention comprises: a combination of ADC-12 and a stabilizing buffer selected from the group consisting of:
  • the concentration of ADC-12 is in the range of 1 mg/ml to 30 mg/ml, preferably 10-20 mg/ml, and most preferably 10 mg/ml.
  • the implementable scheme may be selected from, but not limited to, the following combinations:
  • anti-ADC-12 30 mg/ml, 60 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.05 mg/ml polysorbate 20, and 10 mM succinate buffer at pH 5.2;
  • anti-ADC-12 1 mg/ml, 50 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 10 mM succinate buffer at pH 5.0;
  • anti-ADC-12 10 mg/ml, 60 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.4 mg/ml polysorbate 20, and pH 5.1 10 mM succinate buffer;
  • anti-ADC-12 15 mg/ml, 50 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.3 mg/ml polysorbate 20, and pH 5.4 20 mM succinate buffer;
  • anti-ADC-12 5 mg/ml, 70 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.1 mg/ml polysorbate 20, and pH 5.3 20 mM succinate buffer;
  • anti-ADC-12 10 mg/ml, 60 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 15 mM succinate buffer at pH 5.2;
  • anti-ADC-12 30 mg/ml, 40 mg/ml sucrose, 0.05 mg/ml polysorbate 20, and pH 5.3 30 mM citrate buffer;
  • anti-ADC-12 20 mg/ml, 60 mg/ml lactose, 0.1 mg/ml polysorbate 20, and pH 5.4 20 mM citrate buffer;
  • anti-ADC-12 10 mg/ml, 70 mg/ml ⁇ , ⁇ -dihydrate trehalose, 0.4 mg/ml polysorbate 80, and 10 mM citrate buffer at pH 5.2;
  • Anti-ADC-12 1 mg/ml, 80 mg/ml maltose, 0.2 mg/ml polyoxyethylene hydrogenated castor oil, and 10 mM citrate buffer pH 5.2.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Dermatology (AREA)
  • Cell Biology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)

Abstract

一种稳定的c-Met抗体药物偶联物（c-Met ADC）制剂及其在医药上的应用，该制剂含有c-Met ADC、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c-Met ADC制剂可以有效抑制抗体的聚集和异构化作用，阻止其中抗体产品的降解，得到稳定的注射药物制剂。

Description

一种含c-Met抗体药物偶联物的药物组合物及其用途
本申请要求申请日为2017年6月6日的中国专利申请CN201710417725.4以及申请日为2017年7月7日的中国专利申请CN201710551046.6的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于药物制剂领域，具体涉及一种包含c-Met抗体药物偶联物的药物组合物，以及其作为药物的用途。
背景技术
c-Met原癌基因位于人类第7号染色体长臂(7q31)，大小超过120kb，编码分子量约150kD的c-Met蛋白前体，经局部糖基化生成一个170kD的糖蛋白，该糖蛋白进一步剪切为α亚基(50kDa)和β亚基(140kDa)，以二硫键相连，形成成熟的c-Met蛋白受体。该异二聚体包含两条链，β链有胞外区、跨膜区(也称膜伸展片段)和胞内区(包含细胞内酪氨酸激酶结合位点)。α链只有胞外部分，但它高度糖基化，通过二硫键附着于β链上。两个亚基的胞外区域是相应配体的识别部位，胞内区域具有酪氨酸激酶活性。
c-Met激活的机制分为三种：一是依赖HGF的激活机制，二是不依赖HGF激活机制，三是经过其他膜途径，例如通过透明质酸膜表面受体的CD44、粘附素以及RON信号传导途径等等。其中最常见的是依赖HGF的激活机制。HGF的N末端与c-Met结合，促进β链上Tyr1234和Tyr1235二聚化和自磷酸化，C-末端附近的Tyr1349和Tyr1356磷酸化产生多个接头蛋白的结合位点，这些接头蛋白诱导了P13K/Akt、Ras/Mapk、c-Src和STAT3/5介导的下游信号的激活，引发不同细胞反应，如细胞生存和活动(与P13K/Akt通路密切相关)，肿瘤转移和细胞增殖(主要由Ras/Mapk介导)。此外，c-Met与其它膜受体存在交联(cross-talk)，现已确知这种交联可促进肿瘤形成及转移，由于c-Met是导致肿瘤形成及转移的许多通路的交叉点，以c-Met为靶标可相对较容易地实现对许多通路的同时干扰，c-Met已成为抗肿瘤生成和转移治疗的一个有希望的靶点。
抗体药物偶联物(antibody drug conjugate，ADC)把单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒素相连，充分利用了抗体对肿瘤细胞特异或高表达抗原结合的特异性和细胞毒素的高效性，避免对正常细胞的毒副作用。这也就意味着，与以往传统的化疗药物相比，抗体药物偶联物能精准地结合肿瘤细胞并降低 将对正常细胞的影响。
ADC药物由抗体(靶向部分)，接头和毒素三部分组成。其中，好的靶向部分决定了ADC药物的特异性，这不仅包括特异靶向结合，还包括有效的内吞。
但是，抗体药物，尤其是ADC与其他的化学药物相比，其分子量更大，结构更复杂，容易降解、聚合或发生不希望发生的化学修饰等而变得不稳定。为了使抗体药物偶联物适合于给药，并且在储存及随后使用过程中能保持稳定性，发挥更好的效果，抗体药物的稳定制剂研究显得尤为重要。
尽管，目前有多家公司在研发包含c-Met抗体或抗体药物偶联物及药物配制剂，如：CN103781493A，CN105050618A，WO2016042412A1等。但对于c-Met ADC，该制剂并不是最适制剂组合物。本发明提供足够稳定且更适于给药的包含c-Met ADC的药物(制剂)组合物。
发明内容
本发明提供一种药物组合物，其包含c-Met抗体药物偶联物，及其他赋形剂。
在一些实施方案中，药物组合物，其包含c-Met抗体药物偶联物，以及缓冲剂，所述缓冲剂优选为琥珀酸盐或柠檬酸盐缓冲剂，更优选为琥珀酸盐缓冲剂。
在一些实施方案中，药物组合物，其中所述c-Met抗体药物偶联物浓度为1mg/ml至30mg/ml，优选为大约1mg/ml至20mg/ml，进一步优选为大约5mg/ml至20mg/ml，最优选为10-20mg/ml，非限制性实施例包括1mg/ml、2mg/ml、3mg/ml、4mg/ml、5mg/ml、6mg/ml、7mg/ml、8mg/ml、9mg/ml、10mg/ml、11mg/ml、12mg/ml、13mg/ml、14mg/ml、15mg/ml、16mg/ml、17mg/ml、18mg/ml、19mg/ml、20mg/ml和30mg/ml。
在一些实施方案中，药物组合物，其pH约为5.0到6.0，优选为大约5.0至5.5，最优选为5.0、5.1、5.2、5.3、5.4、5.5。
在一些实施方案中，药物组合物，其中所述缓冲剂浓度为大约5mM至30mM，优选为5mM至20mM，进一步优选为大约10mM至20mM，更优选为大约10mM至15mM，最优选为10mM。
在一些实施方案中，药物组合物中还包含其糖。“糖”包含常规组合物(CH2O) n及其衍生物，包括单糖，二糖，三糖，多糖，糖醇，还原性糖，非还原性糖等等。可选自葡萄糖，蔗糖，海藻糖，乳糖，果糖，麦芽糖，右旋糖苷，甘油，右旋糖苷，赤藻糖醇，丙三醇，阿拉伯糖醇，sylitol，山梨糖醇，甘露醇，密里二糖，松三糖，蜜三糖，甘露三糖，水苏糖，麦芽糖，乳果糖，麦芽酮糖，山梨醇，麦芽糖醇，乳糖醇，异-麦芽酮糖等等。 优选的糖是非还原性二糖，更优选为海藻糖或蔗糖。药物组合物，其中还包括二糖，所述二糖优选自海藻糖或蔗糖，最优选为海藻糖。
在一些实施方案中，药物组合物，其中所述糖的浓度为约40mg/ml至约80mg/ml，优选为50mg/ml至约70mg/ml，更优选为55mg/ml至约65mg/ml，非限制性实施例包括55mg/ml、57mg/ml、59mg/ml、60mg/ml、61mg/ml、63mg/ml、65mg/ml。
在一些实施方案中，药物组合物，其中还包括表面活性剂，可选自聚山梨醇酯20、聚山梨醇酯80、聚羟亚烃、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基-肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇、乙烯与丙烯二醇的共聚物等等。优选的表面活性剂是聚山梨醇酯80或聚山梨醇酯20，更优选为聚山梨醇酯20。
在一些实施方案中，药物组合物中表面活性剂的浓度为约0.05mg/ml至1.0mg/ml，进一步优选为0.05mg/ml至0.4mg/ml，更优选为0.1mg/ml至0.4mg/ml，最优选为0.1mg/ml至0.2mg/ml，非限制性实施例包括0.1mg/ml、0.2mg/ml、0.3mg/ml、0.4mg/ml。
本文还提供一种药物组合物，其包含：
(a)1-20mg/ml的c-Met抗体药物偶联物；
(b)10-20mM的琥珀酸盐缓冲剂，pH5.0-5.5；
(c)40-80mg/ml的α,α-二水合海藻糖；
(d)0.05-0.4mg/ml的聚山梨醇酯20。
在一些实施方案中，所述药物组合物包含：(a)1-20mg/ml的c-Met抗体药物偶联物；(b)10-20mM的琥珀酸盐缓冲剂，pH5.0-5.5；(c)60mg/ml的α,α-二水合海藻糖；(d)0.05-0.4mg/ml的聚山梨醇酯20。
在一些实施方案中，所述药物组合物包含：(a)5-20mg/ml的c-Met抗体药物偶联物；(b)10-20mM的琥珀酸盐缓冲剂，pH5.0-5.5；(c)50-70mg/ml的α,α-二水合海藻糖；(d)0.1-0.2mg/ml的聚山梨醇酯20。
在一些实施方案中，药物组合物，其中c-Met抗体药物偶联物中的抗体为Ab-10，所述Ab-10抗体重链序列如WO2016/165580A1中SEQ ID NO：24所示，Ab-10抗体轻链 序列如WO2016/165580A1中SEQ ID NO：27所示。
在一些实施方案中，药物组合物，其中c-Met抗体药物偶联物为ADC-12，具有如下式所示的结构：
Figure PCTCN2018089955-appb-000001
，其中y的范围为1-8，优选2-5；y可以为小数。
在一些实施方案中，所述药物组合物包含：(a)1-30mg/ml的ADC-12；(b)10-30mM的琥珀酸盐缓冲剂或柠檬酸盐缓冲液，pH5.0-5.5；(c)40-80mg/ml的α,α-二水合海藻糖；(d)0.05-0.4mg/ml的聚山梨醇酯20。
在一些实施方案中，所述药物组合物包含：(a)1-20mg/ml的ADC-12；(b)10-20mM的琥珀酸盐缓冲剂，pH5.0-5.5；(c)40-80mg/ml的α,α-二水合海藻糖；(d)0.05-0.4mg/ml的聚山梨醇酯20。
在一些实施方案中，所述药物组合物包含：(a)1-20mg/ml的ADC-12；(b)10-20mM的琥珀酸盐缓冲剂，pH5.0-5.5；(c)60mg/ml的α,α-二水合海藻糖；(d)0.05-0.4mg/ml的聚山梨醇酯20。
在一些实施方案中，所述药物组合物包含：(a)5-20mg/ml的ADC-12；(b)10-20mM的琥珀酸盐缓冲剂，pH5.0-5.5；(c)50-70mg/ml的α,α-二水合海藻糖；(d)0.1-0.2mg/ml的聚山梨醇酯20。
在一些实施方案中，所述药物组合物包含：1mg/ml的ADC-12，20mM琥珀酸盐缓冲剂，pH为5.5或6.0。
在一些实施方案中，所述药物组合物包含：1mg/ml的ADC-12，20mM柠檬酸盐缓冲剂，pH为5.0、5.5或6.0。
在一些实施方案中，所述药物组合物包含：20mg/ml的ADC-12，10mM琥珀酸盐缓冲剂或柠檬酸盐缓冲剂，60mg/ml的蔗糖，0.2mg/ml聚山梨醇酯20，pH5.5。
在一些实施方案中，所述药物组合物包含：20mg/ml的ADC-12，10mM琥珀酸盐缓冲剂，60mg/ml的α,α-二水合海藻糖，0.05-0.4mg/ml聚山梨醇酯20，pH5.0-5.5。
在一些实施方案中，所述药物组合物包含：20mg/ml的ADC-12，10mM琥珀酸盐缓冲剂，60mg/ml的α,α-二水合海藻糖，0.2mg/ml聚山梨醇酯20，pH5.0-5.5。
在一些实施方案中，所述药物组合物包含：20mg/ml的ADC-12，10mM琥珀酸盐缓冲剂，60mg/ml的α,α-二水合海藻糖，0.2mg/ml聚山梨醇酯20，pH5.3。
本文还提供一种制备含c-Met抗体药物偶联物的冷冻干燥制剂的方法，其中包括将前述的药物组合物经冷冻干燥的步骤。
在一些实施方案中，制备含c-Met抗体药物偶联物的冷冻干燥制剂的方法，其中所述冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。预冻目的是冷冻产品，获得结晶固体。预冻温度和预冻速度是两个重要工艺参数，本发明预冻温度最优设定为-45℃，预冻速度设定为1℃/min(自-5℃开始)。一次干燥也称为主干燥，是样品冷冻干燥的主要阶段。目的是移除产品里冰的同时，保持产品形状，最小程度的减少对产品的破坏。若一次干燥的温度和真空度选择不当，会导致制品的塌陷。较高的温度和真空度均会加快冻干效率，但同时也会增加制品塌陷的风险。本发明所述一次干燥的温度可为本领域常规的温度，例如-27--20℃，优选为-20℃。本发明所述一次干燥的温度优选为0.1mbar。二次干燥也称为解析干燥，是通过抽极限真空(0.01mbar)和升高温度(25-40℃)除去制品中结合水的主要步骤。由于大多数生物制品对温度比较敏感，所以二次干燥温度选择在温度范围的低点，即25℃。另外，在实际生产过程中，冷冻干燥条件可以随制剂和样品容纳容器的大小和类型以及液体的体积变化。
本文还提供一种前述的方法制备的含c-Met抗体药物偶联物的冷冻干燥制剂。
在一些实施方案中，还提供一种冷冻干燥制剂，其中所述冷冻干燥制剂经复溶后可形成前述的任意一种药物组合物，优选使用注射用水进行复溶。
本文还提供前述的药物组合物或冷冻干燥制剂在制备用于治疗c-Met相关的疾病或病症的药物中的用途，其中所述的疾病或病症优选为癌症；更优选为表达c-Met的癌症；更优选为表达c-Met的胃癌、胰腺癌、肺癌(例如非小细胞肺癌)、成胶质细胞瘤、肉瘤、结肠直肠癌、肾癌、肝细胞癌、黑素瘤和乳腺癌；最优选为胃癌、胰腺癌、非小细胞肺癌和肾癌。
本文还提供一种治疗和预防c-Met相关的疾病或病症的方法，包括给予所需患者治疗有效量的根据前述药物组合物或冷冻干燥制剂；其中所述的疾病优选为癌症；更优选为表达c-Met的癌症；更优选为表达c-Met的胃癌、胰腺癌、肺癌(例如非小细胞肺癌)、成胶质细胞瘤、肉瘤、结肠直肠癌、肾癌、肝细胞癌、黑素瘤和乳腺癌；最优选为胃癌、胰腺癌、非小细胞肺癌和肾癌。
本文还提供一种制品，其包括容器，该容器中装有前述的药物组合物或冷冻干燥制剂。所述容器优选为玻璃瓶、储液袋或316L不锈钢罐。
在本发明的其他实施方案中，药物组合物中所述c-Met抗体药物偶联物中的抗体包含和如WO2016/165580A1中SEQ ID NO：24所示的氨基酸序列具有85％以上的序列同一性，如具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列同一性的重链序列，优选地具有95％以上的序列同一性；和如WO2016/165580A1中SEQ ID NO：27所示的氨基酸序列具有85％以上的序列同一性，如具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列同一性的轻链序列，优选地具有95％以上的序列同一性。
在一些实施方案中，该配制剂于2-8℃稳定至少3个月，至少6个月，至少12个月，至少18个月或至少24个月。在一些实施方案中，该配制剂于40℃稳定至少7天，至少14天或至少28天。
要理解，可以组合本文所述各个实施方案的一项、一些或所有特性以形成本发明的其它实施方案。本发明的这些和其它方面对于本领域技术人员会变得显然。本发明的这些和其它实施方案通过下面的详述进一步描述。
具体实施方式
一、术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
“缓冲剂”指通过其酸-碱共轭组分的作用而耐受pH变化的缓冲剂。本发明的缓冲剂pH约为4.5到6.0，优选为大约5.0至6.0，更优选为大约5.0至5.5，最优选为5.3。将pH控制在此范围中的缓冲剂的例子包括醋酸盐缓冲剂、琥珀酸盐缓冲剂、葡萄糖酸盐缓冲剂、组氨酸缓冲剂、草酸盐缓冲剂、乳酸盐缓冲剂、磷酸盐缓冲剂、柠檬酸盐缓冲剂、酒石酸盐缓冲剂、延胡索酸盐缓冲剂、甘氨酰甘氨酸和其它有机酸缓冲剂。本发明优选的缓冲剂是琥珀酸盐缓冲剂或柠檬酸盐缓冲剂，更优选为琥珀酸盐缓冲剂。
“琥珀酸盐缓冲剂”是包括琥珀酸根离子的缓冲剂。琥珀酸盐缓冲剂的实例包括琥珀酸-琥珀酸钠、琥珀酸组氨酸盐、琥珀酸-琥珀酸钾、琥珀酸-琥珀酸钙盐等。本发明优选的琥珀酸盐缓冲剂是琥珀酸-琥珀酸钠。
“柠檬酸盐缓冲剂”是包括柠檬酸根离子的缓冲剂。柠檬酸盐缓冲剂的实例包括柠檬酸-柠檬酸钠、柠檬酸组氨酸盐、柠檬酸-柠檬酸钾、柠檬酸-柠檬酸钙、柠檬酸-柠檬酸镁 等。本发明优选的柠檬酸盐缓冲剂是柠檬酸-柠檬酸钠。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。本文中，“药物组合物”和“制剂”并不互相排斥。
本发明所述的药物组合物能够达到一种稳定的效果：其中的抗体在贮藏后基本上保留其物理稳定性和/或化学稳定性和/或生物学活性的药物组合物，优选地，药物组合物在贮藏后基本上保留其物理和化学稳定性以及其生物学活性。贮藏期一般基于药物组合物的预定保存期来选择。目前有多种测量蛋白质稳定性的分析技术，可测量在选定温度贮藏选定时间段后的稳定性。
“冷冻干燥制剂”表示液体或溶液形式的药物组合物或液体或溶液制剂真空经冷冻干燥步骤之后获得的制剂。
稳定的药物制剂是在下述情况下没有观察到显著变化的制剂：在冷藏温度(2-8℃)保存至少3个月、优选6个月、更优选1年，且甚至更优选地最多达2年。另外，稳定的液体制剂包括这样的液体制剂：其在包括25℃和40℃在内的温度保存包括1个月、3个月、6个月在内的时段后表现出期望的特征。稳定性的典型的可接受的标准如下：通过SEC-HPLC测得，通常不超过约10％、优选不超过约5％的抗体单体发生降解。通过视觉分析，药物液体制剂是无色的，或澄清至稍微乳白色。所述制剂的浓度、pH和重量克分子渗透压浓度具有不超过±10％变化。通常观察到不超过约10％、优选不超过约5％的截短。通常形成不超过约10％、优选不超过约5％的聚集。
如果在目检颜色和/或澄清度后，或者通过UV光散射、尺寸排阻色谱法(SEC)和动态光散射(DLS)测得，抗体没有显示出显著的聚集增加、沉淀和/或变性，那么所述抗体在药物制剂中“保留它的物理稳定性”。蛋白构象的变化可以通过荧光光谱法(其确定蛋白三级结构)和通过FTIR光谱法(其确定蛋白二级结构)来评价。
如果抗体没有显示出显著的化学改变，那么所述抗体在药物制剂中“保留它的化学稳定性”。通过检测和定量化学上改变的形式的蛋白，可以评估化学稳定性。经常改变蛋白化学结构的降解过程包括水解或截短(通过诸如尺寸排阻色谱法和SDS-PAGE等方法来评价)、氧化(通过诸如与质谱法或MALDI/TOF/MS结合的肽谱法等方法来评价)、脱酰胺作用(通过诸如离子交换色谱法、毛细管等电聚焦、肽谱法、异天冬氨酸测量等方法来评价)和异构化(通过测量异天冬氨酸含量、肽谱法等来评价)。
如果抗体在给定时间的生物活性是在制备药物制剂时表现出的生物活性的预定范围 内，那么所述抗体在药物制剂中“保留它的生物活性”。抗体的生物活性可以例如通过抗原结合测定来确定。
“抗体药物偶联物(antibody drug conjugate，ADC)”是把单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒素或具有细胞杀伤活性的小分子药物相连，充分利用了抗体对肿瘤细胞特异或高表达抗原结合的特异性和细胞毒素的高效性，避免对正常细胞的毒副作用。这也就意味着，与以往传统的化疗药物相比，抗体药物偶联物能精准地结合肿瘤细胞并降低将对正常细胞的影响。
“c-Met抗体药物偶联物”是指以c-Met为靶点的抗体经化学接头与细胞毒素或小分子药物连接形成的抗体药物偶联物(ADC)。包括但不限于本发明的ADC-12。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
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。
在本发明中，本发明所述的抗体轻链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。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本发明所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDE2-3)，或者符合kabat和chothia的编号规则(HCDR1)。
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“鼠源抗体”在本发明中为根据本领域知识和技能制备的对抗原的单克隆抗体。制备时用抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
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，然后从小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，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。在本发明一个优选的实施方案中，所述的c-Met嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的c-Met嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG4。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的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小鼠蛋白成分，从而诱导的强烈的抗体可变抗体反应。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。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本发明的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。
术语“同一性”也称“一致性”或“相似性”，是指序列比对过程中检测序列和目标序列之间相同碱基或氨基酸残基序列所占比例的大小。
本发明中所述的“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab‘片段，F(ab’)2片段，以及与人c-Met结合的ScFv片段，以及其他利用了能与人c-Met结合的抗体的VH和VL区形成的可结合人c-Met的片段；其包含本发明所述抗体的选自SEQ ID NO:1至SEQ ID NO:2中的一个或多个CDR区。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。本发明的术语“与c-Met结合”，指能与人c-Met相互作用。本发明的术语“抗原结合位点”指抗原上连续或不连续的，由c-Met抗体或抗原结合片段识别的三维空间位点。
“c-Met抗体”为与c-Met特异性结合的抗体，包括但不限于WO2016/165580A1所公开的c-Met抗体。
“Ab-10”为WO2016/165580A1所公开的c-Met抗体Ab-10，其重链氨基酸序列为QVQLVESGGGVVQPGRSLRLSCAASGFSLSNYGVHWVRQAPGKGLEWLAVIWSGGSTNYAAAFVSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARNHDNPYNYAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:24)
轻链氨基酸序列为
Figure PCTCN2018089955-appb-000002
(SEQ ID NO:27)
“ADC-12”为Ab-10经化学接头与小分子毒素连接形成的c-Met抗体药物偶联物。具有下列ADC-12所示的结构
Figure PCTCN2018089955-appb-000003
其中y的范围为1-8，优选2-5；y可以为小数。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和 轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人c-Met特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲剂的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
氨基酸序列“同一性”是指两个蛋白或多肽之间的序列相似性。当两个比较序列中的位置均被相同氨基酸残基占据时，例如如果两个多肽的一个位置都被同一个氨基酸残基占据时，那么所述分子在该位置是一致的。适于确定序列同一性百分比和序列相似性百分比的算法的实例是BLAST和BLAST2.0算法，它们分别描述于Altschul et al.(1990)J.Mol.Biol.215:403-410和Altschul et al.(1977)Nucleic Acids Res.25:3389-3402。用于执行BLAST分析的软件可在美国国家生物技术信息中心公开获取(http://www.ncbi.nlm.nih.gov/)。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本发明的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。 通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本发明的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“Tm值”是指蛋白质热变性温度，即一半蛋白去折叠时的温度，此时蛋白的空间结构被破坏，所以Tm值越高，蛋白热稳定性越高。
二、实施例和测试例
以下结合实施例进一步描述本发明，但这些实施例并非限制着本发明的范围。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1、抗c-Met抗体Ab-10偶联毒素制备ADC-12
1、毒素的制备
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
Figure PCTCN2018089955-appb-000004
Figure PCTCN2018089955-appb-000005
第一步
(S)-叔丁酯2-氨基-3-(2-氟苯基)丙酸
将原料((S)-2-氨基-3-(2-氟苯基)丙酸12a(400mg，2.18mmol，上海恒创生物科技有限公司，CAT#F2202)溶于10Ml乙酸叔丁酯，加入高氯酸(300mg(70％)，3.3mmol)，于室温下搅拌16小时。反应完毕后加入6Ml水，分液，有机相用饱和碳酸氢钠溶液(5Ml)洗涤。水相用饱和碳酸氢钠溶液调节至Ph＝8，二氯甲烷(5Ml×3)萃取，合并有机相，依次用水(3Ml)，饱和氯化钠溶液(5Ml)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品标题产物(S)-叔丁酯2-氨基-3-(2-氟苯基)丙酸12b(390mg，黄色油状物)，产品不经纯化直接进行下一步反应。
第二步
(1S,3S,5S)-叔丁酯3-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙基-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸
将原料(2R,3R)-3-((1S,3S,5S)-2-(叔丁氧羰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酸12e(100mg，0.334mmol)溶于6Ml二氯甲烷和二甲基甲酰胺(V/V＝5:1)混合 溶剂中，加入反应物粗品(S)-叔丁酯2-氨基-3-(2-氟苯基)丙酸12b(80mg，0.334mmol)。再加入N,N-二异丙基乙基胺(0.29Ml，1.67mmol)和2-(7-偶氮苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(152.3mg，0.40mmol)。反应体系在氩气氛下，于室温搅拌1小时。反应结束后，加10Ml水搅拌，分层，二氯甲烷层用饱和氯化钠溶液(10Ml)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(1S,3S,5S)-叔丁酯3-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙基-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸12c(173mg，无色液体)，收率99.5％。
MS m/z(ESI):521.2[M+1]
第三步
(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将原料(1S,3S,5S)-叔丁酯3-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙基-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸12c(173mg，0.33mmol)溶于2Ml二氧六环中，加入5.6M的氯化氢二氧六环溶液(0.21Ml，1.16mmol)，氩气氛下，于室温搅拌1小时，置于0℃冰箱内12小时。反应结束后，将反应液减压浓缩，加入5Ml二氯甲烷稀释，加入10Ml饱和碳酸氢钠溶液，搅拌10分钟。体系分层，水层用二氯甲烷萃取(5Ml×3)。合并二氯甲烷层，用饱和氯化钠溶液(10Ml)洗涤，无水硫酸钠干燥。过滤，滤液减压浓缩，得到粗品标题产品(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12d(77mg，黄色液体)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):421.2[M+1]
第四步
(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧-4,7,10-三氮杂十四烷基-14-酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将粗品(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12d(77mg，0.183mmol)，(5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷-14-羧酸12i(116.8mg，0.183mmol,采用专利申请“WO 2013072813”公开的方法制备而得，具体见说明书第115-119页的化合物#8的合成步骤)溶于6Ml二氯甲烷和二 甲基甲酰胺(V/V＝5:1)混合溶剂中，加入N,N-二异丙基乙基胺(0.16Ml，0.915mmol)和2-(7-偶氮苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(84mg，0.22mmol)。反应体系在氩气氛下，于室温下搅拌1小时。反应结束后，加入10Ml水搅拌，分层。二氯甲烷层用饱和氯化钠溶液(10Ml)洗涤，无水硫酸钠干燥。过滤，滤液减压浓缩。用硅胶柱色谱法以洗脱剂体系B纯化残留物，得到标题产品(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12e(190.5mg，黄色粘稠物)，收率100％。
MS m/z(ESI):1040.6[M+1]
第五步
(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将原料(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12e(190.5mg，0.183mmol)溶于1.5Ml二氯甲烷中，加入2Ml二乙胺。反应体系在氩气氛下，于室温搅拌3小时。反应结束后，将反应液减压浓缩，得到粗品标题产品(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12f(150mg，黄色粘稠物)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):818.5[M+1]
第六步
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将粗品(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12f(150mg，0.183mmol)溶于1Ml二氧六环中，加入5.6M的氯化氢二氧六环溶液3Ml，氩气氛下，于室温搅拌12小时。反应结 束后，将反应液减压浓缩，用乙醚带旋溶剂。所得残余物用高效液相色谱法纯化得标题产品(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12g(28mg，白色粉末固体)，收率20％。
MS m/z(ESI):762.7[M+1]
1H NMR(400MHz,CD 3OD):δ7.38-7.18(m,2H),7.13-7.01(m,2H),4.80-4.67(m,2H),4.30-4.15(m,1H),4.13-4.01(m,1H),3.96-3.83(m,2H),3.75-3.60(m,2H),3.42-3.11(m,9H),3.06-2.95(m,1H),2.70-2.58(m,4H),2.28-2.01(m,4H),1.88-1.70(m,3H),1.57-1.25(m,4H),1.22-0.95(m,18H),0.92-0.80(m,4H),0.78-0.65(m,1H).
2、毒素中间体的制备
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
Figure PCTCN2018089955-appb-000006
将原料(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12g(25mg，0.033mmol)溶于3mL二氯甲烷中，加入N,N-二异丙基乙基胺(0.029mL，0.164mmol)，反应体系在氩气氛下，冰浴下滴加预制的6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯4b(11.3mg，0.049mmol)的二氯甲烷溶液，于室温反应3小时。反应结束后，加入5mL水，搅拌20分钟，分液，有机层用无水硫酸钠干燥，过滤，滤液减压浓缩，残留物用高效液相色谱法纯化得标题产物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12h(7mg，黄色粘稠 物)，收率22.4％。
MS m/z(ESI):955.4[M+1]
1H NMR(400MHz,CD 3OD):δ7.36-7.30(m,1H),7.29-7.21(m,1H),7.17-7.02(m,2H),6.83-6.79(m,2H),4.81-4.71(m,2H),4.69-4.55(m,2H),4.25-4.15(m,1H),4.13-4.04(m,1H),3.96-3.85(m,2H),3.70-3.61(m,1H),3.55-3.46(m,3H),3.40-3.21(m,4H),3.18-3.10(m,2H),3.07-2.96(m,4H),2.67-2.56(m,2H),2.54-2.34(m,3H),2.29-2.17(m,2H),2.10-1.99(m,1H),1.89-1.57(m,7H),1.52-1.28(m,6H),1.21-1.11(m,4H),1.07-0.96(m,6H),0.95-0.81(m,12H),0.80-0.69(m,1H).
3、抗体毒素偶联物的制备
Figure PCTCN2018089955-appb-000007
将化合物12h(1.2mg，1.2μmol)溶解于0.3mL乙腈中，加入Ab-10单抗-丙硫醇1c溶液(6.17mg/mL，3.0mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物ADC-12的PBS缓冲液(3.3mg/mL，5.0mL)，于4℃冷冻储存。
Ab-10单抗的制备方法同WO2016/165580A1中实施例1～3、5～6所公开的Ab-10单 抗的制备方法。
Q-TOF LC/MS:特征峰：148119.6(M Ab+0D)、149150.5(M Ab+1D)、150221.1(M Ab+2D)、151265.1(M Ab+3D)、152314.3(M Ab+4D)。
平均值：y＝1.6。
ADC的稳定制剂制备工艺如下：
第一步：ADC-12原液过滤后中控取样检测无菌。将原液过0.22μm PVDF滤芯，收集滤液。所述ADC-12是抗c-Met抗体ADC，其中的c-MET抗体为Ab-10，具有如WO2016/165580A1中SEQ ID NO：24所示的重链和SEQ ID NO：27所示的轻链。
第二步：调节装量至4.2ml，将滤液灌装于15ml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，进行冷冻干燥工艺。冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。冻干程序结束后，真空加塞。示例性的冻干参数如下：
Figure PCTCN2018089955-appb-000008
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。
以ADC-12制剂的缓冲体系、缓冲剂浓度、pH值、糖种类以及糖浓度进行实验设计，利用DSC技术测定样品的Tm值，初步筛选制剂的处方。
以缓冲体系、缓冲剂浓度、pH值、糖种类以及糖浓度为因子，以Tm值为响应值设计试验，生成设计表，根据设计表实验组进行实验，测定Tm值。
实施例中冷冻干燥使用东富龙LYO-3(SIP、CIP)真空冷冻干燥机。SE-HPLC的检测使用安捷伦1200DAD高压液相色谱仪(Waters Xbridge Protein BEH SEC
Figure PCTCN2018089955-appb-000009
色谱柱)。CE-SDS的检测使用贝克曼PA800plus毛细管电泳仪(SDS-Gel MW Analysis Kit)。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2
在下列缓冲液中，配置浓度为1mg/ml的ADC-12制剂：
1)20mM醋酸(醋酸钠)，pH5.0
2)20mM醋酸(醋酸钠)，pH5.5
3)20mM琥珀酸(琥珀酸钠)，pH5.5
4)20mM琥珀酸(琥珀酸钠)，pH6.0
5)20mM柠檬酸(柠檬酸钠)，pH5.0
6)20mM柠檬酸(柠檬酸钠)，pH5.5
7)20mM柠檬酸(柠檬酸钠)，pH6.0
8)20mM组氨酸(盐酸)，pH5.5
9)20mM组氨酸(盐酸)，pH6.0
10)20mM磷酸氢二钠(磷酸二氢钠)，pH6.0
用差示扫描量热法(differential scanning calorimetry,DSC)测量ADC-12在每种制剂中的热稳定性(检测结果见表1)。
表1.ADC-12缓冲体系-pH值DSC筛选结果
Figure PCTCN2018089955-appb-000010
注：N/A表示未添加该成分。
结果显示，组氨酸(盐酸)缓冲体系明显低于其余几组。醋酸盐缓冲体系在冻干时可能由于挥发会导致pH值偏移。磷酸盐缓冲剂的缓冲范围(pH6.0～8.0)与ADC-12的等电点范围过于重叠，不宜采用。因此选择了Tm onset和Tm均相对较高的琥珀酸盐、柠 檬酸盐两种缓冲体系。
以pH 5.5的10mM琥珀酸(琥珀酸钠)或柠檬酸(柠檬酸钠)为缓冲液配制ADC-12制剂，含60mg/ml蔗糖，0.2mg/ml聚山梨醇酯20，ADC-12终浓度为20mg/ml。以4mL/瓶填充入15mL西林瓶中冻干，并用冻干胶塞封口。将冻干品放置于25℃考察。结果显示，琥珀酸(琥珀酸钠)体系略优于柠檬酸(柠檬酸钠)体系。
表2.ADC-12不同缓冲体系25℃稳定性结果
Figure PCTCN2018089955-appb-000011
注：M0表示第0个月，D15表示第15天，M1表示第1个月，M3表示第3个月，M6表示第6个月。
实施例3
以pH 4.8–5.8的含10mM琥珀酸-琥珀酸钠为缓冲液配制ADC-12制剂，含60mg/mlα,α-二水合海藻糖，0.2mg/ml聚山梨醇酯20的制剂，ADC-12终浓度为20mg/ml。过滤每种制剂并以4mL/瓶填充入15mL中性硼硅玻璃管制注射剂瓶中进行冻干，所述玻璃瓶用胶塞封口。将冻干品保存于25℃进行稳定性分析。不同pH下25℃,0-6个月ADC-12的稳定性结果如表3所示，结果显示ADC-12在pH 5.0–5.5均非常稳定。
表3.ADC-12不同pH 25℃稳定性结果
Figure PCTCN2018089955-appb-000012
Figure PCTCN2018089955-appb-000013
注：M0表示第0个月，M3表示第3个月，M6表示第6个月。
实施例4
以pH 5.5的60mg/ml的α,α-二水合海藻糖或蔗糖为缓冲液配制ADC-12制剂，ADC-12终浓度为20mg/ml，含10mM琥珀酸-琥珀酸钠，0.2mg/ml聚山梨醇酯20。将每种制剂过滤并以4mL/瓶填充入15mL西林瓶中，冻干并用冻干胶塞封口。将冻干品保存于25℃和2-8℃进行稳定性分析。结果显示ADC-12在海藻糖体系中更为稳定。
表4.ADC-12冻干成品糖筛选25℃稳定性结果
Figure PCTCN2018089955-appb-000014
注：M0表示第0个月，M3表示第3个月，M6表示第6个月。
表5.ADC-12冻干成品糖筛选2～8℃稳定性结果
Figure PCTCN2018089955-appb-000015
注：M0表示第0个月，M3表示第3个月，M6表示第6个月。
实施例5
以含下列不同浓度表面活性剂的pH5.5的缓冲液配制ADC-12制剂，制备ADC-12终浓度为20mg/ml，含10mM琥珀酸-琥珀酸钠，60mg/mlα,α-二水合海藻糖，的ADC-12制剂：
1)不含表面活性剂
2)0.05mg/ml聚山梨醇酯20
3)0.1mg/ml聚山梨醇酯20
4)0.2mg/ml聚山梨醇酯20
5)0.4mg/ml聚山梨醇酯20
样品制备完成后将样品放置在-35℃冰箱冷冻保存12h，然后转移至2-8℃12h，此为一个冻融循环。一共重复5个循环。稳定性结果表明，0.05-0.4mg/ml聚山梨醇酯20，有效防止了ADC-12在冻融过程中的聚集。
表6
Figure PCTCN2018089955-appb-000016
Figure PCTCN2018089955-appb-000017
实施例6
以pH 5.3的10mM琥珀酸(钠)为缓冲液配制ADC-12制剂，含60mg/mlα,α-二水合海藻糖，0.2mg/ml聚山梨醇酯20，ADC-12终浓度为20mg/ml。将抗体以4mL/瓶填充入15mL西林瓶中，分别以-27℃、-20℃和-15℃的一次干燥温度进行冻干，并用冻干胶塞封口后进行检测。结果表明，-20℃为冻干工艺最佳的一次干燥温度。
表7.用不同一次干燥工艺制备的ADC-12制剂检测
Figure PCTCN2018089955-appb-000018
Figure PCTCN2018089955-appb-000019
实施例7
以pH 5.3的10mM琥珀酸(钠)为缓冲液配制ADC-12制剂，含60mg/mlα,α-二水合海藻糖，0.2mg/ml聚山梨醇酯20，ADC-12终浓度为20mg/ml。将制剂分别填充在玻璃瓶、储液袋和316L不锈钢罐中，2-8℃放置24小时。蛋白含量和纯度分析表明(见表8)，ADC-12在24小时内是稳定的。该制剂与316L不锈钢罐、玻璃瓶和储液袋都是可以相容的。以pH 5.3的10mM琥珀酸(钠)为缓冲液配制ADC-12制剂，含60mg/mlα,α-二水合海藻糖，0.2mg/ml聚山梨醇酯20，ADC-12终浓度为10mg/ml，1mg/ml，也具有良好的稳定性。
表8.在不同接触材料中ADC-12的稳定性
Figure PCTCN2018089955-appb-000020
注：N/A表示未检测。
实施例8其它可选择制剂配方
本发明提供的稳定的药物制剂包含：ADC-12和任选自以下的稳定缓冲液的组合：
(i)60mg/mlα,α-二水合海藻糖，和pH5.3的10mM琥珀酸盐缓冲液；
(ii)60mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨醇酯20，和pH5.3的10mM琥珀酸盐缓冲液；
(iii)50mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨醇酯20，和pH5.2的20mM琥珀酸盐缓冲液；
(iv)60mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨醇酯20，和pH5.0的20mM琥珀酸盐缓冲液；
(v)70mg/mlα,α-二水合海藻糖，0.1mg/ml的聚山梨醇酯20，和pH5.2的20mM琥珀酸盐缓冲液；
(vi)60mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨醇酯20，和pH5.2的10mM琥珀酸盐缓冲液；
(vii)60mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨醇酯20，和pH5.0的10mM琥珀酸盐缓冲液；
(viii)60mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨醇酯20，和pH5.2的30mM柠檬酸盐缓冲液；或者
(ix)60mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨醇酯20，和pH5.5的10mM柠檬酸盐缓冲液。
以上实施例中，ADC-12的浓度在1mg/ml至30mg/ml的范围内，优选10-20mg/ml，最优选10mg/ml。可实施的方案可选自，但不限于以下组合：
(1)抗ADC-12 30mg/ml，60mg/mlα,α-二水合海藻糖，0.05mg/ml的聚山梨醇酯20，和pH5.2的10mM琥珀酸盐缓冲液；
(2)抗ADC-12 1mg/ml，50mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨醇酯20，和pH5.0的10mM琥珀酸盐缓冲液；
(3)抗ADC-12 10mg/ml，60mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨醇酯20，和pH5.1的10mM琥珀酸盐缓冲液；
(4)抗ADC-12 15mg/ml，50mg/mlα,α-二水合海藻糖，0.3mg/ml的聚山梨醇酯20，和pH5.4的20mM琥珀酸盐缓冲液；
(5)抗ADC-12 5mg/ml，70mg/mlα,α-二水合海藻糖，0.1mg/ml的聚山梨醇酯20，和pH5.3的20mM琥珀酸盐缓冲液；
(6)抗ADC-12 10mg/ml，60mg/mlα,α-二水合海藻糖，0.2mg/ml的聚山梨醇酯20，和pH5.2的15mM琥珀酸盐缓冲液；
(7)抗ADC-12 30mg/ml，40mg/ml蔗糖，0.05mg/ml的聚山梨醇酯20，和pH5.3 的30mM柠檬酸盐缓冲液；
(8)抗ADC-12 20mg/ml，60mg/ml乳糖，0.1mg/ml的聚山梨醇酯20，和pH5.4的20mM柠檬酸盐缓冲液；
(9)抗ADC-12 10mg/ml，70mg/mlα,α-二水合海藻糖，0.4mg/ml的聚山梨醇酯80，和pH5.2的10mM柠檬酸盐缓冲液；
(10)抗ADC-12 1mg/ml，80mg/ml麦芽糖，0.2mg/ml的聚氧乙烯氢化蓖麻油，和pH5.2的10mM柠檬酸盐缓冲液。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (18)

  1. 一种药物组合物，其包含c-Met抗体药物偶联物，以及缓冲剂，所述缓冲剂优选为琥珀酸盐或柠檬酸盐缓冲剂，更优选为琥珀酸盐缓冲剂。
  2. 如权利要求1所述的药物组合物，其中所述c-Met抗体药物偶联物浓度为大约1mg/ml至30mg/ml，优选为大约1mg/ml至20mg/ml，进一步优选为5mg/ml至20mg/ml，最优选为10-20mg/ml。
  3. 如权利要求1或2所述的药物组合物，其pH约为5.0至6.0，优选为大约5.0至5.5，最优选为5.3。
  4. 如权利要求1至3任一项所述的药物组合物，其中所述缓冲剂浓度为大约5mM至30mM，优选为5mM至20mM，进一步优选为大约10mM至20mM，更优选为大约10mM至15mM，最优选为10mM。
  5. 如权利要求1至4任一项所述的药物组合物，其中还包括二糖，所述二糖优选自海藻糖或蔗糖，最优选为海藻糖。
  6. 如权利要求5所述的药物组合物，其中所述糖浓度为大约40mg/ml至80mg/ml，优选为大约50mg/ml至70mg/ml，更优选为55mg/ml至约65mg/ml，最优为60mg/ml。
  7. 如权利要求1至6任一项所述的药物组合物，其中还包括表面活性剂，所述表面活性剂优选为聚山梨醇酯，更优选为聚山梨醇酯20。
  8. 如权利要求7所述的药物组合物，其中表面活性剂的浓度从大约为0.05mg/ml至1.0mg/ml，优选为0.05mg/ml至0.4mg/ml，更优选为0.1mg/ml至0.2mg/ml，最优选为0.2mg/ml。
  9. 如权利要求1至8任一项所述的药物组合物，其包含：
    (a)1-20mg/ml的c-Met抗体药物偶联物；
    (b)10-20mM的琥珀酸盐缓冲剂，pH5.0-5.5；
    (c)40-80mg/ml的α,α-二水合海藻糖；
    (d)0.05-0.4mg/ml的聚山梨醇酯20；
    优选地，所述药物组合物包含：(a)1-20mg/ml的c-Met抗体药物偶联物；(b)10-20mM的琥珀酸盐缓冲剂，pH5.0-5.5；(c)60mg/ml的α,α-二水合海藻糖；(d)0.05-0.4mg/ml的聚山梨醇酯20；或，(a)5-20mg/ml的c-Met抗体药物偶联物；(b)10-20mM的琥珀酸盐缓冲剂，pH5.0-5.5；(c)50-70mg/ml的α,α-二水合海藻糖；(d)0.1-0.2mg/ml的聚山梨醇酯20。
  10. 如权利要求1至9任一项所述的药物组合物，其中c-Met抗体药物偶联物中的 c-Met抗体的重链与Ab-10的抗体重链氨基酸序列具有95％以上的序列同一性，所述c-Met抗体的轻链氨基酸序列与Ab-10的抗体轻链具有95％以上的序列同一性；
    所述Ab-10的抗体重链氨基酸序列为：
    Figure PCTCN2018089955-appb-100001
    所述Ab-10的抗体轻链氨基酸序列为：
    Figure PCTCN2018089955-appb-100002
  11. 如权利要求1至10任一项所述的药物组合物，其中c-Met抗体药物偶联物为ADC-12，具有如下所示的结构：
    Figure PCTCN2018089955-appb-100003
    其中y的范围为1-8，优选2-5。
  12. 制备含c-Met抗体药物偶联物的冷冻干燥制剂的方法，其中包括将如权利要求1至11任一项所述的药物组合物经冷冻干燥的步骤。
  13. 如权利要求12所述制备含c-Met抗体药物偶联物的冷冻干燥制剂的方法，其中所述冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。
  14. 一种由如权利要求12或13所述的方法制备的含c-Met抗体药物偶联物的冷冻干燥制剂。
  15. 一种冷冻干燥制剂，其中所述冷冻干燥制剂经复溶后可形成如权利要求1至11任一项所述的药物组合物，优选所述复溶使用注射用水。
  16. 如权利要求1-11任一项所述的药物组合物或权利要求14或15所述的冷冻干燥制剂在制备用于治疗c-Met相关的疾病或病症的药物中的用途，其中所述的疾病或病症优选为癌症；更优选为表达c-Met的癌症；更优选为表达c-Met的胃癌、胰腺癌、肺癌(例如非小细胞肺癌)、成胶质细胞瘤、肉瘤、结肠直肠癌、肾癌、肝细胞癌、黑素瘤和乳腺癌；最优选为胃癌、胰腺癌、非小细胞肺癌和肾癌。
  17. 一种治疗和预防c-Met相关的疾病或病症的方法，包括给予所需患者治疗有效量的如权利要求1-11任一项所述的药物组合物或权利要求14或15所述的冷冻干燥制剂；其中所述的疾病优选为癌症；更优选为表达c-Met的癌症；更优选为表达c-Met的胃癌、胰腺癌、肺癌(例如非小细胞肺癌)、成胶质细胞瘤、肉瘤、结肠直肠癌、肾癌、肝细胞癌、黑素瘤和乳腺癌；最优选为胃癌、胰腺癌、非小细胞肺癌和肾癌。
  18. 一种制品，其包括容器，该容器中装有权利要求1-11任一项所述的药物组合物或权利要求14或15所述的冷冻干燥制剂。
PCT/CN2018/089955 2017-06-06 2018-06-05 一种含c-Met抗体药物偶联物的药物组合物及其用途 WO2018223958A1 (zh)

Priority Applications (7)

Application Number Priority Date Filing Date Title
CA3064470A CA3064470A1 (en) 2017-06-06 2018-06-05 Pharmaceutical composition comprising c-met antibody-drug conjugate and use thereof
KR1020197038495A KR20200012937A (ko) 2017-06-06 2018-06-05 c-Met 항체 약물 접합체를 포함하는 약학 조성물 및 그의 용도
CN201880011419.3A CN110382008A (zh) 2017-06-06 2018-06-05 一种含c-Met抗体药物偶联物的药物组合物及其用途
AU2018280485A AU2018280485A1 (en) 2017-06-06 2018-06-05 Pharmaceutical composition comprising c-Met antibody-drug conjugate and use thereof
MX2019014666A MX2019014666A (es) 2017-06-06 2018-06-05 Composicion farmaceutica que comprende un conjugado de anticuerpo contra c-met-farmaco y uso del mismo.
US16/618,974 US20200129633A1 (en) 2017-06-06 2018-06-05 Pharmaceutical composition comprising c-met antibody-drug conjugate and use thereof
BR112019025591-5A BR112019025591A2 (pt) 2017-06-06 2018-06-05 Composição farmacêutica compreendendo conjugado de fármaco-anticorpo c-met e uso do mesmo

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201710417725 2017-06-06
CN201710417725.4 2017-06-06
CN201710551046.6 2017-07-07
CN201710551046 2017-07-07

Publications (1)

Publication Number Publication Date
WO2018223958A1 true WO2018223958A1 (zh) 2018-12-13

Family

ID=64566475

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/089955 WO2018223958A1 (zh) 2017-06-06 2018-06-05 一种含c-Met抗体药物偶联物的药物组合物及其用途

Country Status (9)

Country Link
US (1) US20200129633A1 (zh)
KR (1) KR20200012937A (zh)
CN (1) CN110382008A (zh)
AU (1) AU2018280485A1 (zh)
BR (1) BR112019025591A2 (zh)
CA (1) CA3064470A1 (zh)
MX (1) MX2019014666A (zh)
TW (1) TW201902518A (zh)
WO (1) WO2018223958A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11142578B2 (en) 2016-11-16 2021-10-12 Regeneron Pharmaceuticals, Inc. Anti-MET antibodies, bispecific antigen binding molecules that bind MET, and methods of use thereof
US11896682B2 (en) 2019-09-16 2024-02-13 Regeneron Pharmaceuticals, Inc. Radiolabeled MET binding proteins for immuno-PET imaging and methods of use thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW202304929A (zh) * 2021-04-29 2023-02-01 美商艾伯維有限公司 抗c-Met抗體藥物結合物
WO2023185907A1 (en) * 2022-03-29 2023-10-05 Genequantum Healthcare (Suzhou) Co., Ltd. A freeze-drying process for an ADC

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013072813A2 (en) 2011-11-17 2013-05-23 Pfizer Inc. Cytotoxic peptides and antibody drug conjugates thereof
CN103781493A (zh) 2011-06-30 2014-05-07 霍夫曼-拉罗奇有限公司 抗c-met抗体配制剂
CN105050618A (zh) 2012-06-21 2015-11-11 索伦托治疗有限公司 与c-Met结合的抗原结合蛋白
CN105163763A (zh) * 2013-03-15 2015-12-16 阿布维德国有限责任两合公司 抗egfr抗体药物偶联物制剂
WO2016042412A1 (en) 2014-09-16 2016-03-24 Symphogen A/S Anti-met antibodies and compositions
WO2016165580A1 (zh) 2015-04-17 2016-10-20 江苏恒瑞医药股份有限公司 抗c-Met抗体和抗c-Met抗体-细胞毒性药物偶联物及其医药用途
CN106188293A (zh) * 2015-04-17 2016-12-07 江苏恒瑞医药股份有限公司 抗c-Met抗体和抗c-Met抗体-细胞毒性药物偶联物及其医药用途
CN106456794A (zh) * 2014-05-22 2017-02-22 斯索恩生物制药有限公司 接头药物与抗体的位点特异性缀合以及所得adc

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2854477A1 (en) * 2011-11-21 2013-05-30 Genentech, Inc. Purification of anti-c-met antibodies

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103781493A (zh) 2011-06-30 2014-05-07 霍夫曼-拉罗奇有限公司 抗c-met抗体配制剂
WO2013072813A2 (en) 2011-11-17 2013-05-23 Pfizer Inc. Cytotoxic peptides and antibody drug conjugates thereof
CN105050618A (zh) 2012-06-21 2015-11-11 索伦托治疗有限公司 与c-Met结合的抗原结合蛋白
CN105163763A (zh) * 2013-03-15 2015-12-16 阿布维德国有限责任两合公司 抗egfr抗体药物偶联物制剂
CN106456794A (zh) * 2014-05-22 2017-02-22 斯索恩生物制药有限公司 接头药物与抗体的位点特异性缀合以及所得adc
WO2016042412A1 (en) 2014-09-16 2016-03-24 Symphogen A/S Anti-met antibodies and compositions
WO2016165580A1 (zh) 2015-04-17 2016-10-20 江苏恒瑞医药股份有限公司 抗c-Met抗体和抗c-Met抗体-细胞毒性药物偶联物及其医药用途
CN106188293A (zh) * 2015-04-17 2016-12-07 江苏恒瑞医药股份有限公司 抗c-Met抗体和抗c-Met抗体-细胞毒性药物偶联物及其医药用途

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1977, pages 3389 - 3402
J. BIOL. CHEM., vol. 243, 1968, pages 3558
KABAT, E A ET AL.: "Sequences of Proteins of Immunological Interest", 1991
WATSON ET AL.: "Molecular Biology of the Gene", 1987, THE BENJARNIN/CUMMINGS PUB. CO., pages: 224

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11142578B2 (en) 2016-11-16 2021-10-12 Regeneron Pharmaceuticals, Inc. Anti-MET antibodies, bispecific antigen binding molecules that bind MET, and methods of use thereof
US11896682B2 (en) 2019-09-16 2024-02-13 Regeneron Pharmaceuticals, Inc. Radiolabeled MET binding proteins for immuno-PET imaging and methods of use thereof

Also Published As

Publication number Publication date
BR112019025591A2 (pt) 2020-06-16
CA3064470A1 (en) 2018-12-13
KR20200012937A (ko) 2020-02-05
MX2019014666A (es) 2020-02-07
CN110382008A (zh) 2019-10-25
TW201902518A (zh) 2019-01-16
US20200129633A1 (en) 2020-04-30
AU2018280485A1 (en) 2020-01-30

Similar Documents

Publication Publication Date Title
JP7436477B2 (ja) ＴＧＦ－β受容体融合タンパク質医薬組成物およびその使用
US11654194B2 (en) PD-L1 antibody pharmaceutical composition and use thereof
WO2018223958A1 (zh) 一种含c-Met抗体药物偶联物的药物组合物及其用途
EP3714901A1 (en) Lag-3 antibody pharmaceutical composition and use thereof
US11498961B2 (en) SOST antibody pharmaceutical composition and uses thereof
CN110960490A (zh) 一种抗egfr抗体偶联药物组合物及其用途
US20230295329A1 (en) Anti-pd-1 antibody pharmaceutical composition and use thereof
CN112839961B (zh) 一种cd40抗体药物组合物及其用途
US20210261663A1 (en) Anti-tim3 antibody pharmaceutical composition and use thereof
EP4257603A1 (en) Pharmaceutical composition comprising anti-connective tissue growth factor antibody
WO2019001560A1 (zh) 一种pcsk-9抗体药物组合物及其用途
CN116688115B (zh) 一种PD-L1/TGF-β双功能融合蛋白制剂及其用途
CN116172947A (zh) 一种含特异性结合vegf和ang2的双特异性抗体的药物组合物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18812741

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3064470

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2019568028

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112019025591

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 20197038495

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2019141616

Country of ref document: RU

ENP Entry into the national phase

Ref document number: 2018812741

Country of ref document: EP

Effective date: 20200107

ENP Entry into the national phase

Ref document number: 2018280485

Country of ref document: AU

Date of ref document: 20180605

Kind code of ref document: A

122 Ep: pct application non-entry in european phase

Ref document number: 18812741

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 112019025591

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20191203

NENP Non-entry into the national phase

Ref country code: JP