WO2018210230A1 - 一种pd-l1抗体药物组合物及其用途 - Google Patents

一种pd-l1抗体药物组合物及其用途 Download PDF

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WO2018210230A1
WO2018210230A1 PCT/CN2018/086866 CN2018086866W WO2018210230A1 WO 2018210230 A1 WO2018210230 A1 WO 2018210230A1 CN 2018086866 W CN2018086866 W CN 2018086866W WO 2018210230 A1 WO2018210230 A1 WO 2018210230A1
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seq
antibody
pharmaceutical composition
antigen
cancer
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PCT/CN2018/086866
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English (en)
French (fr)
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WO2018210230A8 (zh
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颜贞
杨健健
颜晓丹
吴珊
刘洵
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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 BR112019023846A priority Critical patent/BR112019023846A2/pt
Priority to US16/614,148 priority patent/US11654194B2/en
Priority to MX2019013751A priority patent/MX2019013751A/es
Priority to CA3062487A priority patent/CA3062487A1/en
Priority to MYPI2019006627A priority patent/MY195465A/en
Priority to EP18801322.1A priority patent/EP3626266A4/en
Priority to KR1020197036735A priority patent/KR102623679B1/ko
Priority to AU2018267843A priority patent/AU2018267843A1/en
Priority to RU2019139093A priority patent/RU2766590C2/ru
Priority to CN201880007923.6A priority patent/CN110198739B/zh
Priority to JP2019563895A priority patent/JP7263256B6/ja
Publication of WO2018210230A1 publication Critical patent/WO2018210230A1/zh
Publication of WO2018210230A8 publication Critical patent/WO2018210230A8/zh

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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
    • 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
    • A61K47/06—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
    • A61K47/26—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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention belongs to the field of pharmaceutical preparations, and in particular relates to a pharmaceutical composition comprising a PD-L1 antibody and an antigen-binding fragment thereof, and the use thereof as an anticancer drug.
  • PD-1 Programmed death-1
  • PD-1 has two ligands, PD-L1 and PD-L2.
  • PD-L1 is mainly expressed on T cells, B cells, macrophages, and dendritic cells (DCs), and expression on cells after activation can be up-regulated.
  • the expression of PD-L2 is relatively limited, mainly expressed on antigen presenting cells, such as activated macrophages and dendritic cells.
  • PD-L1 inhibits the immune system by binding to PD-1 and B7-1, and many tumor cells and tumor tissue microenvironmental immune cells express PD-L1.
  • New research found high PD-L1 protein in human tumor tissues such as breast, lung, stomach, bowel, kidney, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer and liver cancer Expression, and the expression level of PD-L1 is closely related to the clinical and prognosis of patients. Since PD-L1 acts as a second signaling pathway to inhibit T cell proliferation, blocking the binding between PD-L1/PD-1 has become a very promising emerging target in the field of tumor immunotherapy.
  • antibody drugs Compared with other chemical drugs, antibody drugs become unstable due to their larger molecular weight, more complicated structure, and easy degradation, polymerization, or undesired chemical modification.
  • formulation studies of antibody drugs are particularly important.
  • compositions containing PD-L1/PD-1 antibodies such as CN105793288A, CN103429264A, and CN105960415A.
  • the present invention provides a pharmaceutical composition comprising a PD-L1 antibody and antigen-binding fragment thereof that is sufficiently stable and suitable for administration.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a PD-L1 antibody and an antigen-binding fragment, and a buffering agent, preferably a succinate or acetate buffer, more preferably a succinate buffer.
  • the concentration of the buffer is from about 5 mM to 50 mM, preferably from about 10 mM to 30 mM, more preferably from 10 mM to 20 mM, non-limiting examples include 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM.
  • the pharmaceutical composition has a pH of from about 4.5 to 6.0, preferably from about 4.8 to 5.7, more preferably from 5.0 to 5.5, and may be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5.
  • the concentration of the antibody in the pharmaceutical composition is from about 30 mg/ml to about 80 mg/ml, preferably from about 40 mg/ml to about 60 mg/ml, more preferably from 45 mg/ml to about 55 mg/ml, Non-limiting examples include 45 mg/ml, 46 mg/ml, 47 mg/ml, 48 mg/ml, 49 mg/ml, 50 mg/ml, 51 mg/ml, 52 mg/ml, 53 mg/ml, 54 mg/ml, 55 mg/ml.
  • the pharmaceutical composition of the present invention further comprises a sugar.
  • the "sugar” of the present invention comprises a conventional composition (CH2O) n and derivatives thereof, 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.
  • the concentration of the sugar in the pharmaceutical composition is from about 30 mg/ml to about 90 mg/ml, preferably from 50 mg/ml to about 70 mg/ml, more preferably from 55 mg/ml to about 65 mg/ml, Restrictive 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 of the present invention further comprises a surfactant.
  • a surfactant May be selected from polysorbate 20, polysorbate 80, polyhydroxyl hydrocarbon, Triton, sodium dodecyl sulfate, sodium lauryl sulfonate, sodium octyl sulphate, lauryl-, myristyl-, sub- Oil-based, stearyl-sulfobetaine, lauryl-, myristyl-, linoleyl-, stearyl-sarcosine, linoleyl-, myristyl-, cetyl-beet Base, lauramide-propyl-, cocamidopropyl-, linoleidopropyl-, myristylpropyl-, palmitoylpropyl-, isostearamidopropyl-betaine , myristylaminopropyl-, palmitoylpropyl-, isosteara
  • the concentration of the surfactant in the pharmaceutical composition is from about 0.1 mg/ml to 1.0 mg/ml, preferably from 0.2 mg/ml to 0.8 mg/ml, more preferably from 0.4 mg/ml to 0.8.
  • the mg/ml non-limiting examples include 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, and 0.8 mg/ml.
  • the antibody or antigen-binding fragment of the pharmaceutical composition comprises any one of the CDR region sequences selected from the following or a mutant sequence thereof: antibody heavy chain variable region HCDR region sequence: SEQ ID NO: 1. -3, SEQ ID NOs: 7-9; and/or, antibody light chain variable region LCDR region sequences: SEQ ID NOs: 4-6, SEQ ID NOs: 10-12;
  • HCDR1 is selected from the group consisting of: NDYWX 1 SEQ ID NO: 1
  • HCDR2 is selected from: YISYTGSTYYNPSLKS SEQ ID NO: 2
  • HCDR3 is selected from: SGGWLAPFDY SEQ ID NO: 3
  • LCDR1 is selected from: KSSQSLFY X 2 SNQK X 3 SLA SEQ ID NO: 4
  • LCDR2 is selected from: GASTRES SEQ ID NO: 5
  • LCDR3 is selected from: QQYYGYPYT SEQ ID NO: 6
  • X 1 is selected from N or T
  • X 2 is selected from R or H
  • X 3 is selected from N or H
  • X 4 is selected from H or G
  • X 5 is selected from G or F.
  • the antibody or antigen-binding fragment thereof described in the pharmaceutical composition comprises a light chain variable selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: a region CDR sequence or a mutated sequence thereof, and a heavy chain variable region CDR sequence selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 or a mutant sequence thereof; more preferably, the antibody Or an antigen-binding fragment thereof comprising the LCDR1, LCDR2 and LCDR3 sequences as set forth in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and SEQ ID NO: 7, SEQ ID NO: 8 and HCDR1, HCDR2 and HCDR3 sequences set forth in SEQ ID NO:9;
  • the antibody or antigen-binding fragment thereof described in the pharmaceutical composition comprises a heavy chain variable region CDR sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a mutation thereof a sequence, and a light chain variable region CDR sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or a mutant sequence thereof; more preferably, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively The LCDR1, LCDR2 and LCDR3 sequences are shown.
  • the antibody or antigen-binding fragment thereof of the pharmaceutical composition comprises at least 85%, 86% of the amino acid sequence: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of light chain variable region CDR sequences
  • amino acid sequence: SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity heavy chain variable region CDR sequences.
  • the antibody or antigen-binding fragment thereof in the pharmaceutical composition may be selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, preferably a humanized antibody.
  • the antibody or antigen-binding fragment thereof of the pharmaceutical composition comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91 of the amino acid sequence of SEQ ID NO: %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of the heavy chain variable region sequence, and at least 85% with the amino acid sequence SEQ ID NO: , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence-consistent light chain Variable sequence.
  • the antibody or antigen-binding fragment thereof of the pharmaceutical composition comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91 of the amino acid sequence of SEQ ID NO: %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of the heavy chain variable region sequence, and at least 85% with the amino acid sequence SEQ ID NO: 17. , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence-consistent light chain sequence .
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a PD-L1 antibody or antigen-binding fragment thereof at a concentration of from about 30 mg/ml to about 80 mg/ml, at a concentration of from about 5 mM to about 50 mM succinate buffer. , pH 5.0-6.0, a disaccharide having a concentration of from about 30 mg/ml to about 90 mg/ml, and a concentration of from about 0.1 mg/ml to about 1.0 mg/ml of polysorbate 80.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 40-60 mg/ml of PD-L1 antibody or antigen-binding fragment, 10-30 mM succinate buffer, pH 5.0-5.5, 40-80 mg/ Mol of sucrose, 0.4-0.8 mg/ml of polysorbate 80.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 45-55 mg/ml of PD-L1 antibody or antigen-binding fragment thereof, 10-20 mM succinate buffer, pH 5.0-5.5, 55-65 mg /ml of sucrose, 0.5-0.7 mg/ml of polysorbate 80.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a PD-L1 antibody or antigen-binding fragment thereof at a concentration of from about 30 mg/ml to about 80 mg/ml, at a concentration of from about 5 mM to about 50 mM acetate buffer. , a pH 5.0-6.0, a disaccharide having a concentration of from about 30 mg/ml to about 90 mg/ml, and a concentration of from about 0.1 mg/ml to about 1.0 mg/ml of polysorbate 80.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of a PD-L1 antibody or antigen-binding fragment, 10 mM of a succinate buffer, pH 5.3, 60 mg/ml of sucrose.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of a PD-L1 antibody or antigen-binding fragment, 10 mM acetate buffer, pH 5, 90 mg/ml of sucrose.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of a PD-L1 antibody or antigen-binding fragment, 30 mM acetate buffer, pH 5, 60 mg/ml of trehalose.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of a PD-L1 antibody or antigen-binding fragment, 30 mM acetate buffer, pH 5, 60 mg/ml of trehalose.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of a PD-L1 antibody or antigen-binding fragment, 30 mM acetate buffer, pH 5.6, 90 mg/ml of sucrose.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of PD-L1 antibody or antigen-binding fragment, 10 mM succinate buffer system, pH 5.0-5.5, 60 mg/ml sucrose, 0.2 mg/ml Polysorbate 20.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of PD-L1 antibody or antigen-binding fragment, 10-20 mM succinate buffer system, pH 5.2, 60 mg/ml sucrose, 0.2 mg/ml Polysorbate 20.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of PD-L1 antibody or antigen-binding fragment, 20 mM acetate buffer, pH 5.2, 60 mg/ml sucrose, 0.1-0.3 mg /ml of polysorbate 20.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of PD-L1 antibody or antigen-binding fragment, 20 mM acetate buffer, pH 5.2, 60 mg/ml sucrose, 0.1-0.3 mg /ml of polysorbate 80.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer, pH 5.2, 60 mg/ml sucrose, 0.2-0.6 Mg/ml of polysorbate 20.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer, pH 5.2, 60 mg/ml sucrose, 0.4-0.8 Mg/ml of polysorbate 80.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer, pH 5.2, 60 mg/ml sucrose, 0.4 mg/ Ml of polysorbate 80.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer, pH 5.2, 60 mg/ml sucrose, 0.6 mg/ Ml of polysorbate 80.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising 50 mg/ml of PD-L1 antibody or antigen-binding fragment thereof, 20 mM succinate buffer, pH 5.2, 60 mg/ml sucrose, 0.8 mg/ Ml of polysorbate 80.
  • the concentration of the succinate buffer in the pharmaceutical composition is from about 5 mM to 50 mM. In some embodiments, the concentration of the succinate buffer in the pharmaceutical composition is from about 10 mM to 30 mM. In some embodiments, the concentration of the succinate buffer in the pharmaceutical composition is about 20 mM.
  • the concentration of the acetate buffer in the pharmaceutical composition is from about 5 mM to 50 mM. In some embodiments, the concentration of the acetate buffer in the pharmaceutical composition is from about 10 mM to 30 mM. In some embodiments, the concentration of the acetate buffer in the pharmaceutical composition is about 20 mM.
  • the pharmaceutical composition has a pH of from about 5.0 to 6.0. In some embodiments, the pharmaceutical composition has a pH of from about 5.0 to about 5.5. In some embodiments, the pharmaceutical composition has a pH of about 5.2 or 5.5.
  • the concentration of the antibody in the pharmaceutical composition is from about 30 mg/ml to about 80 mg/ml. In some embodiments, the concentration of the antibody in the pharmaceutical composition is from about 40 mg/ml to about 60 mg/ml. In some embodiments, the concentration of the antibody in the pharmaceutical composition is about 50 mg/ml.
  • the pharmaceutical composition comprises the disaccharide at a concentration of from about 30 mg/ml to about 90 mg/ml. In some embodiments, the concentration of the disaccharide in the pharmaceutical composition is from about 40 mg/ml to about 80 mg/ml. In some embodiments, the concentration of the disaccharide in the pharmaceutical composition is about 60 mg/ml.
  • the polysorbate in the pharmaceutical composition is polysorbate 20 or polysorbate 80. In some embodiments, the polysorbate in the pharmaceutical composition is polysorbate 80. In some embodiments, the concentration of the polysorbate in the pharmaceutical composition is from about 0.1 mg/ml to 1.0 mg/ml. In some embodiments, the concentration of the polysorbate in the pharmaceutical composition is from about 0.4 mg/ml to 0.8 mg/ml. In some embodiments, the concentration of the polysorbate in the pharmaceutical composition is about 0.6 mg/ml.
  • 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.
  • the invention further provides an article or kit comprising a container containing any of the stable pharmaceutical compositions described herein.
  • the container is a glass bottle that is a neutral borosilicate glass controlled injection bottle.
  • the invention further provides a method of preparing the pharmaceutical composition of any of the above, comprising mixing a PD-L1 antibody or antigen-binding fragment thereof with a pharmaceutically acceptable excipient.
  • the invention further provides the use of a pharmaceutical composition according to any of the above, in the manufacture of a medicament for the treatment of a PD-L1-mediated disease or condition, wherein the disease or condition is preferably cancer; more preferably expression of PD- The cancer of L1; most preferably breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer; further preferably non-small cell lung cancer, melanoma, bladder cancer and kidney cancer.
  • the disease or condition is preferably cancer; more preferably expression of PD- The cancer of L1; most preferably breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer; further preferably non-small cell lung cancer, melanoma, bladder cancer and kidney cancer.
  • the invention further provides a method of treating and preventing a PD-L1 mediated disease or condition, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a PD-L1 antibody or antigen-binding fragment thereof;
  • the disease is preferably cancer; more preferably a cancer expressing PD-L1; the cancer is most preferably breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, bladder cancer; most preferably Non-small cell lung cancer, melanoma, bladder cancer and kidney cancer.
  • Figure 1 Schematic diagram of primer design during humanized cloning.
  • Figure 2 Schematic diagram of vector construction of humanized clone construction.
  • FIG. 3 Main effect map of Tm factors (including buffer system, buffer concentration, pH of the pharmaceutical composition, sugar concentration, and type of sugar).
  • 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 5.2.
  • Examples of the buffer which controls the pH in this range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, Glycyl glycine and other organic acid buffers.
  • a preferred buffering agent of the invention is a succinate buffer or acetate buffer, more preferably a succinate buffer.
  • succinate buffer is a buffer that includes succinic acid 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.
  • Acetate buffer is a buffer that includes acetate ions.
  • examples of the acetate buffer include acetic acid-sodium acetate, acetic acid histidine, acetic acid-potassium acetate, calcium acetate acetate, acetic acid-magnesium acetate, and the like.
  • a preferred acetate buffer of the invention is acetic acid-sodium acetate.
  • “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.
  • 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.
  • a stable pharmaceutical antibody preparation is one in which no significant change is observed in the following conditions: storage at a refrigerating temperature (2-8 ° C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years.
  • 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 antibody 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.
  • the "antibody” as used in the present invention 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 immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain, respectively. , ⁇ chain, and ⁇ chain.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either a kappa chain or a lambda chain by the constant region.
  • Each class Ig of the five classes of Ig may have a kappa chain or a lambda chain.
  • 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 human PD-L1 prepared according to the knowledge and skill in the art.
  • the test subject is injected with the PD-L1 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional property is isolated.
  • the murine PD-L1 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa, a lambda chain or a variant thereof, or further comprising a murine IgG1 , heavy chain constant region of IgG2, IgG3 or variants 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 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 PD-L1 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the PD-L1 chimeric antibody further comprises a heavy chain constant region of human IgG1, 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.
  • the "antigen-binding fragment” as used in the present invention refers to a Fab fragment having antigen-binding activity, a Fab' fragment, an F(ab')2 fragment, and an Fv fragment ScFv fragment which binds to human PD-L1;
  • the antibody is selected from one or more of the CDR regions of SEQ ID NO: 1 to SEQ ID NO: 12.
  • 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 PD-L1 means that it can interact with human PD-L1.
  • antigen binding site refers to a three-dimensional spatial site that is discrete on an antigen and is recognized by an antibody or antigen-binding fragment of the present invention.
  • a mouse can be immunized with human PD-L1 or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method.
  • the antigen-binding fragment can also be prepared by a conventional method.
  • 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.
  • IMGT ImMunoGeneTics
  • the engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods.
  • a cDNA sequence encoding a heavy chain and a light chain can be cloned and recombined into a GS expression vector.
  • 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 PD-L1. 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. For example, 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.
  • Identity refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in both comparison sequences are occupied by the same base or amino acid monomer subunit, for example if each position of the two DNA molecules is occupied by adenine, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of matches or consistent positions shared by the two sequences divided by the number of positions compared ⁇ 100. For example, in the optimal alignment of sequences, if there are 6 matches or coincidences in 10 of the two sequences, then the two sequences are 60% identical. In general, comparisons are made when the two sequences are aligned to obtain the highest percent identity.
  • 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.
  • the human PD-L1 full-length gene (Hypothesis Shenzhou Biotechnology Co., Ltd., HG10084-M) of UniProt Programmed Cell Death1 Ligand1 (PD-L1) isoform1 (SEQ ID NO: 19) was used as a template for the PD-L1 of the present invention.
  • a gene sequence encoding the antigen of the present invention and a protein for detection is obtained, optionally recombined with an antibody heavy chain Fc fragment (such as human IgG1), and cloned into a pTT5 vector (Biovector, Cat#: 102762) or a pTargeT vector (promega, respectively).
  • the human PD-1 gene was purchased from ORIGENE, catalog number SC117011, NCBI Reference Sequence: NM_005018.1.
  • the double-crossed line is the signal peptide (Signal peptide: 1-18); the horizontal line is the PD-L1 extracellular domain (Extracellular domain: 19-238), of which 19-127 is the Ig-like V-type Domain 133-225 is an Ig-like C2-type Domain; the dotted line portion is a transmembrane domain (Transmembrane domain: 239-259); and the italicized portion is a Cytoplasmic domain (260-290).
  • PD-L1 with His and PADRE tags PD-L1 (Extra Cellular Domain, ECD for short)-PADRE-His6
  • the cross-hatched portion is the PD-L1 extracellular region; the dotted line portion is the PADRE marker; the italicized portion is the His6-tag marker.
  • the cross-hatched portion is the PD-L1 extracellular region; the dotted line portion is the FLAG-Tag marker; the italicized portion is the His6-tag marker.
  • Fc fusion protein of PD-L1 PD-L1 (ECD)-Fc, which is used as an immunological antigen or detection reagent of the present invention.
  • the cross-hatched portion is the extracellular region of PD-L1; the italic portion is the human IgG1 Fc portion.
  • Fc fusion protein of PD-1 PD-1 (ECD)-Fc, used for performance testing of the antibody of the present invention.
  • the cross-hatched portion is the extracellular region of PD-1 (ECD); the italic portion is the hFC (human IgG1) portion.
  • PD-L1 with His, PADRE tag purification step of recombinant protein of PD-L1 (ECD)-PADRE-His6 (SEQ ID NO: 20)
  • the cell expression supernatant sample was centrifuged at high speed to remove impurities, and the buffer was exchanged for PBS, and imidazole was added to a final concentration of 5 mM.
  • the nickel column was equilibrated with PBS solution containing 5 mM imidazole and rinsed 2-5 column volumes.
  • the displaced supernatant sample was loaded on a Ni column (GE, 17-5318-01).
  • the column was washed with PBS containing 5 mM imidazole until the A280 reading dropped to baseline.
  • the column was washed with PBS + 10 mM imidazole, the non-specifically bound heteroprotein was removed, and the effluent was collected.
  • the protein of interest was eluted with PBS containing 300 mM imidazole, and the eluted peak was collected.
  • the collected eluate was concentrated and further purified by gel chromatography Superdex 200 (GE), and the mobile phase was PBS. Depolymerized peaks were collected and the eluted peaks were collected.
  • the obtained protein was identified by electrophoresis, peptide map (Agilent, 6530 Q-TOF), LC-MS (Agilent, 6530Q-TOF) as the correct post-equipment.
  • PD-L1 with a His, PADRE tag: PD-L1 (ECD)-PADRE-His6 (SEQ ID NO: 2) was obtained for the immunogen of the antibody of the present invention.
  • the sample was centrifuged at high speed to remove impurities and concentrated to an appropriate volume.
  • the protein peak eluted as above on the IMAC column was loaded onto a 0.5 x PBS equilibrated flag affinity column (Sigma, A2220) and washed 2-5 column volumes.
  • the supernatant-expressed cell expression supernatant sample was loaded on the column.
  • the column was rinsed with 0.5 x PBS until the A 280 reading dropped to baseline.
  • the column was washed with PBS containing 0.3 M NaCl, and the protein was washed and collected.
  • the protein of interest was eluted with 0.1 M acetic acid (pH 3.5-4.0) and collected to adjust the pH to neutral.
  • the collected eluate was concentrated and further purified by gel chromatography Superdex 200 (GE), and the mobile phase was PBS. Depolymerization peaks were collected, and the eluted peaks were collected. The samples were collected by electrophoresis, peptide mapping, and LC-MS.
  • PD-L1:PD-L1(ECD)-Flag-His6 SEQ ID NO: 3 with His-tag and Flag tag was obtained for performance testing of the antibody of the present invention.
  • the cell expression supernatant samples were centrifuged at high speed to remove impurities, concentrated to an appropriate volume, and loaded on a Protein A column (GE, 17-5438-01). The column was rinsed with PBS until the A 280 reading dropped to baseline. The protein of interest was eluted with 100 mM sodium acetate pH 3.0. The protein neutralized by 1 M TrisHCl was further purified by PBS-balanced gel chromatography Superdex 200 (GE). Deaggregate the peak, collect the elution peak, and use it. This method was used to purify PD-L1 (ECD)-Fc (SEQ ID NO: 4) and PD-1 (ECD)-Fc (SEQ ID NO: 5). PD-L1 (ECD)-Fc can be used as an immunizing antigen or detecting reagent of the present invention, and PD-1 (ECD)-Fc is used for performance testing of the antibody of the present invention.
  • ECD Protein A column
  • Anti-human PD-L1 monoclonal antibody was produced by immunizing mice, using SJL white mice, female, 6 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012- 0001). Feeding environment: SPF level. After the mice were purchased, the laboratory environment was kept for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 ° C; humidity 40-60%. Mice that have adapted to the environment are immunized in two regimens (Scheme A and Protocol B), 6-10 per group.
  • the immunizing antigen was PD-L1 with a His, PADRE tag: PD-L1 (ECD)-PADRE-His6 (SEQ ID NO: 20).
  • Protocol A was emulsified with Freund's adjuvant (sigma Lot Num: F5881/F5506): the first use of Freund's complete adjuvant (CFA), and the rest of the booster with Freund's incomplete adjuvant (IFA).
  • CFA Freund's complete adjuvant
  • IFA Freund's incomplete adjuvant
  • the ratio of antigen to adjuvant is 1:1, 100ug/only (first exempt), 50ug/only (enhanced immunization).
  • IP intraperitoneal
  • Scheme B was cross-immunized with Titermax (sigma Lot Num: T2684) and Alum (Thremo Lot Num: 77161).
  • the ratio of antigen to adjuvant (titermax) was 1:1, and the ratio of antigen to adjuvant (Alum) was 3:1, 10-20 ug/only (first escaping), and 5 ug/only (boosting).
  • Splenocytes were fused to mice with high antibody titers in serum and titers to the platform. The selected mice were vaccinated 72 hours before fusion and injected intraperitoneally. Spleen lymphocytes and myeloma cell Sp2/0 cells were optimized using an optimized PEG-mediated fusion step ( CRL-8287 (TM ) was fused to obtain hybridoma cells. The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI) and dispensed into 96-well cell culture plates (1 ⁇ 10 5 /150 ul). /well), incubated at 37 ° C, 5% CO 2 .
  • HAT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI
  • HAT complete medium On day 5 after fusion, HAT complete medium was added, 50 ul/well, and incubated at 37 ° C, 5% CO 2 . From the 7th to 8th day after the fusion, according to the cell growth density, the whole medium was changed, and the medium was HT complete medium (RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI), 200 ul/well, Incubate at 37 ° C, 5% CO 2 .
  • HT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
  • ELISA method for binding to PD-L1 was performed according to cell growth density.
  • the positive well cells combined with ELISA were subjected to blocking ELISA for PD-L1/PD-1 binding, and the positive wells were changed, and expanded into 24-well plates according to cell density.
  • the cell line transferred into the 24-well plate was subjected to retesting and then subjected to seed conservation and first subcloning.
  • the first subcloning screen was positive for conservation and the second subcloning was performed.
  • the second subcloning was positive for conservation and protein expression. Multiple fusions resulted in hybridoma cells that blocked the binding of PD-L1 and PD-1.
  • Hybridoma clones 1 and 2 were screened by blocking assay and binding assay, and antibodies were further prepared by ascites method or by serum-free cell culture, and the antibodies were purified according to the purification examples for use in the test examples.
  • variable region sequences of the murine antibody were obtained by sequencing, wherein the CDR variable region sequences of the hybridoma clones were determined as shown in Table 1 below:
  • X1 is selected from N or T
  • X2 is selected from R or H
  • X3 is selected from N or H
  • X4 is selected from H or G
  • X5 is selected from G or F.
  • the CDRs of murine antibody 1 and 2 were separately transplanted by comparing the IMGT human antibody heavy light chain variable region germline gene database and MOE software, and selecting the homologous heavy and light chain variable region germline genes as templates.
  • the affinity region is matured to form a variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the humanized light chain template of murine antibody 1 is IGKV4-1*01 and hjk4.1
  • the humanized heavy chain template is IGHV4-30-4*01 and hjh2
  • the humanized variable region sequence is as follows:
  • the humanized light chain template of murine antibody 2 is IGKV7-3*01 and hjk2.1, and the humanized heavy chain template is IGHV1-46*01 and hjh6.1.
  • the sequence of humanized variable region is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • the primers were designed to construct the VH/VK gene fragment of each humanized antibody, and then homologously recombined with the expression vector pHr (with signal peptide and constant region gene (CH1-FC/CL) fragment) to construct the full-length antibody expression vector VH- CH1-FC-pHr/VK-CL-pHr.
  • Primer design use the online software DNAWorks(v3.2.2) (http://helixweb.nih.gov/dnaworks/) to design multiple primers to synthesize VH/VK gene fragments containing recombinant: 5'-30bp signal peptide + VH/VK+30bpCH1/CL-3'.
  • Primer design principle The target gene 2 is different from the target gene 1 by 2 aa, and the primer of the mutation site is also set, as shown in Fig. 1.
  • the expression vector pHr (with signal peptide and constant region gene (CH1-FC/CL) fragment) was constructed by using some special restriction enzymes, such as BsmBI, and the recognition sequence was different from the restriction site. 2 is shown.
  • the vector was digested with BsmBI, and the gel was recovered for use.
  • VH/VK contains the gene fragment required for recombination and BsmBI digestion and expression vector pHr (with signal peptide and constant region gene (CH1-FC/CL) fragment) is added to DH5H competent cells in a molar ratio of 3:1, 0 °C ice bath for 30 min, heat shock at 42 °C for 90 s, add 5 volumes of LB medium, incubate for 45 min at 37 ° C, coat LB-Amp plate, incubate overnight at 37 ° C, pick monoclonal and send to sequencing to obtain each clone.
  • pHr signal peptide and constant region gene
  • Humanized PD-L1 antibodies 1 and 2 were constructed into phagemid vectors in scFv mode (VH-3 GGGGS-VL), respectively, as wild-type sequences (ie, mutant sequences screened for affinity maturation, as Original or starting sequence).
  • VH, (GGGGS)3 linker, VL were spliced by over-lap PCR, and phagemid vector was ligated using NcoI and NotI restriction sites.
  • each codon in the mutation region has 50% wild-type codon and 50% NNK (reverse primer is MNN). Mutations were constructed in all CDR regions to construct a mutant library. The PCR fragment was digested with NcoI and NotI, ligated into a phagemid vector, and finally electrotransformed into E. coli TG1. An independent library was constructed for each codon-based primer, in which antibody 1 was divided into 7 libraries and antibody 2 was divided into 8 libraries.
  • the biotinylated human PD-L1 (ECD) antigen and streptavidin magnetic beads were used for liquid chromatography, and each round of screening was compared to the previous one. Both rounds reduce the antigen concentration.
  • antibody 1 and antibody 2 were picked up for 250 clones for phage ELISA to detect binding activity, and positive clones were sequenced.
  • IG affinity purification using BIAcore TM X-100 instrument (GE Life Sciences) for affinity assays.
  • variable region sequences of the humanized antibody 2 after affinity maturation are as follows:
  • X 4 of CDR2 is G and X 5 is F.
  • the ACE sequence is in italics in the sequence; the underlined is the CDR sequence, and the double underlined site is the site obtained after affinity maturation screening.
  • variable region sequences of humanized antibody 1 after affinity maturation are as follows:
  • CDR1 X 2 is H and X 3 is H.
  • the affinity-obtained clone was converted to the IgG4 type, and the core hinge region was selected to contain the S228P mutant IgG4 to obtain an antibody without ADCC and CDC, wherein the antibody obtained from the antibody 2 was named HRP00052.
  • the last three nucleotides "TGA" of the following gene sequences SEQ ID NO: 16 and 18 are stop codons and do not encode any amino acids.
  • the underlined portion is the variable region sequence of the antibody heavy or light chain, or the nucleotide sequence encoding the same; the unlined portion is the antibody constant region sequence and its corresponding coding nucleotide sequence.
  • An expression plasmid for expressing the PD-L1 antibody HRP00052 was constructed, and the nucleotide sequences encoding the heavy and light chains and their respective promoter and polyadenylation signal sequences were confirmed by DNA sequence analysis, and then the expression was used.
  • the vector was transfected into a CHO cell line. Clones expressing the antibody are selected based on growth and production stability, a master seed bank is prepared, and then the seed bank is used to prepare antibodies and generate a master cell bank.
  • the cells from the master cell bank are propagated in shake flasks, culture bags, and seed bioreactors, and the resulting seed cells are used to produce antibody products using a bioreactor.
  • the resulting antibody is further purified by protein A affinity chromatography, cation exchange chromatography and anion exchange chromatography, as well as low pH virus inactivation and filtration steps to remove the virus.
  • the specific purification steps were as follows: The cell expression supernatant sample was loaded on a PBS buffer-balanced Protein A column (Merck, 175118824), and the column was washed with PBS until the A280 reading was lowered to the baseline, and then the column was washed with PB buffer and washed away.
  • the protein was eluted with 50 mM sodium citrate pH 3.5 and the elution peak was collected. Purification was carried out using an anion chromatography column (GE, 17-5316-10) equilibrated with PB buffer on 1 M Tris-neutralized protein, and the flow-through peak was collected and adjusted back to pH 5.0 with 1 M citric acid. After anion chromatography, the protein was further purified on a cation chromatography column (Merck, 1.168882) with a balanced citrate buffer (pH 5.0), and eluted with a citrate buffer (pH 5.0) containing 0.18 M sodium chloride. The protein was collected and eluted and used.
  • the PD-L1 preparation (1 mg/ml) buffer system, buffer concentration, pH value, sugar type and sugar concentration were used for experimental design, and the Tm value of the sample was determined by DSC technique, and the formulation 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.
  • the concentration of sugar is in g/100mL.
  • the prescriptions preliminarily selected from the main effect diagrams of Tm factors are: buffer system, acetic acid (sodium) is better, followed by succinic acid (sodium); buffer concentration is 20-30 mM.
  • the Tm value was higher; pH 5-5.6 had no significant effect on the Tm value; the sugar concentration was 6%.
  • Anti-PD-L1 antibody (HRP00052) was prepared as a preparation containing 10 mM succinic acid (sodium), acetic acid (sodium), 60 mg/ml sucrose, 0.2 mg/ml polysorbate 20 at pH 5.0-5.5, protein concentration It is 50 mg/ml. Each preparation was filtered and filled in a neutral borosilicate glass controlled injection bottle, which was sealed with a bromobutyl rubber stopper for the long-term stability of 2 to 8 ° C. The stability of the sample was shown in Table 3. The various features shown are illustrated.
  • the color, appearance and clarity of the sample were determined by visual inspection of the sample under a white fluorescent light at room temperature on a black background.
  • the purity of the samples was further evaluated by high performance size exclusion chromatography (HP-SEC), where the percentage of monomer and the percentage of high molecular weight species (possibly aggregates) and late eluting peaks (possibly degradation products) were determined. Purity was assessed by revealing the presence of acidic or basic variants using high performance ion exchange chromatography (HP-IEX) and the results are expressed as a percentage of the total observed material.
  • HP-SEC high performance size exclusion chromatography
  • Samples were analyzed by CE-SDS technique in which proteins were denatured with sodium dodecyl sulfate (SDS) under reducing and non-reducing conditions and separated using capillary electrophoresis (CE). The proteins are separated based on their apparent molecular weight. Under non-reducing conditions, all substances except the main IgG peak are classified as impurities. Under reducing conditions, IgG is split into heavy and light chains, and all other substances are classified as impurities.
  • SDS sodium dodecyl sulfate
  • CE capillary electrophoresis
  • the anti-PD-L1 antibody was prepared as a preparation of 10 mM and 20 mM succinic acid (sodium), pH 5.2, 60 mg/ml sucrose, 0.2 mg/ml polysorbate 20, and the protein concentration was 50 mg/ml.
  • Each of the preparations was filtered and filled in a neutral borosilicate glass controlled injection bottle sealed with a bromobutyl rubber stopper for accelerated acceleration at 40 ° C and long-term stability study at 2 to 8 ° C. The results showed that the anti-PD-L1 antibody was very stable in the 10-20 mM succinate buffer system.
  • PD-L1 antibody HRP00052
  • concentration of 50 mg/ml containing 20 mM acetic acid (sodium), pH 5.2, 60 mg/ml sucrose, anti-PD-L1 antibody preparation containing different kinds and concentrations of surfactant, filtering and filling
  • a neutral borosilicate glass controlled injection bottle sealed with a bromobutyl rubber stopper placed on a 25 ° C constant temperature shaker, and shaken at 200 rpm.
  • DLS Dynamic Light Scattering
  • PDI particle dispersion index

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Abstract

本发明提供了一种PD-L1抗体药物组合物及其用途。具体而言，本发明提供了一种药物组合物，其包含琥珀酸盐缓冲剂中的PD-L1抗体或其抗原结合片段。除此之外，该药物组合物还可含有糖和非离子表面活性剂。

Description

一种PD-L1抗体药物组合物及其用途
本申请要求申请日为2017年5月16日的中国专利申请CN201710341680.7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PD-L1抗体及其抗原结合片段的药物组合物，以及其作为抗癌药物的用途。
背景技术
程序性死亡分子1(programmed death-1,PD-1)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-1有两个配体，分别为PD-L1和PD-L2。PD-L1主要表达于T细胞、B细胞、巨噬细胞和树突状细胞(dendritic cell,DC)上，在活化后细胞上的表达能够进行上调。而PD-L2的表达相对较局限，主要表达在抗原呈递细胞上，如活化的巨噬细胞和树突状细胞。
PD-L1通过和PD-1及B7-1的结合抑制免疫系统，很多肿瘤细胞及肿瘤组织微环境的免疫细胞表达PD-L1。新的研究发现乳腺癌、肺癌、胃癌、肠癌、肾癌、黑色素瘤、非小细胞肺癌、结肠癌、膀胱癌、卵巢癌、胰腺癌及肝癌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。
抗体药物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。为了使抗体分子适合于给药，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，抗体药物的制剂研究显得尤为重要。
目前有多家跨国制药公司在研发包含PD-L1/PD-1抗体的药物组合物，相关专利如：CN105793288A、CN103429264A、CN105960415A。
本发明提供足够稳定且适于给药的包含PD-L1抗体及其抗原结合片段的药物组合物。
发明内容
本发明提供一种药物组合物，其包含PD-L1抗体及抗原结合片段，以及缓冲剂，所 述缓冲剂优选为琥珀酸盐或醋酸盐缓冲剂，更优选为琥珀酸盐缓冲剂。
在本发明的实施方案中，缓冲剂的浓度为约5mM至50mM，优选为大约10mM至30mM，更优选为10mM至20mM，非限制性实施例包括10mM、12mM、14mM、16mM、18mM、20mM。
在本发明的实施方案中，药物组合物的pH值约为4.5到6.0，优选约为4.8到5.7，更优选为5.0到5.5，可为5.0、5.1、5.2、5.3、5.4、5.5。
在本发明的实施方案中，药物组合物中抗体的浓度为约30mg/ml至约80mg/ml，优选为约40mg/ml至约60mg/ml，更优选为45mg/ml至约55mg/ml，非限制性实施例包括45mg/ml、46mg/ml、47mg/ml、48mg/ml、49mg/ml、50mg/ml、51mg/ml、52mg/ml、53mg/ml、54mg/ml、55mg/ml。
进一步的，本发明的药物组合物还包含糖。本发明的“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。
在本发明的实施方案中，药物组合物中糖的浓度为约30mg/ml至约90mg/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。
进一步的，本发明的药物组合物还包含表面活性剂。可选自聚山梨酯20、聚山梨酯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-、肉豆蔻基-、亚油基-、硬脂基-磺基甜菜碱、月桂基-、肉豆蔻基-、亚油基-、硬脂基-肌氨酸、亚油基-、肉豆蔻基-、鲸蜡基-甜菜碱、月桂酰胺基丙基-、柯卡酰胺基丙基-、亚油酰胺基丙基-、肉豆蔻酰胺基丙基-、棕榈酰胺基丙基-、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-、棕榈酰胺基丙基-、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酯80或聚山梨酯20，更优选为聚山梨酯80。
在本发明的实施方案中，药物组合物中表面活性剂的浓度为约0.1mg/ml至1.0mg/ml，优选为0.2mg/ml至0.8mg/ml，更优选为0.4mg/ml至0.8mg/ml，非限制性实施例包括0.1mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/ml、0.5mg/ml、0.6mg/ml、0.7mg/ml和0.8mg/ml。
在本发明的实施方案中，药物组合物中所述抗体或抗原结合片段包含任意1个选自以下的CDR区序列或其突变序列：抗体重链可变区HCDR区序列：SEQ ID NO：1-3，SEQ ID NO：7-9；和/或，抗体轻链可变区LCDR区序列：SEQ ID NO：4-6，SEQ ID NO：10-12；
具体如下：
HCDR1选自：NDYWX 1                 SEQ ID NO：1
或         SYWMH                  SEQ ID NO：7，
HCDR2选自：YISYTGSTYYNPSLKS       SEQ ID NO：2
或         RI X 4PNSG X 5TSYNEKFKN  SEQ ID NO：8，和/或
HCDR3选自：SGGWLAPFDY             SEQ ID NO：3
或         GGSSYDYFDY             SEQ ID NO：9；和/或
LCDR1选自：KSSQSLFY X 2 SNQK X 3SLA SEQ ID NO：4
或         RASESVSIHGTHLMH        SEQ ID NO：10，
LCDR2选自：GASTRES                SEQ ID NO：5
或         AASNLES                SEQ ID NO：11，和/或
LCDR3选自：QQYYGYPYT              SEQ ID NO：6
或         QQSFEDPLT              SEQ ID NO：12；
其中X 1选自N或T，X 2选自R或H，X 3选自N或H，X 4选自H或G，X 5选自G或F。
在本发明的实施方案中，优选地，药物组合物中所述的抗体或其抗原结合片段包含选自：SEQ ID NO：10，SEQ ID NO：11，SEQ ID NO：12的轻链可变区CDR序列或其突变序列，和选自：SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：9的重链可变区CDR序列或其突变序列；更优选地，所述的抗体或其抗原结合片段包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1、LCDR2和LCDR3序列，以及分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：9所示的HCDR1、HCDR2和HCDR3序列；
或，优选地，药物组合物中所述的抗体或其抗原结合片段包含选自：SEQ ID NO：1，SEQ ID NO：2，SEQ ID NO：3的重链可变区CDR序列或其突变序列，和选自：SEQ ID NO：4，SEQ ID NO：5和SEQ ID NO：6的轻链可变区CDR序列或其突变序列；更优选地，所述的抗体或其抗原结合片段包含分别如SEQ ID NO：1，SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3序列，以及分别如SEQ ID NO：4，SEQ ID  NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3序列。
在本发明的实施方案中，药物组合物中所述抗体或其抗原结合片段包含与氨基酸序列：SEQ ID NO：10，SEQ ID NO：11，SEQ ID NO：12具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的轻链可变区CDR序列，和与氨基酸序列：SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：9具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的重链可变区CDR序列。
在本发明的实施方案中，药物组合物中所述抗体或其抗原结合片段可选自鼠源抗体、嵌合抗体、人源化抗体，人抗体，优选人源化抗体。
在本发明的实施方案中，药物组合物中所述抗体或其抗原结合片段包含与氨基酸序列SEQ ID NO：13具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的重链可变区序列，和与氨基酸序列SEQ ID NO：14具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的轻链可变区序列。
在本发明的实施方案中，药物组合物中所述抗体或其抗原结合片段包含与氨基酸序列SEQ ID NO：15具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的重链可变区序列，和与氨基酸序列SEQ ID NO：17具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的轻链序列。
本发明进一步提供一种药物组合物，该药物组合物包含浓度为约30mg/ml至约80mg/ml的PD-L1抗体或其抗原结合片段，浓度为约5mM至约50mM的琥珀酸盐缓冲剂，pH5.0-6.0，浓度为约30mg/ml至约90mg/ml的二糖，浓度为约0.1mg/ml至约1.0mg/ml的聚山梨酯80。
本发明进一步提供一种药物组合物，该药物组合物包含40-60mg/ml的PD-L1抗体或抗原结合片段，10-30mM的琥珀酸盐缓冲剂，pH5.0-5.5，40-80mg/ml的蔗糖，0.4-0.8mg/ml的聚山梨酯80。
本发明进一步提供一种药物组合物，该药物组合物包含45-55mg/ml的PD-L1抗体或其抗原结合片段，10-20mM的琥珀酸盐缓冲剂，pH5.0-5.5，55-65mg/ml的蔗糖，0.5-0.7mg/ml的聚山梨酯80。
本发明进一步提供一种药物组合物，该药物组合物包含浓度为约30mg/ml至约80mg/ml的PD-L1抗体或其抗原结合片段，浓度为约5mM至约50mM的醋酸盐缓冲剂， pH5.0-6.0，浓度为约30mg/ml至约90mg/ml的二糖，浓度为约0.1mg/ml至约1.0mg/ml的聚山梨酯80。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或抗原结合片段，10mM的琥珀酸盐缓冲剂，pH5.3，60mg/ml的蔗糖。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或抗原结合片段，10mM的醋酸盐缓冲剂，pH5，90mg/ml的蔗糖。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或抗原结合片段，30mM的醋酸盐缓冲剂，pH5，60mg/ml的海藻糖。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或抗原结合片段，30mM的醋酸盐缓冲剂，pH5，60mg/ml的海藻糖。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或抗原结合片段，30mM的醋酸盐缓冲剂，pH5.6，90mg/ml的蔗糖。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或抗原结合片段，10mM琥珀酸盐缓冲体系，pH5.0-5.5，60mg/ml蔗糖，0.2mg/ml的聚山梨醇酯20。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或抗原结合片段，10-20mM琥珀酸盐缓冲体系，pH5.2，60mg/ml蔗糖，0.2mg/ml的聚山梨醇酯20。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或抗原结合片段，20mM的醋酸盐缓冲剂，pH5.2，60mg/ml的蔗糖，0.1-0.3mg/ml的聚山梨酯20。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或抗原结合片段，20mM的醋酸盐缓冲剂，pH5.2，60mg/ml的蔗糖，0.1-0.3mg/ml的聚山梨酯80。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或其抗原结合片段，20mM的琥珀酸盐缓冲剂，pH5.2，60mg/ml的蔗糖，0.2-0.6mg/ml的聚山梨酯20。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或其抗原结合片段，20mM的琥珀酸盐缓冲剂，pH5.2，60mg/ml的蔗糖，0.4-0.8mg/ml的聚山梨酯80。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或其 抗原结合片段，20mM的琥珀酸盐缓冲剂，pH5.2，60mg/ml的蔗糖，0.4mg/ml的聚山梨酯80。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或其抗原结合片段，20mM的琥珀酸盐缓冲剂，pH5.2，60mg/ml的蔗糖，0.6mg/ml的聚山梨酯80。
本发明进一步提供一种药物组合物，该药物组合物包含50mg/ml的PD-L1抗体或其抗原结合片段，20mM的琥珀酸盐缓冲剂，pH5.2，60mg/ml的蔗糖，0.8mg/ml的聚山梨酯80。
在一些实施方案中，该药物组合物中的琥珀酸盐缓冲剂的浓度为约5mM至50mM。在一些实施方案中，该药物组合物中的琥珀酸盐缓冲剂的浓度为约10mM至30mM。在一些实施方案中，该药物组合物中的琥珀酸盐缓冲剂的浓度为约20mM。
在一些实施方案中，该药物组合物中的醋酸盐缓冲剂的浓度为约5mM至50mM。在一些实施方案中，该药物组合物中的醋酸盐缓冲剂的浓度为约10mM至30mM。在一些实施方案中，该药物组合物中的醋酸盐缓冲剂的浓度为约20mM。
在一些实施方案中，该药物组合物的pH值约为5.0到6.0。在一些实施方案中，该药物组合物的pH值约为5.0到5.5。在一些实施方案中，该药物组合物的pH值约为5.2或5.5。
在一些实施方案中，该药物组合物中抗体的浓度为约30mg/ml至约80mg/ml。在一些实施方案中，该药物组合物中抗体的浓度为约40mg/ml至约60mg/ml。在一些实施方案中，该药物组合物中抗体的浓度为约50mg/ml。
在一些实施方案中，该药物组合物中包含所述二糖的浓度为约30mg/ml至约90mg/ml。在一些实施方案中，该药物组合物中的所述二糖的浓度为约40mg/ml至约80mg/ml。在一些实施方案中，该药物组合物中的所述二糖的浓度为约60mg/ml。
在一些实施方案中，该药物组合物中所述聚山梨酯为聚山梨酯20或聚山梨酯80。在一些实施方案中，该药物组合物中所述聚山梨酯为聚山梨酯80。在一些实施方案中，该药物组合物中所述聚山梨酯的浓度为约0.1mg/ml至1.0mg/ml。在一些实施方案中，该药物组合物中所述聚山梨酯的浓度为约0.4mg/ml至0.8mg/ml。在一些实施方案中，该药物组合物中所述聚山梨酯的浓度为约0.6mg/ml。
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。
本发明进一步提供一种制品或试剂盒，其包含装有本文所述任何稳定的药物组合物的容器。在一些实施方案中，该容器为玻璃瓶，该玻璃瓶为中性硼硅玻璃管制注射剂瓶。
本发明进一步提供一种制备以上任一项所述的药物组合物的方法，包括将PD-L1抗体或其抗原结合片段与药学上可接受的赋形剂混合。
本发明进一步提供以上任一项所述药物组合物在制备用于治疗PD-L1介导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PD-L1的癌症；最优选为乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤、非小细胞肺癌；进一步优选为非小细胞肺癌、黑素瘤、膀胱癌和肾癌。
本发明进一步提供一种治疗和预防PD-L1介导的疾病或病症的方法，该方法包括给予所需患者有效量的包含PD-L1抗体或其抗原结合片段的药物组合物；其中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、非小细胞肺癌、膀胱癌；最优选为非小细胞肺癌、黑素瘤、膀胱癌和肾癌。
如本领域技术人员所熟知的，本公开中所述各个实施方案的一项、一些或所有特性可以进一步组合以形成本发明的其它实施方案。本发明的以上实施方案和通过组合得到的其他实施方案通过下面的详述进一步说明。
附图说明
图1：人源化克隆构建时引物设计示意图。
图2：人源化克隆构建的载体构建示意图。
图3：Tm各因子主效应图(包括缓冲体系、缓冲液浓度、药物组合物的pH、糖浓度和糖的种类)。
具体实施方式
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5.2。将pH控制在此范围中的缓冲剂的例子包括醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸、草 酸盐、乳酸盐、磷酸盐、柠檬酸盐、酒石酸盐、延胡索酸盐、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醋酸盐缓冲剂，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。
“醋酸盐缓冲剂”是包括醋酸根离子的缓冲剂。醋酸盐缓冲剂的实例包括醋酸-醋酸钠、醋酸组氨酸盐、醋酸-醋酸钾、醋酸醋酸钙、醋酸-醋酸镁等。本发明优选的醋酸盐缓冲剂是醋酸-醋酸钠。
“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。
稳定的药物抗体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，药物抗体制剂是无色的，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。
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。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
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人PD-L1的单克隆 抗体。制备时用PD-L1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。在本发明一个优选的实施方案中，所述的鼠源PD-L1抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
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PD-L1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PD-L1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，1991 Sequences 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。
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PD-L1结合的Fv片段ScFv片段；其包含本发明所述抗体的选自SEQ ID NO:1至SEQ ID NO:12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PD-L1结合”，指能与人PD-L1相互作用。本发明的术语“抗原结合位点”指抗原上不连续的，由本发明抗体或抗原结合片段识别的三维空间位点。
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。例如，老鼠可以用人PD-L1或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。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PD-L1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。
“一致性”是指两个多核苷酸序列之间或两个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碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是一致的。两个序列之间的一致性百分率是两个序列共有的匹配或一致位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或一致，那么两个序列为60％一致性。 一般而言，当比对两个序列而得到最大的一致性百分率时进行比较。
“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。
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。
实施例中SEC-HPLC和IEC-HPLC的检测使用安捷伦-HPLC1260高压液相色谱仪(Waters
Figure PCTCN2018086866-appb-000001
BEH
Figure PCTCN2018086866-appb-000002
SEC 3.5μm 7.8*300mm色谱柱和Thermo ProPac TM WCX-10BioLC TM,250×4mm色谱柱)。还原CE-SDS及非还原CE-SDS的检测使用Beckman公司 的PA 800plus毛细管电泳仪。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 PD-L1抗体的制备
(1)PD-L1抗原及检测用蛋白的制备
以UniProt Programmed Cell Death1 Ligand1(PD-L1)isoform1(SEQ ID NO：19)的人PD-L1全长基因(义翘神州生物技术有限公司，HG10084-M)，作为本发明PD-L1的模板，获得编码本发明抗原及检测用蛋白的基因序列，可选地与抗体重链Fc片段(如human IgG1)重组，分别克隆到pTT5载体上(Biovector，Cat#：102762)或pTargeT载体上(promega,A1410)，在293F细胞(Invitrogen，R79007)瞬转表达或CHO-S细胞(Invitrogen,k9000-20)稳定表达，纯化，获得编码本发明抗原及检测用蛋白。人PD-1基因购自ORIGENE，货号SC117011，NCBI Reference Sequence:NM_005018.1。
1、人PD-L1全长氨基酸序列
Figure PCTCN2018086866-appb-000003
                                                                  SEQ ID NO：19
注释：双横线部分为信号肽(Signal peptide:1-18)；划横线部分为PD-L1胞外区(Extracellular domain：19-238)，其中19-127为Ig-like V-type Domain，133-225为Ig-like C2-type Domain；点划线部分为跨膜区部分(Transmembrane domain:239-259)；斜体部分为胞内区(Cytoplasmic domain:260-290)。
2、免疫原：带His、PADRE标签的PD-L1：PD-L1(Extra Cellular Domain，简称ECD)-PADRE-His6
Figure PCTCN2018086866-appb-000004
                                                                  SEQ ID NO：20
注释：划横线部分为PD-L1胞外区；点划线部分为PADRE标记；斜体部分为His6-tag标记。
3、得到带FLAG、HIS标签的PD-L1：PD-L1(ECD)-Flag-His6，用于本发明抗体的性能测试。
Figure PCTCN2018086866-appb-000005
                                                                  SEQ ID NO：21
注释：划横线部分为PD-L1胞外区；点划线部分为FLAG-Tag标记；斜体部分为His6-tag标记。
4、PD-L1的Fc融合蛋白：PD-L1(ECD)-Fc，用作本发明的免疫抗原或检测试剂。VKL-PD-L1(ECD)-Fc(human IgG1)
Figure PCTCN2018086866-appb-000006
                                                                  SEQ ID NO：22
注释：划横线部分为PD-L1胞外区；斜体部分为human IgG1 Fc部分。
5、PD-1的Fc融合蛋白：PD-1(ECD)-Fc，用于本发明抗体的性能测试。
Figure PCTCN2018086866-appb-000007
                                                                  SEQ ID NO：23
注释：划横线部分为PD-1(ECD)胞外区；斜体部分为hFC(human IgG1)部分。
(2)PD-L1、PD-1重组蛋白以及杂交瘤抗体、重组抗体的纯化
1、“带His、PADRE标签的PD-L1”：PD-L1(ECD)-PADRE-His6(SEQ ID NO：20)重组蛋白的纯化步骤
将细胞表达上清样品高速离心去除杂质，并将缓冲液换置换为PBS，加入咪唑至终浓度为5mM。用含有5mM咪唑的PBS溶液平衡镍柱，冲洗2-5倍柱体积。将置换后的上清样品装载于Ni柱(GE，17-5318-01)上。用含有5mM咪唑的PBS溶液冲洗柱子，至A 280读数降至基线。后用PBS+10mM咪唑冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液。再用含有300mM咪唑的PBS溶液洗脱目的蛋白，并收集洗脱峰。收集的洗脱液浓缩后用凝胶层析Superdex200(GE)进一步纯化，流动相为PBS。去聚体峰，收集洗脱峰。所得到的蛋白经电泳、肽图(安捷伦，6530 Q-TOF)、LC-MS(安捷伦，6530Q-TOF)鉴定为正确后分装备用。得到带His、PADRE标签的PD-L1：PD-L1(ECD)-PADRE-His6(SEQ ID NO：2)用于本发明抗体的免疫原。
2、带His标签和Flag标签的PD-L1(ECD)-Flag-His6(SEQ ID NO：21)重组蛋白的纯化步骤
将样品高速离心去除杂质，并浓缩至适当体积。将如上IMAC柱洗出的蛋白峰装载于0.5×PBS平衡的flag亲和柱(Sigma，A2220)上，冲洗2-5倍柱体积。将除杂后的细胞表达上清样品装载于柱上。用0.5×PBS冲洗柱子，至A 280读数降至基线。用含有0.3MNaCl的PBS冲洗柱子，冲洗杂蛋白，并收集。用0.1M乙酸(pH3.5-4.0)洗脱目的蛋白，并收集，调节pH至中性。收集的洗脱液浓缩后用凝胶层析Superdex200(GE)进一步纯化，流动相为PBS。去聚体峰，收集洗脱峰收集样品经电泳、肽图、LC-MS鉴定正确后分装备用。得到带His标签和Flag标签的PD-L1：PD-L1(ECD)-Flag-His6(SEQ ID NO：3)，用于本发明抗体的性能测试。
3、PD-L1和PD-1的Fc融合蛋白的纯化步骤
将细胞表达上清样品高速离心去除杂质，浓缩至适当体积后装载于Protein A柱(GE，17-5438-01)上。用PBS冲洗柱子，至A 280读数降至基线。用100mM醋酸钠pH3.0洗脱目的蛋白。1M TrisHCl中和后的蛋白上PBS平衡好的凝胶层析Superdex200(GE)进一步纯化。去聚体峰，收集洗脱峰后，分装备用。此方法用来纯化PD-L1(ECD)-Fc(SEQ ID NO：4)和PD-1(ECD)-Fc(SEQ ID NO：5)。PD-L1(ECD)-Fc可用作本发明的免疫抗原或检测试剂，PD-1(ECD)-Fc用于本发明抗体的性能测试。
(3)抗人PD-L1杂交瘤单克隆抗体的制备
1、免疫
抗人PD-L1单克隆抗体通过免疫小鼠产生，实验用SJL白小鼠，雌性，6周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按两种方案(方案A和方案B)免疫，每组6-10只。免疫抗原为带His、PADRE标签的PD-L1：PD-L1(ECD)-PADRE-His6(SEQ ID NO：20)。
方案A用弗氏佐剂(sigma Lot Num：F5881/F5506)乳化：首免用弗氏完全佐剂(CFA)，其余加强免疫用弗氏不完全佐剂(IFA)。抗原与佐剂比例为1:1，100ug/只(首免)，50ug/只(加强免疫)。第0天腹膜内(IP)注射100ug/只的乳化后抗原，首免后每两周一次，共6-8周。
方案B用Titermax(sigma Lot Num：T2684)与Alum(Thremo Lot Num：77161)交叉免疫。抗原与佐剂(titermax)比例为1:1，抗原与佐剂(Alum)比例为3:1，10-20ug/只(首免)，5ug/只(加强免疫)。第0天腹膜内(IP)注射20/10μg/只的乳化后抗原，首免后每周一次，Titermax和Alum交替使用，共6-11周。免疫四周后，根据背部结块和腹部肿胀情况，选择背部或腹膜内注射抗原。
2、细胞融合
选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合，融合前72小时冲刺免疫所选小鼠，腹腔注射。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2018086866-appb-000008
CRL-8287 TM)进行融合得到杂交瘤细胞。融合好的杂交瘤细胞用HAT完全培养基(含20％FBS、1×HAT和1×OPI的RPMI-1640培养基)重悬，分装于96孔细胞培养板中(1×10 5/150ul/孔)，37℃、5％CO 2孵育。融合后的第5天加入HAT完全培养基，50ul/孔，37℃、5％CO 2孵育。融合后第7天～8天，根据细胞生长密度，全换液，培养基为HT完全培养基(含20％FBS、1×HT和1×OPI的RPMI-1640培养基)，200ul/孔，37℃、5％CO 2孵育。
3、杂交瘤细胞筛选
融合后第10-11天，根据细胞生长密度，进行结合PD-L1的ELISA方法检测。并将结合ELISA检测的阳性孔细胞进行PD-L1/PD-1结合的阻断ELISA检测，阳性孔换液，并根据细胞密度及时扩大至24孔板中。移入24孔板的细胞株经过复测后进行保种和第一次亚克隆。第一次亚克隆筛选为阳性的进行保种，并进行第二次亚克隆。第二次亚克隆为阳性的进行保种和蛋白表达。多次融合获得有阻断PD-L1和PD-1结合效果的杂交 瘤细胞。
通过阻断实验和结合实验筛选得到杂交瘤克隆1和2，用腹水法或用无血清细胞培养法进一步制备抗体，按纯化实例纯化抗体，供在检测例中使用。
测序获得鼠源抗体的可变区序列，其中测得杂交瘤克隆的CDR可变区序列如下表1所示：
表1
Figure PCTCN2018086866-appb-000009
其中X1选自N或T，X2选自R或H，X3选自N或H，X4选自H或G，X5选自G或F。
(4)抗人PD-L1杂交瘤单克隆抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，挑选同源性高的重轻链可变区种系基因作为模板，将鼠源抗体1和2的CDR分别移植到相应的人源模板中，经亲和力成熟后形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。
鼠源抗体1的人源化轻链模板为IGKV4-1*01和hjk4.1，人源化重链模板为IGHV4-30-4*01和hjh2，人源化可变区序列如下：
>1 hVH-CDR graft
Figure PCTCN2018086866-appb-000010
                                                                  SEQ ID NO：24
>1 hVL CDR graft
Figure PCTCN2018086866-appb-000011
                                                                  SEQ ID NO：25
鼠源抗体2的人源化轻链模板为IGKV7-3*01和hjk2.1，人源化重链模板为IGHV1-46*01和hjh6.1，人源化可变区序列如下：
>2-hVH.1
Figure PCTCN2018086866-appb-000012
                                                                  SEQ ID NO：26
>2-hVL.1
Figure PCTCN2018086866-appb-000013
                                                                  SEQ ID NO：27
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
(5)人源化克隆构建
设计引物PCR搭建各人源化抗体VH/VK基因片段，再与表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)进行同源重组，构建抗体全长表达载体VH-CH1-FC-pHr/VK-CL-pHr。
1、引物设计：利用在线软件DNAWorks(v3.2.2)(http://helixweb.nih.gov/dnaworks/)设计多条引物合成VH/VK含重组所需基因片段：5’-30bp信号肽+VH/VK+30bpCH1/CL-3’。引物设计原则：目的基因2与目的基因1有2个aa不一样，则另设突变位点所在引物，如图1所示。
2、片段拼接：按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，分两步PCR扩增得到VH/VK含重组所需基因片段。
3、表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)构建及酶切
利用一些特殊的限制性内切酶，如BsmBI，识别序列与酶切位点不同的特性设计构建表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)，构建示意图如图2所示。BsmBI酶切载体，切胶回收备用。
4、重组构建表达载体VH-CH1-FC-pHr/VK-CL-pHr
VH/VK含重组所需基因片段与BsmBI酶切回收表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)按摩尔比3:1比例分别加入DH5H感受态细胞中，0℃冰浴30min，42℃热击90s，加入5倍体积LB介质，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
(6)抗-PD-L1人源化抗体亲和力成熟
1、构建人源化PD-L1抗体1和2噬菌粒载体
人源化后的PD-L1抗体1和2分别以scFv模式(VH-3个GGGGS-VL)构建到噬菌粒载体中，作为野生型序列(即相对于亲和力成熟筛选得到的突变序列，作为原始或起始序列)。利用over-lap PCR拼接VH、(GGGGS)3 linker、VL，采用NcoI和NotI酶切位点连接入噬菌粒载体。
2、构建噬菌体展示文库
利用构建好的野生型scFv为模板，采用codon-based引物，在引物合成过程中，突变区域每个密码子都有50％野生型的密码子和50％的NNK(反向引物为MNN))在所有CDR区引入突变构建突变文库。PCR片段经过NcoI和NotI酶切，连接到噬菌粒体载体中，最后电转化大肠杆菌TG1。每条codon-based引物建立一个独立的文库，其中抗体1分为7个文库，抗体2分为8个文库。
3、文库淘筛
文库经过拯救包装出淘筛用的噬菌体颗粒后，利用生物素化的人PD-L1(ECD)抗原和链霉亲和素磁珠进行液相法淘筛，并且每一轮筛选相对于上一轮都降低抗原浓度。三轮淘筛之后，抗体1和抗体2分别挑取250克隆进行噬菌体ELISA检测结合活性，阳性克隆进行测序。
4、表面等离子体共振(SPR)检测亲合力
经过对测序克隆进行比对分析，去除冗余序列之后，将非冗余序列转换成全长IG(γ1,κ)进行哺乳动物细胞表达。亲和纯化之后的全长IG采用BIAcore TM X-100 instrument(GE Life Sciences)进行亲合力测定。
亲和力成熟后的人源化抗体2的可变区序列如下：
重链可变区
Figure PCTCN2018086866-appb-000014
                                                                  SEQ ID NO：13
其中，CDR2的X 4为G，X 5为F。
轻链可变区
Figure PCTCN2018086866-appb-000015
                                                                  SEQ ID NO：14
注：序列中斜体为FR序列；下划线为CDR序列，其中双下划线的位点为亲和力成熟筛选后得到的位点。
亲和力成熟后的人源化抗体1的可变区序列如下：
重链可变区
Figure PCTCN2018086866-appb-000016
                                                                  SEQ ID NO：28
其中，CDR1的X 1为T。
轻链可变区
Figure PCTCN2018086866-appb-000017
                                                                  SEQ ID NO：29
其中，CDR1 X 2为H，X 3为H。
将亲合力成熟得到的克隆转换成IgG4类型，选择核心铰链区包含S228P突变的IgG4以得到无ADCC和CDC的抗体，其中由抗体2得到的抗体命名为HRP00052。
下述基因序列SEQ ID NO:16和18的最后三位核苷酸“TGA”为终止密码子，不编码任何氨基酸。
HRP00052抗体重链序列
Figure PCTCN2018086866-appb-000018
Figure PCTCN2018086866-appb-000019
                                                                  SEQ ID NO：15
HRP00052抗体重链序列编码基因序列
Figure PCTCN2018086866-appb-000020
                                                                  SEQ ID NO：16
HRP00052抗体轻链序列
Figure PCTCN2018086866-appb-000021
Figure PCTCN2018086866-appb-000022
                                                                  SEQ ID NO：17
HRP00052抗体轻链序列编码基因序列：
Figure PCTCN2018086866-appb-000023
                                                                  SEQ ID NO：18
注：划线部分为抗体重链或轻链的可变区序列，或编码其的核苷酸序列；未划线部分为抗体恒定区序列及其相应的编码核苷酸序列。
构建用于表达PD-L1抗体HRP00052的表达质粒，通过DNA序列分析，确认了编码重链和轻链的核苷酸序列以及它们各自的启动子和聚腺苷酸信号序列，随后使用所述表达载体转染CHO细胞系。基于生长和生产稳定性选择表达抗体的克隆，制备主种子库，然后使用该种子库来制备抗体和产生主细胞库。
在摇瓶、培养袋和种子生物反应器中繁殖来自主细胞库的细胞，将所得的种细胞利用生物反应器生产抗体产物。得到的抗体进一步通过蛋白A亲和色谱法、阳离子交换色谱法和阴离子交换色谱法纯化，以及低pH病毒灭活和过滤步骤清除病毒。具体纯化步骤如下：将细胞表达上清样品装载于PBS缓冲液平衡的Protein A柱(Merck，175118824)上，用PBS冲洗柱子，至A280读数降至基线，后用PB缓冲液冲洗柱子，洗去杂蛋白，再用50mM柠檬酸钠pH3.5洗脱目的蛋白并收集洗脱峰。用1M Tris中和后的蛋白上PB缓冲液平衡好的阴离子层析柱(GE，17-5316-10)进行纯化，收集流穿峰，用1M柠檬 酸回调至pH5.0。阴离子层析后蛋白上柠檬酸缓冲液(pH5.0)平衡好的阳离子层析柱(Merck，1.16882)进一步纯化，用含0.18M氯化钠的柠檬酸缓冲液(pH5.0)洗脱目的蛋白并收集洗脱峰，分装备用。
实施例2
以PD-L1制剂(1mg/ml)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。
表2.以Tm值为响应值的DSC结果
Figure PCTCN2018086866-appb-000024
以Tm值为响应值，根据实验结果，对模型进行拟合，R 2为0.999877，调整R 2为0.997913，方差分析P＝0.0348＜0.05，模型有效，结果可靠。
注：糖的浓度单位为g/100mL。
根据Tm值最大化原则，由Tm各因子主效应图(图3)初步筛选出的处方为：缓冲体系，醋酸(钠)较好，其次是琥珀酸(钠)；缓冲剂浓度20-30mM时，Tm值较高；pH5-5.6对Tm值无显著影响；糖浓度6％最好。
实施例3
将抗PD-L1抗体(HRP00052)分别配制为pH5.0-5.5的含10mM琥珀酸(钠)、醋酸(钠)，60mg/ml蔗糖，0.2mg/ml聚山梨醇酯20的制剂，蛋白浓度为50mg/ml。过滤并灌装每种制剂于中性硼硅玻璃管制注射剂瓶中，所述玻璃瓶用注射液用溴化丁基橡胶塞封口，进行2～8℃长期稳定性考察，样品稳定性通过表3所示的多种特征进行说明。
对样品于室温下以黑色背景在白色荧光灯下通过目测检查确定样品颜色，外观和清澈度。通过高效尺寸排阻色谱法(HP-SEC)进一步评估样品的纯度，其中测定单体的百分比以及高分子量物质(可能为聚集体)和晚期洗脱峰(可能为降解产物)的百分比。使用高效离子交换色谱法(HP-IEX)，通过揭示酸性或碱性变体的存在来评估纯度，将结果表示为总观察物质的百分比。通过CE-SDS技术分析样品，其中在还原和非还原条件下用十二烷基硫酸钠(SDS)将蛋白变性，并使用毛细管电泳(CE)进行分离。所述蛋白基于它们的表观分子量而分离。在非还原条件下，除了主要IgG峰以外的所有物质被归类为杂质。在还原条件下，IgG被拆分为重链和轻链，所有其它物质被归类为杂质。
结果显示抗PD-L1抗体在琥珀酸缓冲盐体系中的稳定性明显优于醋酸盐缓冲体系；抗PD-L1抗体在pH5.0–5.5均非常稳定。
表3.pH及缓冲盐体系对抗PD-L1抗体的2～8℃长期稳定性的影响
Figure PCTCN2018086866-appb-000025
Figure PCTCN2018086866-appb-000026
实施例4
将抗PD-L1抗体分别配制为10mM及20mM琥珀酸(钠)，pH5.2，60mg/ml蔗糖，0.2mg/ml聚山梨醇酯20的制剂，蛋白浓度为50mg/ml。过滤并灌装每种制剂于中性硼硅玻璃管制注射剂瓶中，所述玻璃瓶用注射液用溴化丁基橡胶塞封口，进行40℃加速和2～8℃长期稳定性研究考察。结果显示抗PD-L1抗体在10～20mM的琥珀酸缓冲体系中均非常稳定。
表4.不同缓冲体系浓度的制剂处方40℃稳定性结果
Figure PCTCN2018086866-appb-000027
表5.不同缓冲体系浓度的制剂处方2～8℃稳定性结果
Figure PCTCN2018086866-appb-000028
Figure PCTCN2018086866-appb-000029
实施例5
制备PD-L1抗体(HRP00052)浓度为50mg/ml，含20mM醋酸(钠)，pH5.2，60mg/ml蔗糖，含不同种类及浓度表面活性剂的抗PD-L1抗体制剂，过滤并灌装每种制剂于中性硼硅玻璃管制注射剂瓶中，所述玻璃瓶用注射液用溴化丁基橡胶塞封口，置于25℃恒温摇床上，以200rpm的速度振摇。
使用DLS(Dynamic Light Scattering)测量平均扩散系数，用于表征溶液中纳米级粒子的粒径大小和粒径分布，PDI(particle dispersion index)分布系数体现了粒子粒径均一程度，PDI越小，则粒径分布越窄，粒径越均一。
稳定性结果表明，0.1-0.3mg/ml聚山梨酯20或聚山梨酯80，有效防止了抗PD-L1抗体的聚集和团聚大颗粒物的形成。
表6.不同种类和浓度的聚山梨酯振摇实验结果
Figure PCTCN2018086866-appb-000030
Figure PCTCN2018086866-appb-000031
实施例6
制备PD-LI抗体(HRP00052)浓度为50mg/ml，含20mM琥珀酸(钠)，pH5.2，60mg/ml蔗糖，含不同种类及浓度表面活性剂的抗PD-L1抗体制剂，过滤并灌装每种制剂于中性硼硅玻璃管制注射剂瓶中，所述玻璃瓶用注射液用溴化丁基橡胶塞封口，置于2～8℃条件下进行稳定性实验。结果表明聚山梨酯80明显优于聚山梨酯20，且各个浓度之间无明显差异。
表7.不同聚山梨酯种类及浓度的制剂处方2～8℃稳定性结果
Figure PCTCN2018086866-appb-000032
Figure PCTCN2018086866-appb-000033

Claims (18)

  1. 一种药物组合物，其包含PD-L1抗体或其抗原结合片段，以及缓冲剂，所述缓冲剂优选为琥珀酸盐或醋酸盐缓冲剂，更优选为琥珀酸盐缓冲剂。
  2. 根据权利要求1所述的药物组合物，其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5.2。
  3. 根据权利要求1或2所述的药物组合物，其中所述缓冲剂浓度为大约5mM至50mM，优选为大约10mM至30mM，更优选为10mM至20mM，最优选为20mM。
  4. 根据权利要求1至3任一项所述的药物组合物，其中所述抗体浓度为大约30mg/ml至80mg/ml，优选为大约40mg/ml至60mg/ml，更优选为大约45mg/ml至55mg/ml，最优选为50mg/ml。
  5. 根据权利要求1至4任一项所述的药物组合物，其中还包括糖，所述糖优选为二糖，更优选自海藻糖或蔗糖，最优选为蔗糖。
  6. 根据权利要求5所述的药物组合物，其中所述糖浓度为大约30mg/ml至90mg/ml，优选为大约40mg/ml至80mg/ml，更优选为55mg/ml至65mg/ml，最优为60mg/ml。
  7. 根据权利要求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酯，更优选为聚山梨酯80或聚山梨酯20。
  8. 根据权利要求7所述的药物组合物，其中表面活性剂的浓度从大约0.1mg/ml至1.0mg/ml，优选为0.4mg/ml至0.8mg/ml，更优选为0.5mg/ml至0.7mg/ml，最优选为0.6mg/ml。
  9. 根据权利要求1至8任一项所述的药物组合物，其包含：
    (a)30-80mg/ml的PD-L1抗体或其抗原结合片段；
    (b)5-50mM的琥珀酸盐缓冲剂，pH5.0-6.0；
    (c)30-90mg/ml的二糖；和，
    (d)0.1-1.0mg/ml的聚山梨酯80；
    优选地，所述药物组合物包含：40-60mg/ml的PD-L1抗体或抗原结合片段，10-30mM的琥珀酸盐缓冲剂，pH5.0-5.5，40-80mg/ml的蔗糖，0.4-0.8mg/ml的聚山梨酯80；
    更优选地，所述药物组合物包含：45-55mg/ml的PD-L1抗体或其抗原结合片段，10-20mM的琥珀酸盐缓冲剂，pH5.0-5.5，55-65mg/ml的蔗糖，0.5-0.7mg/ml的聚山梨酯80。
  10. 根据权利要求9所述的药物组合物，其可选自以下组合物中的一种：
    药物组合物A，包含：50mg/ml的PD-L1抗体或抗原结合片段，20mM的琥珀酸盐缓冲剂，pH5.2，60mg/ml的蔗糖，0.4mg/ml的聚山梨酯80；
    药物组合物B，包含：50mg/ml的PD-L1抗体或其抗原结合片段，20mM的琥珀酸盐缓冲剂，pH5.2，60mg/ml的蔗糖，0.6mg/ml的聚山梨酯80；
    药物组合物C，包含：50mg/ml的PD-L1抗体或其抗原结合片段，20mM的琥珀酸盐缓冲剂，pH5.2，60mg/ml的蔗糖，0.8mg/ml的聚山梨酯80；
    药物组合物D，包含：50mg/ml的PD-L1抗体或其抗原结合片段，20mM的琥珀酸盐缓冲剂，pH5.5，60mg/ml的蔗糖，0.6mg/ml的聚山梨酯80；
    药物组合物E，包含：50mg/ml的PD-L1抗体或抗原结合片段，20mM的琥珀酸盐缓冲剂，pH5.8，60mg/ml的蔗糖；0.6mg/ml的聚山梨酯80。
  11. 根据权利要求1至10任一项所述的药物组合物，其中所述的抗体或其抗原结合片段包含任意1个选自以下的CDR区序列或其突变序列：抗体重链可变区HCDR区序列：SEQ ID NO：1-3以及SEQ ID NO：7-9；和/或，抗体轻链可变区LCDR区序列：SEQ ID NO：4-6以及SEQ ID NO：10-12；优选地，
    HCDR1选自：NDYWX1                    SEQ ID NO：1
    或         SYWMH                     SEQ ID NO：7，
    HCDR2选自：YISYTGSTYYNPSLKS          SEQ ID NO：2
    或         RI X4PNSGX5TSYNEKFKN     SEQ ID NO：8，和/或
    HCDR3选自：SGGWLAPFDY               SEQ ID NO：3
    或         GGSSYDYFDY               SEQ ID NO：9；和/或
    LCDR1选自：KSSQSLFYX2SNQKX3SLA      SEQ ID NO：4
    或         RASESVSIHGTHLMH          SEQ ID NO：10，
    LCDR2选自：GASTRES                  SEQ ID NO：5
    或         AASNLES                  SEQ ID NO：11，和/或
    LCDR3选自：QQYYGYPYT                SEQ ID NO：6
    或         QQSFEDPLT                SEQ ID NO：12；
    其中X1选自N或T，X2选自R或H，X3选自N或H，X4选自H或G，X5选自G或F。
  12. 根据权利要求11所述的药物组合物，其中所述的抗体或其抗原结合片段包含选自：SEQ ID NO：10，SEQ ID NO：11，SEQ ID NO：12的轻链可变区CDR序列或其突变序列，和选自：SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：9的重链可变区CDR 序列或其突变序列；优选地，所述的抗体或其抗原结合片段包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1、LCDR2和LCDR3序列，以及分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：9所示的HCDR1、HCDR2和HCDR3序列；
    或，所述的抗体或其抗原结合片段包含选自：SEQ ID NO：1，SEQ ID NO：2，SEQ ID NO：3的重链可变区CDR序列或其突变序列，和选自：SEQ ID NO：4，SEQ ID NO：5和SEQ ID NO：6的轻链可变区CDR序列或其突变序列；优选地，所述的抗体或其抗原结合片段包含分别如SEQ ID NO：1，SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3序列，以及分别如SEQ ID NO：4，SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3序列。
  13. 根据权利要求1至12任一项所述的药物组合物，其中所述的抗体或其抗原结合片段选自鼠源抗体、嵌合抗体、人源化抗体，人抗体，优选人源化抗体。
  14. 根据权利要求12所述的药物组合物，其中所述的抗体或其抗原结合片段的重链可变区序列为SEQ ID NO：13，轻链可变区序列为SEQ ID NO：14。
  15. 根据权利要求12所述的药物组合物，其中所述的抗体或其抗原结合片段重链序列为SEQ ID NO：15，轻链序列为SEQ ID NO：17。
  16. 制备权利要求1至15任一项所述的药物组合物的方法，包括将PD-L1抗体或抗原结合片段与药学上可接受的赋形剂混合。
  17. 根据权利要求1至15任一项所述的药物组合物在制备用于治疗PD-L1介导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PD-L1的癌症；最优选为乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤、非小细胞肺癌；进一步优选为非小细胞肺癌、黑素瘤、膀胱癌和肾癌。
  18. 一种治疗和预防PD-L1介导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权利要求1至15任一项所述的药物组合物；其中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、非小细胞肺癌、膀胱癌；最优选为非小细胞肺癌、黑素瘤、膀胱癌和肾癌。
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CN113518823A (zh) * 2019-01-07 2021-10-19 托马斯杰斐逊大学 多功能的融合蛋白及其用途
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WO2023131213A1 (zh) * 2022-01-05 2023-07-13 上海迈晋生物医药科技有限公司 一种包含抗FXI/FXIa抗体的药物组合物及其用途

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