WO2019120269A1 - Lag-3抗体药物组合物及其用途 - Google Patents

Lag-3抗体药物组合物及其用途 Download PDF

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WO2019120269A1
WO2019120269A1 PCT/CN2018/122534 CN2018122534W WO2019120269A1 WO 2019120269 A1 WO2019120269 A1 WO 2019120269A1 CN 2018122534 W CN2018122534 W CN 2018122534W WO 2019120269 A1 WO2019120269 A1 WO 2019120269A1
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Prior art keywords
seq
variable region
chain variable
antibody
lag
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PCT/CN2018/122534
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English (en)
French (fr)
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吴婷婷
李皓
刘洵
付雅媛
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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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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to AU2018391217A priority Critical patent/AU2018391217A1/en
Priority to CN201880059038.2A priority patent/CN111356476B/zh
Priority to RU2020118313A priority patent/RU2771384C2/ru
Priority to US16/954,176 priority patent/US20220031842A1/en
Priority to CA3085656A priority patent/CA3085656A1/en
Priority to JP2020534482A priority patent/JP2021506922A/ja
Priority to EP18892762.8A priority patent/EP3714901A4/en
Priority to BR112020011664-5A priority patent/BR112020011664A2/pt
Priority to KR1020207018644A priority patent/KR20200104314A/ko
Publication of WO2019120269A1 publication Critical patent/WO2019120269A1/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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K47/14—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
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • 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
    • 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
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention is in the field of pharmaceutical preparations, and in particular, the present invention relates to a pharmaceutical composition comprising a LAG-3 antibody and an antigen-binding fragment thereof, and the use thereof as a medicament.
  • Lymphocyte activation gene-3 (also known as LAG-3, CD215) is a member of the immunoglobulin superfamily, which negatively regulates the functions and life cycle of immune cells. Studies have shown that LAG-3 plays an important role in viral infection, autoimmune diseases, and immune system dysfunction caused by tumors. By affecting the function of LAG-3, immune dysfunction can be improved during the evolution of these diseases. Improve the prognosis of the disease.
  • LAG-3 As a member of the immunoglobulin superfamily, LAG-3 is composed of three parts: the extracellular region, the transmembrane region and the cytoplasmic region.
  • the mature LAG-3 molecule consists of 470 amino acids with a relative molecular weight of 70 kDa, first discovered by Triebel et al. in 1990 (J Exp Med, 1990, 171(5): 1393-405). The study found that LAG-3, like CTLA-4 and PD-1, is a negative costimulatory molecule whose activation can negatively regulate lymphocyte function.
  • LAG-3 is structurally closely related to CD4, but has a diametrically opposite function to CD4, showing a high similarity between LAG-3 and CD4 molecules, and can be compared with MHC-II (major histocompatibility complex). Class of molecular binding. However, LAG-3 binds more closely to MHC class II molecules, thereby interfering with TCR activation of CD4 + T lymphocytes and inhibiting T lymphocyte activation (Curr Opin Immunol, 2009, 21(2): 179-86; Eur J Immunol, 2003, 33(4): 970-9).
  • LAG-3 can inhibit antigen-induced T lymphocyte proliferative response, while blocking LAG-3, T lymphocyte activation and proliferation, type 1 T helper cells (Th1) secretion
  • Th1 type 1 T helper cells
  • anti-LAG-3 antibodies also maintain CD4 + and CD8 + T lymphocyte homeostasis, and the ability of CD8 + T lymphocytes to kill tumor cells after LAG-3 is significantly enhanced (J Clin Invest, 2007, 117(11) :3383-92).
  • LAG-3 plays an important role in regulating the development of disease.
  • Singer et al. confirmed that the expression level of T lymphocyte LAG-3 in human lymphoma tissues is associated with T lymphocyte dysfunction. Clearing LAG-3 + T lymphocytes can significantly enhance lymphocyte tumor clearance (Blood, 2006, 108 (7) ): 2280-9).
  • the results show that LAG-3 is an important inhibitory molecule on the surface of immune cells and can produce significant negative regulation of T lymphocytes.
  • LAG-3 is mainly expressed on T lymphocytes, B lymphocytes, NK cells, Treg cells, and DCs (Proc Natl Acad Sci USA, 1997, 94(11): 5744-9. Eur J Immunol, 2005, 35 ( 7): 2081-8; J Immunol, 2009, 182(4): 1885-91).
  • LAG-3 is a class of immunosuppressive molecules and is one of the co-receptor components of TCR. It interferes with TCR activation of T lymphocytes and plays a negative regulatory role in T lymphocyte activation. In some diseases, LAG-3 expression is elevated and corresponding immunosuppression occurs.
  • a number of multinational pharmaceutical companies such as BMS and Novartis are currently developing monoclonal antibodies against LAG-3, which stimulate the antigen-specific T cell response and enhance the anti-tumor effect of T cells, thereby maximizing the patient's own
  • the tumor's immune system responds to the purpose of killing tumor cells.
  • antibody drugs are unstable due to their large molecular weight, complex structure, easy degradation, polymerization, or undesired chemical modification.
  • stable formulation studies of antibody drugs are particularly important.
  • LAG3 antibodies and their preparations such as: WO2018204374, WO2010019570, WO2014008218, WO9530750, WO2004078928, WO2008132601, WO2014140180, WO2015138920, etc., but there are not many studies on the new LAG3 antibody preparation, so still There is a need to develop pharmaceutical compositions (formulations) comprising LAG3 that are more suitable for administration.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a LAG3 antibody or antigen-binding fragment thereof, and a buffer selected from the group consisting of acetate buffer, histidine buffer, citrate buffer, succinate Buffer or Tris buffer.
  • the acetate buffer described in the pharmaceutical composition is selected from the group consisting of acetic acid-sodium acetate buffer, acetic acid-potassium acetate buffer, acetate-histidine buffer, and acetic acid-calcium acetate buffer.
  • Agent acetic acid-magnesium acetate buffer, preferably acetic acid-sodium acetate buffer.
  • the histidine buffer described in the pharmaceutical composition is selected from the group consisting of histidine-hydrochloric acid buffer, histidine-acetate buffer, histidine-phosphate buffer, histidine- Sulfuric acid buffer, preferably histidine-hydrochloric acid buffer.
  • the citrate buffer described in the pharmaceutical composition is selected from the group consisting of citric acid-sodium citrate buffer; the succinate buffer is selected from the group consisting of sodium succinate-succinate buffer.
  • the concentration of the LAG-3 antibody or antigen-binding fragment thereof in the pharmaceutical composition is from about 1 mg/ml to 90 mg/ml, preferably from about 10 mg/ml to 90 mg/ml, preferably from about 20 mg/ml to 90 mg/ml, preferably from about 30 mg/ml to 90 mg/ml, preferably from about 40 mg/ml to 90 mg/ml, preferably from about 50 mg/ml to 90 mg/ml, preferably from about 60 mg/ml to 90 mg/ml, preferably from about 70 mg/ml to 90 mg/ml, preferably from about 80 mg/ml to 90 mg/ml, preferably from about 10 mg/ml to 80 mg/ml, preferably from about 20 mg/ml to 80 mg/ml, preferably from about 30 mg/ml to 80 mg/ml, preferably from about 40 mg/ml to 80 mg/ml, preferably from about 50 mg/ml to 80 mg/ml, preferably from about 60 mg/ml,
  • the concentration of the LAG-3 antibody or antigen-binding fragment thereof is about 40 mg/ml, 41 mg/ml, 42 mg/ml, 43 mg/ml, 44 mg/ml, 45 mg/ml, 46 mg/ml, 47 mg. /ml, 48mg/ml, 49mg/ml, 50mg/ml, 51mg/ml, 52mg/ml, 53mg/ml, 54mg/ml, 55mg/ml, 56mg/ml, 57mg/ml, 58mg/ml, 59mg/ml Or 60 mg/ml, most preferably 50 mg/ml.
  • the concentration of the buffer is from about 5 mM to 30 mM, preferably from about 10 mM to 30 mM, preferably from about 15 mM to 30 mM, preferably from about 20 mM to 30 mM, preferably from about 25 mM to 30 mM, preferably about 5 mM.
  • Up to 25 mM preferably about 10 mM to 25 mM, preferably about 15 mM to 25 mM, preferably about 20 mM to 25 mM, preferably about 5 mM to 20 mM, preferably about 10 mM to 15 mM; a non-limiting example of the buffer concentration is About 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, 26 mM, 28 mM or 30 mM, most preferably 10 mM.
  • the pH of the buffer in the pharmaceutical composition is from about 5.0 to 7.5, preferably from about 5.5 to 7.5, preferably from about 6.0 to 7.5, preferably from about 6.5 to 7.5, preferably about 7.0.
  • To 7.5 preferably about 5.0 to 7.0, preferably about 5.5 to 7.0, preferably about 6.0 to 7.0, preferably about 6.5 to 7.0, preferably about 5.0 to 6.5, preferably about 5.5 to 6.5, preferably about 6.0 to 6.5.
  • the non-limiting embodiment of the buffer pH may alternatively be about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, more preferably from about 5.5 to 6.0, still more preferably 5.5 or 6.0.
  • the pharmaceutical composition further comprises an excipient selected from one or more of a sugar and a surfactant.
  • the sugar is a disaccharide, preferably trehalose, sucrose, mannitol or sorbitol, more preferably sucrose.
  • the concentration of the sugar in the pharmaceutical composition is from about 30 mg/ml to 90 mg/ml, preferably from about 60 mg/ml to 90 mg/ml, from 35 mg/ml to 90 mg/ml, preferably about 40 mg/ml.
  • Up to 90 mg/ml preferably from about 45 mg/ml to 90 mg/ml, preferably from about 50 mg/ml to 90 mg/ml, preferably from about 55 mg/ml to 90 mg/ml, preferably from about 60 mg/ml to 90 mg/ml, preferably From about 65 mg/ml to 90 mg/ml, preferably from about 70 mg/ml to 90 mg/ml, preferably from about 75 mg/ml to 90 mg/ml, preferably from about 80 mg/ml to 90 mg/ml, preferably from about 85 mg/ml to 90 mg/ml, preferably from about 30 mg/ml to 85 mg/ml, preferably from about 35 mg/ml to 85 mg/ml, preferably from about 40 mg/ml to 85 mg/ml, preferably from about 45 mg/ml to 85 mg/ml, preferably about From 50 mg/ml to 85 mg/ml, preferably from about 55 mg/ml to 85 mg/ml, preferably from about 60 mg/
  • Up to 70 mg/ml preferably from about 35 mg/ml to 70 mg/ml, preferably from about 40 mg/ml to 70 mg/ml, preferably from about 45 mg/ml to 70 mg/ml, preferably from about 50 mg/ml to 70 mg/ml, preferably From about 55 mg/ml to 70 mg/ml, preferably from about 60 mg/ml to 70 mg/ml, preferably from about 65 mg/ml to 70 mg/ml, preferably from about 30 mg/ml to 65 mg/ml, preferably from about 35 mg/ml to 65 mg/ml, preferably from about 40 mg/ml to 65 mg/ml, preferably from about 45 mg/ml to 65 mg/ml, preferably from about 50 mg/ml to 65 mg/ml, preferably from about 55 mg/ml to 65 mg/ml, preferably about From 60 mg/ml to 65 mg/ml, preferably from about 30 mg/ml to 60 mg/ml, preferably from about 35 mg/
  • the pharmaceutical composition further comprises a surfactant.
  • the surfactant may be selected from the group consisting of polysorbate 20, polysorbate 80, polyhydroxyl hydrocarbon, Triton, sodium dodecyl sulfate, sodium lauryl sulfonate, sodium octyl sulphate, lauryl-sulfo Betaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleic-muscle Acid, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, laurylpropyl-betaine, cocamidopropyl-betaine , linoleidopropyl-betaine, myristylpropyl-betaine, palmitoyl
  • the concentration of the surfactant in the pharmaceutical composition is from about 0.02 mg/ml to about 0.8 mg/ml, preferably from about 0.1 mg/ml to about 0.8 mg/ml, preferably from about 0.2 mg/ml to about 0.8 mg.
  • /ml preferably from about 0.3 mg/ml to 0.8 mg/ml, preferably from about 0.4 mg/ml to 0.8 mg/ml, preferably from about 0.5 mg/ml to 0.8 mg/ml, preferably from about 0.6 mg/ml to 0.8 mg/ml
  • it is from about 0.7 mg/ml to 0.8 mg/ml, preferably from about 0.02 mg/ml to 0.7 mg/ml, preferably from about 0.1 mg/ml to 0.7 mg/ml, preferably from about 0.2 mg/ml to 0.7 mg/ml
  • it is from about 0.3 mg/ml to 0.7 mg/ml, preferably from about 0.4 mg/ml to 0.7 mg/ml, preferably from about 0.5 mg/ml to 0.7 mg/ml, preferably from about 0.6 mg/ml to 0.7 mg/ml, preferably about It is from 0.02 mg/ml to 0.6 mg/ml, preferably from about 0.1 mg/
  • From mg/ml to 0.6 mg/ml preferably from about 0.5 mg/ml to 0.6 mg/ml, preferably from about 0.02 mg/ml to 0.5 mg/ml, preferably from about 0.1 mg/ml to 0.5 mg/ml, preferably about 0.2 mg /ml to 0.5 mg/ml, preferably from about 0.3 mg/ml to 0.5 mg/ml, preferably from about 0.4 mg/ml to 0.5 mg/ml, preferably from about 0.02 mg/ml to 0.4 mg/ml, preferably about 0.1 mg/ Ml to 0.4 mg/ml, preferably from about 0.2 mg/ml to 0.4 mg/ml, preferably about 0.3 mg/ Mol to 0.4 mg/ml, preferably about 0.02 mg/ml to 0.3 mg/ml, preferably about 0.1 mg/ml to 0.3 mg/ml, preferably about 0.2 mg/ml to 0.3 mg/ml, preferably about 0.02 mg/ml to 0.3 mg/
  • the pharmaceutical composition comprises the components shown in i) or) ii) below:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the LAG3 antibody or antigen-binding fragment thereof described in the pharmaceutical composition comprises the heavy chain HCDR1, HCDR2 and SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively.
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • a LAG-3 antibody or antigen-binding fragment thereof from 50 mg/ml to 60 mg/ml, (b) a 10 mM to 30 mM acetic acid-sodium acetate buffer, and a pH of about 5.2-5.8.
  • the pharmaceutical composition comprises:
  • the LAG-3 antibody or antigen-binding fragment thereof in the above pharmaceutical composition is a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen thereof Combine the fragments.
  • the LAG3 murine antibody of the above pharmaceutical composition comprises the heavy chain variable region set forth in SEQ ID NO: 5 and the light chain variable region set forth in SEQ ID NO: .
  • the LAG-3 humanized antibody of the above pharmaceutical composition comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region amino acid sequence is SEQ ID An amino acid sequence of NO: 21, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or having at least 85% sequence identity to it;
  • the light chain variable region is an amino acid sequence of any one of SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28 or having at least 85% sequence identity thereto.
  • the light chain variable region of the LAG-3 antibody in the pharmaceutical composition is set forth in SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28
  • the light chain amino acid variable region sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or 100% sequence identity
  • the antibody heavy chain variable region shown at 25 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or 100% sequence identity.
  • the LAG3 humanized antibody described in the above pharmaceutical composition comprises a combination of a heavy chain variable region and a light chain variable region selected from the group consisting of:
  • the LAG-3 humanized antibody of the above pharmaceutical composition comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is preferably as set forth in SEQ ID NO:38
  • the light chain constant region is preferably as set forth in SEQ ID NO:39.
  • the heavy chain of the LAG-3 humanized antibody described in the above pharmaceutical composition is selected from the sequence set forth in SEQ ID NO: 40 or has at least 95% sequence identity amino acid sequence thereto.
  • the light chain is selected from the sequence set forth in SEQ ID NO: 41 or has at least 95% sequence identity amino acid sequence therewith.
  • the heavy chain of the LAG-3 antibody in the pharmaceutical composition has at least 95%, 96%, 97%, 98%, 99% of the heavy chain amino acid sequence set forth in SEQ ID NO:40. Or 100% sequence identity, the light chain amino acid sequence of the LAG-3 antibody having at least 95%, 96%, 97%, 98%, 99% or 100% of the antibody light chain set forth in SEQ ID NO:41 Sequence identity.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu229-013, 10 mM acetic acid-sodium acetate buffer, pH 5.5.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu229-013, 10 mM sodium succinate-succinate buffer, pH 6.0.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu229-013, 10 mM histidine-hydrochloric acid buffer, pH 6.0.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu229-013, 10 mM sodium succinate-succinate, pH 6.0 and 0.1 mg/ml polysorbate 80.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu229-013, 10 mM sodium succinate-succinate, pH 6.0, and 70 mg/ml sucrose.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu229-013, 10 mM acetic acid-sodium acetate, pH 5.5, 60 mg/ml sucrose, and 0.4 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu229-013, 10 mM histidine-acetic acid, pH 6.0, 60 mg/ml sucrose, and 0.4 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 1 mg/ml LAG3 antibody Hu229-013, 10-30 mM histidine-acetic acid, pH 5.5, 75 mg/ml sucrose, and 0.2 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu229-013, 10-30 mM acetic acid-sodium acetate, pH 5.2-5.8, 75 mg/ml sucrose, and 0.2 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 60 mg/ml LAG3 antibody Hu229-013, 10 mM acetic acid-sodium acetate, pH 5.5, 60 mg/ml sucrose, and 0.4 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 50-60 mg/ml of LAG3 antibody Hu229-013, 10 mM acetic acid-sodium acetate, pH 5.5, 30-90 mg/ml sucrose, and 0.4-0.5 mg/mL polysorbate 80. .
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu229-013, 10 mM acetic acid-sodium acetate, pH 5.5, 75 mg/ml sucrose, and 0.4 mg/mL polysorbate 80.
  • the LAG-3 antibody or antigen-binding fragment thereof of the pharmaceutical composition comprises HCDR1, HCDR2 as set forth in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively. And the HCDR3 region and the LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively.
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the LAG-3 antibody or antigen-binding fragment thereof described in the above pharmaceutical composition is a murine antibody or fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding thereof Fragment.
  • the LAG3 murine antibody of the above pharmaceutical composition comprises the heavy chain variable region set forth in SEQ ID NO: 7 and the light chain variable region set forth in SEQ ID NO: .
  • the LAG-3 humanized antibody of the above pharmaceutical composition comprises:
  • a heavy chain variable region sequence selected from any one of SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33 or an amino acid sequence and selection thereof having at least 85% sequence identity a light chain variable region sequence as set forth in any one of SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37 or having at least 85% sequence identity thereto Amino acid sequence.
  • the light chain variable region of the LAG-3 antibody in the pharmaceutical composition is SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ
  • the light chain amino acid variable region sequence set forth in ID37 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or 100% sequence identity
  • the antibody heavy chain variable region set forth in SEQ ID NO: 33 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% or 100% sequence identity.
  • the LAG3 humanized antibody described in the above pharmaceutical composition comprises a combination of a heavy chain variable region and a light chain variable region selected from the group consisting of:
  • the chimeric antibody or humanized antibody heavy chain in the above pharmaceutical composition further comprises a heavy chain constant region of human IgGl, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a human source
  • the heavy chain constant region of IgG4 or a variant thereof most preferably the heavy chain constant region set forth in SEQ ID NO: 38
  • the chimeric antibody or humanized antibody light chain further comprises human ⁇ , ⁇ chain or variant thereof
  • the light chain constant region of the body is preferably a light chain constant region as set forth in SEQ ID NO:39.
  • the heavy chain of the LAG-3 humanized antibody described in the above pharmaceutical composition is selected from the sequence set forth in SEQ ID NO: 42 or has at least 95% sequence identity amino acid sequence thereto.
  • the light chain is selected from the sequence set forth in SEQ ID NO: 43 or has at least 95% sequence identity amino acid sequence therewith.
  • the heavy chain of the LAG-3 antibody in the pharmaceutical composition has at least 95%, 96%, 97%, 98%, 99% of the heavy chain amino acid sequence set forth in SEQ ID NO:42. Or 100% sequence identity, the light chain amino acid sequence of the LAG-3 antibody having at least 95%, 96%, 97%, 98%, 99% or 100% of the antibody light chain set forth in SEQ ID NO:43 Sequence identity.
  • the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu303-005, 10 mM histidine-hydrochloric acid buffer, pH 6.0.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu303-005, 10 mM histidine-hydrochloric acid buffer, pH 5.0.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu303-005, 10 mM histidine-hydrochloric acid buffer, pH 6.5.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu303-005, 10 mM acetic acid-sodium acetate buffer, pH 5.5.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu303-005, 10 mM acetic acid-sodium acetate buffer, pH 5.5, 75 mg/ml sucrose, and 0.2 mg/ml polysorbate 80.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu303-005, 10 mM acetic acid-sodium acetate buffer, pH 5.5, 75 mg/ml trehalose, and 0.2 mg/ml polysorbate 80.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu303-005, 10 mM acetic acid-sodium acetate buffer, pH 5.5, and 0.4 mg/ml polysorbate 80.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu303-005, 10 mM histidine-hydrochloric acid buffer, pH 6.0, 75 mg/ml sucrose, and 0.4 mg/ml polysorbate 80.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu303-005, 10 mM histidine-HCl buffer, pH 6.5, 75 mg/ml sucrose, and 0.4 mg/ml polysorbate 80.
  • the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu303-005, 10 mM histidine-hydrochloric acid buffer, pH 5.5, 75 mg/ml sucrose, and 0.4 mg/ml polysorbate 80.
  • the pharmaceutical composition comprises: 45-60 mg/ml of LAG3 antibody Hu303-005, 10 mM histidine-hydrochloric acid buffer, pH 5.5, 75 mg/ml sucrose, and 0.2-0.6 mg/ml polysorbate 80.
  • the pharmaceutical composition comprises: about 50 mg/ml of LAG3 antibody Hu303-005, about 10 mM histidine-hydrochloric acid buffer, about pH 6.0, about 75 mg/ml sucrose, and about 0.3 mg/ml poly Sorbate 80.
  • the invention also provides a method of preparing a pharmaceutical composition comprising a LAG-3 antibody comprising mixing a LAG-3 antibody or antigen-binding fragment with a pharmaceutically acceptable excipient.
  • the present invention also provides a method of preparing a lyophilized preparation containing a LAG-3 antibody, which comprises the step of freeze-drying the aforementioned pharmaceutical composition.
  • the method of preparing a lyophilized formulation comprising a LAG-3 antibody comprises the steps of pre-freezing, primary drying, and secondary drying, in sequence.
  • the method of preparing a lyophilized formulation comprising a LAG-3 antibody has a primary drying temperature of from -5 °C to -20 °C, preferably -10 °C.
  • the present invention also provides a lyophilized preparation containing the LAG-3 antibody prepared by the aforementioned method for preparing a lyophilized preparation containing a LAG-3 antibody.
  • the lyophilized 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 lyophilized formulation is stable at 40 ° C for at least 7 days, at least 14 days or at least 28 days.
  • the present invention also provides a lyophilized preparation containing the LAG-3 antibody prepared by the above lyophilization method.
  • the present invention also provides a lyophilized preparation containing a LAG3 antibody, characterized in that the above pharmaceutical composition can be formed after the preparation is reconstituted.
  • the present invention also provides a method for preparing a reconstituted solution of a lyophilized preparation containing a LAG-3 antibody, which comprises the step of reconstituting the aforementioned lyophilized preparation, wherein the solution for reconstitution is selected from, but not limited to, water for injection, physiological saline or Glucose solution.
  • the present invention also provides a reconstituted solution of the obtained lyophilized preparation containing the LAG-3 antibody by the method of reconstituting a lyophilized preparation containing the LAG-3 antibody.
  • the present invention also provides a reconstituted solution comprising a LAG3 antibody, which further comprises the following components:
  • the present invention also provides a reconstituted solution comprising a LAG3 antibody, which further comprises the following components:
  • the present invention also provides a reconstituted solution comprising a LAG3 antibody, which further comprises the following components:
  • the present invention also provides a reconstituted solution comprising a LAG3 antibody, which further comprises the following components:
  • the present invention also provides a reconstituted solution comprising a LAG3 antibody, which further comprises the following components:
  • the invention further provides an article or kit comprising a container containing any of the stable pharmaceutical compositions described herein.
  • the vial is a neutral borosilicate glass controlled injection vial.
  • the reconstituted solution of the aforementioned pharmaceutical composition or lyophilized preparation or lyophilized preparation of the present invention can be used as a medicament.
  • the present invention also provides the use of the aforementioned pharmaceutical composition or a reconstituted solution of a lyophilized preparation or a lyophilized preparation for the preparation of a medicament for treating a LAG-3 related disease or condition, wherein the disease or condition is pathogenic A related disease or condition in which a sexual T cell is involved, preferably a cancer;
  • the cancer includes, but is not limited to, ovarian cancer, melanoma, prostate cancer, intestinal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, Uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain cancer, testicular cancer, skin cancer, thyroid cancer, and hematological malignancies, including hematological malignancies including myeloma and chronic and acute leukemia.
  • the invention also provides a method of treating and preventing a disease or condition associated with LAG-3 comprising administering to a subject in need thereof a therapeutically effective amount of a reconstituted solution of the aforementioned pharmaceutical composition or lyophilized formulation or lyophilized formulation, wherein said
  • the disease or condition is a related disease or condition in which pathogenic T cells are involved, preferably a cancer; the cancer includes, but is not limited to, ovarian cancer, melanoma, prostate cancer, intestinal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer , kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain cancer, testicular cancer, skin cancer, thyroid cancer, and hematological malignancy, wherein the hematological malignant tumor includes bone marrow Tumors and chronic and acute leukemia.
  • the present invention also provides an article comprising a container containing the aforementioned pharmaceutical composition or a reconstituted solution of a lyophilized preparation or a lyophilized preparation.
  • FIG. 1 Effect of humanized anti-LAG-3 antibody samples on secretion of IL-2 cytokines by SEB-stimulated activated T lymphocytes.
  • the data showed that the LAG-3 humanized candidate antibodies Hu229-013 and Hu303-005 could enhance the secretion of cytokine IL-2 by activated T lymphocytes to varying degrees, and there was a drug concentration dose effect.
  • FIG. 2 Effect of humanized anti-LAG-3 antibody on tumor volume of U-87MG tumor-bearing mice. The results showed that after 14 days of administration, LAG-3 antibodies Hu229-013 6mpk and Hu303-005 6mpk had certain anti-tumor effects, and the tumor inhibition rates were 27.25% (p ⁇ 0.05) and 34.94% (p ⁇ 0.01), respectively. Significantly different from the control group (p ⁇ 0.001 vs hIGg)
  • Figure 3 Trend profile of Hu229-013 antibody at 40 ° C CE purity.
  • Figure 4 Trend of the IEC neutral peak of the Hu229-013 antibody at 40 °C.
  • Figure 5 Trend diagram of non-reduced CE of Hu303-005 antibody at 40 °C.
  • Figure 6 Trend diagram of the main peak of 40°CiCE of Hu303-005 antibody.
  • Figure 7 Trend graph of Hu303-005 shaking SEC results.
  • Figure 8 Fitting results of Hu303-005 antibody with IEC 0 °C and 40 °C data.
  • Figure 9 Fitted map of Hu303-005 antibody with CE purity at 0 °C and 40 °C data.
  • Figure 10 Results of the 25°C and 40°CiCE/CE/DLS fitting of the Hu303-005 antibody preparation.
  • Buffer refers to a buffer that is resistant to changes in pH by the action of its acid-base conjugate component.
  • Examples of the buffer which controls the pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, Glycyl glycine and other organic acid buffers.
  • a "histidine buffer” is a buffer containing a histidine ion.
  • the histidine buffer include a histidine-hydrochloride, a histidine-acetate, a histidine-phosphate, a histidine-sulfate buffer, and the like, preferably a histidine-hydrochloride buffer.
  • Agent. The histidine-hydrochloride buffer is prepared by histidine and hydrochloric acid or histidine and histidine hydrochloride.
  • citrate buffer is a buffer that includes citrate ions.
  • the citrate buffer include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like.
  • a preferred citrate buffer is a citric acid-sodium citrate buffer.
  • a “succinate buffer” is a buffer that includes succinic acid ions.
  • the succinate buffer include sodium succinate-succinate, potassium succinate-succinate, calcium succinate-succinate, and the like.
  • a preferred succinate buffer is a sodium succinate-succinate buffer.
  • a “phosphate buffer” is a buffer that includes phosphate ions.
  • the phosphate buffer include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and the like.
  • a preferred phosphate buffer is dihydrogen phosphate-sodium dihydrogen phosphate buffer.
  • Acetate buffer is a buffer that includes acetate ions.
  • examples of the acetate buffer include acetic acid-sodium acetate, acetic acid-histidine salt, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like.
  • a preferred acetate buffer is an acetic acid-sodium acetate buffer.
  • Tris buffer refers to a buffer solution containing tris(hydroxymethyl)aminomethane, also known as Tris base, Trizma, Trisamine, THAM, tromethamine, tromethamine, and tromethamine.
  • the effective buffering range of the Tris buffer is between pH 7.0 and 9.2, and the pH of the aqueous solution of the Tris base is about 10.5.
  • hydrochloric acid is added to adjust the pH to a desired value to obtain a buffer of the pH.
  • the "sugar” of the present invention comprises a conventional composition (CH 2 O) 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 sucrose.
  • Viscosity modifier is a conventional pharmaceutical material added to adjust the viscosity of the preparation.
  • the viscosity modifier herein refers mainly to inorganic salts and amino acid salts, wherein the inorganic salts are preferably selected from sodium chloride, calcium chloride, magnesium chloride. Calcium acetate, the amino acid salt is preferably selected from the group consisting of arginine hydrochloride, histidine hydrochloride, glycine hydrochloride, histidine acetate and the like.
  • “Pharmaceutical composition” means a mixture comprising one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiological/pharmaceutically acceptable Carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • “pharmaceutical composition” and “formulation” are not mutually exclusive.
  • the solution form of the pharmaceutical composition of the present invention is water unless otherwise specified.
  • Freeze-dried formulation means a formulation or pharmaceutical composition obtained after a vacuum freeze-drying step of a pharmaceutical or solution formulation in liquid or solution form.
  • Freeze drying of the present disclosure includes pre-freezing, primary drying, and secondary drying.
  • the purpose of pre-freezing is to freeze the product to obtain a crystalline solid.
  • the pre-freezing temperature and the pre-freezing speed are two important process parameters.
  • the pre-freezing temperature is set to -45 ° C
  • the pre-freezing speed is set to 1 ° C / min.
  • One drying, also known as main drying is the main stage of sample freeze drying. The goal is to remove the ice from the product while maintaining the shape of the product, minimizing damage to the product. If the temperature and vacuum of one drying are not properly selected, it will cause the product to collapse. Higher temperatures and vacuums will accelerate lyophilization efficiency, but at the same time increase the risk of product collapse.
  • the temperature of the primary drying of the present invention may be a temperature conventional in the art, for example, -30 ° C to 0 ° C.
  • Secondary drying also known as analytical drying, is the primary step in removing bound water from an article by pumping an ultimate vacuum (0.01 mbar) and elevated temperature (20-40 °C). Since most biologics are sensitive to temperature, the secondary drying temperature is chosen to be at the low end of the temperature range, ie 25 °C.
  • the freeze-drying time is related to the freezer, the lyophilized formulation dose, and the lyophilized drug container. Such adjustments in time are well known to those skilled in the art.
  • the term "about” as used herein is an index of values within an acceptable tolerance of a particular value as determined by one of ordinary skill in the art, depending in part on how the measurement or measurement is made (ie, the limits of the measurement system). For example, “about” in each implementation of the art may mean a standard deviation of one or more than one. Alternatively, “about” or “substantially encompasses” may mean a range of up to 20%, for example, a pH of about 5.5 means pH 5.5 ⁇ 1.1. Moreover, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. The meaning of "about” or “substantially encompasses” should be assumed to be within the acceptable tolerance of the particular value, unless stated otherwise.
  • the pharmaceutical composition of the present invention is capable of achieving a stable effect in which the antibody substantially retains its physical stability and/or chemical stability and/or biological activity after storage, preferably, the pharmaceutical composition is stored. Substantially retains its physical and chemical stability as well as its biological activity.
  • the shelf life is generally selected based on the predetermined shelf life of the pharmaceutical composition. There are currently a number of analytical techniques for measuring protein stability that measure stability after storage for a selected period of time at a selected temperature.
  • 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 formulations include liquid formulations that exhibit desirable characteristics after storage including storage at 25 ° C for 1 month, 3 months, 6 months, or at 40 ° C for 1 month.
  • Typical acceptable criteria for stability are as follows: typically no more than about 5%, 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 ⁇ 5%.
  • a truncation of no more than about 5%, preferably no more than about 5%, is generally observed, typically forming no more than about 5%, 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.
  • LAG-3 refers to lymphocyte activating gene 3.
  • LAG-3 encompasses variants, isoforms, homologs, orthologs, and paralogs.
  • human LAG-3 refers to the human sequence LAG-3, for example the complete amino acid sequence of human LAG-3 with Uniprot number: P18627.
  • LAG-3 such as CD215, is also known in the art.
  • the human LAG-3 sequence differs from the human LAG-3 of Uniprot No.: P18627 in that it has, for example, a conservative mutation or a mutation in a non-conserved region, and LAG-3 and the human LAG-3 of Uniprot No.: P18627 have substantial The same biological function.
  • the biological function of human LAG-3 is to have an epitope in the extracellular domain of LAG-3 that is specifically bound by the antibodies of the present disclosure, or the biological function of human LAG-3 is to bind to MHC class II molecules.
  • the specific human LAG-3 sequence is typically at least 90% identical in amino acid sequence to human LAG-3 of Uniprot No.: P18627 and contains a human amino acid sequence when compared to the LAG-3 amino acid sequence of other species (eg, murine). Amino acid residues.
  • human LAG-3 may be at least 85% or even at least 95%, 96%, 97%, 98% or 99% identical to the LAG-3 of Uniprot number: P18627 in the amino acid sequence.
  • the human LAG-3 sequence shows no more than 10 amino acid differences from the LAG-3 sequence of Uniprot No.: P18627.
  • human LAG-3 may exhibit no more than 5 or even no more than 4, 3, 2 or 1 amino acid differences than the LAG-3 sequence of Uniprot No.: P18627. Percent identity can be determined as set forth herein.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the antibody light chain of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising IgG1, IgG2, IgG3, IgG4 or a variant thereof of human or murine origin.
  • variable region The sequence of about 15 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region.
  • 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).
  • 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 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 LAG-3 prepared according to the knowledge and skill in the art.
  • the test subject is injected with the LAG-3 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the mouse hybridoma cell, and the mouse variable region gene is cloned as needed, and the mouse variable region is cloned.
  • the gene is ligated into a human vector after being ligated into a human chimeric gene, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the antibody light chain of the LAG-3 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the LAG-3 chimeric antibody further comprises a heavy chain constant region of human IgGl, IgG2, IgG3, IgG4 or variants thereof.
  • 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.
  • anti-LAG-3 antibody means an antibody which can bind with sufficient affinity
  • the LAG-3 antibody is such that the antibody can be used as a diagnostic and/or therapeutic agent in targeting LAG-3.
  • binding to LAG-3 in the present invention means that it can interact with human LAG-3.
  • the term “specifically binds” refers to, for example, by techniques available in the art, such as competition ELISA, Determination or The measurement was measured.
  • the term also applies when, for example, an antigen binding domain of an antibody of the invention is specific for a particular epitope carried by a plurality of antigens, in which case an antibody carrying an antigen binding domain is capable of specifically binding to a plurality of antigens carrying the epitope. .
  • competitive binding refers to an antibody that recognizes the same epitope (also referred to as an antigenic determinant) or a portion of the same epitope on the extracellular region of human LAG-3 and binds to the antigen with the monoclonal antibody of the present invention.
  • An antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of human LAG-3 recognized by the monoclonal antibody of the present invention.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.
  • an antibody of the invention binds to LAG-3 with a dissociation equilibrium constant (KD) of less than about 10 -7 M, such as less than about 10 -8 M, 10 -9 M or 10 -10 M or less, for example, Determined using a surface plasmon resonance (SPR) technique in a BIACORE instrument.
  • SPR surface plasmon resonance
  • the "antigen-binding fragment” as used in the present invention refers to a Fab fragment having antigen-binding activity, a Fab' fragment, a F(ab')2 fragment, and a scFv fragment which binds to human LAG-3 and others utilizing energy and human a fragment of human LAG-3 formed by the VH and VL regions of a LAG-3-bound antibody; comprising an antibody of the invention selected from the group consisting of SEQ ID NOs: 9, 10, 11, 15, 16, 17, and 12, One or more of the CDR regions of 13, 14, 18, 19 and 20.
  • 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, called a single chain antibody or a single chain Fv (sFv).
  • epitope refers to a portion of a molecule that is capable of being recognized and bound by an antibody in the antigen binding region of one or more antibodies.
  • Constantly modified refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • Amino acid sequence "identity” refers to sequence similarity between two proteins or polypeptides. When the positions in both comparison sequences are occupied by the same amino acid residue, for example if one position of both polypeptides is occupied by the same amino acid residue, then the molecules are identical at that position.
  • Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-45 and Altschul et al, respectively. (1977) Nucleic Acids Res. 25: 3389-3402. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region.
  • the human FR germline sequence can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN 014441351. obtain.
  • the engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human LAG-3. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by a conventional technique.
  • purification is carried out using an A or G Sepharose FF column containing an adjusted buffer.
  • the non-specifically bound components are washed away.
  • the bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange.
  • the resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
  • 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 clinically measurable extent.
  • 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, the patient's health, the patient's behavior, and the patient's diet.
  • 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 present invention provides a pharmaceutical composition (formulation) which stably comprises a LAG3 antibody or antigen-binding fragment thereof, acetate buffer or histidine salt, sucrose and polysorbate 80, and the pharmaceutical composition (formulation) is more suitable For administration.
  • the amino acid sequence of the antigen and the detection protein of the present invention is designed, and the LAG-3 protein base is optional.
  • Different tags were fused and cloned into pHr vector (self-produced) or pTT5 vector (Biovector, Cat#: 102762) or pTargeT vector (promega, A1410), transient expression in 293 cells or stable expression of CHO-S Purification, obtaining the antigen encoding the present invention and the protein for detection.
  • the following LAG-3 antigens are not specifically described as human LAG-3.
  • LAG-3 extracellular domain with Flag tag LAG-3-Flag, used to immunize mice
  • the horizontal line is the signal peptide and the italic part is the Flag-tag label.
  • Full-length LAG-3 used to construct LAG-3 overexpressing cell lines, immunized mice and tested
  • Fusion protein of LAG-3 extracellular domain and hIgG1 Fc LAG-3-Fc, for detection
  • the cross-hatched portion is the signal peptide
  • the double-lined portion is the linker
  • the italicized portion is the Fc.
  • Fusion protein of LAG-3 extracellular domain and mIgG2a Fc LAG-3-mFc, for detection
  • the cross-hatched portion is the signal peptide
  • the double-lined portion is the linker
  • the italicized portion is the mFc.
  • the sample was centrifuged at high speed to remove impurities and concentrated to an appropriate volume.
  • the flag affinity column was equilibrated with 0.5 x PBS and washed 2-5 column volumes.
  • the supernatant cells were subjected to supernatant analysis and the supernatant samples were applied to the column.
  • the column was rinsed with 0.5 x PBS until the A280 reading dropped to baseline. Rinse the column with PBS, rinse the heteroprotein, and collect.
  • the protein of interest was eluted with 100 mM glycine, pH 3.0. and collected for subsequent in vitro activation and further purification.
  • the cell expression supernatant samples were centrifuged at high speed to remove impurities, and the hybridoma expression supernatant was purified by Protein G column, and the recombinant antibody and Fc fusion protein expression supernatant were purified using a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline.
  • the protein of interest was eluted with 100 mM acetic acid pH 3.0 and neutralized with 1 M Tris-HCl, pH 8.0.
  • the eluted sample was appropriately concentrated and further purified by PBS-balanced gel chromatography Superdex 200 (GE). The peak of the depolymerized product was collected and used.
  • Anti-human LAG-3 monoclonal antibodies are produced by immunizing mice.
  • Experimental 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 according to the following protocol.
  • the immunizing antigen is the Flag tagged human LAG-3 extracellular domain (SEQ ID NO: 1).
  • Immunization program A use Gold Adjuvant (Sigma Cat No. T2684) and Thermo Alum (Thermo Cat No. 77161) was cross-immunized.
  • the antigen was emulsified and inoculated for 0, 7, 14, 21, 28, 35, and 42 days. On day 0, subcutaneous (SC) multiple injections of 50 ⁇ g/only of the emulsified antigen. On day 7, 25 ⁇ g/head was injected intraperitoneally (IP).
  • SC subcutaneous
  • IP intraperitoneally
  • the back or intraperitoneal injection of antigen was selected according to the agglomeration of the back and the swelling of the abdomen.
  • Blood was taken on days 21, 35, and 49, and antibody titers in the serum of the mice were determined by ELISA.
  • spleen cell fusion was performed in mice with high antibody titers in serum and titers that tended to plate.
  • the immunization was boosted 3 days before the splenocyte fusion, and an antigen solution prepared by intraperitoneal (IP) injection of 50 ⁇ g/vial of physiological saline was administered.
  • IP intraperitoneal
  • Immunization Protocol B Mice were immunized with QuickAntibody-Mouse 5W (KX0210041). The ratio of antigen to adjuvant was 1:1, 25 ⁇ g/mouse/time (first exempt/enhanced immunization). The antigen and the adjuvant were quickly and thoroughly mixed and inoculated for 0, 21, and 35 days. On day 0, the mouse calf muscle (IM) was injected with 25 ⁇ g/antigen. On the 21st and 35th day, 25 ⁇ g/mouse was injected in the same manner (depending on the titer, whether or not the third exemption was performed). Blood was taken on the 28th and 42nd day, and the antibody titer in the serum of the mouse was determined by ELISA.
  • IM mouse calf muscle
  • Splenocyte fusion was performed in mice with high antibody titers in serum and titers that tended to plate.
  • the immunization was boosted 3 days before the splenocyte fusion, and an antigen solution prepared by intraperitoneal (IP) injection of 50 ⁇ g/vial of physiological saline was injected.
  • IP intraperitoneal
  • 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 complete medium (DMEM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI) at a density of 0.5-1 ⁇ 10 6 /ml, and 100 ⁇ l/well in 96-well plates. After incubating for 3-4 days at 37 ° C, 5% CO 2 , HAT complete medium was supplemented with 100 ⁇ l/well, and culture was continued for 3-4 days until a needle-like clone was formed.
  • complete medium DMEM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI
  • HT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
  • RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI was added, and cultured at 37 ° C, 5% CO 2 for 3 days, and then subjected to ELISA.
  • Hybridoma culture supernatants were detected by binding ELISA according to the growth density of hybridoma cells.
  • the cell supernatant assay was performed in combination with the positive well cell supernatant detected by ELISA.
  • the well cells that were positive for both binding and blocking were expanded in a timely manner for cryopreservation and two to three subclones until a single cell clone was obtained.
  • the hybridoma clones were screened by the above experiments, and the antibody was further prepared by serum-free cell culture, and the antibody was purified according to the purification example for use in the test examples.
  • the sequence of cloning from a positive hybridoma is as follows. Hybridoma cells in logarithmic growth phase were collected, using Trizol (Invitrogen, Cat No.15596-018) according to kit instructions an RNA extraction step, with PrimeScript TM Reverse Transcriptase reverse transcription kit (Takara, Cat No.2680A). The cDNA obtained by reverse transcription was subjected to PCR amplification using a mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503), and sent to a sequencing company for sequencing. The amino acid sequences corresponding to the DNA sequences of the heavy and light chains of the obtained hybridoma clone mAb229 are represented by SEQ ID NO: 5, 6 and SEQ ID NO: 7, 8.
  • the obtained positive clones were subjected to an ELISA assay for binding to human LAG-3 (the results are shown in Table 2 for protein level binding activity EC50 values), and an ELISA assay for binding human LAG-3 overexpressing CHO-s cells (the results are shown in Table 2 for cell levels). Binding activity EC50 values) and blocking LAG-3 antigen binding to Daudi cells (results see blocking EC50 values in Table 2) and detecting their affinity for human LAG-3 protein (see Table 3 for results).
  • the data in Table 2 shows that the LAG-3 antibodies mAb229 and mAb303 have good binding activity to human LAG-3 protein.
  • the LAG-3 antibodies mAb229 and mAb303 have good binding activity to CHO-S cells overexpressing the human LAG-3 full-length protein. Both the LAG-3 antibodies mAb229 and mAb303 significantly blocked the binding of human LAG-3 antigen to Daudi cells.
  • the data in Table 3 indicates that the LAG-3 antibodies mAb229 and mAb303 of the present invention have strong binding activity and affinity to human LAG-3 protein.
  • the heavy and light chain variable region germline genes with high homology to mAb229 were selected as templates, and the CDRs of the murine antibody were respectively determined.
  • the amino acid residues are determined and annotated by the Kabat numbering system.
  • the humanized light chain template of the murine antibody mAb229 is IGKV1-39*01 and hjk4.1, and the humanized heavy chain template is IGHV7-4-1*02 and hjh6.1.
  • the sequence of the 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.
  • I48V indicates that 48-bit I is mutated back to V according to the Kabat numbering system.
  • Grafted represents the murine antibody CDRs implanted into the human germline FR region sequence.
  • Hu229-005 indicates that there are two mutations in the light chain HumAb229_VL.1A and heavy chain HumAb229_VH.1 on the humanized mouse antibody Hu229-005. Other analogies.
  • Hu229VH.1 (same as Hu229VH-CDR graft)
  • Hu229VL.1 (same as Hu229VL-CDR graft)
  • the heavy and light chain variable region germline genes with high homology to mAb303 were selected as templates, and the CDRs of the murine antibody were respectively determined.
  • the amino acid residues are determined and annotated by the Kabat numbering system.
  • the humanized light chain templates of the murine antibody mAb303 are IGKV1-39*01 and hjk4.1, and the humanized heavy chain templates are IGHV1-3*01 and hjh6.1.
  • the humanized variable region sequences are 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.
  • L46R indicates that 46-bit L is mutated back to R according to the Kabat numbering system. Grafted represents the murine antibody CDRs implanted into the human germline FR region sequence.
  • Hu303-005 indicates that there are two mutations in the light chain HumAb303_VL.1A and heavy chain HumAb303_VH.1 on the humanized mouse antibody Hu303-005. Other analogies.
  • Hu303_VH.1 (same as Hu303VH-CDR graft)
  • Hu303_VL.1 (same as Hu303VL-CDR graft)
  • the antibody uses the constant region of the human heavy chain IgG4/light chain kappa in combination with each variable region, and the S228P mutation is made in the Fc segment to increase the stability of the IgG4 antibody, and other mutations known in the art can be used to increase its performance.
  • the heavy chain amino acid sequence of Hu229-013 is:
  • the light chain amino acid sequence of Hu229-013 is:
  • the heavy chain amino acid sequence of Hu303-005 is:
  • the light chain amino acid sequence of Hu303-005 is:
  • the positive antibody molecule obtained by hybridoma screening is sequenced to obtain a variable region coding gene sequence.
  • the primers were designed to sequence the primers, and the sequencing gene was used as a template.
  • the VH/VK gene fragment of each antibody was constructed by PCR, and then the expression vector pHr (with signal peptide and hIgG4/hkappa constant region gene (CH1-FC/CL) fragment).
  • the homologous recombination was carried out to construct a recombinant antibody full-length expression plasmid VH-CH1-FC-pHr/VL-CL-pHr.
  • the antibody sequence after human design was codon-optimized to generate the coding sequence of the human codon-preferred gene.
  • the primers were designed to construct the VH/VK gene fragment of each antibody, and then the expression vector pHr (with signal peptide and hIgG4/hkappa constant region).
  • the gene (CH1-FC/CL) fragment was subjected to homologous recombination to construct a humanized antibody full-length expression plasmid VH-CH1-FC-pHr/VL-CL-pHr.
  • the plasmid expressing the light heavy chain of the antibody was transfected into HEK293E cells at a ratio of 1:1.2. After 6 days, the expression supernatant was collected, and the impurities were removed by high-speed centrifugation and purified by a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline. The protein of interest was eluted with an acidic eluent of pH 3.0 - pH 3.5 and neutralized with 1 M Tris-HCl, pH 8.0-9.0. The eluted sample was appropriately concentrated, and further purified by PBS-balanced gel chromatography Superdex 200 (GE) to remove the aggregate, collect the monomer peak, and equilibrate the device.
  • PBS-balanced gel chromatography Superdex 200 GE
  • Example 6 ELISA assay of LAG-3 antibody binding to human LAG-3 (human LAG-3) protein
  • the binding capacity of the anti-LAG-3 antibody was detected by an ELISA assay of the antibody with the human LAG-3 protein.
  • the LAG-3 fusion protein with Fc or mFc tag is immobilized into a 96-well microtiter plate by binding to an anti-Fc or mFc antibody coated in a ELISA plate, and the signal intensity after antibody addition is used to judge the antibody.
  • the binding activity to LAG-3 is as follows.
  • Goat anti-human Fc antibody Jackson Immuno Research, Cat No. 109-005-008
  • goat anti-mouse Fc antibody Sigma, Cat No.
  • PBS pH 7.4 Sigma, Cat No. P4417-100TAB
  • M3534-1ML was diluted to a concentration of 2 ⁇ g/ml, and added to a 96-well microtiter plate in a volume of 50 ⁇ l/well, and placed in an incubator at 37 ° C for 2 hours.
  • LAG-3-Fc fusion protein internal production, SEQ ID NO: 3
  • LAG-3-mFc fusion protein internal production, SEQ ID NO: 4
  • incubate at 37 ° C incubator for 1 hour or 4 ° C Leave it overnight. After the incubation, discard the reaction solution in the plate, wash the plate with PBST for 6 times, and add 50 ⁇ l/well of the antibody to be tested (hybridoma purified antibody or humanized antibody) diluted with the sample dilution. Incubate for 1 hour at 37 ° C in the incubator.
  • the NOVOStar microplate reader reads the absorbance at a wavelength of 450 nm and calculates the binding EC50 value of the LAG-3 antibody to human LAG-3.
  • the results are shown in Table 8. The data indicate that the humanized antibody screened by the present invention has high binding activity to human LAG-3 protein.
  • Hu229-011 0.201 Hu229-012 0.268 Hu229-013 0.106 Hu229-014 0.153 Hu229-015 0.156 Hu229-016 0.154 Hu229-017 0.048 Hu229-019 0.068 mAb303 0.172 Hu303-004 0.278 Hu303-005 0.309 Hu303-006 0.288 Hu303-007 0.135 Hu303-008 0.140 Hu303-009 0.316 Hu303-010 0.137 Hu303-011 0.314 Hu303-012 0.164 Hu303-013 0.166 Hu303-014 0.232 Hu303-015 0.172 Hu303-016 0.161 Hu303-017 0.168 Hu303-018 0.244 Hu303-019 0.277 Hu303-020 0.140 Hu303-021 0.170 Hu303-022 0.145 Hu303-023 0.152
  • Example 7 Binding experiment of LAG-3 antibody with human LAG-3 overexpressing CHO-S cells
  • the binding ability of the anti-LAG-3 antibody was detected by binding experiments of the antibody to CHO-S cells overexpressing the LAG-3 protein.
  • the LAG-3 full-length plasmid (internal production, SEQ ID NO: 2) was transfected into CHO-S cells by electroporation, and after two weeks of press screening, the expression level of LAG-3 was examined. After the overexpressing cells were fixed in the 96-well plate bottom, the signal intensity after the antibody was added was used to judge the binding activity of the antibody and LAG-3 overexpressing CHO-S cells, and the specific experimental methods were as follows.
  • the cells were seeded in a 96-well plate at a density of 4 ⁇ 10 5 /ml, 100 ⁇ l/well, and cultured overnight. The supernatant was discarded, washed three times with PBS, and fixed at 100 ⁇ l/well 4% PFA for half an hour, and washed three times with PBS. After discarding the liquid, 200 ⁇ l/well of a 5% skim milk (bright skimmed milk powder) blocking solution diluted with PBS was added, and the mixture was incubated at 37 ° C for 2.5 hours for blocking.
  • a 5% skim milk (bright skimmed milk powder) blocking solution diluted with PBS was added, and the mixture was incubated at 37 ° C for 2.5 hours for blocking.
  • PBST buffer pH 7.4 PBS containing 0.05% tweeen-20
  • 50 ⁇ l/well of different concentrations of the test antibody diluted with the sample dilution (hybridoma purified antibody). Or humanized antibody), incubated in a 37 ° C incubator for 1 hour. After the incubation, the plate was washed 5 times with PBST, and 100 ⁇ l/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, Cat No. 115-035-003) or goat anti-human secondary antibody (Jackson) diluted with the sample dilution was added.
  • HRP-labeled goat anti-mouse secondary antibody Jackson Immuno Research, Cat No. 115-035-003
  • goat anti-human secondary antibody (Jackson) diluted with the sample dilution was added.
  • 109-035-003 incubated for 1 hour at 37 °C. After washing the plate 6 times with PBST, add 50 ⁇ l/well TMB chromogenic substrate (KPL, Cat No. 52-00-03), incubate for 5-15 min at room temperature, and add 50 ⁇ l/well 1 M H 2 SO 4 to terminate the reaction.
  • the NOVOStar plate reader reads the absorbance at a wavelength of 450 nm and calculates the binding EC50 value of the LAG-3 antibody to LAG-3 overexpressing CHO-S cells.
  • Daudi cells human leukemia cells, purchased from the Chinese Academy of Sciences cell bank
  • a 96-well culture plate at a number of 3 ⁇ 10 5 /well, centrifuged at 1000 rpm, and the supernatant was discarded, and fixed at 4% PFA for 30 minutes at room temperature. After discarding the fixative, it was washed 4 times with PBS buffer, and added to a blocking solution of 5% skim milk (bright skim milk powder) diluted with PBS at 200 ⁇ l/well, and incubated at 37 ° C for 2.5 hours to block.
  • 5% skim milk (bright skim milk powder) diluted with PBS at 200 ⁇ l/well
  • the NOVOStar microplate reader reads the absorbance at a wavelength of 450 nm and calculates the blocking effect of the LAG-3 antibody on the binding of the antigen to the daudi cell.
  • the results are shown in Table 9, and the data indicate that the humanized antibodies screened by the present invention can significantly block the binding of human LAG-3 antigen to Daudi cells.
  • PBMC peripheral blood mononuclear cells
  • the results are shown in Figure 1.
  • the LAG-3 humanized candidate antibodies Hu229-013 and Hu303-005 can enhance the secretion of cytokine IL-2 by activated T lymphocytes to varying degrees, and have a drug concentration dose effect.
  • Example 11 Effect of LAG-3 antibody on inhibition of U-87MG subcutaneous xenografts
  • mice 100 ⁇ l of human glioma U-87MG cells (3.5 ⁇ 10 6 ) were inoculated subcutaneously into the right flank of NOD-SCID (purchased from Changzhou Cavans Laboratory Animal Co., Ltd.), and the tumor grew to about 10-14 days. After 40 mm 3 , the body weight was removed, the tumor was too large and too small.
  • the mice were randomly divided into control group Isotype matched hIgG, LAG-3 humanized candidate antibody Hu229-013 group and Hu303-005 group (3 groups). Grouping and dosage are shown in Table 11), 8 (D0) per group.
  • the CD3 antibody-stimulated PBMC was injected into the tumor tissue in an amount of 5 ⁇ 10 5 cells / 60 ⁇ l, and the antibody was intraperitoneally injected three times a week for a total of 6 times.
  • the tumor volume was measured twice a week and data was recorded.
  • the tumor volume (V) is calculated as:
  • Tumor volume (TV) 1/2 ⁇ L length ⁇ L short 2
  • the tumor volume of each group was expressed by mean ⁇ standard deviation (Mean ⁇ SEM) and plotted with Graphpad Prism 5 software. Statistical analysis was performed using two way ANOVA, and the tumor inhibition rate was calculated. The formula is:
  • Tumor proliferation rate (T/C%) (TT 0 /CC 0 ) ⁇ 100%
  • D0 first dosing time; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 vs hIGg, analyzed by two way ANOVA.
  • ICR mice Eighteen ICR mice, male, weighing 18-22 g, were purchased from Sipple-Beikai Experimental Animal Co., Ltd. Free feed and water during feeding, laboratory environment adaptive feeding for not less than 3 days, 12/12 hours light/dark cycle adjustment, temperature 16-26 ° C, relative humidity 40-70%.
  • ICR mice were numbered and randomized into groups of 3 each.
  • the two groups of mice were intravenously injected with humanized candidate antibody (Hu229-013) at doses of 3 mg/kg and 10 mg/kg; each group of mice was injected with humanized candidate antibody intravenously (Hu303- 005), the doses administered were 3 mg/kg and 10 mg/kg.
  • the intravenous injection volume was 20 ml/kg.
  • the time of blood collection after administration was 15 min, 8 h, 1 d, 2 d, 4 d, 7 d, 10 d, 14 d, 21 d, 28 d, 35 d.
  • About 0.1 ml of whole blood was taken each time, no anticoagulant was added. After taking blood, it was placed at 4 ° C for 30 min, centrifuged at 1000 g for 15 min, and the supernatant was placed in an EP tube and stored at -80 ° C.
  • the blood concentration in the serum was measured by ELISA, and the T1/2 and its main parameters of the test drug were calculated using Winnolin software.
  • the main pharmacokinetic parameters obtained are shown in Table 12:
  • LAG-3 humanized antibodies Hu229-013 and Hu303-005 were similar, and the exposure, peak concentration and dose growth of the two antibodies at 3,10 mg/kg were linear. Has linear dynamic characteristics.
  • the first step the prescription LAG-3 antibody stock solution passed through a 0.22 ⁇ m PVDF filter element, filtered, and then sampled and tested for sterility, and the filtrate was collected.
  • the second step adjust the loading to 5.3ml, fill the filtrate in a 6ml vial, and add the plug, respectively, at the beginning of filling, in the middle of filling, at the end of filling, sampling and control the difference in loading.
  • the third step open the capping machine, add the aluminum cover, and carry out the capping.
  • the fourth step visual inspection, to confirm that the product is not loaded and other defects. Print and paste the vial label; print the tray label, fold the tray, box, and sticker box labels.
  • the first step the prescription consists of Hu303-005 stock solution over 0.22 ⁇ m PVDF filter element. After filtration, it is sampled and tested for sterility, and the filtrate is collected.
  • the second step adjust the loading to 5.3ml, fill the filtrate in a 20ml vial, add a freeze-dried plug, lyophilize the original solution in the vial, and seal the rubber stopper.
  • the third step open the capping machine, add the aluminum cover, and carry out the capping.
  • the fourth step visual inspection, to confirm that the product has no defects such as freeze-drying collapse.
  • Print and paste the vial label print the tray label, fold the tray, box, and sticker box labels.
  • a LAG-3 antibody Hu229-013 or Hu303-005 preparation having a protein concentration of 50 mg/mL was prepared in a series of buffers of 10 mM pH 5.0-7.5, and each preparation was filter-filled, stoppered, capped, and sealed.
  • the sample was subjected to forced degradation experiments such as 40 ° C high temperature, shaking, etc., by appearance, size exclusion chromatography (SEC), non-reduced sodium lauryl sulfate (CE-SDS)-capillary electrophoresis and ion exchange chromatography (IEC). Or full-column imaging capillary isoelectric focusing electrophoresis (iCIEF) was used as an evaluation index.
  • SEC size exclusion chromatography
  • CE-SDS non-reduced sodium lauryl sulfate
  • IEC ion exchange chromatography
  • iCIEF full-column imaging capillary isoelectric focusing electrophoresis
  • D means day; N/A means not detected.
  • Hu229-013 antibody has the best appearance in acetic acid-sodium acetate (AA) system, followed by sodium succinate-succinate (SA) and histidine-hydrochloric acid (His-HCl) system, acetic acid-sodium acetate (AA) ), pH 5.5, sodium succinate-succinate (SA), pH 6.0, citric acid-sodium citrate (CA), pH 6.0, histidine-hydrochloric acid (His), pH 6.0 system 40 ° CCE IEC purity is high, considering the appearance and CE and IEC results, LAG-3 antibody Hu229-013 is stable in AA (pH 5.5), SA (pH 6.0), His-HCl (pH 6.0) systems. See Figure 3 and Figure 4.
  • the Hu229-013 antibody preparation was better than the PS20 by shaking the PS80 group; after freezing and thawing, a small amount of particles appeared in the sucrose group, and a large amount of particles appeared in the trehalose group; there was no difference between other data groups; therefore, the adjuvant was preferably polysorbate. 80 and sucrose.
  • Each preparation was filter-filled, stoppered, capped, and sealed. After the sample preparation was completed, it was placed at 25 ° C or 4 ° C to examine the stability.
  • the detection items are appearance, SEC, IEC or iCE, CE-SDS (non-restore).
  • M indicates month and N/A indicates no detection.
  • LAG-3 antibody Hu229-013 was stable in 10 mM AA pH 5.5, 10 mM His-AA pH 6.0 system.
  • M1 means one month and D means day.
  • LAG-3 antibody in 10-30 mM acetic acid-sodium acetate (AA) buffer or histidine-acetate buffer (His-AA), pH 5.2-5.8 system
  • the stability of Hu229-013 is good, and the buffer system is preferably 10-30 mM acetic acid-sodium acetate (AA), pH 5.5.
  • a LAG-3 Hu229-013 preparation with a protein concentration of 60 mg/mL, 60 mg/ml sucrose, 0.4 mg/mL polysorbate 80 was prepared in 10 mM acetic acid-sodium acetate pH 5.5 buffer:
  • the preparation is filtered, filled, stoppered, capped and sealed. After the sample preparation was completed, it was placed at 4 ° C to examine the stability.
  • the detection items are appearance, SEC, IEC, CE-SDS (non-restore).
  • sucrose and polysorbate 80 In order to further optimize the concentration of protein, sucrose and polysorbate 80, the buffer was set to 10 mM acetic acid-sodium acetate, pH 5.5, and the DOP experiment design was carried out using JMP software. A series of prescriptions were obtained by using the RSM model, and the forced degradation method was adopted. The results were statistically analyzed by least squares method using IEC and CE (non-reduction). The DOE parameters are shown in Table 20. The test results are shown in Table 21. The statistical analysis results are shown in Figure 8, Figure 9, and Table 21.
  • the difference between the IEC 0 and 40 ° C data is fitted, R 2 >0.98, P ⁇ 0.06, and the model is valid.
  • the results are shown in Fig. 8. Fitting with the difference between CE purity 0 and 40 °C data, R 2 >0.99, P ⁇ 0.05, the model is effective, the results are shown in Figure 9.
  • the 40 °C IEC fitting results show that the better prescription is: protein concentration 40-60mg /ml, sugar concentration 30-90mg/ml, PS80 concentration 0.4-0.5mg/ml;
  • 40 °CCE fitting results show that the preferred prescription is: protein concentration 50-80mg/ml, sugar concentration 30-90mg/ml, PS80 concentration 0.1-0.5 mg/ml. Therefore, the optimal range is: protein concentration 50-60 mg/ml, sucrose concentration 30-90 mg/ml, and PS80 concentration 0.4-0.5 mg/ml.
  • the CE data was optimal at pH 6.0, and combined with the previous experimental results (10 mM His pH 6.0). Stable), determine the optimal pH is 6.0; 40 degree CE data shows that the protein concentration is 45-60mg/ml, so the optimal concentration is 50mg/ml; the effect of polysorbate concentration on protein stability under various conditions Results and polysorbate concentration screening results in other test cases (optimal at concentrations greater than 0.2 mg/ml), PS80 concentration was set at 0.3 mg/ml; for Hu303-005 antibody liquid preparation, in this and other examples In the middle and high conditions, iCIEF showed a significant decrease, and it was considered to be developed as a lyophilized preparation. In order to ensure good moldability and suitable osmotic pressure of the lyophilized preparation, the sucrose concentration was determined to be 75 mg/ml.
  • a lyophilized preparation of Hu303-005 having a protein content of 50 mg/ml, 10 mM histidine-hydrochloric acid, pH 5.5 or 6.0, 75 mg/ml sucrose, and 0.4 mg/ml PS80 was prepared.
  • the lyophilization procedure is as follows:
  • the stability of the complex solution of the Hu229-013 antibody was determined after lyophilization.
  • the concentration of the LAG-3 antibody was 50 mg/ml and 75 mg/ml sucrose with a buffer containing 10 mM acetic acid-sodium acetate at pH 5.5.
  • Hu303-005 antibody content 50 mg/ml, 10 mM histidine-hydrochloric acid, pH 6.0, 75 mg/ml sucrose, 0.4 mg/ml PS80 was prepared and lyophilized.
  • the collapse temperature of Hu303-005 measured by freeze-drying microscope is about -19 °C.
  • the primary drying temperature is an important parameter of the freeze-drying process. Therefore, the temperature of the separator in a drying process is carefully optimized.
  • the freeze-drying parameters are shown in Table 27.
  • the results are shown in Table 28.
  • the appearance of the lyophilized powder cake at each temperature met the requirements, but a small amount of particles appeared in the appearance after reconstitution in the -5 °C group, so the temperature of the primary drying separator was set to -10 °C.
  • the final freeze-drying process is as follows:
  • the stable pharmaceutical preparation provided by the present invention may also be a stable combination selected from the following:
  • LAG-3 antibody Hu229-013 90 mg / ml, 80 mg / ml sucrose, 0.4 mg / ml polysorbate 80, 15 mM acetic acid - sodium acetate buffer, pH 5.5;
  • LAG-3 antibody Hu229-013 90 mg / ml, 80 mg / ml sucrose, 0.4 mg / ml of polysorbate 80, 15 mM acetic acid - sodium acetate buffer, pH 6.5;
  • LAG-3 antibody Hu229-013 90 mg / ml, 50 mg / ml sucrose, 0.4 mg / ml polysorbate 80, 25 mM acetic acid - sodium acetate buffer, pH 5.5;
  • LAG-3 antibody Hu229-013 70 mg / ml, 50 mg / ml sucrose, 0.3 mg / ml polysorbate 80, 10 mM acetic acid - sodium acetate buffer, pH 5.5;
  • LAG-3 antibody Hu229-013 70 mg / ml, 80 mg / ml sucrose, 0.3 mg / ml of polysorbate 80, 15 mM acetic acid - sodium acetate buffer, pH 5.2;
  • LAG-3 antibody Hu229-013 40 mg/ml, 75 mg/ml sucrose, 0.4 mg/ml polysorbate 80, 10 mM acetic acid-sodium acetate buffer, pH 6.0;
  • LAG-3 antibody Hu229-013 55 mg / ml, 75 mg / ml sucrose, 0.4 mg / ml of polysorbate 80, 10 mM acetic acid - sodium acetate buffer, pH 5.7;
  • LAG-3 antibody Hu229-013 30 mg / ml, 70 mg / ml sucrose, 0.5 mg / ml of polysorbate 80, 10 mM acetic acid - sodium acetate buffer, pH 5.5;
  • LAG-3 antibody Hu229-013 20 mg / ml, 70 mg / ml sucrose, 0.2 mg / ml polysorbate 80, 10 mM acetic acid - sodium acetate buffer, pH 5.4;
  • LAG-3 antibody Hu229-013 15 mg / ml, 85 mg / ml sucrose, 0.1 mg / ml polysorbate 80, 25 mM acetic acid - sodium acetate buffer, pH 5.6;
  • LAG-3 antibody Hu229-013 50 mg / ml, 85 mg / ml sucrose, 0.3 mg / ml of polysorbate 80, 25 mM acetic acid - sodium acetate buffer, pH 5.8;
  • LAG-3 antibody Hu229-013 50 mg / ml, 75 mg / ml sucrose, 0.4 mg / ml polysorbate 80, 25 mM acetic acid - sodium acetate buffer, pH 6.0;
  • LAG-3 antibody Hu229-013 55mg / ml, 90mg / ml sucrose, 0.4mg / ml of polysorbate 80, 10mM acetic acid - sodium acetate buffer, pH 5.3;
  • LAG-3 antibody Hu229-013 50 mg / ml, 90 mg / ml sucrose, 0.4 mg / ml of polysorbate 80, 10 mM acetic acid - sodium acetate buffer, pH 5.0;
  • LAG-3 antibody Hu303-005 90mg/ml, 75mg/ml sucrose, 0.3mg/ml polysorbate 80, 30mM histidine-hydrochloric acid buffer, pH6.0;
  • LAG-3 antibody Hu303-005 90mg/ml, 75mg/ml sucrose, 0.3mg/ml polysorbate 80, 30mM histidine-hydrochloric acid buffer, pH 5.5;
  • LAG-3 antibody Hu303-005 75mg/ml, 75mg/ml sucrose, 0.3mg/ml polysorbate 80, 30mM histidine-hydrochloric acid buffer, pH5.0;
  • LAG-3 antibody Hu303-005 1mg/ml, 75mg/ml sucrose, 0.3mg/ml polysorbate 80, 5mM histidine-hydrochloric acid buffer, pH6.0;
  • LAG-3 antibody Hu303-005 50mg/ml, 60mg/ml trehalose, 0.3mg/ml polysorbate 80, 10mM histidine-hydrochloric acid buffer, pH6.0;
  • LAG-3 antibody Hu303-005 50 mg/ml, 90 mg/ml trehalose, 0.3 mg/ml polysorbate 80, 10 mM histidine-hydrochloric acid buffer, pH 6.0.

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Abstract

本发明公开了一种LAG-3抗体药物组合物及其用途。所述药物组合物包含在醋酸盐缓冲剂或组氨酸盐缓冲剂中的LAG-3抗体或其抗原结合片段。该药物组合物还可以含有糖、非离子型表面活性剂和其他辅料。

Description

一种LAG-3抗体药物组合物及其用途 技术领域
本发明属于药物制剂领域，具体地本发明涉及一种包含LAG-3抗体及其抗原结合片段的药物组合物，以及其作为药物的用途。
背景技术
淋巴细胞活化基因3(lymphocyteactivationgene-3，也称LAG-3，CD215)是免疫球蛋白超家族的一员，可负性调节免疫细胞的各项功能和生存周期。已有研究表明，LAG-3在病毒感染、自身免疫疾病以及肿瘤所致的免疫系统功能紊乱中发挥着重要作用，通过影响LAG-3的功能可以改善这些疾病演进过程中免疫功能失常状态，从而改善疾病的预后。
LAG-3作为免疫球蛋白超家族的一员，由胞外区、跨膜区和胞质区3个部分组成。成熟的LAG-3分子包括470个氨基酸，相对分子量为70kDa，1990年由Triebel等首次发现(J Exp Med,1990,171(5):1393-405)。研究发现，LAG-3与CTLA-4、PD-1一样，是一个负性共刺激分子，其激活可负向调控淋巴细胞功能。LAG-3在结构上与CD4密切相关，但却有着与CD4截然相反的功能，表现在LAG-3与CD4分子具有较高的相似性，可与MHC-II(主要组织相容性复合体)类分子结合。但是，LAG-3与MHC-II类分子的结合更为紧密，从而干预CD4 +T淋巴细胞TCR激活，抑制T淋巴细胞激活(Curr Opin Immunol,2009,21(2):179-86；Eur J Immunol,2003,33(4):970-9)。体外研究显示，LAG-3可抑制抗原诱导的T淋巴细胞增殖反应，而阻断了LAG-3之后，T淋巴细胞的活化和增殖、1型辅助性T细胞(type1 T helper cells,Th1)分泌细胞因子的情况均有所改善；Huang等研究表明，活化的CD4 +Treg细胞表面LAG-3水平显著升高，且LAG-3是CD4 +Tregs发挥最大免疫抑制作用的必需条件(Immunity,2004,21(4):503-13)。此外，抗LAG-3抗体还可维持CD4 +和CD8 +T淋巴细胞稳态，阻断LAG-3后CD8 +T淋巴细胞杀伤肿瘤细胞的能力显著增强(J Clin Invest,2007,117(11):3383-92)。一些疾病相关研究也发现，LAG-3在调控疾病发生发展中具有重要作用。Gandhi等证实人类淋巴瘤组织中T淋巴细胞LAG-3的表达水平与T淋巴细胞功能障碍相关，清除LAG-3 +T淋巴细胞可显著增强淋巴细胞的肿瘤清除能力(Blood,2006,108(7):2280-9)。研究结果显示LAG-3是免疫细胞表面重要的抑制性分子，并且对T淋巴细胞可产生显著的负向调控作用。
LAG-3主要表达在T淋巴细胞、B淋巴细胞、NK细胞、Treg细胞以及DC等细胞上(Proc Natl Acad Sci U S A,1997,94(11):5744-9.Eur J Immunol,2005,35(7):2081-8；J Immunol,2009,182(4):1885-91)。LAG-3是一类免疫抑制性分子，是TCR的共受体组成部分之一，它干预T淋巴细胞TCR激活，在T淋巴细胞激活中 发挥负性调控功能。在一些疾病中，LAG-3表达均会升高，并且会出现相应的免疫抑制。Gandhi等发现霍奇金淋巴瘤患者血液和肿瘤组织中，淋巴细胞高表达LAG-3；肿瘤组织中特异性CD8 +T细胞的功能明显受损，如果去除LAG-3阳性的T细胞，其抗肿瘤功能可以恢复，细胞因子分泌增加。据此推测，LAG-3的表达与特异性T细胞的免疫负调节功能相关，抑制LAG-3分子功能可以增强T细胞的抗肿瘤作用，该分子有可能是一个潜在的肿瘤免疫治疗靶点(Blood,2006,108(7):2280-9)。
目前有多家跨国制药公司如BMS公司和Novartis公司在研发针对LAG-3的单克隆抗体，它通过刺激抗原特异性T细胞应答，增强T细胞的抗肿瘤作用，从而最大限度的提高患者自身对肿瘤的免疫系统反应，达到对肿瘤细胞进行杀伤的目的。
但是，抗体药物由于分子量大、结构复杂，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。为了使抗体适合于给药，并且在储存及随后使用过程中能保持稳定性，发挥更好的效果，抗体药物的稳定制剂研究显得尤为重要。
尽管，目前有多家公司在研发包含LAG3抗体及其制剂，如：WO2018204374、WO2010019570、WO2014008218、WO9530750、WO2004078928、WO2008132601、WO2014140180、WO2015138920等，但对于新的LAG3抗体制剂的研究并不多，因此仍需要研制更适于给药的包含LAG3的药物组合物(制剂)。
发明内容
本发明提供一种药物组合物，其包含LAG3抗体或其抗原结合片段，以及缓冲剂，所述缓冲剂选自醋酸盐缓冲剂、组氨酸缓冲剂、柠檬酸盐缓冲剂、琥珀酸盐缓冲剂或Tris缓冲剂。
在可选的实施方案中，药物组合物中所述的醋酸盐缓冲剂选自醋酸-醋酸钠缓冲剂、醋酸-醋酸钾缓冲剂，醋酸-组氨酸盐缓冲剂、醋酸-醋酸钙缓冲剂、醋酸-醋酸镁缓冲剂，优选醋酸-醋酸钠缓冲剂。
在可选的实施方案中，药物组合物中所述的组氨酸缓冲剂选自组氨酸-盐酸缓冲剂、组氨酸-醋酸缓冲剂、组氨酸-磷酸缓冲剂、组氨酸-硫酸缓冲剂，优选组氨酸-盐酸缓冲剂。
在可选的实施方案中，药物组合物中所述的柠檬酸盐缓冲剂选自柠檬酸-柠檬酸钠缓冲剂；所述的琥珀酸盐缓冲剂选自琥珀酸-琥珀酸钠缓冲剂。
在可选的实施方案中，药物组合物中所述LAG-3抗体或其抗原结合片段浓度为约1mg/ml至90mg/ml，优选约10mg/ml至90mg/ml，优选约20mg/ml至90mg/ml，优选约30mg/ml至90mg/ml，优选约40mg/ml至90mg/ml，优选约50mg/ml至90mg/ml，优选约60mg/ml至90mg/ml，优选约70mg/ml至90mg/ml，优选约80mg/ml至90mg/ml，优选约10mg/ml至80mg/ml，优选约20mg/ml至80mg/ml，优选约 30mg/ml至80mg/ml，优选约40mg/ml至80mg/ml，优选约50mg/ml至80mg/ml，优选约60mg/ml至80mg/ml，优选约70mg/ml至80mg/ml，优选约10mg/ml至70mg/ml，优选约20mg/ml至70mg/ml，优选约30mg/ml至70mg/ml，优选约40mg/ml至70mg/ml，优选约50mg/ml至70mg/ml，优选约60mg/ml至70mg/ml，优选约10mg/ml至60mg/ml，优选约20mg/ml至60mg/ml，优选约30mg/ml至60mg/ml，优选约40mg/ml至60mg/ml，优选约50mg/ml至60mg/ml，优选约10mg/ml至50mg/ml，优选约20mg/ml至50mg/ml，优选约30mg/ml至50mg/ml，优选约40mg/ml至50mg/ml，优选约10mg/ml至40mg/ml，优选约20mg/ml至40mg/ml，优选约30mg/ml至40mg/ml，优选约10mg/ml至30mg/ml，优选约20mg/ml至30mg/ml。作为非限制性实施例，所述LAG-3抗体或其抗原结合片段浓度为约40mg/ml、41mg/ml、42mg/ml、43mg/ml、44mg/ml、45mg/ml、46mg/ml、47mg/ml、48mg/ml、49mg/ml、50mg/ml、51mg/ml、52mg/ml、53mg/ml、54mg/ml、55mg/ml、56mg/ml、57mg/ml、58mg/ml、59mg/ml或60mg/ml，最优选为50mg/ml。
在可选的实施方案中，缓冲剂的浓度约为5mM至30mM，优选约为10mM至30mM，优选约为15mM至30mM，优选约为20mM至30mM，优选约为25mM至30mM，优选约为5mM至25mM，优选约为10mM至25mM，优选约为15mM至25mM，优选约为20mM至25mM，优选约为5mM至20mM，优选约为10mM至15mM；所述缓冲剂浓度的非限制性实施例为约10mM、12mM、14mM、16mM、18mM、20mM、22mM、24mM、26mM、28mM或30mM，最优选为10mM。
在可选的实施方案中，药物组合物中所述缓冲剂的pH值约为5.0到7.5，优选约为5.5到7.5，优选约为6.0到7.5，优选约为6.5到7.5，优选约为7.0到7.5，优选约5.0至7.0，优选约为5.5到7.0，优选约为6.0到7.0，优选约为6.5到7.0，优选约5.0至6.5，优选约为5.5到6.5，优选约为6.0到6.5，优选约5.0至6.0，优选约为5.5到6.0，优选约5.0至5.5，所述缓冲剂pH值的非限制性实施例还可选约为5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.0、6.1、6.2、6.3、6.4、6.5，更优选为约5.5-6.0，更优选为5.5或6.0。
进一步地，在可选的实施方案中，药物组合物还包括辅料，所述辅料选自糖和表面活性剂中的一种或多种。
在可选实施方案中，其中所述的糖为二糖，优选为海藻糖、蔗糖、甘露醇或山梨醇，更优选为蔗糖。在可选的实施方案中，药物组合物中糖的浓度约为30mg/ml至90mg/ml，优选约为60mg/ml至90mg/ml，35mg/ml至90mg/ml，优选约为40mg/ml至90mg/ml，优选约为45mg/ml至90mg/ml，优选约为50mg/ml至90mg/ml，优选约为55mg/ml至90mg/ml，优选约为60mg/ml至90mg/ml，优选约为65mg/ml至90mg/ml，优选约为70mg/ml至90mg/ml，优选约为75mg/ml至90mg/ml，优选约为80mg/ml至90mg/ml，优选约为85mg/ml至90mg/ml，优选约为30mg/ml至85mg/ml，优选约为35mg/ml至85mg/ml，优选约为40mg/ml至85mg/ml，优选约 为45mg/ml至85mg/ml，优选约为50mg/ml至85mg/ml，优选约为55mg/ml至85mg/ml，优选约为60mg/ml至85mg/ml，优选约为65mg/ml至85mg/ml，优选约为70mg/ml至85mg/ml，优选约为75mg/ml至85mg/ml，优选约为80mg/ml至85mg/ml，优选约为30mg/ml至80mg/ml，优选约为35mg/ml至80mg/ml，优选约为40mg/ml至80mg/ml，优选约为45mg/ml至80mg/ml，优选约为50mg/ml至80mg/ml，优选约为55mg/ml至80mg/ml，优选约为60mg/ml至80mg/ml，优选约为65mg/ml至80mg/ml，优选约为70mg/ml至80mg/ml，优选约为75mg/ml至80mg/ml，优选约为30mg/ml至75mg/ml，优选约为35mg/ml至75mg/ml，优选约为40mg/ml至75mg/ml，优选约为45mg/ml至75mg/ml，优选约为50mg/ml至75mg/ml，优选约为55mg/ml至75mg/ml，优选约为60mg/ml至75mg/ml，优选约为65mg/ml至75mg/ml，优选约为70mg/ml至75mg/ml，优选约为30mg/ml至70mg/ml，优选约为35mg/ml至70mg/ml，优选约为40mg/ml至70mg/ml，优选约为45mg/ml至70mg/ml，优选约为50mg/ml至70mg/ml，优选约为55mg/ml至70mg/ml，优选约为60mg/ml至70mg/ml，优选约为65mg/ml至70mg/ml，优选约为30mg/ml至65mg/ml，优选约为35mg/ml至65mg/ml，优选约为40mg/ml至65mg/ml，优选约为45mg/ml至65mg/ml，优选约为50mg/ml至65mg/ml，优选约为55mg/ml至65mg/ml，优选约为60mg/ml至65mg/ml，优选约为30mg/ml至60mg/ml，优选约为35mg/ml至60mg/ml，优选约为40mg/ml至60mg/ml，优选约为45mg/ml至60mg/ml，优选约为50mg/ml至60mg/ml，优选约为55mg/ml至60mg/ml，优选约为30mg/ml至55mg/ml，优选约为35mg/ml至55mg/ml，优选约为40mg/ml至55mg/ml，优选约为45mg/ml至55mg/ml，优选约为50mg/ml至55mg/ml，优选约为30mg/ml至50mg/ml，优选约为35mg/ml至50mg/ml，优选约为40mg/ml至50mg/ml，优选约为45mg/ml至50mg/ml，优选约为30mg/ml至45mg/ml，优选约为35mg/ml至45mg/ml，优选约为40mg/ml至45mg/ml，优选约为30mg/ml至40mg/ml，优选约为35mg/ml至40mg/ml，优选约为30mg/ml至35mg/ml；作为非限制性实施例，药物组合物中糖的浓度约为包括60mg/ml、65mg/ml、70mg/ml、75mg/ml、80mg/ml、85mg/ml或90mg/ml，更优选约为70mg/ml-80mg/ml，最优选75mg/ml。
在可选的实施方案中，药物组合物还包含表面活性剂。所述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-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基-肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、异硬脂酰胺基丙基-二甲基胺、甲基 可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇、乙烯与丙烯二醇的共聚物等等。优选的表面活性剂为聚山梨酯80或聚山梨酯20，更优选为聚山梨酯80。
在可选的实施方案中，药物组合物中表面活性剂的浓度约为0.02mg/ml至0.8mg/ml，优选约0.1mg/ml至0.8mg/ml，优选约0.2mg/ml至0.8mg/ml，优选约0.3mg/ml至0.8mg/ml，优选约0.4mg/ml至0.8mg/ml，优选约0.5mg/ml至0.8mg/ml，优选约0.6mg/ml至0.8mg/ml，优选约0.7mg/ml至0.8mg/ml，优选约为0.02mg/ml至0.7mg/ml，优选约0.1mg/ml至0.7mg/ml，优选约0.2mg/ml至0.7mg/ml，优选约0.3mg/ml至0.7mg/ml，优选约0.4mg/ml至0.7mg/ml，优选约0.5mg/ml至0.7mg/ml，优选约0.6mg/ml至0.7mg/ml，优选约为0.02mg/ml至0.6mg/ml，优选约0.1mg/ml至0.6mg/ml，优选约0.2mg/ml至0.6mg/ml，优选约0.3mg/ml至0.6mg/ml，优选约0.4mg/ml至0.6mg/ml，优选约0.5mg/ml至0.6mg/ml，优选约为0.02mg/ml至0.5mg/ml，优选约0.1mg/ml至0.5mg/ml，优选约0.2mg/ml至0.5mg/ml，优选约0.3mg/ml至0.5mg/ml，优选约0.4mg/ml至0.5mg/ml，优选约为0.02mg/ml至0.4mg/ml，优选约0.1mg/ml至0.4mg/ml，优选约0.2mg/ml至0.4mg/ml，优选约0.3mg/ml至0.4mg/ml，优选约为0.02mg/ml至0.3mg/ml，优选约0.1mg/ml至0.3mg/ml，优选约0.2mg/ml至0.3mg/ml，优选约为0.02mg/ml至0.2mg/ml，优选约0.1mg/ml至0.2mg/ml，优选约为0.02mg/ml至0.1mg/ml，作为非限制性实施例，所述药物组合物中表面活性剂的浓度约为0.2mg/ml、0.25mg/ml、0.3mg/ml、0.35mg/ml、0.4mg/ml、0.45mg/ml或0.5mg/ml，更优选约为0.3mg/ml-0.5mg/ml。
在一种实施方案中，所述的药物组合物包含如下i)或)ii)所示的组分：
i)(a)1mg/ml至90mg/ml的LAG-3抗体或其抗原结合片段，(b)5mM至30mM的醋酸-醋酸钠缓冲剂，pH约为5.0-6.5，(c)30mg/ml至90mg/ml的蔗糖，和(d)0.02mg/ml至0.8mg/ml的聚山梨酯80；优选的，所述药物组合物包含：
(e)40mg/ml至80mg/ml的LAG-3抗体或其抗原结合片段，(f)10mM至30mM的醋酸-醋酸钠缓冲剂，pH约为5.2-5.8，(g)70mg/ml至80mg/ml的蔗糖，和(h)0.4mg/ml至0.5mg/ml的聚山梨酯80；或
ii)(a)1mg/ml至90mg/ml的LAG-3抗体或其抗原结合片段，(b)5mM至30mM的组氨酸-盐酸缓冲剂，pH约为5.0-6.5，(c)30mg/ml至90mg/ml的蔗糖或海藻糖，和(d)0.05mg/ml至0.6mg/ml的聚山梨酯80，优选的，所述药物组合物包含：
(e)45mg/ml至60mg/ml的LAG-3抗体或其抗原结合片段，(f)10mM至30mM的组氨酸-盐酸缓冲剂，pH约为5.5-6.0，(g)60mg/ml至90mg/ml的蔗糖，和(h)0.2mg/ml至0.6mg/ml的聚山梨酯80。
在一种实施方案中，药物组合物中所述的LAG3抗体或其抗原结合片段包含分别如SEQ ID NO:9，SEQ ID NO:10和SEQ ID NO:11所示的重链HCDR1、HCDR2和HCDR3和分别如SEQ ID NO:15，SEQ ID NO:16和SEQ ID NO:17所示的轻链LCDR1、LCDR2和LCDR3。
在可选的实施方案中，所述药物组合物包含：
(a)1mg/ml至90mg/ml的LAG-3抗体或其抗原结合片段，(b)5mM至30mM的醋酸盐缓冲剂，pH优选约为5.0至6.5，(c)30mg/ml至90mg/ml的蔗糖，和(d)0.02mg/ml至0.8mg/ml的聚山梨酯80。
在可选的实施方案中，所述药物组合物包含：
(a)40mg/ml至80mg/ml的LAG-3抗体或其抗原结合片段，(b)10mM至30mM的醋酸盐缓冲剂，pH为约5.0-6.0，(c)60mg/ml至90mg/ml的蔗糖，和(d)0.1mg/ml至0.5mg/ml的聚山梨酯80。
在可选的实施方案中，所述药物组合物包含：
(a)50mg/ml至60mg/ml的LAG-3抗体或其抗原结合片段，(b)10mM至30mM的醋酸-醋酸钠缓冲剂，且pH约为5.2-5.8。(c)70mg至80mg/ml的蔗糖，和(d)0.4mg/ml至0.5mg/ml的聚山梨酯80。
在可选的实施方案中，所述药物组合物包含：
(a)约50mg/ml的LAG-3抗体或其抗原结合片段，(b)10mM的醋酸-醋酸钠缓冲剂，pH约为5.5，(c)约75mg/ml的蔗糖，和(d)约0.4mg/ml的聚山梨酯80。
在可选的实施方案中，上述药物组合物中所述LAG-3抗体或其抗原结合片段为鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、人源化抗体或其抗原结合片段。
在可选的实施方案中，上述药物组合物中所述的LAG3鼠源抗体包含如SEQ ID NO:5所示的重链可变区和如SEQ ID NO:6所示的轻链可变区。
在可选的实施方案中，上述药物组合物中所述的LAG-3人源化抗体包含重链可变区和轻链可变区，其中所述的重链可变区氨基酸序列如SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:24或SEQ ID NO:25任一序列所示或与其具有至少85％序列同一性的氨基酸序列；
其中所述的轻链可变区如SEQ ID NO:22、SEQ ID NO:26、SEQ ID NO:27或SEQ ID NO:28任一所示或与其具有至少85％序列同一性的氨基酸序列。
在可选的实施方案中，药物组合物中所述LAG-3抗体的轻链可变区与SEQ ID NO:22、SEQ ID NO:26、SEQ ID NO:27或SEQ ID NO:28所示的轻链氨基酸可变区序列具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同一性，所述LAG-3抗体的重链氨可变区基酸序列与SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:24或SEQ ID NO:25所示的抗体重链可变区具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同一性。
在可选的实施方案中，上述药物组合物中所述的LAG3人源化抗体包含选自以下的重链可变区和轻链可变区的组合：
1)SEQ ID NO:21的重链可变区和SEQ ID NO:22的轻链可变区；
2)SEQ ID NO:21的重链可变区和SEQ ID NO:26的轻链可变区；
3)SEQ ID NO:21的重链可变区和SEQ ID NO:27的轻链可变区；
4)SEQ ID NO:21的重链可变区和SEQ ID NO:28的轻链可变区；
5)SEQ ID NO:23的重链可变区和SEQ ID NO:22的轻链可变区；
6)SEQ ID NO:23的重链可变区和SEQ ID NO:26的轻链可变区；
7)SEQ ID NO:23的重链可变区和SEQ ID NO:27的轻链可变区；
8)SEQ ID NO:23的重链可变区和SEQ ID NO:28的轻链可变区；
9)SEQ ID NO:24的重链可变区和SEQ ID NO:22的轻链可变区；
10)SEQ ID NO:24的重链可变区和SEQ ID NO:26的轻链可变区；
11)SEQ ID NO:24的重链可变区和SEQ ID NO:27的轻链可变区；
12)SEQ ID NO:24的重链可变区和SEQ ID NO:28的轻链可变区；
13)SEQ ID NO:25的重链可变区和SEQ ID NO:22的轻链可变区；
14)SEQ ID NO:25的重链可变区和SEQ ID NO:26的轻链可变区；
15)SEQ ID NO:25的重链可变区和SEQ ID NO:27的轻链可变区；和
16)SEQ ID NO:25的重链可变区和SEQ ID NO:28的轻链可变区。
在可选的实施方案中，上述药物组合物中所述的LAG-3人源化抗体包含重链恒定区和轻链恒定区，其中所述的重链恒定区优选如SEQ ID NO：38所示；其中所述的轻链恒定区优选如SEQ ID NO：39所示。
在可选的实施方案中，上述药物组合物中所述的LAG-3人源化抗体的重链选自SEQ ID NO:40所示的序列或与其具有至少95％的序列同一性氨基酸序列，轻链选自SEQ ID NO:41所示的的序列或与其具有至少95％的序列同一性氨基酸序列。
在可选的实施方案中，药物组合物中所述LAG-3抗体的重链与SEQ ID NO:40所示的重链氨基酸序列具有至少95％，96％，97％，98％，99％或100％的序列同一性，所述LAG-3抗体的轻链氨基酸序列与SEQ ID NO:41所示的抗体轻链具有至少95％，96％，97％，98％，99％或100％的序列同一性。
在一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu229-013，10mM醋酸-醋酸钠缓冲液，pH 5.5。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu229-013，10mM琥珀酸-琥珀酸钠缓冲液，pH 6.0。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu229-013，10mM组氨酸-盐酸缓冲液，pH 6.0。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu229-013，10mM琥珀酸-琥珀酸钠，pH6.0和0.1mg/ml聚山梨酯80。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu229-013，10mM琥珀酸-琥珀酸钠，pH6.0，和70mg/ml蔗糖。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu229-013， 10mM醋酸-醋酸钠，pH 5.5，60mg/ml蔗糖和0.4mg/mL聚山梨酯80。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu229-013，10mM组氨酸-醋酸，pH 6.0，60mg/ml蔗糖和0.4mg/mL聚山梨酯80。
在一些实施方案中，药物组合物包含：1mg/ml的LAG3抗体Hu229-013，10-30mM组氨酸-醋酸，pH 5.5，75mg/ml蔗糖和0.2mg/mL聚山梨酯80。
在又一些实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu229-013，10-30mM醋酸-醋酸钠，pH 5.2-5.8，75mg/ml蔗糖和0.2mg/mL聚山梨酯80。
在又一种实施方案中，药物组合物包含：60mg/ml的LAG3抗体Hu229-013，10mM醋酸-醋酸钠，pH 5.5，60mg/ml蔗糖和0.4mg/mL聚山梨酯80。
在又一些实施方案中，药物组合物包含：50-60mg/ml的LAG3抗体Hu229-013，10mM醋酸-醋酸钠，pH 5.5，30-90mg/ml蔗糖和0.4-0.5mg/mL聚山梨酯80。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu229-013，10mM醋酸-醋酸钠，pH 5.5，75mg/ml蔗糖和0.4mg/mL聚山梨酯80。
在另一些实施方案中，药物组合物中所述的LAG-3抗体或其抗原结合片段具包含分别如SEQ ID NO:12，SEQ ID NO:13和SEQ ID NO:14所示的HCDR1、HCDR2和HCDR3区和分别如SEQ ID NO:18，SEQ ID NO:19和SEQ ID NO:20所示的LCDR1、LCDR2和LCDR3区。
在可选实施方案中，所述的药物组合物包含：
(a)1mg/ml至90mg/ml的LAG-3抗体或其抗原结合片段，(b)5mM至30mM的组氨酸-盐酸缓冲剂，pH约为5.0-6.5，(c)30mg/ml至90mg/ml的蔗糖或海藻糖，和(d)0.05mg/ml至0.6mg/ml的聚山梨酯80。
在可选实施方案中，所述的药物组合物包含：
(a)45mg/ml至60mg/ml的LAG-3抗体或其抗原结合片段，(b)约10mM至30mM的组氨酸-盐酸钠缓冲剂，pH约为5.0-6.0，(c)60mg至90mg/ml的蔗糖或海藻糖，和(d)0.2mg/ml至0.6mg/ml的聚山梨酯80。
在可选的实施方案中，所述的药物组合物包含：
(a)约50mg/ml的LAG-3抗体或其抗原结合片段，(b)10mM的组氨酸-盐酸缓冲剂，pH约为5.0，(c)约75mg/ml的蔗糖或海藻糖，和(d)约0.3mg/ml的聚山梨酯80。
在可选的实施方案中，上述药物组合物中所述的LAG-3抗体或其抗原结合片段为鼠源抗体或其片段、嵌合抗体或其抗原结合片段、人源化抗体或其抗原结合片段。
在可选的实施方案中，上述药物组合物中所述的LAG3鼠源抗体包含如SEQ ID NO:7所示的重链可变区和如SEQ ID NO:8所示的轻链可变区。
在可选的实施方案中，上述药物组合物中所述的LAG-3人源化抗体包含：
选自SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:32或SEQ ID NO:33任意 一个所示的重链可变区序列或与其具有至少85％序列同一性的氨基酸序列和选自SEQ ID NO:30、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36或SEQ ID NO:37任意一个所示的轻链可变区序列或与其具有至少85％序列同一性的氨基酸序列。
在可选的实施方案中，药物组合物中所述LAG-3抗体的轻链可变区与SEQ ID NO:30、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36或SEQ ID37所示的轻链氨基酸可变区序列具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同一性，所述LAG-3抗体的重链氨可变区基酸序列与SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:32或SEQ ID NO:33所示的抗体重链可变区具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同一性。
在可选的实施方案中，上述药物组合物中所述的LAG3人源化抗体包含选自以下的重链可变区和轻链可变区的组合：
1)SEQ ID NO:29的重链可变区和SEQ ID NO:30的轻链可变区；
2)SEQ ID NO:29的重链可变区和SEQ ID NO:34的轻链可变区；
3)SEQ ID NO:29的重链可变区和SEQ ID NO:35的轻链可变区；
4)SEQ ID NO:29的重链可变区和SEQ ID NO:36的轻链可变区；
5)SEQ ID NO:29的重链可变区和SEQ ID NO:37的轻链可变区；
6)SEQ ID NO:31的重链可变区和SEQ ID NO:30的轻链可变区；
7)SEQ ID NO:31的重链可变区和SEQ ID NO:34的轻链可变区；
8)SEQ ID NO:31的重链可变区和SEQ ID NO:35的轻链可变区；
9)SEQ ID NO:31的重链可变区和SEQ ID NO:36的轻链可变区；
10)SEQ ID NO:31的重链可变区和SEQ ID NO:37的轻链可变区；
11)SEQ ID NO:32的重链可变区和SEQ ID NO:30的轻链可变区；
12)SEQ ID NO:32的重链可变区和SEQ ID NO:34的轻链可变区；
13)SEQ ID NO:32的重链可变区和SEQ ID NO:35的轻链可变区；
14)SEQ ID NO:32的重链可变区和SEQ ID NO:36的轻链可变区；
15)SEQ ID NO:32的重链可变区和SEQ ID NO:37的轻链可变区；
16)SEQ ID NO:33的重链可变区和SEQ ID NO:30的轻链可变区；
17)SEQ ID NO:33的重链可变区和SEQ ID NO:34的轻链可变区；
18)SEQ ID NO:33的重链可变区和SEQ ID NO:35的轻链可变区；
19)SEQ ID NO:33的重链可变区和SEQ ID NO:36的轻链可变区；和
20)SEQ ID NO:33的重链可变区和SEQ ID NO:37的轻链可变区。
在可选的实施方案中，上述药物组合物中所述嵌合抗体或人源化抗体重链进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源 IgG4或其变体的重链恒定区，最优选如SEQ ID NO:38所示的重链恒定区；所述嵌合抗体或人源化抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区，优选如SEQ ID NO:39所示的轻链恒定区。
在可选的实施方案中，上述药物组合物中所述的LAG-3人源化抗体的重链选自SEQ ID NO:42所示的序列或与其具有至少95％的序列同一性氨基酸序列，轻链选自SEQ ID NO:43所示的的序列或与其具有至少95％的序列同一性氨基酸序列。
在可选的实施方案中，药物组合物中所述LAG-3抗体的重链与SEQ ID NO:42所示的重链氨基酸序列具有至少95％，96％，97％，98％，99％或100％的序列同一性，所述LAG-3抗体的轻链氨基酸序列与SEQ ID NO:43所示的抗体轻链具有至少95％，96％，97％，98％，99％或100％的序列同一性。
在一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu303-005，10mM组氨酸-盐酸缓冲液，pH6.0。
在一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu303-005，10mM组氨酸-盐酸缓冲液，pH5.0。
在一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu303-005，10mM组氨酸-盐酸缓冲液，pH6.5。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu303-005，10mM醋酸-醋酸钠缓冲液，pH5.5。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu303-005，10mM醋酸-醋酸钠缓冲液，pH5.5，75mg/ml蔗糖和0.2mg/ml聚山梨酯80。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu303-005，10mM醋酸-醋酸钠缓冲液，pH5.5，75mg/ml海藻糖和0.2mg/ml聚山梨酯80。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu303-005，10mM醋酸-醋酸钠缓冲液，pH5.5，和0.4mg/ml聚山梨酯80。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu303-005，10mM组氨酸-盐酸缓冲液，pH6.0，75mg/ml蔗糖和0.4mg/ml聚山梨酯80。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu303-005，10mM组氨酸-盐酸缓冲液，pH6.5，75mg/ml蔗糖和0.4mg/ml聚山梨酯80。
在又一种实施方案中，药物组合物包含：50mg/ml的LAG3抗体Hu303-005，10mM组氨酸-盐酸缓冲液，pH5.5，75mg/ml蔗糖和0.4mg/ml聚山梨酯80。
在一些实施方案中，药物组合物包含：45-60mg/ml的LAG3抗体Hu303-005，10mM组氨酸-盐酸缓冲液，pH5.5，75mg/ml蔗糖和0.2-0.6mg/ml聚山梨酯80。
在又一种实施方案中，药物组合物包含：约50mg/ml的LAG3抗体Hu303-005，约10mM组氨酸-盐酸缓冲液，约pH6.0，约75mg/ml蔗糖和约0.3mg/ml聚山梨酯80。
本发明还提供一种制备含LAG-3抗体的药物组合物的方法，包括将LAG-3抗体或抗原结合片段与药学上可接受的赋形剂混合。
本发明还提供一种制备含LAG-3抗体的冻干制剂的方法，其中包括将前述药物组合物经冷冻干燥的步骤。
在可选的实施方案中，制备含LAG-3抗体的冻干制剂的方法中所述冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。
在可选的实施方案中，制备含LAG-3抗体的冻干制剂的方法中所述一次干燥温度为-5℃至-20℃，优选为-10℃。
本发明还提供一种经前述制备含LAG-3抗体的冻干制剂的方法制备所得含LAG-3抗体的冻干制剂。
在一些实施方案中，该冻干制剂于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，该冻干制剂于40℃稳定至少7天，至少14天或至少28天。
本发明还提供一种由上述冻干方法制备所得含LAG-3抗体的冻干制剂。
本发明还提供一种一种含LAG3抗体的冻干制剂，其特征在于所述制剂复溶后可形成上述药物组合物。
本发明还提供制备含有LAG-3抗体的冻干制剂的复溶溶液的方法，其中包括将前述冻干制剂经复溶的步骤，其复溶所用溶液选自但不限于注射用水、生理盐水或葡萄糖溶液。
本发明还提供一种经前述含有LAG-3抗体的冻干制剂的复溶溶液的方法制备所得含有LAG-3抗体的冻干制剂的复溶溶液。
本发明还提供一种包含LAG3抗体的复溶溶液，其还含有如下成分：
(a)1至90mg/ml的LAG-3抗体或其抗原结合片段，(b)5至30mM的醋酸-醋酸钠缓冲剂，pH约为5.0-6.5，(c)30至90mg/ml的蔗糖，(d)0.02至0.8mg/ml的聚山梨酯80。
本发明还提供一种包含LAG3抗体的复溶溶液，其还含有如下成分：
(a)40至80mg/ml的LAG-3抗体或其抗原结合片段，(b)10至30mM的醋酸-醋酸钠缓冲剂，pH约为5.2-5.8，(c)70至80mg/ml的蔗糖，(d)0.4至0.5mg/ml的聚山梨酯80。
本发明还提供一种包含LAG3抗体的复溶溶液，其还含有如下成分：
(a)50mg/ml的LAG-3抗体或其抗原结合片段，(b)10mM的醋酸-醋酸钠缓冲剂，pH约为5.5，(c)75mg/ml的蔗糖，(d)0.4mg/ml的聚山梨酯80。
(a)1至90mg/ml的LAG-3抗体或其抗原结合片段，(b)5至30mM的组氨酸-盐酸缓冲剂，pH约为5.0-6.5，(c)30至90mg/ml的蔗糖，(d)0.05至0.6mg/ml的聚山梨酯80。
本发明还提供一种包含LAG3抗体的复溶溶液，其还含有如下成分：
(a)45至60mg/ml的LAG-3抗体或其抗原结合片段，(b)10至30mM的组氨酸-盐酸缓冲剂，pH约为5.5-6.0，(c)60至80mg/ml的蔗糖，(d)0.2至0.6mg/ml的聚山梨酯80。
本发明还提供一种包含LAG3抗体的复溶溶液，其还含有如下成分：
(a)50mg/ml的LAG-3抗体或其抗原结合片段，(b)10mM的组氨酸-盐酸缓冲剂，pH约为6.0，(c)75mg/ml的蔗糖，(d)0.3mg/ml的聚山梨酯80。
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，该玻璃瓶为中性硼硅玻璃管制注射剂瓶。
本发明前述的药物组合物或冻干制剂或冻干制剂的复溶溶液可用作药物。
本发明还提供前述的药物组合物或冻干制剂或冻干制剂的复溶溶液在制备用于治疗LAG-3相关的疾病或病症的药物中的用途，其中所述的疾病或病症为致病性T细胞参与的相关疾病或病症，优选为癌症；所述癌症包括，但不限于卵巢癌、黑色素瘤、前列腺癌、肠癌、胃癌、食管癌、乳腺癌、肺癌、肾癌、胰腺癌、子宫癌、肝癌、膀胱癌、子宫颈癌、口腔癌、脑癌、睾丸癌、皮肤癌、甲状腺癌以及血液学恶性肿瘤，其中所述的血液学恶性肿瘤包括包括骨髓瘤和慢性和急性白血病。
本发明还提供一种治疗和预防LAG-3相关的疾病或病症的方法，包括给予所需患者治疗有效量的前述的药物组合物或冻干制剂或冻干制剂的复溶溶液，其中所述的疾病或病症为致病性T细胞参与的相关疾病或病症，优选为癌症；所述癌症包括，但不限于卵巢癌、黑色素瘤、前列腺癌、肠癌、胃癌、食管癌、乳腺癌、肺癌、肾癌、胰腺癌、子宫癌、肝癌、膀胱癌、子宫颈癌、口腔癌、脑癌、睾丸癌、皮肤癌、甲状腺癌以及血液学恶性肿瘤，其中所述的血液学恶性肿瘤包括包括骨髓瘤和慢性和急性白血病。
本发明还提供一种制品，其包括容器，该容器中装有前述的药物组合物或冻干制剂或冻干制剂的复溶溶液。
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：人源化抗LAG-3抗体样品增强对SEB刺激激活的T淋巴细胞分泌IL-2细胞因子的作用。数据结果显示，LAG-3人源化候选抗体Hu229-013和Hu303-005能够不同程度增强激活的T淋巴细胞分泌细胞因子IL-2,并且有药物浓度剂量效应。
图2：人源化抗LAG-3抗体对U-87MG荷瘤小鼠肿瘤体积的影响。结果显示，给药14天后，LAG-3抗体Hu229-013 6mpk和Hu303-005 6mpk均有一定的抑瘤 效果，抑瘤率分别为27.25％(p<0.05)及34.94％(p<0.01)；与对照组有显著差异(p<0.001vs hIGg)
图3：Hu229-013抗体40℃CE纯度趋势图。
图4：Hu229-013抗体40℃IEC中性峰趋势图。
图5：Hu303-005抗体40℃非还原CE趋势图。
图6：Hu303-005抗体40℃iCE主峰趋势图。
图7：Hu303-005振摇SEC结果趋势图。
图8：Hu303-005抗体IEC 0℃时与40℃数据差值拟合结果。
图9：Hu303-005抗体CE纯度0℃时与40℃数据差值拟合图。
图10：Hu303-005抗体制剂25℃和40℃iCE/CE/DLS拟合结果。
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。将pH控制在适当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。
“组氨酸缓冲剂”是包含组氨酸离子的缓冲剂。组氨酸缓冲剂的实例包括组氨酸-盐酸盐，组氨酸-醋酸盐，组氨酸-磷酸盐，组氨酸-硫酸盐等缓冲剂，优选组氨酸-盐酸盐缓冲剂。组氨酸-盐酸盐缓冲剂是组氨酸与盐酸或组氨酸与组氨酸盐酸盐配制而成。
“柠檬酸盐缓冲剂”是包括柠檬酸根离子的缓冲剂。柠檬酸盐缓冲剂的实例包括柠檬酸-柠檬酸钠、柠檬酸-柠檬酸钾、柠檬酸-柠檬酸钙、柠檬酸-柠檬酸镁等。优选的柠檬酸盐缓冲剂为柠檬酸-柠檬酸钠缓冲剂。
“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-琥珀酸钾、琥珀酸-琥珀酸钙盐等。优选的琥珀酸盐缓冲剂为琥珀酸-琥珀酸钠缓冲剂。
“磷酸盐缓冲剂”是包括磷酸离子的缓冲剂。磷酸盐缓冲剂的实例包括磷酸氢二钠酸-磷酸二氢钠、磷酸氢二钠酸-磷酸二氢钾等。优选的磷酸盐缓冲剂为磷酸氢二钠酸-磷酸二氢钠缓冲剂。
“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。优选的醋酸盐缓冲剂为醋酸-醋酸钠缓冲剂。
“Tris缓冲剂”是指包含三(羟甲基)氨基甲烷的缓冲溶液，Tris缓冲剂也被 称为Tris碱、Trizma、Trisamine、THAM、氨丁三醇、氨基丁三醇和缓血酸胺。Tris缓冲剂的有效缓冲范围在pH7.0到9.2之间，Tris碱的水溶液pH在10.5左右，一般加入盐酸以调节pH值至所需值，即可获得该pH值的缓冲剂。
本发明的“糖”包含常规组合物(CH 2O) 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。可选自葡萄糖，蔗糖，海藻糖，乳糖，果糖，麦芽糖，右旋糖苷，甘油，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。优选的糖为非还原性二糖，更优选为蔗糖。
“粘度调节剂”是为调节制剂的粘度而加入的常规药用物料，此处所指的粘度调节剂主要是指无机盐和氨基酸盐，其中无机盐优选自氯化钠、氯化钙、氯化镁、醋酸钙，所述氨基酸盐优选自精氨酸盐酸盐，组氨酸盐酸盐，甘氨酸盐酸盐，组氨酸醋酸盐等。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。本文中，“药物组合物”和“制剂”并不互相排斥。
本发明中所述药物组合物的溶液形式，若无特殊说明，其中的溶剂均为水。
“冻干制剂”表示液体或溶液形式的药物组合物或溶液制剂经过真空冷冻干燥步骤之后获得的制剂或药物组合物。
本公开的冷冻干燥包括预冻，一次干燥，二次干燥。预冻目的是冷冻产品，获得结晶固体。预冻温度和预冻速度是两个重要工艺参数，本发明中将预冻温度设定为-45℃，预冻速度设定为1℃/min。一次干燥也称为主干燥，是样品冷冻干燥的主要阶段。目的是移除产品里冰的同时，保持产品形状，最小程度的减少对产品的破坏。若一次干燥的温度和真空度选择不当，会导致制品的塌陷。较高的温度和真空度均会加快冻干效率，但同时也会增加制品塌陷的风险。本发明所述一次干燥的温度可为本领域常规的温度，例如-30℃-0℃。二次干燥也称为解析干燥，是通过抽极限真空(0.01mbar)和升高温度(20-40℃)除去制品中结合水的主要步骤。由于大多数生物制品对温度比较敏感，所以二次干燥温度选择在温度范围的低点，即25℃。冷冻干燥的时间与冷冻机，冻干制剂剂量，冻干药剂的容器有关。这种时间的调整是在本领域技术人员所熟知的。
本文所用术语“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围，例如，约5.5的pH意指pH5.5±1.1。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值 的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
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，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℃保存包括1个月、3个月、6个月或在40℃保存1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5％、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±5％变化。通常观察到不超过约5％、优选不超过约5％的截短，通常形成不超过约5％、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。
术语“LAG-3”是指淋巴细胞活化基因3。术语“LAG-3”包含变体、同等型(isoform)、同源物、直系同源体(ortholog)及旁系同源体(paralog)。术语“人LAG-3”指人序列LAG-3，例如具有Uniprot号:P18627的人LAG-3的完整氨基酸序列。本领域中亦已知LAG-3，例如CD215。人LAG-3序列与Uniprot号:P18627的人LAG-3的不同之处可在于具有例如保守突变或在非保守区中的突变，且LAG-3 与Uniprot号:P18627的人LAG-3具有实质上相同的生物功能。举例而言，人LAG-3的生物功能是在LAG-3的胞外域中具有表位，该表位被本公开的抗体特异性结合，或人LAG-3的生物功能是结合MHCII类分子。
特定人LAG-3序列在氨基酸序列中通常与Uniprot号:P18627的人LAG-3至少90％相同，且含有在与其他物种(例如鼠类)的LAG-3氨基酸序列相比时鉴别为人氨基酸序列的氨基酸残基。在某些情形下，人LAG-3在氨基酸序列中可与Uniprot号:P18627的LAG-3至少85％或甚至至少95％、96％、97％、98％或99％相同。在某些实施方案中，人LAG-3序列较Uniprot号:P18627的LAG-3序列显示不超过10个氨基酸差异。在某些实施方案中，人LAG-3可较Uniprot号:P18627的LAG-3序列显示不超过5或甚至不超过4、3、2或1个氨基酸差异。可如本文所阐述的测定百分比同一性。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本发明所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。
在本发明中，本发明所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本发明中，本发明所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
抗体重链和轻链靠近N端的约15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LAG-3的单克隆抗体。制备时用LAG-3抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。在本发明一个优选的实施方案中，所述的LAG-3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LAG-3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。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的抗体可变抗体反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网 www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本发明的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。
本发明中术语“抗LAG-3抗体”、“抗LAG-3”、“LAG-3抗体”或“结合LAG-3的抗体”是指这样的抗体，所述抗体能够以足够的亲合力结合LAG-3抗体以致所述抗体可以用作靶向LAG-3中的诊断剂和/或治疗剂。
本发明中术语“与LAG-3结合”，指能与人LAG-3相互作用。
术语“特异性结合”指如通过本领域可用的技术，例如竞争ELISA、
Figure PCTCN2018122534-appb-000001
测定或
Figure PCTCN2018122534-appb-000002
测定所测定的。该术语还适用于当例如本发明抗体的抗原结合结构域对许多抗原携带的特定表位特异的情况，在该情况下携带抗原结合结构域的抗体能够特异性结合携带该表位的多种抗原。
术语“竞争结合”是指与本发明的单克隆抗体识别人LAG-3的胞外区上的相同表位(也称为抗原决定簇)或相同表位的一部分并与所述抗原结合的抗体。与本发明的单克隆抗体结合相同表位的抗体是指识别并结合于本发明的单克隆抗体所识别的人LAG-3的氨基酸序列的抗体。
术语“KD”或“Kd”是指特定抗体-抗原相互作用的解离平衡常数。通常，本发明的抗体以小于大约10 -7M，例如小于大约10 -8M、10 -9M或10 -10M或更小的解离平衡常数(KD)结合LAG-3，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。
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LAG-3结合的scFv片段及其他利用了能与人LAG-3结合的抗体的VH和VL区形成的可结合人LAG-3的片段；其包含本发明所述抗体的选自SEQ ID NO:9、10、11、15、16、17和12、13、14、18、19和20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)。
术语“表位”指在一个或更多个抗体的抗原结合区中能够被抗体识别并结合的分子部分。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
氨基酸序列“同一性”是指两个蛋白或多肽之间的序列相似性。当两个比较序列中的位置均被相同氨基酸残基占据时，例如如果两个多肽的一个位置都被同一个氨基酸残基占据时，那么所述分子在该位置是一致的。适于确定序列同一性百分比和序列相似性百分比的算法的实例是BLAST和BLAST2.0算法，它们分别描述于Altschul et al.(1990)J.Mol.Biol.215:403-45和Altschul et al.(1977)Nucleic Acids Res.25:3389-3402。用于执行BLAST分析的软件可在美国国家生物技术信息中心公开获取(http://www.ncbi.nlm.nih.gov/)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人LAG-3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。
“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可测量的程度。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，患者的健康状况、患者的行为、患者的饮食、给药时间、给药方式、排泄的速率、药物的组合等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。
发明详述：
本发明获得了一种稳定包含LAG3抗体或其抗原结合片段、醋酸盐缓冲剂或组氨酸盐、蔗糖和聚山梨酯80的药物组合物(制剂)，该药物组合物(制剂)更适于给药。
具体实施方式
实施例
通过以下实施例进一步详细说明本发明。这些实施例仅用于说明性目的，而并不用于限制本发明的范围。
本发明实施例中未注明具体条件的实验方法，通常按照常规条件；或按照原 料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.LAG-3抗原抗体的制备
1、蛋白设计及表达
以UniProt Lymphocyte activation gene 3 protein(人LAG-3，Uniprot号：P18627)作为本发明LAG-3的模板，设计本发明涉及的抗原及检测用蛋白的氨基酸序列，可选的在LAG-3蛋白基础上融合不同的标签，分别克隆到pHr载体上(自产)或pTT5载体上(Biovector，Cat#:102762)或pTargeT载体上(promega,A1410)，在293细胞瞬转表达或CHO-S稳定表达纯化，获得编码本发明抗原及检测用蛋白。以下LAG-3抗原未特殊说明的均指人LAG-3。
带Flag标签的LAG-3胞外区：LAG-3-Flag，用于免疫小鼠
Figure PCTCN2018122534-appb-000003
                                                           SEQ ID NO:1
注释：划横线部分为信号肽，斜体部分为Flag-tag标签。
全长LAG-3：用于构建LAG-3过表达细胞株，免疫小鼠和检测
Figure PCTCN2018122534-appb-000004
                                                           SEQ ID NO:2
注释：
Figure PCTCN2018122534-appb-000005
LAG-3胞外区和hIgG1 Fc的融合蛋白：LAG-3-Fc，用于检测
Figure PCTCN2018122534-appb-000006
                                                           SEQ ID NO:3
注释：划横线部分为信号肽，双划线部分为接头，斜体部分为Fc。
LAG-3胞外区和mIgG2a Fc的融合蛋白：LAG-3-mFc，用于检测
Figure PCTCN2018122534-appb-000007
                                                           SEQ ID NO:4
注释：划横线部分为信号肽，双划线部分为接头，斜体部分为mFc。
2、LAG-3相关重组蛋白的纯化，以及杂交瘤抗体、重组抗体的纯化
1)带Flag标签的LAG-3-Flag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。利用0.5×PBS平衡flag亲和柱，冲洗2-5倍柱体积。将除杂后的细胞表达上清样品上柱。用0.5×PBS冲洗柱子，至A280读数降至基线。用PBS冲洗柱子，冲洗杂蛋白，并收集。用100mM甘氨酸，pH 3.0.洗脱目的蛋白，并收集，以备后续体外激活和进一步纯化。
2)杂交瘤、重组抗体、Fc融合蛋白的纯化
将细胞表达上清样品高速离心去除杂质，杂交瘤表达上清用Protein G柱，重组抗体、Fc融合蛋白表达上清用Protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用100mM乙酸pH3.0洗脱目的蛋白，用1M Tris-HCl,pH8.0中和。洗脱样品适当浓缩后利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，去聚体的峰收集好后分装备用。
实施例2.抗人LAG-3杂交瘤单克隆抗体的制备
1.免疫
抗人LAG-3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以下方案免疫。免疫抗原为带Flag标签的人LAG-3胞外区(SEQ ID NO:1)。
免疫方案A：用
Figure PCTCN2018122534-appb-000008
Gold Adjuvant(Sigma Cat No.T2684)与Thermo
Figure PCTCN2018122534-appb-000009
Alum(Thermo Cat No.77161)交叉免疫。抗原与佐剂(
Figure PCTCN2018122534-appb-000010
Gold Adjuvant)比例为1:1，抗原与佐剂(Thermo
Figure PCTCN2018122534-appb-000011
Alum)比例为3:1，50μg/只/次(首免)，25μg/只/次(加强免疫)。抗原乳化后进行接种，时间为第0、7、14、21、28、35、42天。第0天皮下(SC)多点注射50μg/只的乳化后抗原。第7天腹膜内(IP)注射25μg/只。第14、28、35、42天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。于第21，35，49天取血，用ELISA方法确定小鼠血清中的抗体滴度。在7免以后，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。在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射50μg/只的生理盐水配制的抗原溶液。
免疫方案B：用QuickAntibody-Mouse5W(KX0210041)对小鼠进行免疫。抗原与佐剂比例为1:1，25μg/只/次(首免/加强免疫)。抗原与佐剂迅速充分混匀后接种，时间为第0、21、35天。第0天小鼠后小腿肌肉(IM)注射25μg/只的抗原。第21，35天按同样方式注射25μg/只(根据滴度决定第3免是否进行)。于第28，42天取血，用ELISA方法确定小鼠血清中的抗体滴度。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。在进行脾细胞融合前3天加强免疫， 腹膜内(IP)注射50μg/只的生理盐水配制的抗原溶液。
2.脾细胞融合
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 PCTCN2018122534-appb-000012
CRL-8287 TM)进行融合得到杂交瘤细胞。融合好的杂交瘤细胞以0.5-1×10 6/ml的密度用完全培养基(含20％FBS、1×HAT、1×OPI的DMEM培养基)重悬，100μl/孔种于96孔板中，37℃，5％CO 2孵育3-4天后，补充HAT完全培养基100μl/孔，继续培养3-4天至形成针尖般克隆。去除上清，加入200μl/well的HT完全培养基(含20％FBS、1×HT和1×OPI的RPMI-1640培养基)，37℃，5％CO 2培养3天后进行ELISA检测。
3.杂交瘤细胞筛选
根据杂交瘤细胞生长密度，用结合ELISA方法进行杂交瘤培养上清检测。并将结合ELISA检测的阳性孔细胞上清进行细胞阻断实验。结合和阻断均为阳性的孔细胞及时进行扩增冻存保种和二到三次亚克隆直至获得单细胞克隆。
每次亚克隆细胞均需进行LAG-3结合ELISA、细胞阻断实验检测。通过以上实验筛选得到杂交瘤克隆，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。
4.杂交瘤阳性克隆序列测定
从阳性杂交瘤中克隆序列过程如下。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,Cat No.15596-018)按照试剂盒说明书步骤提取RNA，用PrimeScript TM Reverse Transcriptase试剂盒反转录(Takara,Cat No.2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen,TB326Rev.B 0503)进行PCR扩增后送测序公司测序。得到的杂交瘤克隆mAb229的重链、轻链的DNA序列对应的氨基酸序列SEQ ID NO:5、6和SEQ ID NO:7、8所示：
mAb229-VH
Figure PCTCN2018122534-appb-000013
                                                     SEQ ID NO：5
mAb229-VL
Figure PCTCN2018122534-appb-000014
                                                     SEQ ID NO：6
mAb303-VH
Figure PCTCN2018122534-appb-000015
                                                     SEQ ID NO：7
mAb303-VL
Figure PCTCN2018122534-appb-000016
                                                  SEQ ID NO：8
表1.各重链及轻链CDR区序列
Figure PCTCN2018122534-appb-000017
对得到的阳性克隆进行结合人LAG-3的ELISA实验(结果见表2中蛋白水平结合活性EC50值)、结合人LAG-3过表达CHO-s细胞的ELISA实验(结果见表2中细胞水平结合活性EC50值)和阻断LAG-3抗原与Daudi细胞结合实验(结果见表2中阻断活性EC50值)，并检测其与人LAG-3蛋白的亲和力(结果见表3)。
表2.LAG-3鼠源抗体体外活性
Figure PCTCN2018122534-appb-000018
表3.LAG-3鼠源抗体的亲和力
Figure PCTCN2018122534-appb-000019
表2数据显示LAG-3抗体mAb229和mAb303与人LAG-3蛋白均有很好的 结合活性。LAG-3抗体mAb229和mAb303与过表达人LAG-3全长蛋白的CHO-S细胞均有很好的结合活性。LAG-3抗体mAb229和mAb303均可显著阻断人LAG-3抗原与Daudi细胞的结合。
表3数据表明，本发明LAG-3抗体mAb229和mAb303对人LAG-3蛋白有较强的结合活性和亲和力。
实施例3.抗人LAG-3鼠杂交瘤单克隆抗体mAb229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，分别挑选与mAb229同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。其中氨基酸残基由Kabat编号系统确定并注释。
1、杂交瘤克隆mAb229人源化构架选择
鼠源抗体mAb229的人源化轻链模板为IGKV1-39*01和hjk4.1，人源化重链模板为IGHV7-4-1*02和hjh6.1，人源化可变区序列如下：
Hu229VH-CDR graft
Figure PCTCN2018122534-appb-000020
                                                      SEQ ID NO：21
Hu229VL-CDR graft
Figure PCTCN2018122534-appb-000021
                                                      SEQ ID NO：22
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。
2、杂交瘤克隆mAb229的模板选择和回复突变设计，见下表4：
表4.mAb229的模板选择和回复突变设计
Figure PCTCN2018122534-appb-000022
Figure PCTCN2018122534-appb-000023
注：如I48V表示依照Kabat编号系统，将48位I突变回V。Grafted代表鼠抗体CDR植入人种系FR区序列。
表5.鼠抗mAb229人源化序列组合
  Hu229_VL.1 Hu229_VL.1A Hu229_VL.1B Hu229_VL.1C
Hu229_VH.1 Hu229-004 LF 229-005 Hu229-006 Hu229-007
Hu229_VH.1A Hu229-008 Hu229-009 Hu229-010 Hu229-011
Hu229_VH.1B Hu229-012 Hu229-013 Hu229-014 Hu229-015
Hu229_VH.1C Hu229-016 Hu229-017 Hu229-018 Hu229-019
注：该表表示各种突变组合所得的序列。如Hu229-005表示，在人源化的鼠抗体Hu229-005上的有轻链HumAb229_VL.1A、重链HumAb229_VH.1两种突变。其它类推。
mAb229人源化具体序列如下：
Hu229VH.1(同Hu229VH-CDR graft)
Figure PCTCN2018122534-appb-000024
SEQ ID NO：21
Hu229VH.1A
Figure PCTCN2018122534-appb-000025
SEQ ID NO：23
Hu229VH.1B
Figure PCTCN2018122534-appb-000026
SEQ ID NO：24
Hu229VH.1C
Figure PCTCN2018122534-appb-000027
SEQ ID NO：25
Hu229VL.1(同Hu229VL-CDR graft)
Figure PCTCN2018122534-appb-000028
                                                     SEQ ID NO：22
Hu229VL.1A
Figure PCTCN2018122534-appb-000029
                                                     SEQ ID NO：26
Hu229VL.1B
Figure PCTCN2018122534-appb-000030
                                                    SEQ ID NO：27
Hu229VL.1C
Figure PCTCN2018122534-appb-000031
                                                    SEQ ID NO：28
实施例4.抗人LAG-3鼠杂交瘤单克隆抗体mAb303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，分别挑选与mAb303同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。其中氨基酸残基由Kabat编号系统确定并注释。
1、杂交瘤克隆mAb303人源化构架选择
鼠源抗体mAb303的人源化轻链模板为IGKV1-39*01和hjk4.1，人源化重链模板为IGHV1-3*01和hjh6.1，人源化可变区序列如下：
Hu303VH-CDR graft
Figure PCTCN2018122534-appb-000032
                                                  SEQ ID NO：29
Hu303VL-CDR graft
Figure PCTCN2018122534-appb-000033
                                                       SEQ ID NO：30
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。
2、杂交瘤克隆mAb303的模板选择和回复突变设计，见下表6：
表6.杂交瘤克隆mAb303人源化回复突变
Figure PCTCN2018122534-appb-000034
注：如L46R表示依照Kabat编号系统，将46位L突变回R。Grafted代表鼠抗体CDR植入人种系FR区序列。
各突变序列组合如下：
表7.鼠抗mAb303人源化序列组合
Figure PCTCN2018122534-appb-000035
注：该表表示各种突变组合所得的序列。如Hu303-005表示，在人源化的鼠抗体Hu303-005上的有轻链HumAb303_VL.1A、重链HumAb303_VH.1两种突变。其它类推。
mAb303人源化具体序列如下：
Hu303_VH.1(同Hu303VH-CDR graft)
Figure PCTCN2018122534-appb-000036
SEQ ID NO：29
Hu303_VH.1A
Figure PCTCN2018122534-appb-000037
SEQ ID NO：31
Hu303_VH.1B
Figure PCTCN2018122534-appb-000038
SEQ ID NO：32
Hu303_VH.1C
Figure PCTCN2018122534-appb-000039
SEQ ID NO：33
Hu303_VL.1(同Hu303VL-CDR graft)
Figure PCTCN2018122534-appb-000040
                                                     SEQ ID NO：30
Hu303_VL.1A
Figure PCTCN2018122534-appb-000041
                                                     SEQ ID NO：34
Hu303_VL.1B
Figure PCTCN2018122534-appb-000042
                                                     SEQ ID NO：35
Hu303_VL.1C
Figure PCTCN2018122534-appb-000043
                                                      SEQ ID NO：36
Hu303_VL.1D
Figure PCTCN2018122534-appb-000044
                                                      SEQ ID NO：37
实施例5.重组以及人源化抗体的制备
抗体选用人重链IgG4/轻链kappa的恒定区与各可变区组合，在Fc段做了S228P突变来增加IgG4抗体的稳定性，也可选用本领域其它已知的突变来增加其性能。
重链恒定区：
Figure PCTCN2018122534-appb-000045
轻链恒定区：
Figure PCTCN2018122534-appb-000046
                                                 SEQ ID NO：39
Hu229-013的重链氨基酸序列为：
Figure PCTCN2018122534-appb-000047
Hu229-013的轻链氨基酸序列为：
Figure PCTCN2018122534-appb-000048
Hu303-005的重链氨基酸序列为：
Figure PCTCN2018122534-appb-000049
Hu303-005的轻链氨基酸序列为：
Figure PCTCN2018122534-appb-000050
SEQ ID NO：43
1.重组抗体的分子克隆
杂交瘤筛选所获得的阳性抗体分子经过测序后，得到可变区编码基因序列。以测序所得序列设计首尾引物，以测序基因为模板，经过PCR搭建各抗体VH/VK基因片段，再与表达载体pHr(带信号肽及hIgG4/hkappa恒定区基因(CH1-FC/CL)片段)进行同源重组,构建重组抗体全长表达质粒VH-CH1-FC-pHr/VL-CL-pHr。
2.人源化抗体的分子克隆
人源设计之后的抗体序列，经过密码子优化后产生人密码子偏好的编码基因序列，设计引物PCR搭建各抗体VH/VK基因片段，再与表达载体pHr(带信号肽及hIgG4/hkappa恒定区基因(CH1-FC/CL)片段)进行同源重组,构建人源化抗体全长表达质粒VH-CH1-FC-pHr/VL-CL-pHr。
3.重组以及人源化抗体的表达与纯化
分别表达抗体轻重链的质粒以1：1.2的比例转染HEK293E细胞，6天后收集表达上清，高速离心去除杂质，用Protein A柱进行纯化。用PBS冲洗柱子， 至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl，pH8.0-9.0中和。洗脱样品适当浓缩后，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，以去除聚体，收集单体峰，分装备用。
以下用生化测试方法验证本发明抗体性能及有益效果。
实施例6：LAG-3抗体结合人LAG-3(human LAG-3)蛋白的ELISA实验
抗LAG-3抗体的结合力通过抗体与human LAG-3蛋白的ELISA实验来检测。用带Fc或mFc标签的LAG-3融合蛋白通过与包被在酶标板中的抗Fc或mFc抗体结合从而固定到96孔酶标板中，抗体加入后信号的强弱被用于判断抗体和LAG-3的结合活性，具体实验方法如下。
用pH7.4的PBS(Sigma，Cat No.P4417-100TAB)缓冲液将羊抗人Fc抗体(Jackson Immuno Research，Cat No.109-005-008)或羊抗鼠Fc抗体(Sigma，Cat No.M3534-1ML)稀释至2μg/ml浓度，以50μl/孔的体积加入96孔酶标板中，于37℃孵育箱中放置2小时。弃去液体后，加入用PBS稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液200μl/孔，37℃孵育箱孵育2.5小时或4℃放置过夜(16-18小时)进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(PH7.4PBS含0.05％tweeen-20)洗板5次后，加入50μl/孔用样品稀释液(PH7.4PBS含1％BSA)稀释至1μg/ml的LAG-3-Fc融合蛋白(内部生产，SEQ ID NO:3)或LAG-3-mFc融合蛋白(内部生产，SEQ ID NO:4)，置37℃孵育箱孵育1小时或4℃放置过夜。孵育结束后，弃去酶标板中的反应液，用PBST洗板6次后，加入50μl/孔用样品稀释液稀释的不同浓度待测抗体(杂交瘤纯化抗体或人源化抗体)，放于37℃孵育箱孵育1小时。孵育结束后用PBST洗板5次，加入100μl/孔用样品稀释液稀释的HRP标记的羊抗鼠二抗(Jackson Immuno Research，Cat No.115-035-003)或羊抗人二抗(Jackson Immuno Research，Cat No.109-035-003)，37℃孵育1小时。用PBST洗板6次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育5-15min，加入50μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在波长450nm处读取吸收值，计算LAG-3抗体对人LAG-3的结合EC50值。结果如表8所示，数据表明，本发明筛选得到的人源化抗体与人LAG-3蛋白均有较高的结合活性。
表8.候选抗体在结合实验中的EC50的测定
候选抗体 结合ELISA EC50(nM)
mAb229 0.129
Hu229-008 0.506
Hu229-009 0.152
Hu229-010 0.174
Hu229-011 0.201
Hu229-012 0.268
Hu229-013 0.106
Hu229-014 0.153
Hu229-015 0.156
Hu229-016 0.154
Hu229-017 0.048
Hu229-019 0.068
mAb303 0.172
Hu303-004 0.278
Hu303-005 0.309
Hu303-006 0.288
Hu303-007 0.135
Hu303-008 0.140
Hu303-009 0.316
Hu303-010 0.137
Hu303-011 0.314
Hu303-012 0.164
Hu303-013 0.166
Hu303-014 0.232
Hu303-015 0.172
Hu303-016 0.161
Hu303-017 0.168
Hu303-018 0.244
Hu303-019 0.277
Hu303-020 0.140
Hu303-021 0.170
Hu303-022 0.145
Hu303-023 0.152
实施例7：LAG-3抗体与human LAG-3过表达CHO-S细胞的结合实验
抗LAG-3抗体的结合力通过抗体与过表达LAG-3蛋白的CHO-S细胞的结合实验来检测。通过电转染的方法将LAG-3全长质粒(内部生产，SEQ ID NO:2)转染进CHO-S细胞中后加压筛选两周后，检测LAG-3的表达量。将过表达细胞固定于96孔板底后，抗体加入后信号的强弱被用于判断抗体和LAG-3过表达 CHO-S细胞的结合活性，具体实验方法如下。
将细胞以4×10 5/ml密度，100μl/孔接种于96孔板中过夜培养。弃上清，用PBS洗三遍后，加入100μl/孔4％PFA室温固定半小时，PBS洗三遍。弃去液体后，加入用PBS稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液200μl/孔，37℃孵育箱孵育2.5小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(PH7.4PBS含0.05％tweeen-20)洗板5次后，加入50μl/孔用样品稀释液稀释的不同浓度待测抗体(杂交瘤纯化抗体或人源化抗体)，放于37℃孵育箱孵育1小时。孵育结束后用PBST洗板5次，加入100μl/孔用样品稀释液稀释的HRP标记的羊抗鼠二抗(Jackson Immuno Research，Cat No.115-035-003)或羊抗人二抗(Jackson Immuno Research，Cat No.109-035-003)，37℃孵育1小时。用PBST洗板6次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育5-15min，加入50μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在波长450nm处读取吸收值，计算LAG-3抗体对LAG-3过表达CHO-S细胞的结合EC50值。
实施例8：抗LAG-3抗体阻断LAG-3抗原和Daudi细胞结合实验
Daudi细胞(人白血病细胞，购自中科院细胞库)以3×10 5/孔的数量接种于96孔培养板中，1000转离心后弃上清，加入4％PFA室温固定30分钟。弃去固定液后用PBS缓冲液洗4遍，加入用PBS稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液200μl/孔，37℃孵育箱孵育2.5小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(PH7.4PBS含0.05％tweeen-20)洗板5次后，加入50μl/孔用样品稀释液(pH7.4PBS含1％BSA)稀释的已预混合孵育1小时的终浓度为0.4μg/ml的生物素(生物素标记试剂盒，东仁化学，Cat No.LK03)标记的LAG-3-Fc融合蛋白(内部生产，SEQ ID NO:3)和梯度浓度的待测抗体的混合液，置37℃孵育箱孵育1小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板5次后，加入50μl/孔用样品稀释液稀释HRP标记的链霉亲和素(Sigma，Cat No.S2438)，37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育5-15min，加入50μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在波长450nm处读取吸收值，计算LAG-3抗体对抗原与daudi细胞结合的阻断作用。结果如表9所示，数据表明，本发明筛选得到的人源化抗体均可显著阻断人LAG-3抗原与Daudi细胞的结合。
表9.候选抗体在结合阻断实验中的IC50的测定
Figure PCTCN2018122534-appb-000051
Figure PCTCN2018122534-appb-000052
实施例9：BIAcore检测LAG-3抗体亲和力实验
1、按照鼠抗捕获试剂盒(Cat.#BR-1008-38，GE)说明书中所述的方法将鼠抗捕获抗体共价偶联于CM5生物传感芯片(Cat.#BR-1000-12，GE)上，从而亲和捕获待测抗体，然后于芯片表面流经LAG-3-Flag(内部生产，SEQ ID NO:1)抗原，利用Biacore仪器实时检测反应信号从而获得结合和解离曲线，通过拟合得到亲和力数值，见上表2。在实验中每个循环解离完成后，用鼠抗捕获试剂盒里配置 的再生溶液将生物芯片洗净再生。结果表明，LAG-3抗体mAb229和mAb303对人LAG-3蛋白有较强的结合活性和亲和力。
2、按照人抗捕获试剂盒(Cat.#BR-1008-39，GE)说明书中所述的方法将人抗捕获抗体共价偶联于CM5生物传感芯片(Cat.#BR-1000-12，GE)上，从而亲和捕获待测抗体，然后于芯片表面流经LAG-3-Flag(内部生产，SEQ ID NO:1)抗原，利用Biacore仪器实时检测反应信号从而获得结合和解离曲线，通过拟合得到亲和力数值，见下表10。在实验中每个循环解离完成后，用人抗捕获试剂盒里配置的再生溶液将生物芯片洗净再生。结果表明，本发明筛选得到的人源化抗体对人LAG-3蛋白有较强的结合活性和亲和力。
表10.抗LAG-3抗体的亲和力
Figure PCTCN2018122534-appb-000053
实施例10：PBMC-T淋巴细胞激活实验
为了研究LAG-3抗体对T淋巴细胞激活，收集和纯化人外周血单核细胞(PBMC)，采用超抗原金黄色葡萄球菌肠毒素B(SEB)体外刺激72小时，检测IL-2细胞因子的分泌水平。实验过程简单描述如下：
新鲜分离纯化的PBMC，接种至96孔细胞培养板，细胞密度约为1×10 5/孔，加入100ng/mL SEB超抗原刺激，同时加入梯度稀释的抗体样品(用培养基稀释)或培养基作为空白对照。37℃，5％CO 2培养箱培养72h后，收集细胞培养上清。 采用ELISA(BD,CAT#550611)方法检测细胞培养上清内IL-2分泌水平。具体操作参考试剂说明书。
结果如图1所示，LAG-3人源化候选抗体Hu229-013，Hu303-005能够不同程度增强激活的T淋巴细胞分泌细胞因子IL-2,并且有药物浓度剂量效应。
实施例11：LAG-3抗体对U-87MG皮下移植瘤抑制的影响
本实验用于人源化抗LAG-3抗体对U-87MG荷瘤小鼠肿瘤体积的影响。
将人脑胶质瘤U-87MG细胞(3.5×10 6个)100μl接种于NOD-SCID(购自常州卡文斯实验动物有限公司)小鼠右肋部皮下，待10-14d肿瘤长至约40mm 3后，去除体重、肿瘤过大和过小的，按肿瘤体积将小鼠随机分为对照组Isotype matched hIgG，LAG-3人源化候选抗体Hu229-013组，Hu303-005组共3组(分组及剂量见表11)，每组8只(D0)。将经CD3抗体刺激的PBMC以5×10 5cells/60μl量注射到肿瘤组织中，并开始腹腔注射抗体，一周三次，共给药6次。每周测2次瘤体积，记录数据。肿瘤体积(V)计算公式为：
肿瘤体积(TV)＝1/2×L 长×L 短 2
各组动物肿瘤体积均用平均值±标准差(Mean±SEM)表示，并用Graphpad Prism 5软件作图，使用two way ANOVA统计分析，并计算抑瘤率，公式为：
肿瘤增殖率(T/C％)＝(T-T 0/C-C 0)×100％
抑瘤率％TGI＝1-T/C％
实验结果如表11及图2所示，给药14天后，LAG-3抗体Hu229-013 6mpk，Hu303-005 6mpk均有一定的抑瘤效果，抑瘤率分别为27.25％(p<0.05)及34.94％(p<0.01)；与对照组有显著差异(p<0.001vs hIGg)。
表11.人源化抗LAG-3抗体对U-87MG小鼠皮下移植瘤的疗效
Figure PCTCN2018122534-appb-000054
注：D0:第一次给药时间；*p<0.05,**p<0.01,***p<0.001vs hIGg，通过two way ANOVA分析。
实施例12.抗LAG-3人源化抗体，Hu229-013和Hu303-005的小鼠PK测定
ICR小鼠18只，雄性，体重18-22g，购自西普尔-必凯实验动物有限公司。饲养期间自由摄取饲料和水，实验室环境适应性饲养不小于3天，12/12小时光/ 暗周期调节，温度16-26℃，相对湿度40-70％。实验开始前一天，对ICR小鼠进行编号，随机分组，每组各3只。实验当天，两组小鼠分别静脉注射人源化候选抗体(Hu229-013)，给药剂量为3mg/kg和10mg/kg；两组每只小鼠分别静脉注射人源化候选抗体(Hu303-005)，给药剂量为3mg/kg和10mg/kg。静脉注射体积为20ml/kg。
给药后采血时间点为15min,8h,1d,2d,4d,7d,10d,14d,21d,28d,35d。每次约取全血0.1ml，不加抗凝剂，取血后在4℃放置30min，1000g离心15min，取上清置于EP管中，-80℃保存。
用ELISA方法检测血清中的血药浓度，用Winnolin软件来计算受试药物的T1/2及其主要参数。所得主要药动学参数见表12：
表12.Hu229-013和Hu303-005在小鼠体内的主要药动学参数表
Figure PCTCN2018122534-appb-000055
LAG-3人源化抗体Hu229-013和Hu303-005在小鼠体内的暴露量相近，且2个抗体在3,10mg/kg剂量下的暴露量、达峰浓度与剂量增长基本呈线性关系，具有线性动力学特征。
示例性抗体药物组合物(制剂)制备工艺
第一步：处方组成LAG-3抗体原液过0.22μm PVDF滤芯，过滤后中控取样检测无菌，收集滤液。
第二步：调节装量至5.3ml，将滤液灌装于6ml西林瓶中，加塞，分别于灌装开始、灌装中间、灌装结束时取样中控检测装量差异。
第三步：开启轧盖机，加铝盖，进行轧盖。
第四步：目检，确认产品无装量不准等缺陷。打印、粘贴西林瓶标签；打印纸盒标签，折叠纸盒，装盒，贴纸盒标签。
示例性的抗体药物组合物制备工艺：
第一步：处方组成Hu303-005原液过0.22μm PVDF滤芯，滤后中控取样检测无菌，收集滤液。
第二步：调节装量至5.3ml，将滤液灌装于20ml西林瓶中，半加冻干塞，将西林瓶中原液冻干，密封胶塞。
第三步：开启轧盖机，加铝盖，进行轧盖。
第四步：目检，确认产品无冻干塌陷等缺陷。打印、粘贴西林瓶标签；打印纸盒标签，折叠纸盒，装盒，贴纸盒标签。
实施例13.LAG-3抗体制剂缓冲体系的筛选
在10mM pH5.0-7.5一系列缓冲剂中，配制蛋白浓度为50mg/mL的LAG-3抗体Hu229-013或Hu303-005制剂，将每种制剂过滤灌装，加塞，轧盖，封口。将样品进行40℃高温、振摇等强制降解实验，以外观、分子排阻色谱法(SEC)、非还原十二烷基硫酸钠(CE-SDS)-毛细管电泳和离子交换色谱法(IEC)或全柱成像毛细管等电聚焦电泳(iCIEF)为评价指标。结果见表13-1和表13-2，统计分析结果见图3-图6。
表13-1.Hu229-013缓冲体系筛选结果
Figure PCTCN2018122534-appb-000056
Figure PCTCN2018122534-appb-000057
Figure PCTCN2018122534-appb-000058
注：振摇D12样品制备时加入0.01mg/ml聚山梨酯80，其他样品不含聚山梨酯80；D表示天数。
表13-2.Hu303-005抗体缓冲体系筛选结果
Figure PCTCN2018122534-appb-000059
Figure PCTCN2018122534-appb-000060
备注：D表示天；N/A表示未检测。
结果表明：
(1)Hu229-013抗体在醋酸-醋酸钠(AA)体系外观最佳，其次为琥珀酸-琥珀酸钠(SA)和组氨酸-盐酸(His-HCl)体系，醋酸-醋酸钠(AA)，pH5.5、琥珀酸-琥珀酸钠(SA)，pH6.0、柠檬酸-柠檬酸钠(CA)，pH6.0、组氨酸-盐酸(His)，pH6.0体系40℃CE、IEC纯度较高，综合考虑外观及CE、IEC结果，LAG-3抗体Hu229-013在AA(pH5.5)、SA(pH6.0)、His-HCl(pH6.0)体系中较为稳定，见图3和图4。
(2)Hu303-005抗体的振摇外观数据显示，pH较低时外观较好，缓冲体系组氨酸-盐酸盐(His-HCl)和醋酸-醋酸钠(AA)较优。在40℃加速条件下CE-SDS(非还原)及iCIEF出现明显下降，其中CE-SDS数据显示，pH 6.0较优，缓冲体系醋酸-醋酸钠、组氨酸-盐酸(His-HCl)和磷酸盐体系较优。iCE数据显示，pH较高时中性峰下降较少，见图5和图6。综合考虑优选缓冲体系为10mM His-HCl pH6.0。
实施例14.LAG-3抗体制剂中辅料的筛选
(1)在含下列不同浓度表面活性剂及糖的缓冲剂中，制备蛋白浓度为50mg/mL，含10mM琥珀酸-琥珀酸钠，pH6.0的LAG-3Hu229-013制剂，结果见表14-1。具体如下：
1)0.1mg/mL聚山梨酯20(PS20)
2)0.1mg/mL聚山梨酯80(PS80)
3)70mg/mL蔗糖
4)70mg/mL海藻糖
5)50mg/mL甘露醇
6)50mg/mL山梨醇
(2)在含下列不同浓度表面活性剂及糖的缓冲液中，制备Hu303-005抗体浓度为50mg/mL，含10mM醋酸(钠)，pH5.5的Hu303-005制剂，结果见图7和表14-2。
1)75mg/mL蔗糖+0.2mg/mL PS80
2)75mg/mL海藻糖+0.2mg/mL PS80
3)0.05mg/mL聚山梨酯20(PS20)
4)0.05mg/mL聚山梨酯80(PS80)
5)0.2mg/mL PS20
6)0.2mg/mL PS80
7)0.4mg/mL PS20
8)0.4mg/mL PS80
将每种制剂过滤灌装，加塞，轧盖，封口。将样品进行40℃高温、反复冻融、振摇等强制降解实验，结果见表14和图7。
表14-1.不同辅料对Hu229-013稳定性影响
Figure PCTCN2018122534-appb-000061
Figure PCTCN2018122534-appb-000062
注：D表示天。
表14-2.不同辅料对Hu303-005稳定性影响
Figure PCTCN2018122534-appb-000063
注：D表示天数。
结果表明：
(1)Hu229-013抗体制剂经振摇PS80组外观优于PS20；冻融1次后，蔗糖组出现少量颗粒，海藻糖组出现大量颗粒；其他数据组间无差异；因此辅料优选聚山梨酯80和蔗糖。
(2)Hu303-005抗体制剂外观结果显示海藻糖略优，但SEC结果显示蔗糖略优，整体蔗糖与海藻糖差异不大。外观及SEC数据显示PS80优于PS20，增加聚山梨酯含量可明显改善外观及SEC稳定性，见图7。因此优选PS80，PS浓度应大于0.2mg/ml。
实施例15.LAG-3抗体稳定性评价
(1).在下列不同缓冲剂中，制备蛋白浓度为50mg/mL，含60mg/ml蔗糖，0.4mg/mL聚山梨酯80的LAG-3抗体Hu229-013制剂：
1)10mM醋酸-醋酸钠(AA)pH5.5；
2)10mM组氨酸-醋酸(His-AA)pH6.0；
3)10mM组氨酸-盐酸(His-HCl)pH6.0；
4)10mM琥珀酸-琥珀酸钠(SA)pH6.0。
(2).在下列不同缓冲液中，制备蛋白含量50mg/ml，含75mg/ml蔗糖，0.4mg/ml PS80的Hu303-005制剂：
1)10mM醋酸-醋酸钠(AA)pH5.5
2)10mM组氨酸-盐酸(His-HCl)pH6.0
3)10mM组氨酸-盐酸(His-HCl)pH6.5
将每种制剂过滤灌装，加塞，轧盖，封口。样品制备完成后将其分别放于25℃或4℃考察稳定性。检测项为外观、SEC、IEC或iCE、CE-SDS(非还原)。
表15-1.Hu229-013稳定性结果
Figure PCTCN2018122534-appb-000064
注：M3.5表示3.5个月
表15-2 Hu303-005 4℃稳定性结果
Figure PCTCN2018122534-appb-000065
注：M表示月，N/A表示未检测。
结果显示：
(1)LAG-3抗体Hu229-013在10mM AA pH5.5、10mM His-AA pH6.0体系中均较稳定。
(2)Hu303-005抗体在4℃放置3个月后，His-HCl(pH6.5)组CE略有下降，AA pH 5.5、His-HCl pH 6.5组iCE略有变化，His-HCl(pH6.0)时最为稳定。
实施例16.LAG-3抗体制剂的优化
为了进一步对缓冲剂的种类、pH、离子强度进行优化，在过固定Hu229-013抗体浓度为50mg/ml后，运用JMP软件进行DOE实验设计，应用RSM模型得到一系列处方，通过强制降解方法，以IEC、CE(非还原)、微流成像(MFI)为评价指标，采用最小二乘法对结果进行统计分析，DOE参数见表16，试验处方及结果见表17、表18。
表16.DOE设计因子及水平
Figure PCTCN2018122534-appb-000066
表17.Hu229-013DOE设计处方
Figure PCTCN2018122534-appb-000067
表18.DOE处方筛选结果
Figure PCTCN2018122534-appb-000068
Figure PCTCN2018122534-appb-000069
注：M1表示一个月，D表示天。
将各强制降解数据进行拟合，结果显示：在10-30mM醋酸-醋酸钠(AA)缓冲液或组氨酸-醋酸缓冲液(His-AA)，pH5.2–5.8体系中LAG-3抗体Hu229-013稳定性均较好，优选缓冲体系为10-30mM醋酸-醋酸钠(AA)，pH5.5。
实施例17.LAG-3抗体制剂稳定性测试
在10mM醋酸-醋酸钠pH5.5缓冲剂中，制备蛋白浓度为60mg/mL，含60mg/ml蔗糖，0.4mg/mL聚山梨酯80的LAG-3Hu229-013制剂：
将制剂过滤灌装，加塞，轧盖，封口。样品制备完成后将其放于4℃考察稳定性。检测项为外观、SEC、IEC、CE-SDS(非还原)。
表19.Hu229-013稳定性结果
Figure PCTCN2018122534-appb-000070
结果显示：4℃时该Hu229-013制剂可稳定保持9个月。
实施例18.LAG-3抗体制剂的优化
(1)Hu229-013制剂成分的优化
为了进一步对蛋白、蔗糖和聚山梨酯80浓度进行优化，设定缓冲液为10mM醋酸-醋酸钠，pH5.5，运用JMP软件进行DOE实验设计，应用RSM模型得到一系列处方，通过强制降解方法，以IEC、CE(非还原)为评价指标，采用最小二乘法对结果进行统计分析，DOE参数见表20，试验结果见表21，统计分析结果见图8、图9和 表21。
表20.DOE设因子及水平
Figure PCTCN2018122534-appb-000071
表21.DOE处方设计及筛选结果
Figure PCTCN2018122534-appb-000072
将各强制降解数据进行拟合，结果如下：
用IEC 0时与40℃数据的差值进行拟合，R 2>0.98、P<0.06，模型有效，结果见图8。用CE纯度0时与40℃数据的差值进行拟合，R 2>0.99、P<0.05，模型有效，结果见图9。40℃IEC拟合结果显示较优处方为：蛋白浓度40-60mg/ml，糖浓度30-90mg/ml，PS80浓度0.4-0.5mg/ml；40℃CE拟合结果显示较优处方为：蛋白浓度50-80mg/ml，糖浓度30-90mg/ml，PS80浓度0.1-0.5mg/ml。因此最优范围为：蛋白浓度50-60mg/ml，蔗糖浓度30-90mg/ml，PS80浓度0.4-0.5mg/ml。
(2).Hu303-005抗体制剂成分的优化
在固定蔗糖浓度为75mg/ml后，以10mM His缓冲液pH、蛋白浓度和聚山梨酯浓度为变量进行DOE实验设计，应用RSM模型得到一系列处方，处方见表22，通过强制降解方法，以iCIEF、CE(非还原)、DLS为评价指标，采用最小二乘法对结 果进行统计分析，结果见表23和图10。
表22.Hu303-005抗体制剂DOE处方筛选实验处方设计
Figure PCTCN2018122534-appb-000073
表23.Hu303-005抗体制剂DOE处方筛选实验结果
Figure PCTCN2018122534-appb-000074
Figure PCTCN2018122534-appb-000075
将各强制降解数据进行拟合，其中25度和40度iCIEF/CE/DLS拟合较好，模型有效，结果如图10所示。
结果显示，高温条件下，pH升高粒径增加，中性峰也增加，iCIEF变化速度随温度降低而减缓，pH6.0时CE数据最优，同时结合前期实验结果(10mM His pH6.0较为稳定)，确定最优pH为6.0；40度CE数据显示蛋白浓度45-60mg/ml时较优，因此最优浓度定为50mg/ml；结合各条件下聚山梨酯浓度对蛋白稳定性的影响结果及其他测试例中的聚山梨酯浓度筛选结果(浓度大于0.2mg/ml时较优)，PS80浓度定为0.3mg/ml；对于Hu303-005抗体液体制剂，在该实施例及其他实施例中，高位条件下iCIEF均出现明显下降，考虑开发为冻干制剂。为保证冻干制剂较好的成型性及适宜的渗透压，蔗糖浓度定为75mg/ml。
实施例19.LAG-3抗体制剂的冻干
制备蛋白含量50mg/ml，10mM组氨酸-盐酸，pH5.5或6.0，75mg/ml蔗糖，0.4mg/ml PS80的Hu303-005冻干制剂。冻干程序如下：
表24.冻干程序
Figure PCTCN2018122534-appb-000076
将样品置于4℃及25℃条件考察稳定性，于不同时间点取样，用适量注射用 水复溶检测，结果见表25和表26，结果显示，Hu303-005制剂25℃加速及4℃长期M3各项指标无显著变化，稳定性良好。
表25.Hu303-005冻干制剂4℃稳定性结果
Figure PCTCN2018122534-appb-000077
表26.Hu303-005冻干制剂25℃稳定性结果
Figure PCTCN2018122534-appb-000078
同时测定了Hu229-013抗体的制剂冻干后复溶液的稳定性，用pH5.5的含10mM醋酸-醋酸钠的缓冲剂，制备LAG-3抗体蛋白浓度为50mg/ml，含75mg/ml蔗糖，0.4mg/ml聚山梨酯80的抗LAG-3抗体制剂。将抗体以1.1mL/瓶填充入2mL西林瓶中，装入冻干箱中，冻干。对冻干样品进行冻干前后对比及稳定性考察。结果表明，Hu229-013抗体冻干前后质量无变化，且冻干制剂在储存过程中稳定性良好。
实施例20.冻干工艺参数的优化
制备Hu303-005抗体含量为50mg/ml，10mM组氨酸-盐酸，pH 6.0，75mg/ml蔗糖，0.4mg/ml PS80的制剂进行冻干。通过冻干显微镜测得Hu303-005的塌陷温度约为-19℃，一次干燥温度是冻干工艺的重要参数，因此对一次干燥过程的隔板温度进行了细致优化，冻干参数见表27，结果见表28，各温度下冻干粉饼外观均符合要求，但-5℃组复溶后外观出现少量颗粒，因此一次干燥隔板温度定为-10℃。
表27.一次干燥隔板温度筛选的冻干工艺参数表
Figure PCTCN2018122534-appb-000079
Figure PCTCN2018122534-appb-000080
表28.不同工艺所得冻干样品复溶前后外观对比结果
Figure PCTCN2018122534-appb-000081
最终冻干工艺如下：
表29.冻干工艺
Figure PCTCN2018122534-appb-000082
*一次干燥及二次干燥时间根据具体批量及压力升测试而定。
实施例21.其它可选择制剂配方
本发明提供的稳定的药物制剂还可以是任选自以下的稳定组合：
(1)LAG-3抗体Hu229-013 90mg/ml，80mg/ml蔗糖，0.4mg/ml的聚山梨酯80，15mM醋酸-醋酸钠缓冲剂，pH5.5；
(2)LAG-3抗体Hu229-013 90mg/ml，80mg/ml蔗糖，0.4mg/ml的聚山梨酯80，15mM醋酸-醋酸钠缓冲剂，pH 6.5；
(3)LAG-3抗体Hu229-013 90mg/ml，50mg/ml蔗糖，0.4mg/ml的聚山梨酯80，25mM醋酸-醋酸钠缓冲剂，pH 5.5；
(4)LAG-3抗体Hu229-013 70mg/ml，50mg/ml蔗糖，0.3mg/ml的聚山梨酯80，10mM醋酸-醋酸钠缓冲剂，pH 5.5；
(5)LAG-3抗体Hu229-013 70mg/ml，80mg/ml蔗糖，0.3mg/ml的聚山梨酯80，15mM醋酸-醋酸钠缓冲剂，pH 5.2；
(6)LAG-3抗体Hu229-013 40mg/ml，75mg/ml蔗糖，0.4mg/ml的聚山梨酯 80，10mM醋酸-醋酸钠缓冲剂，pH 6.0；
(7)LAG-3抗体Hu229-013 55mg/ml，75mg/ml蔗糖，0.4mg/ml的聚山梨酯80，10mM醋酸-醋酸钠缓冲剂，pH5.7；
(8)LAG-3抗体Hu229-013 30mg/ml，70mg/ml蔗糖，0.5mg/ml的聚山梨酯80，10mM醋酸-醋酸钠缓冲剂，pH 5.5；
(9)LAG-3抗体Hu229-013 20mg/ml，70mg/ml蔗糖，0.2mg/ml的聚山梨酯80，10mM醋酸-醋酸钠缓冲剂，pH 5.4；
(5)LAG-3抗体Hu229-013 15mg/ml，85mg/ml蔗糖，0.1mg/ml的聚山梨酯80，25mM醋酸-醋酸钠缓冲剂，pH 5.6；
(11)LAG-3抗体Hu229-013 50mg/ml，85mg/ml蔗糖，0.3mg/ml的聚山梨酯80，25mM醋酸-醋酸钠缓冲剂，pH 5.8；
(12)LAG-3抗体Hu229-013 50mg/ml，75mg/ml蔗糖，0.4mg/ml的聚山梨酯80，25mM醋酸-醋酸钠缓冲剂，pH 6.0；
(13)LAG-3抗体Hu229-013 55mg/ml，90mg/ml蔗糖，0.4mg/ml的聚山梨酯80，10mM醋酸-醋酸钠缓冲剂，pH 5.3；
(14)LAG-3抗体Hu229-013 50mg/ml，90mg/ml蔗糖，0.4mg/ml的聚山梨酯80，10mM醋酸-醋酸钠缓冲剂，pH 5.0；
(15)LAG-3抗体Hu303-005 90mg/ml，75mg/ml蔗糖，0.3mg/ml聚山梨酯80，30mM组氨酸-盐酸缓冲剂，pH6.0；
(16)LAG-3抗体Hu303-005 90mg/ml，75mg/ml蔗糖，0.3mg/ml聚山梨酯80，30mM组氨酸-盐酸缓冲剂，pH5.5；
(17)LAG-3抗体Hu303-005 75mg/ml，75mg/ml蔗糖，0.3mg/ml聚山梨酯80，30mM组氨酸-盐酸缓冲剂，pH5.0；
(18)LAG-3抗体Hu303-005 1mg/ml，75mg/ml蔗糖，0.3mg/ml聚山梨酯80，5mM组氨酸-盐酸缓冲剂，pH6.0；
(19)LAG-3抗体Hu303-005 70mg/ml，30mg/ml蔗糖，0.3mg/ml聚山梨酯80，5mM组氨酸-盐酸缓冲剂，pH6.0；
(20)LAG-3抗体Hu303-005 50mg/ml，60mg/ml海藻糖，0.3mg/ml聚山梨酯80，10mM组氨酸-盐酸缓冲剂，pH6.0；
(21)LAG-3抗体Hu303-005 50mg/ml，90mg/ml海藻糖，0.3mg/ml聚山梨酯80，10mM组氨酸-盐酸缓冲剂，pH6.0。

Claims (34)

  1. 一种药物组合物，其包含LAG-3抗体或其抗原结合片段，以及缓冲剂，所述缓冲剂选自醋酸盐缓冲剂、组氨酸缓冲剂、柠檬酸盐缓冲剂、琥珀酸盐缓冲剂或Tris缓冲剂。
  2. 如权利要求1所述的药物组合物，其中所述的醋酸盐缓冲剂选自醋酸-醋酸钠缓冲剂；所述的组氨酸缓冲剂选自组氨酸-盐酸缓冲剂、组氨酸-醋酸缓冲剂；所述的柠檬酸盐缓冲剂选自柠檬酸-柠檬酸钠缓冲剂；所述的琥珀酸盐缓冲剂选自琥珀酸-琥珀酸钠缓冲剂；优选为组氨酸-盐酸缓冲剂、醋酸-醋酸钠缓冲剂。
  3. 如权利要求1或2所述的药物组合物，其中所述LAG-3抗体或其抗原结合片段的浓度为1mg/ml至90mg/ml，优选为40mg/ml至60mg/ml，更优选为50mg/ml。
  4. 如权利要求1至3任一项所述的药物组合物，其中所述缓冲剂的pH约为5.0至6.5，优选约为5.0至6.0。
  5. 如权利要求1至4任一项所述的药物组合物，其中所述缓冲剂浓度为5mM至30mM，优选为10mM至30mM。
  6. 如权利要求1至5任一项所述的药物组合物，其中还包括辅料，所述辅料选自糖和表面活性剂中的一种或多种。
  7. 如权利要求6所述的药物组合物，其中所述的糖为二糖，优选为海藻糖、蔗糖、甘露醇或山梨醇，更优选为蔗糖。
  8. 如权利要求7所述的药物组合物，其中所述的蔗糖浓度为30mg/ml至90mg/ml，优选为60mg/ml至90mg/ml，更优选75mg/ml。
  9. 如权利要求6所述的药物组合物，其中所述的表面活性剂为聚山梨酯，其中所述的聚山梨酯选自聚山梨酯80或聚山梨酯20，优选为聚山梨酯80。
  10. 如权利要求9所述的药物组合物，其中所述的表面活性剂的浓度为0.02mg/ml至0.8mg/ml，优选为0.2mg/ml至0.6mg/ml，更优选为0.3mg/ml至0.5mg/ml。
  11. 如权利要求1至10任一项所述的药物组合物，其包含如下i)或)ii)所 示的组分：
    i)(a)1mg/ml至90mg/ml的LAG-3抗体或其抗原结合片段，(b)5mM至30mM的醋酸-醋酸钠缓冲剂，pH约为5.0-6.5，(c)30mg/ml至90mg/ml的蔗糖和(d)0.02mg/ml至0.8mg/ml的聚山梨酯80；或
    ii)(a)1mg/ml至90mg/ml的LAG-3抗体或其抗原结合片段，(b)5mM至30mM的组氨酸-盐酸缓冲剂，pH约为5.0-6.5，(c)30mg/ml至90mg/ml的蔗糖或海藻糖和(d)0.05mg/ml至0.6mg/ml的聚山梨酯80，
    优选地，所述药物组合物包含：
    (e)40mg/ml至80mg/ml的LAG-3抗体或其抗原结合片段，(f)10mM至30mM的醋酸-醋酸钠缓冲剂，pH约为5.2-5.8，(g)70mg/ml至80mg/ml的蔗糖和(h)0.4mg/ml至0.5mg/ml的聚山梨酯80；或所述药物组合物包含：
    (e)45mg/ml至60mg/ml的LAG-3抗体或其抗原结合片段，(f)10mM至30mM的组氨酸-盐酸缓冲剂，pH约为5.5-6.0，(g)60mg/ml至90mg/ml的蔗糖或海藻糖和(h)0.2mg/ml至0.6mg/ml的聚山梨酯80。
  12. 如权利要求1至11任一项所述的药物组合物，其中所述LAG-3人源化抗体包含重链可变区和轻链可变区，其中所述的重链可变区氨基酸序列如SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:24或SEQ ID NO:25任一序列所示或与其具有至少85％序列同一性的氨基酸序列；
    其中所述的轻链可变区如SEQ ID NO:22、SEQ ID NO:26、SEQ ID NO:27或SEQ ID NO:28任一所示或与其具有至少85％序列同一性的氨基酸序列。
  13. 如权利要求12所述的药物组合物，其包含：
    (a)1至90mg/ml的LAG-3抗体或其抗原结合片段，(b)5mM至30mM的醋酸-醋酸盐缓冲剂，pH约为5.0-6.5，(c)30mg/ml至90mg/ml的蔗糖，和(d)0.02mg/ml至0.8mg/ml的聚山梨酯80；优选的，所述药物组合物包含：
    (e)40mg/ml至80mg/ml的LAG-3抗体或其抗原结合片段，(f)10mM至30mM的醋酸-醋酸钠缓冲剂，pH约为5.2-5.8，(g)70mg/ml至80mg/ml的蔗糖，和(h)0.4mg/ml至0.5mg/ml的聚山梨酯80；更优选的，所述的药物组合物包含：
    (i)约50mg/ml的LAG-3抗体或其抗原结合片段，(j)10mM的醋酸-醋酸钠缓冲剂，pH约为5.5，(k)约75mg/ml的蔗糖，(l)约0.4mg/ml的聚山梨醇酯80。
  14. 如权利要求13所述的药物组合物，其中所述LAG3人源化抗体包含选自如下所示的重链可变区和轻链可变区的组合：
    1)SEQ ID NO:21的重链可变区和SEQ ID NO:22的轻链可变区；
    2)SEQ ID NO:21的重链可变区和SEQ ID NO:26的轻链可变区；
    3)SEQ ID NO:21的重链可变区和SEQ ID NO:27的轻链可变区；
    4)SEQ ID NO:21的重链可变区和SEQ ID NO:28的轻链可变区；
    5)SEQ ID NO:23的重链可变区和SEQ ID NO:22的轻链可变区；
    6)SEQ ID NO:23的重链可变区和SEQ ID NO:26的轻链可变区；
    7)SEQ ID NO:23的重链可变区和SEQ ID NO:27的轻链可变区；
    8)SEQ ID NO:23的重链可变区和SEQ ID NO:28的轻链可变区；
    9)SEQ ID NO:24的重链可变区和SEQ ID NO:22的轻链可变区；
    10)SEQ ID NO:24的重链可变区和SEQ ID NO:26的轻链可变区；
    11)SEQ ID NO:24的重链可变区和SEQ ID NO:27的轻链可变区；
    12)SEQ ID NO:24的重链可变区和SEQ ID NO:28的轻链可变区；
    13)SEQ ID NO:25的重链可变区和SEQ ID NO:22的轻链可变区；
    14)SEQ ID NO:25的重链可变区和SEQ ID NO:26的轻链可变区；
    15)SEQ ID NO:25的重链可变区和SEQ ID NO:27的轻链可变区；和
    16)SEQ ID NO:25的重链可变区和SEQ ID NO:28的轻链可变区。
  15. 如权利要求12至14任一项所述的药物组合物，其中所述LAG-3人源化抗体包含重链恒定区和轻链恒定区，其中所述的重链恒定区优选如SEQ ID NO：38所示；其中所述的轻链恒定区优选如SEQ ID NO：39所示。
  16. 如权利要求15所述的药物组合物，其中所述LAG-3人源化抗体的重链氨基酸序列如SEQ ID NO:40所示或与其具有至少95％的序列同一性，轻链氨基酸序列如SEQ ID NO:41所示或与其具有至少95％的序列同一性。
  17. 如权利要求1至11任一项所述的药物组合物，其中所述LAG-3人源化抗体包含重链可变区和轻链可变区，其中重链可变区氨基酸序列选自SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:32或SEQ ID NO:33任一所示的序列或与其具有至少85％序列同一性的序列，和
    其中所述的轻链可变区氨基酸序列选自SEQ ID NO:30、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36或SEQ ID NO:37任一所示的序列或与其具有至少85％序列同一性的序列。
  18. 如权利要求17所述的药物组合物，其包含：
    (a)1mg/ml至90mg/ml的LAG-3抗体或其抗原结合片段，(b)5mM至30mM的组氨酸-盐酸缓冲剂，pH约为5.0-6.5，(c)30mg/ml至90mg/ml的蔗糖或海藻糖，和(d)0.05mg/ml至0.6mg/ml的聚山梨酯80；优选的，所述药物组合物包含：
    (e)45mg/ml至60mg/ml的LAG-3抗体或其抗原结合片段，(f)约10mM至30mM的组氨酸-盐酸缓冲剂，pH约为5.5-6.0，(g)60mg/ml至90mg/ml的蔗糖或 海藻糖，和(h)0.2mg/ml至0.6mg/ml的聚山梨酯80；
    更优选的，所述的药物组合物包含：
    (i)约50mg/ml的LAG-3抗体或其抗原结合片段，(j)10mM的组氨酸-盐酸缓冲剂，pH约为6.0，(k)约75mg/ml的蔗糖，(l)约0.3mg/ml的聚山梨醇酯80。
  19. 如权利要求18所述的药物组合物，其中所述LAG3人源化抗体包含选自以下的重链可变区和轻链可变区的组合：
    1)SEQ ID NO:29的重链可变区和SEQ ID NO:30的轻链可变区；
    2)SEQ ID NO:29的重链可变区和SEQ ID NO:34的轻链可变区；
    3)SEQ ID NO:29的重链可变区和SEQ ID NO:35的轻链可变区；
    4)SEQ ID NO:29的重链可变区和SEQ ID NO:36的轻链可变区；
    5)SEQ ID NO:29的重链可变区和SEQ ID NO:37的轻链可变区；
    6)SEQ ID NO:31的重链可变区和SEQ ID NO:30的轻链可变区；
    7)SEQ ID NO:31的重链可变区和SEQ ID NO:34的轻链可变区；
    8)SEQ ID NO:31的重链可变区和SEQ ID NO:35的轻链可变区；
    9)SEQ ID NO:31的重链可变区和SEQ ID NO:36的轻链可变区；
    10)SEQ ID NO:31的重链可变区和SEQ ID NO:37的轻链可变区；
    11)SEQ ID NO:32的重链可变区和SEQ ID NO:30的轻链可变区；
    12)SEQ ID NO:32的重链可变区和SEQ ID NO:34的轻链可变区；
    13)SEQ ID NO:32的重链可变区和SEQ ID NO:35的轻链可变区；
    14)SEQ ID NO:32的重链可变区和SEQ ID NO:36的轻链可变区；
    15)SEQ ID NO:32的重链可变区和SEQ ID NO:37的轻链可变区；
    16)SEQ ID NO:33的重链可变区和SEQ ID NO:30的轻链可变区；
    17)SEQ ID NO:33的重链可变区和SEQ ID NO:34的轻链可变区；
    18)SEQ ID NO:33的重链可变区和SEQ ID NO:35的轻链可变区；
    19)SEQ ID NO:33的重链可变区和SEQ ID NO:36的轻链可变区；和
    20)SEQ ID NO:33的重链可变区和SEQ ID NO:37的轻链可变区。
  20. 如权利要求17至19任一项所述的药物组合物，其中所述LAG-3人源化抗体包含重链恒定区和轻链恒定区，其中所述的重链恒定区优选如SEQ ID NO：38所示；其中所述的轻链恒定区优选如SEQ ID NO：39所示。
  21. 如权利要求20所述的药物组合物，其中所述LAG-3人源化抗体的重链氨基酸序列选自SEQ ID NO:42所示的序列或与其具有至少95％的序列同一性序列，轻链氨基酸序列选自SEQ ID NO:43所示的序列或与其具有至少95％的序列同一性的序列。
  22. 制备权利要求1至21任一项所述的药物组合物的方法，包括将LAG-3抗体或其抗原结合片段与缓冲剂混合，所述缓冲剂选自醋酸-醋酸钠缓冲剂、组氨酸-盐酸缓冲剂、柠檬酸-柠檬酸钠缓冲剂、琥珀酸-琥珀酸钠缓冲剂或Tris缓冲剂，优选为醋酸-醋酸钠缓冲剂和组氨酸-盐酸缓冲剂。
  23. 制备含LAG-3抗体或其抗原结合片段的冻干制剂的方法，其包括将权利要求1至21任一项所述的药物组合物经冷冻干燥的步骤。
  24. 如权利要求23所述制备含LAG-3抗体或其抗原结合片段的冻干制剂的方法，其中所述冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。
  25. 如权利要求24所述制备含LAG-3抗体或其抗原结合片段的冻干制剂的方法，其中所述的一次干燥温度为-5℃至-20℃，优选为-10℃。
  26. 一种由权利要求23至25任一项所述的方法制备所得含LAG-3抗体或其抗原结合片段的冻干制剂。
  27. 一种含LAG3抗体或其抗原结合片段的冻干制剂，其特征在于所述冻干制剂复溶后可形成权利要求1至21任一项所述的药物组合物。
  28. 制备含有LAG-3抗体或其抗原结合片段的复溶溶液的方法，其中包括将权利要求26或27所述的冻干制剂经复溶的步骤，其复溶所用溶剂优选为注射用水。
  29. 一种由权利要求28所述的方法制备所得含有LAG-3抗体或其抗原结合片段的复溶溶液。
  30. 如权利要求29所述的LAG3抗体或其抗原结合片段的复溶溶液，其包含如下成分：
    i)(a)40mg/ml至80mg/ml的LAG-3抗体或其抗原结合片段，(b)10mM至30mM的醋酸-醋酸钠缓冲剂，pH约为5.2-5.8，(c)70mg/ml至80mg/ml的蔗糖，和(d)0.4mg/ml至0.5mg/ml的聚山梨酯80；或
    ii)(a)45mg/ml至60mg/ml的LAG-3抗体或其抗原结合片段，(b)10mM至30mM的组氨酸-盐酸缓冲剂，pH约为5.5-6.0，(c)60mg/ml至90mg/ml的蔗糖或海藻糖，和(d)0.2mg/ml至0.6mg/ml的聚山梨酯80。
  31. 权利要求1至21任一项所述的药物组合物或权利要求26或27所述的冻干制剂或权利要求29或30所述的复溶溶液用作药物。
  32. 权利要求1至21任一项所述的药物组合物或权利要求26或27所述的冻干制剂或权利要求29或30所述的复溶溶液在制备用于治疗LAG-3相关的疾病或病症的药物中的用途，其中所述的疾病或病症为致病性T细胞参与的相关疾病或病症，优选为癌症；所述癌症选自卵巢癌、黑色素瘤、前列腺癌、肠癌、胃癌、食管癌、乳腺癌、肺癌、肾癌、胰腺癌、子宫癌、肝癌、膀胱癌、子宫颈癌、口腔癌、脑癌、睾丸癌、皮肤癌、甲状腺癌以及血液学恶性肿瘤，其中所述的血液学恶性肿瘤包括骨髓瘤、慢性白血病和急性白血病。
  33. 一种治疗和预防LAG-3相关的疾病或病症的方法，包括给予所需患者治疗有效量的如权利要求1至21任一项所述的药物组合物或权利要求26或27所述的冻干制剂或权利要求29或30所述的复溶溶液，其中所述的疾病或病症为致病性T细胞参与的相关疾病或病症，优选为癌症；所述癌症选自卵巢癌、黑色素瘤、前列腺癌、肠癌、胃癌、食管癌、乳腺癌、肺癌、肾癌、胰腺癌、子宫癌、肝癌、膀胱癌、子宫颈癌、口腔癌、脑癌、睾丸癌、皮肤癌、甲状腺癌以及血液学恶性肿瘤，其中所述的血液学恶性肿瘤包括包括骨髓瘤、慢性白血病和急性白血病。
  34. 一种制品，其包括容器，该容器中装有权利要求1至21任一项所述的药物组合物或权利要求26或27所述的冻干制剂或权利要求29或30所述的复溶溶液。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11634485B2 (en) 2019-02-18 2023-04-25 Eli Lilly And Company Therapeutic antibody formulation

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114617961B (zh) * 2020-12-10 2022-12-02 北京东方百泰生物科技股份有限公司 一种抗lag-3单克隆抗体的注射制剂
WO2023040935A1 (zh) * 2021-09-15 2023-03-23 江苏恒瑞医药股份有限公司 一种含抗pvrig/tigit双特异性抗体的药物组合物
CN114874324B (zh) * 2022-05-13 2023-02-03 苏州旭光科星抗体生物科技有限公司 一种检测可溶性lag-3蛋白含量的酶联免疫检测试剂盒及应用
CN117159703B (zh) * 2023-11-02 2024-04-02 正大天晴(广州)医药有限公司 含抗lag-3抗体的药物组合物及其用途

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995030750A2 (fr) 1994-05-06 1995-11-16 Institut Gustave Roussy Fractions polypeptidiques solubles de la proteine lag-3; procede de production; composition therapeutique; anticorps anti-idiotype
WO2004078928A2 (en) 2003-02-28 2004-09-16 The Johns Hopkins University T cell regulation
WO2008132601A1 (en) 2007-04-30 2008-11-06 Immutep Cytotoxic anti-lag-3 monoclonal antibody and its use in the treatment or prevention of organ transplant rejection and autoimmune disease
WO2010019570A2 (en) 2008-08-11 2010-02-18 Medarex, Inc. Human antibodies that bind lymphocyte activation gene-3 (lag-3), and uses thereof
WO2014008218A1 (en) 2012-07-02 2014-01-09 Bristol-Myers Squibb Company Optimization of antibodies that bind lymphocyte activation gene-3 (lag-3), and uses thereof
WO2014140180A1 (en) 2013-03-15 2014-09-18 Glaxosmithkline Intellectual Property Development Limited Anti-lag-3 binding proteins
WO2015138920A1 (en) 2014-03-14 2015-09-17 Novartis Ag Antibody molecules to lag-3 and uses thereof
WO2017037203A1 (en) * 2015-09-02 2017-03-09 Immutep S.A.S. Anti-LAG-3 Antibodies
CN108472349A (zh) * 2016-06-23 2018-08-31 江苏恒瑞医药股份有限公司 Lag-3抗体、其抗原结合片段及其医药用途
WO2018204374A1 (en) 2017-05-02 2018-11-08 Merck Sharp & Dohme Corp. Formulations of anti-lag3 antibodies and co-formulations of anti-lag3 antibodies and anti-pd-1 antibodies

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6267958B1 (en) * 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation
US6171586B1 (en) * 1997-06-13 2001-01-09 Genentech, Inc. Antibody formulation
AU2014347004B2 (en) * 2013-11-05 2019-08-08 Bavarian Nordic A/S Combination therapy for treating cancer with a poxvirus expressing a tumor antigen and an antagonist and/or agonist of an immune checkpoint inhibitor
US10934550B2 (en) * 2013-12-02 2021-03-02 Phio Pharmaceuticals Corp. Immunotherapy of cancer
WO2015198312A1 (en) * 2014-06-24 2015-12-30 Ccam Therapeutics Ltd. Compositions comprising antibodies to ceacam-1 and lag-3 for cancer therapy
JO3663B1 (ar) * 2014-08-19 2020-08-27 Merck Sharp & Dohme الأجسام المضادة لمضاد lag3 وأجزاء ربط الأنتيجين
US9902772B2 (en) * 2015-07-22 2018-02-27 Sorrento Therapeutics, Inc. Antibody therapeutics that bind LAG3
LT3458478T (lt) * 2016-05-18 2021-02-10 Boehringer Ingelheim International Gmbh Antikūnai prieš pd-1 ir lag-3, skirti vėžio gydymui

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995030750A2 (fr) 1994-05-06 1995-11-16 Institut Gustave Roussy Fractions polypeptidiques solubles de la proteine lag-3; procede de production; composition therapeutique; anticorps anti-idiotype
WO2004078928A2 (en) 2003-02-28 2004-09-16 The Johns Hopkins University T cell regulation
WO2008132601A1 (en) 2007-04-30 2008-11-06 Immutep Cytotoxic anti-lag-3 monoclonal antibody and its use in the treatment or prevention of organ transplant rejection and autoimmune disease
WO2010019570A2 (en) 2008-08-11 2010-02-18 Medarex, Inc. Human antibodies that bind lymphocyte activation gene-3 (lag-3), and uses thereof
CN107474137A (zh) * 2008-08-11 2017-12-15 施贵宝有限责任公司 结合淋巴细胞活化基因3（lag‑3）之人类抗体及其用途
WO2014008218A1 (en) 2012-07-02 2014-01-09 Bristol-Myers Squibb Company Optimization of antibodies that bind lymphocyte activation gene-3 (lag-3), and uses thereof
WO2014140180A1 (en) 2013-03-15 2014-09-18 Glaxosmithkline Intellectual Property Development Limited Anti-lag-3 binding proteins
WO2015138920A1 (en) 2014-03-14 2015-09-17 Novartis Ag Antibody molecules to lag-3 and uses thereof
WO2017037203A1 (en) * 2015-09-02 2017-03-09 Immutep S.A.S. Anti-LAG-3 Antibodies
CN108472349A (zh) * 2016-06-23 2018-08-31 江苏恒瑞医药股份有限公司 Lag-3抗体、其抗原结合片段及其医药用途
WO2018204374A1 (en) 2017-05-02 2018-11-08 Merck Sharp & Dohme Corp. Formulations of anti-lag3 antibodies and co-formulations of anti-lag3 antibodies and anti-pd-1 antibodies

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 45
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1977, pages 3389 - 3402
BLOOD, vol. 108, no. 7, 2006, pages 2280 - 9
CURR OPIN IMMUNOL, vol. 21, no. 2, 2009, pages 179 - 86
EUR J IMMUNOL, vol. 33, no. 4, 2003, pages 970 - 9
EUR J IMMUNOL, vol. 35, no. 7, 2005, pages 2081 - 8
IMMUNITY, vol. 21, no. 4, 2004, pages 503 - 13
J CLIN INVEST, vol. 117, no. 11, 2007, pages 3383 - 92
J IMMUNOL, vol. 182, no. 4, 2009, pages 1885 - 91
J. BIOL. CHEM., vol. 243, 1968, pages 3558
KABAT, E A ET AL.: "Sequences of Proteins of Immunological Interest", 1991
PROC NATL ACAD SCI USA, vol. 94, no. 11, 1997, pages 5744 - 9
See also references of EP3714901A4
THE IMMUNOGLOBULIN FACTSBOOK, 2001, ISBN: 012441351
TRIEBEL ET AL., J EXP MED, vol. 171, no. 5, 1990, pages 1393 - 405
WATSON ET AL.: "Molecular Biology of the Gene", 1987, THE BENJAMIN/CUMMINGS PUB. CO., pages: 224

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11634485B2 (en) 2019-02-18 2023-04-25 Eli Lilly And Company Therapeutic antibody formulation

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