WO2021147846A1 - 抗btla抗体药物组合物及其用途 - Google Patents

抗btla抗体药物组合物及其用途 Download PDF

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WO2021147846A1
WO2021147846A1 PCT/CN2021/072660 CN2021072660W WO2021147846A1 WO 2021147846 A1 WO2021147846 A1 WO 2021147846A1 CN 2021072660 W CN2021072660 W CN 2021072660W WO 2021147846 A1 WO2021147846 A1 WO 2021147846A1
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Prior art keywords
cancer
buffer
sodium chloride
antibody
histidine
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PCT/CN2021/072660
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English (en)
French (fr)
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刘洪川
刘沛想
张静
周岳华
刘辉
陈雪茹
王静
冯辉
姚盛
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上海君实生物医药科技股份有限公司
苏州君盟生物医药科技有限公司
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Application filed by 上海君实生物医药科技股份有限公司, 苏州君盟生物医药科技有限公司 filed Critical 上海君实生物医药科技股份有限公司
Priority to AU2021211795A priority Critical patent/AU2021211795A1/en
Priority to CA3165369A priority patent/CA3165369A1/en
Priority to KR1020227026032A priority patent/KR20220129564A/ko
Priority to EP21744312.6A priority patent/EP4095158A4/en
Priority to BR112022013292A priority patent/BR112022013292A2/pt
Priority to CN202180009885.XA priority patent/CN114981307A/zh
Priority to JP2022543548A priority patent/JP2023510928A/ja
Publication of WO2021147846A1 publication Critical patent/WO2021147846A1/zh
Priority to US17/866,867 priority patent/US20230192854A1/en

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    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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    • A—HUMAN NECESSITIES
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    • 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
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of therapeutic pharmaceutical compositions.
  • the present invention relates to the field of pharmaceutical preparations.
  • the pharmaceutical composition contains a humanized antibody that specifically binds to B and T lymphocyte attenuator (BTLA).
  • BTLA B and T lymphocyte attenuator
  • T cell receptors TCR
  • TCR T cell receptors
  • the prototype T cell costimulatory molecule CD28 When interacting with B7.1 or B7.2 on the surface of antigen presenting cells (APC), the prototype T cell costimulatory molecule CD28 sends out signals that promote T cell proliferation and differentiation in response to the participation of TCR, but the CD28 homolog is cytotoxic T Lymphocyte antigen-4 (CTLA-4) mediates the inhibition of T cell proliferation and effector functions (Chambers et al., Ann. Rev. Immunol., 19:565-594, 2001; Egen et al., Nature Immunol, 3:611- 618, 2002).
  • CTLA-4 cytotoxic T Lymphocyte antigen-4
  • B and T lymphocyte attenuators are members of the CD28 family, which also includes CD28, ICOS, CTLA-4 and PD-1. According to the functional effect of the addition of monoclonal antibodies on improving the proliferation of T cells, it was found that CD28 and ICOS, the first members of the family, have immune activation effects (Hutloff et al., 1999). And BTLA, CTLA-4 and PD-1 are described as negative regulatory proteins. Several in vivo studies have confirmed the inhibitory effect of BTLA in lymphocyte response. BTLA-deficient mice prepared by Murphy and colleagues (Washington University St. Louis) showed a 3-fold increase in IgG production in response to T-dependent antigens.
  • T cells and B cells isolated from BTLA- mouse showed greater proliferative responses to antigen-receptor stimulation using CD3- and anti-IgM, respectively (Watanabe, 2003).
  • BTLA was found to associate with B cell receptor complexes and with T cell receptors. Consistent with the results of this study, in BTLA-deficient lymphocytes, the use of ConA (T cells) or LPS (B cells) for antigen-receptor independent stimulation is not affected, and the use of anti-BTLA antibodies cannot be regulated.
  • T cells ConA
  • B cells LPS
  • BTLA knockout mice develop spontaneous autoimmune diseases over time and shorten their lifespan (Oya, 2008).
  • BTLA knockout mice showed increased disease severity in autoimmune encephalomyelitis (EAE) and allergic airway inflammation models, both of which depend on T cell activity (Watanabe, 2005; Deppong, 2006).
  • HVEM herpes virus entry mediator
  • BTLA a ligand for BTLA
  • CDRs extracellular cysteine-rich regions
  • BTLA and HVEM mainly regulate the functions of T cells and APC through dynamic expression on the cell surface.
  • the combination of BTLA and ligand not only inhibits T cell proliferation, down-regulates T cell activation marker CD25, but also inhibits the production of IFN- ⁇ , IL-2, IL-4, and IL-10, but cannot induce cell apoptosis.
  • Antibodies can be used as therapeutic drugs. Certain antibodies can cause unwanted antibody immunogenicity when used as therapeutic drugs in the body. Because most monoclonal antibodies are derived from rodents, repeated use in humans results in an immune response against therapeutic antibodies (for example, human anti-mouse antibodies or HAMA). This type of immune response leads at least to the loss of therapeutic efficacy, and at the highest to potentially lethal allergic reactions.
  • One method of reducing the immunogenicity of rodent antibodies involves the production of chimeric antibodies, in which the mouse variable region (Fv) is fused with the human constant region (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84 : 3439-43). However, mice injected with a hybrid of human variable region and mouse constant region developed a strong antibody response against the human variable region, which indicates that the retention of the complete rodent Fv region in this chimeric antibody can still be used in patients. Causes harmful immunogenicity in.
  • CDR loops of rodent variable domains onto human frameworks have been used to further minimize rodent sequences. Jones et al. (1986) Nature 321:522; Verhoeyen et al. (1988) Science 239:1534.
  • CDR loop exchange still cannot uniformly produce antibodies with the same binding properties as the starting antibody.
  • FR framework residues
  • sequences of known antibodies are already being used, or more commonly the sequences of antibodies with known X-ray crystal structures such as antibodies NEW and KOL are used. See, for example, Jones et al., supra; Verhoeyen et al., supra; and Gorman et al., supra. The exact sequence information of a few humanized constructs has been reported.
  • anti-BTLA antibodies particularly anti-BTLA monoclonal antibodies
  • human disorders such as inflammatory disorders, autoimmune disorders, and proliferative disorders.
  • Such antibodies may preferably have low immunogenicity in human subjects, allowing repeated administration without adverse immune responses.
  • the pharmaceutical composition of the present invention is a highly stable pharmaceutical composition containing human antibodies specifically binding to BTLA.
  • the present invention found that the combination of trehalose and sodium chloride can significantly improve the stability of the pharmaceutical composition.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (1) a buffer; and (2) an anti-BTLA antibody or an antigen-binding fragment thereof.
  • the above-mentioned anti-BTLA antibody or antigen-binding fragment thereof has an amino acid sequence of HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and an amino acid sequence of HCDR1, HCDR2, and HCDR3, respectively, as shown in The LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.
  • the concentration of the anti-BTLA antibody or antigen-binding fragment thereof in the pharmaceutical composition is about 1-200 mg/mL, preferably about 5-100 mg/mL, preferably about 10-50 mg/mL; more preferably The concentration of the aforementioned anti-BTLA antibody or its antigen-binding fragment is about 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL or 50 mg/mL, preferably about 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL or 50 mg/mL It is 10mg/mL, 20mg/mL, 40mg/mL.
  • the above-mentioned buffer is selected from one or more of acetate buffer, citrate buffer, and histidine buffer.
  • the above-mentioned buffer is a histidine buffer, preferably, the histidine buffer is selected from histidine-hydrochloride buffer or histidine-acetate buffer, preferably histidine Acid-hydrochloride buffer.
  • the aforementioned histidine-hydrochloride buffer is composed of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride.
  • the histidine buffer is made of 1-20 mM L-histidine and 1-20 mM L-histidine monohydrochloride.
  • the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1:4.
  • the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:1.
  • the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:3.
  • the histidine preparation is: a histidine buffer with a pH of 5.5 made of 4.5 mM L-histidine and 15.5 mM L-histidine monohydrochloride.
  • the histidine preparation is: a histidine buffer with a pH of 6.0 made of 15 mM histidine and 15 mM histidine hydrochloride.
  • the above-mentioned histidine buffer is a histidine-acetate buffer, preferably, the molar ratio of the two is 1:1 to 1.5:1, and preferably, the pH of such a buffer is 5.5 ⁇ 0.3 , Preferably about 5.5, preferably, this kind of buffer contains 15-20 mM histidine and 12-15 mM acetic acid.
  • the above-mentioned buffer is an acetate buffer.
  • the acetate buffer is an acetic acid-sodium acetate buffer or an acetic acid-potassium acetate buffer, preferably an acetic acid-sodium acetate buffer.
  • the above-mentioned buffer is a citric acid buffer, and preferably, the citric acid buffer is a citric acid-sodium citrate buffer.
  • the concentration of the above-mentioned buffer is about 1-100 mM, preferably about 5-50 mM, preferably about 10-30 mM, preferably about 20-30 mM; preferably about 10-20 mM, and the above-mentioned buffer concentration is not limited Typical examples are about 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM or 100mM or any two values in these ranges
  • the range formed by the end point is preferably 10 mM, 20 mM, or 30 mM.
  • the pH of the aforementioned buffer is about 5.0-6.5, preferably about 5.0-6.0, preferably about 5.5-6.5, preferably about 5.0-5.5, preferably about 5.5-6.0, preferably about 6.0-6.5 ,
  • a non-limiting example of the pH of the above buffer is about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, preferably about 5.0, 5.5 or 6.0.
  • the aforementioned pharmaceutical composition further includes a stabilizer selected from one or more of sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose.
  • a stabilizer selected from one or more of sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose.
  • the stabilizer is a combination of trehalose and sodium chloride.
  • the concentration of the aforementioned stabilizer is about 20 mM to 300 mM, preferably 50 mM to 300 mM, more preferably 120 mM to 250 mM.
  • the stabilizer is sodium chloride at a concentration of about 50-200 mM; or the stabilizer is mannitol at a concentration of about 100-300 mM; or the stabilizer is sorbitol at a concentration of about 100-300 mM; or The stabilizer is sucrose with a concentration of about 100-300mM; or the stabilizer is trehalose with a concentration of about 100-300mM; or the stabilizer is about 30-100mM sodium chloride and about 50-200mM mannitol
  • the stabilizer is a combination of about 30-100mM sodium chloride and about 50-200mM sorbitol; the stabilizer is a combination of about 30-100mM sodium chloride and about 50-200mM sucrose;
  • the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM trehalose.
  • the aforementioned stabilizer is sodium chloride.
  • the aforementioned stabilizer is sodium chloride at a concentration of about 50-200 mM, and the concentration of the aforementioned sodium chloride is preferably about 100-190 mM, preferably about 120-180 mM, preferably about 130-170 mM, preferably about 130. -150mM, non-limiting examples of the above sodium chloride concentration are about 100mM, 110mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 170mM, 180mM, 190mM, 200mM, preferably 135mM or 140mM .
  • the aforementioned stabilizer is mannitol.
  • the aforementioned stabilizer is mannitol at a concentration of about 100-300 mM, the concentration of the aforementioned mannitol is preferably about 150-300 mM, preferably about 200-280 mM, and a non-limiting example of the aforementioned mannitol concentration is about 200 mM. , 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240mM.
  • the aforementioned stabilizer is sorbitol.
  • the aforementioned stabilizer is sorbitol at a concentration of about 100-300 mM, the concentration of the aforementioned sorbitol is preferably about 150-300 mM, preferably about 200-280 mM, and a non-limiting example of the aforementioned sorbitol concentration is about 200 mM. , 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240mM.
  • the aforementioned stabilizer is sucrose. In some embodiments, the aforementioned stabilizer is sucrose at a concentration of about 100-300 mM. The concentration of the aforementioned sucrose is preferably about 150-300 mM, preferably about 200-280 mM. A non-limiting example of the aforementioned sucrose concentration is about 200 mM, 210 mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 220mM.
  • the aforementioned stabilizer is trehalose.
  • the aforementioned stabilizer is trehalose with a concentration of about 100-300 mM.
  • the concentration of the aforementioned trehalose is preferably about 150-300 mM, preferably about 200-280 mM.
  • a non-limiting example of the aforementioned trehalose concentration is about 180mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 220mM.
  • the aforementioned stabilizer is a combination of sodium chloride and mannitol. In some aspects, the aforementioned stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM mannitol, preferably a combination of about 40-80 mM sodium chloride and about 100-180 mM mannitol, preferably about A combination of 40-60 mM sodium chloride and about 120-160 mM mannitol, a non-limiting example of the above stabilizer is a combination of about 54 mM sodium chloride and about 144 mM mannitol, and about 50 mM sodium chloride and A combination of about 140 mM mannitol.
  • the aforementioned stabilizer is a combination of sodium chloride and sorbitol. In some aspects, the aforementioned stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM sorbitol, preferably a combination of about 40-80 mM sodium chloride and about 100-180 mM sorbitol, preferably about A combination of 40-60 mM sodium chloride and about 120-160 mM sorbitol, a non-limiting example of the above stabilizer is a combination of about 54 mM sodium chloride and about 144 mM sorbitol, and about 40 mM sodium chloride and A combination of about 160 mM sorbitol.
  • the aforementioned stabilizer is a combination of sodium chloride and sucrose. In some aspects, the aforementioned stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM sucrose, preferably a combination of about 40-80 mM sodium chloride and about 100-180 mM sucrose, preferably about 40-180 mM sodium chloride.
  • a combination of 60 mM sodium chloride and about 120-160 mM sucrose, a non-limiting example of the above stabilizer is a combination of about 54 mM sodium chloride and about 132 mM sucrose, about 50 mM sodium chloride and about 150 mM sucrose The combination.
  • the aforementioned stabilizer is a combination of sodium chloride and trehalose.
  • the aforementioned stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM trehalose, preferably a combination of about 40-80 mM sodium chloride and about 100-180 mM trehalose, preferably about A combination of 40-60 mM sodium chloride and about 120-160 mM trehalose, a non-limiting example of the above stabilizer is a combination of about 54 mM sodium chloride and about 132 mM trehalose, and about 50 mM sodium chloride and A combination of about 140mM trehalose, a combination of about 60mM sodium chloride and about 120mM trehalose, preferably a combination of about 54mM sodium chloride and about 132mM trehalose, or a combination of about 50mM sodium chloride and about 140mM The combination of trehalose.
  • the above-mentioned pharmaceutical composition further includes a surfactant, and the surfactant is selected from polysorbate 80, polysorbate 20, or poloxamer 188.
  • the above-mentioned surfactant is selected from polysorbate 80.
  • the above-mentioned surfactant is selected from polysorbate 20.
  • the concentration of the above-mentioned surfactant is about 0.001%-0.1%, preferably about 0.01%-0.05%, preferably about 0.02%-0.04%; as a non-limiting example, the above-mentioned surfactant concentration
  • the concentration of the surfactant is about 0.02%, 0.03% or 0.04%, preferably 0.02%.
  • the aforementioned anti-BTLA antibody or antigen-binding fragment thereof is selected from murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.
  • the aforementioned anti-BTLA antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 8.
  • the above-mentioned anti-BTLA antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 9.
  • the aforementioned anti-BTLA antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 10.
  • the aforementioned anti-BTLA antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence as shown in SEQ ID NO: 11 and a light chain amino acid sequence as shown in SEQ ID NO: 12.
  • the above-mentioned pharmaceutical composition comprises the components shown in any one of the following (1)-(8), or consists of the components shown in any one of the following (1)-(8); wherein
  • the anti-BTLA antibody or antigen-binding fragment thereof is as described in any of the embodiments of the present invention:
  • the above-mentioned pharmaceutical composition comprises the components shown in any one of the following (9)-(13), or consists of the components shown in any one of the following (9)-(13);
  • the pharmaceutical composition is a liquid formulation or a lyophilized formulation.
  • the pharmaceutical composition is a liquid formulation, such as an aqueous formulation.
  • the above-mentioned liquid formulation or 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.
  • the above-mentioned aqueous solution or 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 the use of the above-mentioned pharmaceutical composition in the preparation of a medicine for treating or preventing BTLA-mediated diseases.
  • the aforementioned diseases include tumors, infectious diseases, inflammatory or autoimmune diseases.
  • the tumors include melanoma, breast cancer, kidney cancer, prostate cancer, colon cancer, lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer , Testicular cancer, esophageal cancer, small intestine cancer, cervical cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, endocrine system cancer, thyroid cancer, adrenal glands, soft tissue cancer, urethral cancer, chronic Or acute leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system Biology, primary central nervous system lymphoma, tumor an
  • the autoimmune diseases include organ-specific autoimmune diseases and systemic autoimmune diseases;
  • the organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, and ulcers Colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebrospinal sclerosis, acute idiopathic polyneuritis Etc.
  • the systemic autoimmune diseases include systemic lupus erythematosus, subhumid arthritis, systemic vasculitis, scleroderma, gastrocnemius, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia , Ulcerative colitis, etc.
  • Figure 1 Comparative experiment of the binding of humanized antibody hu 17 with different species of BTLA.
  • Figure 2 The effect of hu18 on the tumor volume of B-hBTLA mice transplanted with MC38-hHVEM cells.
  • Figure 3 The effect of the test substance on the body weight of B-hBTLA mice transplanted with MC38-hHVEM cells.
  • pharmaceutical composition or “preparation” means a mixture containing one or more of the compounds described herein or their pharmaceutically acceptable salts or prodrugs and other components, such as physiologically pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • liquid formulation refers to a formulation in a liquid state, and is not intended to refer to a lyophilized formulation that is resuspended.
  • the liquid formulation of the present invention is stable during storage, and its stability does not depend on lyophilization (or other state change methods, such as spray drying).
  • aqueous liquid preparation refers to a liquid preparation using water as a solvent.
  • the aqueous liquid formulation is a formulation that does not require lyophilization, spray drying, and/or freezing to maintain stability (e.g., chemical and/or physical stability and/or biological activity).
  • excipient refers to an agent that can be added to a formulation to provide desired characteristics (e.g., consistency, increased stability) and/or to adjust osmotic pressure.
  • desired characteristics e.g., consistency, increased stability
  • excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.
  • the "about” used in this application when referring to a measurable value is intended to cover a variation of ⁇ 20% or ⁇ 10% relative to a specific value, including ⁇ 5%, ⁇ 1%, and ⁇ 0.1 %, because these changes are suitable for carrying out the disclosed method.
  • buffer pH is about 5.0-6.5
  • the buffer used in the formulation of the present invention may have a pH in the range of about 5.0 to about 6.5, or a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.0 to about 6.0.
  • examples of the "buffer” for controlling the pH within this range include acetate (such as sodium acetate), succinate (such as sodium succinate), gluconic acid, histidine, histidine hydrochloride , Methionine, citrate, phosphate, citrate/phosphate, imidazole, acetic acid, acetate, citrate, combinations thereof and other organic acid buffers.
  • Hetidine buffer is a buffer containing histidine ions.
  • histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate and histidine sulfate, etc., such as containing histidine
  • the histidine buffer solution of acid and histidine hydrochloride; the histidine buffer solution of the present invention also includes histidine buffer solution containing histidine and acetate (such as sodium salt or potassium salt).
  • citrate buffer is a buffer that includes citrate ions.
  • citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like.
  • the preferred citrate buffer is citric acid-sodium citrate buffer.
  • Acetate buffer is a buffer that includes acetate ions.
  • acetate buffers include acetic acid-sodium acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like.
  • the preferred acetate buffer is acetic acid-sodium acetate buffer.
  • stabilizer means a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and application.
  • Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, Arginine, glycine, alanine ( ⁇ -alanine, ⁇ -alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxy Proline, sarcosine, ⁇ -aminobutyric acid (GABA), opines, alanine, octopine, glycinine (strombine) and trimethylamine N-oxide (TMAO) , Human serum albumin (hsa), bovine serum albumin (bsa), and tri
  • Some stabilizers such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc. can also play a role in controlling osmotic pressure.
  • the stabilizer specifically used in the present invention is selected from one or two or more of polyols, salts, and sugars.
  • the preferred salt is sodium chloride
  • the preferred sugars are sucrose and trehalose
  • the preferred polyols are sorbitol and mannitol.
  • Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose and sodium chloride-trehalose, more preferably Sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose and sodium chloride-trehalose, more preferably sodium chloride-trehalose.
  • surfactant generally includes agents that protect proteins, such as antibodies, from stress induced by the air/solution interface, solution/surface-induced stress to reduce antibody aggregation or minimize the formation of particulates in the formulation.
  • exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, poly Ethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ether, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene- Polyoxypropylene copolymer (Pluronic), sodium dodecyl sulfate (SDS).
  • nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers,
  • isotonic means that the preparation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. The isotonicity can be measured using a vapor pressure or freezing point drop osmometer.
  • stable formulation is a formulation in which the antibody substantially maintains its physical stability and/or chemical stability and/or biological activity during the manufacturing process and/or during storage. Even if the contained antibody fails to maintain 100% of its chemical structure or biological function after a certain period of storage, the pharmaceutical preparation can be stable. In some cases, after a certain period of storage, it can maintain about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the structure or function of the antibody, which can also be considered as " stable”.
  • Various analytical techniques for measuring protein stability are available in this technical field and are reviewed in "Peptide and Protein Drug Delivery” 247-301, Vincent Lee, Editor-in-Chief, Marcel Dekker, Inc. ., New York, NY, Pubs. (1991)), and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90 (both are incorporated by reference).
  • the stability of the protein is determined by the percentage of monomeric protein in a solution that has a low percentage of degradation (eg, fragmentation) and/or aggregated protein.
  • the formulation can be stored stably at room temperature, about 25-30°C, or 40°C for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or Longer, no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% antibody in aggregate form at most.
  • an acceptable degree of stability means that when the formulation is stored at a certain temperature for a certain period of time, the acidic form of the antibody that can be detected therein does not exceed about 49%, 45%, 40%, 35.
  • the certain period of storage before measuring stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer.
  • a certain temperature that allows the storage of the pharmaceutical preparation can be any temperature in the range of about -80°C to about 45°C, for example, storage at about -80°C, about -30°C, about -20°C, or about 0°C , About 2-8°C, about 5°C, about 25°C, or about 40°C.
  • the antibody does not substantially show signs of aggregation, precipitation, and/or denaturation during visual inspection of color and/or clarity or by UV light scattering or measurement by pore exclusion chromatography, the antibody is in the drug combination
  • the substance "maintains its physical stability”. Aggregation is the process by which single molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the point where visible precipitates are formed.
  • the stability of the formulation can be evaluated by methods known in the art, including measuring the apparent extinction (absorbance or optical density) of the sample. Such extinction measurement is related to the turbidity of the formulation.
  • the turbidity of a formulation is partly an inherent property of the protein dissolved in the solution, and is usually measured by turbidimetric method and measured in turbidimetric turbidity units (NTU).
  • the level of turbidity that varies with, for example, the concentration of one or more components in the solution (eg, protein and/or salt concentration) is also referred to as the "opaqueness" or “opaque appearance" of the formulation.
  • the turbidity level can be calculated with reference to a standard curve generated using a suspension of known turbidity.
  • the reference standard used to determine the turbidity level of the pharmaceutical composition can be based on the "European Pharmacopoeia” standard ("European Pharmacopoeia”, fourth edition, "Directorate for the Quality of Medicine” of the Council of Europe (EDQM), France).
  • a clear solution is defined as a solution with a turbidity lower than or equal to the turbidity of a reference suspension of about 3 according to the "European Pharmacopoeia” standard.
  • Turbidity measurement by turbidimetry can detect Rayleigh scattering in the absence of association or non-ideal effects, which usually varies linearly with concentration. Other methods for assessing physical stability are well known in the art.
  • the antibody "retains its chemical stability" in the pharmaceutical composition.
  • the chemical stability can be assessed by, for example, detecting or quantifying the form of the chemical change of the antibody.
  • Chemical changes can include size changes (e.g., cropping), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS).
  • Other types of chemical changes include charge changes (for example, occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.
  • the antibody in the pharmaceutical composition is biologically active for its intended purpose, the antibody "retains its biological activity" in the pharmaceutical composition.
  • the binding affinity of the anti-BTLA antibody contained in the preparation to BTLA is the binding affinity of the antibody before the storage At least 90%, 95% or more of the content, the formulation of the present invention can be considered to be stable.
  • the binding affinity can also be determined using techniques such as ELISA or plasmon resonance.
  • the "therapeutically effective amount” or “effective amount” of the antibody refers to an amount effective in the prevention or treatment or alleviation of symptoms of disorders that the antibody can effectively treat.
  • the "therapeutically effective dose” or “therapeutically effective dose” of the drug is any amount of drug that protects the subject from the onset of disease or promotes the regression of the disease when used alone or in combination with another therapeutic agent. The regression of the disease is evidenced by a reduction in the severity of the symptoms of the disease, an increase in the frequency and duration of the asymptomatic period of the disease, or the prevention of injury or disability caused by the pain of the disease.
  • a therapeutically effective amount of a drug includes a "prophylactically effective amount", that is, any amount that inhibits the development or recurrence of the disease when administered to a subject at risk of disease or a subject who has relapsed, alone or in combination with other therapeutic drugs medicine.
  • subject or "patient” is intended to include mammalian organisms.
  • subjects/patients include humans and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the subject is a human.
  • Anti-PD-1 antibody administration routes include intravenous, intramuscular, subcutaneous, peritoneal, spinal, or other parenteral administration routes, such as injection or infusion.
  • Parenteral administration refers to administration methods usually by injection other than enteral or local administration, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, and intrasaccular , Intra-frame, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspine, intradural and intrasternal injection and infusion, and intracorporeal electroporation.
  • antibody as used herein should be understood to include intact antibody molecules and antigen-binding fragments thereof.
  • antigen-binding portion or “antigen-binding fragment” (or simply “antibody portion” or “antibody fragment") of an antibody refers to the antibody that retains human BTLA (B and T lymphocyte attenuator) or One or more fragments of its epitope-specific binding ability.
  • full-length antibody or “complete antibody molecule” as used herein refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70kDa at full length) and two light (L) chains (Approximately 25kDa at full length) are connected to each other by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region (abbreviated as CH herein).
  • the heavy chain constant region is composed of three structural domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • VH and VL regions can be further subdivided into complementarity determining regions (CDR) with high variability and more conservatively spaced regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors (including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system).
  • CDR refers to the complementarity determining region within the variable sequence of an antibody.
  • HCDR1, HCDR2, and HCDR3 or LCDR1, LCDR2, and LCDR3 are 3 CDRs in each variable region of the heavy chain and light chain.
  • the precise boundaries of these CDRs have different definitions according to different systems.
  • variable region CDRs of the antibodies of the present invention can be determined using any of many well-known schemes, including Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al.
  • antigen-binding fragment includes fragments of antibodies or derivatives thereof, usually including at least one fragment of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody, which retains at least some of the parent antibody Binding specificity.
  • antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments And multispecific antibodies.
  • the binding fragment or derivative thereof When the binding activity of an antibody is expressed on a molar concentration basis, the binding fragment or derivative thereof generally retains at least 10% of the antigen binding activity of the parent antibody.
  • the binding fragment or derivative thereof retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also expected that the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as “conservative variants” or “functionally conservative variants” of the antibody).
  • the anti-BTLA antibody or antigen-binding fragment thereof of the present invention includes any one of the anti-BTLA antibodies described in the application number CN201810870514.0, and the entire content disclosed herein is incorporated herein by way of introduction.
  • the CDR sequences of the antibodies used in the methods and compositions of the present invention include the CDR sequences from the antibody hu18 described in CN201810870514.0.
  • the CDR sequences of the antibodies used in the methods and compositions of the present invention include the CDR sequences from the antibody hu17 described in CN201810870514.0.
  • the CDR sequences of the antibodies used in the methods and compositions of the present invention include the CDR sequences from the antibody hu19 described in CN201810870514.0.
  • the non-limiting and exemplary antibodies used in the examples herein are selected from the hu17, hu18, and hu19 described in CN201810870514.0, all of which are fully humanized antibodies that specifically bind to human BTLA, wherein the antibodies hu17, hu18 and hu19 has amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, HCDR1, HCDR2 and HCDR3, and amino acid sequences such as SEQ ID NO: 4, SEQ ID NO: 5 and The LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 6; preferably, the antibody hu17 has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7 and an amino acid sequence as shown in SEQ ID NO: 8 The variable region of the chain; antibody hu18 has the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 7 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 9; antibody
  • the pharmaceutical composition of the present invention is a highly stable pharmaceutical composition containing human antibodies specifically binding to BTLA.
  • the present invention found that the combination of trehalose and sodium chloride can significantly improve the stability of the pharmaceutical composition.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (1) a buffer; and (2) an anti-BTLA antibody or an antigen-binding fragment thereof.
  • the antibody in the pharmaceutical composition of the present invention may be a murine antibody, a chimeric antibody, a humanized antibody, preferably a humanized antibody, which may have SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • the antibody in the pharmaceutical composition of the present invention has a heavy chain variable region as shown in SEQ ID NO: 7 and a light chain variable region as shown in SEQ ID NO: 8; or has a variable region as shown in SEQ ID NO: respectively.
  • the antibody in the pharmaceutical composition of the present invention has a heavy chain amino acid sequence as shown in SEQ ID NO: 11 and a light chain amino acid sequence as shown in SEQ ID NO: 12, respectively.
  • the concentration of the anti-BTLA antibody or antigen-binding fragment thereof is about 1-200 mg/mL, preferably about 5-100 mg/mL, preferably about 10-50 mg/mL, more preferably 15-25 mg/mL .
  • the concentration of anti-BTLA antibody or its antigen-binding fragment is about 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL or 50mg/mL; preferably about 10mg/mL, 20mg/mL or 40mg/mL.
  • the buffer in the pharmaceutical composition of the present invention can be selected from acetate buffer, citrate buffer, and histidine buffer to provide the pharmaceutical composition of the present invention with a pH of 5.0 to 6.5, preferably 5.0 to 6.0.
  • the pH of the buffer used in the pharmaceutical composition of the present invention may be 5.0-6.5, preferably 5.0-6.0.
  • a particularly preferred buffer in the pharmaceutical composition of the present invention is histidine buffer.
  • the pH of the histidine buffer used in the present invention is 5.0-6.0, more preferably 5.5 ⁇ 0.3, preferably about 5.5.
  • the histidine buffer includes histidine-hydrochloride buffer or histidine-acetate buffer, preferably histidine-hydrochloride buffer. More preferably, the histidine-hydrochloride buffer is composed of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some protocols, the histidine buffer is made of 1-20 mM L-histidine and 1-20 mM L-histidine monohydrochloride.
  • the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1:4. In some schemes, the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:1. In some schemes, the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:3. In some schemes, the histidine preparation is: a histidine buffer with a pH of 5.5 made of 4.5 mM L-histidine and 15.5 mM L-histidine monohydrochloride. In some schemes, the histidine preparation is: a histidine buffer with a pH of 6.0 made of 15 mM histidine and 15 mM histidine hydrochloride.
  • the histidine buffer in the pharmaceutical composition of the present invention can be a histidine-acetate buffer, preferably, the molar ratio of the two is 1:1 to 1.5:1, preferably, the pH of such a buffer It is 5.5 ⁇ 0.3, preferably about 5.5.
  • this type of buffer contains 15-20 mM histidine and 12-15 mM acetic acid.
  • the pharmaceutical composition of the present invention may contain: a histidine-histidine hydrochloride buffer with a pH of 5.0-6.0, and a concentration of 10-30 mM in the pharmaceutical composition; and 15-25 mg/mL
  • a histidine-histidine hydrochloride buffer with a pH of 5.0-6.0, and a concentration of 10-30 mM in the pharmaceutical composition and 15-25 mg/mL
  • the anti-BTLA antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, especially hu17, hu18 or hu19 or the antigen-binding fragment thereof described herein.
  • the pharmaceutical composition of the present invention also contains a stabilizer.
  • the stabilizer is selected from one or more of sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol and trehalose.
  • the stabilizer in the pharmaceutical composition contains at least sodium chloride, and optionally contains one or more of mannitol, sorbitol, sucrose and trehalose.
  • the pharmaceutical composition may contain sodium chloride and mannitol, sodium chloride and sorbitol, sodium chloride and sucrose, or sodium chloride and trehalose.
  • the concentration of the stabilizer in the pharmaceutical composition is about 20 mM to 300 mM, preferably 50 mM to 300 mM, more preferably 120 mM to 250 mM.
  • the stabilizer is sodium chloride at a concentration of about 50-200 mM; or the stabilizer is mannitol at a concentration of about 100-300 mM; or the stabilizer is sorbitol at a concentration of about 100-300 mM; or The stabilizer is sucrose with a concentration of about 100-300mM; or the stabilizer is trehalose with a concentration of about 100-300mM; or the stabilizer is a combination of about 30-100mM sodium chloride and about 50-200mM mannitol.
  • the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM sorbitol; the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM sucrose; The stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM trehalose.
  • the pharmaceutical composition of the present invention contains: a histidine-histidine hydrochloride buffer with a pH of 5.0-6.0, the concentration of which in the pharmaceutical composition is 10-30 mM; 15- 25mg/mL of the anti-BTLA antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, especially hu17, hu18 or hu19 or antigen-binding fragment thereof as described herein; and a stabilizer of 20mM-300mM, preferably,
  • the stabilizer includes at least sodium chloride, optionally including one of mannitol, sorbitol, sucrose and trehalose, preferably 50-150mM sodium chloride, or 40-80mM sodium chloride and 120-150mM Mannitol, or 40-80mM sodium chloride and 120-150mM sorbitol, or 40-80mM sodium chloride and 120-150mM sucrose, or 40-80mM sodium chloride and 120-150mM sucrose, or 40
  • the pharmaceutical composition of the present invention also includes a surfactant.
  • Preferred surfactants are selected from polysorbate 80, polysorbate 20 and poloxamer 188.
  • the most preferred surfactant is polysorbate 80.
  • the concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.001%-0.1%, preferably about 0.01%-0.05%, preferably about 0.02%-0.04%.
  • the concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.02%, 0.03% or 0.04%, preferably 0.02%.
  • the pharmaceutical composition of the present invention contains: a histidine-histidine hydrochloride buffer with a pH of 5.0-6.0, the concentration of which in the pharmaceutical composition is 10-30 mM; 15- 25mg/mL of the anti-BTLA antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, especially hu17, hu18 or hu19 or antigen-binding fragment thereof as described herein; a stabilizer of 20mM-300mM, preferably, the The stabilizer is trehalose, or at least sodium chloride, optionally including one of mannitol, sorbitol, sucrose and trehalose, preferably 200-300mM trehalose, or 50-150mM sodium chloride, or 40-80mM sodium chloride and 120-150mM mannitol, or 40-80mM sodium chloride and 120-150mM sorbitol, or 40-80mM sodium chloride and 120-150mM sucitol, or 40
  • the pharmaceutical composition of the present invention contains or consists of the following components: a histidine-histidine hydrochloride buffer with a pH of 5.5 ⁇ 0.3, and a concentration of 20 ⁇ 5mM; 40-80mM sodium chloride; 120-150mM trehalose, 0.02-0.04% polysorbate 80 in w/v; 15-25mg/mL antibody hu18 or its antigen-binding fragment.
  • the pharmaceutical composition of the present invention may be a liquid preparation or a lyophilized preparation. It should be understood that for liquid preparations, in addition to the buffers, stabilizers, antibodies or antigen-binding fragments thereof, and surfactants described herein, they also contain water for formulating the pharmaceutical composition.
  • the present invention also provides a pharmaceutical composition according to any embodiment of the present invention for the treatment or prevention of BTLA-mediated diseases.
  • the pharmaceutical composition according to any embodiment of the present invention is used to treat or prevent BTLA-mediated diseases.
  • BTLA-mediated diseases refer to diseases that participate in the occurrence and development of diseases in BTLA, including but not limited to tumors, infectious diseases, and inflammatory or autoimmune diseases.
  • tumors suitable for the treatment and prevention of the pharmaceutical composition of the present invention include melanoma, breast cancer, kidney cancer, prostate cancer, colon cancer, lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, and uterine cancer.
  • Cancer ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, esophageal cancer, small intestine cancer, cervical cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, endocrine system cancer, Thyroid cancer, adrenal glands, soft tissue cancer, urethral cancer, chronic or acute leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, Renal or ureteral cancer, renal pelvis cancer, central nervous system neoplasms, primary central nervous system lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid Carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer
  • Autoimmune diseases suitable for the treatment and prevention of the pharmaceutical composition of the present invention include organ-specific autoimmune diseases and systemic autoimmune diseases;
  • the organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, Insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebral spinal cord Sclerosis, acute idiopathic polyneuritis, etc.
  • the systemic autoimmune diseases include systemic lupus erythematosus, wet arthritis, systemic vasculitis, scleroderma, gastric sores, dermatomyositis, mixed Connective tissue disease, autoimmune hemolytic anemia, ulcerative colitis, etc.
  • the buffer system and pH closely affect the stability of the antibody, and each antibody with unique physical and chemical properties has the most suitable buffer type and pH.
  • This example aims to screen an optimal buffer system and pH, so that the anti-BTLA antibody disclosed in the present invention has the best stability for clinical application.
  • This example was performed with antibody hu18 at concentrations of approximately 10 mg/mL, 20 mg/mL, and 40 mg/mL.
  • the sample was concentrated and exchanged by ultrafiltration using VIVAFlow200. After the liquid exchange, the sample was in the corresponding prescription, and the sample was placed in a closed centrifuge tube for buffer screening. Screened acetate buffer, citrate buffer and histidine buffer, pH from 5.0 to 6.5 (as shown in Table 1). The samples were placed in a 40°C environment, and were taken out for analysis and testing in the 0th week, the 2nd week, and the 4th week.
  • the main pathway of protein degradation is the formation of aggregates, lysate products and charged variants.
  • SEC-HPLC Size exclusion chromatography
  • CEX-HPLC cation exchange chromatography
  • a histidine buffer with a protein concentration of 20 mg/ml and a pH of 5.5 or 6.0 was selected for the prescription screening of excipients (stabilizers).
  • Surfactants added to liquid formulations are often used to protect proteins such as antibodies from the stress induced by the air/solution interface and the stress induced by the solution/surface during storage to reduce the aggregation of antibodies or minimize the formation of particulate matter in the formulation.
  • Reagent which is conducive to the stability of the physical and chemical properties of the antibody.
  • 20mM histidine buffer the molar ratio of histidine to histidine hydrochloride is 1:1, and the pH is 6.0
  • the antibody hu18 of 20mg/ml different concentrations of polysorbate 20 are added respectively.
  • polysorbate 80 placed at 40°C for 4 weeks and then analyzed and tested. The results are shown in Table 6.
  • Liquid pharmaceutical products containing therapeutic antibodies usually need to be stored at 2-8°C, so it is very important for the formulation to maintain high stability during long-term storage. Based on the above screening results, we designed No. 27 prescription on the basis of the previous 26 prescriptions to study the long-term stability of the preparation.
  • the histidine buffer is configured with a molar ratio of histidine to histidine hydrochloride of 1:3.
  • NA in the table means that it is a non-key inspection item at this point in time, and no test is done at this sampling point, and NR means that it has not arrived at this point in time, the same below.
  • prescription No. 27 has higher stability against protein degradation, and the degradation kinetic parameters measured at 25 ⁇ 2° C. meet the requirement of storage at room temperature for up to 12 months.
  • NA in the table means that it is a non-key inspection item at this point in time, and no test is done at this sampling point, and NR means that this point in time has not been reached.
  • Example 6 ELISA to detect the binding of humanized antibody to hBTLA
  • the binding specificity of humanized antibodies hu17, hu18 and hu19 with hBTLA was detected by conventional ELISA detection methods. Coat the 96-well microtiter plate with 0.5 ⁇ g/ml hBTLA and incubate at 37°C for 60 minutes. Then the solution in the well was discarded, washed 3 times with washing buffer, and blocked with PBS solution containing 2% BSA for 60 minutes.
  • Table 10 Specific binding data of humanized antibody combination hu17, hu18 and hu19 to hBTLA
  • Example 7 FACS detects the binding of humanized antibody to hBTLA on 293F cells
  • FACS Cell-based flow cytometry
  • the EC 50 value of each antibody is shown in Table 11 below. The results showed that humanized antibodies hu17, hu18, and hu19 can effectively bind to hBTLA on 293F cells.
  • Example 8 FACS detection of the blocking effect of humanized antibodies on blocking the binding of BTLA and HVEM
  • FACS flow cytometry
  • the washed cells were resuspended in a 4°C buffer containing propidium iodide (PI) and 0.02% sodium azide to prevent receptor internalization, and then analyzed by flow cytometry. According to the exclusion of PI-positive cells from the FSC/SSC gate, live cells were gated and their geometric mean fluorescence was measured. The data was analyzed using the S-shaped dose-response model in PrismTM software.
  • PI propidium iodide
  • the IC 50 value of each antibody is shown in Table 12 below. The results show that humanized antibodies hu17, hu18, and hu19 can effectively block the binding of BTLA to HVEM on the cell surface.
  • Example 10 Affinity of humanized anti-BTLA antibody to hBTLA
  • the Biacore T200 instrument of GE Healthcare Life Sciences was used for the detection experiment, and the Series S CM5 chip was loaded on the instrument.
  • the system buffer used was HBS-EP+ (10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA, 0.05% surface activity) Agent P20).
  • the antibody was diluted 2-fold with Biacore system buffer (the ingredients in the diluent except for the antibody are the same as the prescription number 27), a total of 6 concentration points.
  • the concentration gradients are 24nM, 12nM, 6nM, 3nM, 1.5nM and 0.75nM, of which 24nM is a repeated test.
  • the data analysis uses the data analysis software Biacore T200 Evaluation Software version 3.0 of GE Healthcare Life Sciences.
  • the data fitting used model is 1:1Binding.
  • the kinetic constant binding rate ka (1/Ms), the dissociation rate kd (1/s), and the affinity constant KD (M) of the binding between the antibody and the antigen were obtained by fitting. The results are shown in Table 14.
  • Example 11 Characterization of the binding kinetics of humanized antibodies with different species of BTLA
  • BTLA human BTLA, cyno BTLA or murine BTLA was coupled to an activated CM5 biosensor chip to achieve approximately 100-200 response units (RU), and then unreacted groups were blocked with IM ethanolamine.
  • RU response units
  • the RU is used to calculate the combined response of BTLA in different provenances.
  • Example 12 Inhibition of humanized antibody (prescription number: 27) on tumor growth in mice
  • the MC38-hHVME cell bank was constructed by electrotransformation of Hxp-hHVEM plasmid on mouse colon cancer (MC38) cells (ATCC), and then the cells were subcloned by limiting dilution method, and single clones were screened out by flow cytometry to obtain MC38-HVEM cells ; Then MC38-hHVEM cells were inoculated subcutaneously on the right side of B-hBTLA humanized female mice at a concentration of 1 ⁇ 10 6 cells/0.1 mL, and when the tumor grew to about 118 mm 3 , they were randomly divided into groups according to the tumor volume, 8 in each group There are 5 groups, namely: G1 0.9% sodium chloride injection solvent control group, G2KLH (anti-keyhole limpet hemocyanin antibody) (10mg/kg) negative control group, G3hu18 (1mg/kg) group, G4hu18( 3mg/kg) group and G5hu18 (10mg/kg)
  • the route of administration for all groups was intraperitoneal injection, twice a week for 7 consecutive administrations, and the experiment ended 4 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the mouse body weight and tumor volume were recorded.
  • Table 15 and Figure 2 show the effect of each group of test products on the tumor volume of MC38-hHVEM cell transplantation in B-hBTLA mice.
  • 21 days after the first administration the average tumor volume of the KLH (10mg/kg) negative control group was 1560 ⁇ 256mm 3 , and the average tumor volume of the other administration groups were 1073 ⁇ 224mm 3 , 747 ⁇ 268mm 3 and 868 ⁇ 211mm 3, respectively .
  • the TGI% of each administration group were 33.7%, 56.4%, and 48.0%, and the P values were 0.175, 0.046, and 0.056, indicating that the tested drug hu18 had a certain inhibitory effect on tumor growth.
  • a mean ⁇ standard error
  • b the tumor volume of the treatment group and the tumor volume of the KLH negative control group are statistically compared 21 days after the treatment, t-test.
  • Table 16 and Figure 3 show the effect of each group of test products on the body weight of MC38-hHVEM cell transplantation B-hBTLA mice. All the experimental animals were in good activity and eating state during the administration period, and the body weight of the animals in each administration group increased to a certain extent. There was no death of experimental animals during the experiment. 21 days after the administration, compared with the weight of the mice in the KLH negative control group, there was no significant change in the body weight of the mice in each administration group (P>0.05), indicating that the experimental animals had a good tolerance to the test product.
  • Table 16 Effect of the test substance on the body weight of MC38-hHVEM cells transplanted in B-hBTLA mice
  • a Mean ⁇ standard error
  • b The weight of the administration group and the weight of the KLH negative control group are statistically compared 21 days after administration, t-test.
  • Example 13 Humanized antibody hu18 (prescription number: 27) has no ADCC effector function
  • ADCC is initiated when the antibody binds to the target protein on the cell surface and then to the Fc ⁇ receptor (Fc ⁇ R) expressed on the effector cell.
  • Fc ⁇ R Fc ⁇ receptor
  • human IgG1 has a significantly higher binding affinity to Fc ⁇ R than IgG4, especially binding to Fc ⁇ R-I and Fc ⁇ R-IIIA, and this affinity is correlated with the strength of IgG1 to activate ADCC.
  • Reminiscent of ADCC CDC is activated when antibodies cross-link cell surface targets and C1q proteins, followed by a cascade of complement complex formation and target cell lysis.
  • the detection of antibody binding to Fc ⁇ R and C1q can serve as a basic indicator of ADCC and CDC. Therefore, the present invention uses biacore T200 (GE) to evaluate the kinetic affinity of the monoclonal antibody to the main Fc ⁇ R binding.
  • GE biacore T200
  • GE's anti-His antibody is immobilized on the sensor chip.
  • Various Fc receptors including recombinant human Fc ⁇ RIIIA (CD16a) V176, recombinant human Fc ⁇ RIIA (CD32a) R167, recombinant human Fc ⁇ RI (CD64) and recombinant human FcRn were captured, and then injected a series of diluted recombinant human anti-BTLA antibodies (ie hu18), to detect and analyze the binding properties of the interaction.
  • hu18 is an IgG4 subtype antibody
  • hu18-IgG1 is an IgG1 subtype, which shares the same Fab with hu18 and serves as a positive control.

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Abstract

提供一种稳定的抗BTLA(B和T淋巴细胞弱化子)抗体的药物组合物及其在医药上的应用。该药物组合物含有抗BTLA抗体、缓冲液，还可以含有至少一种稳定剂，任选的还可以含有表面活性剂。

Description

抗BTLA抗体药物组合物及其用途 技术领域
本发明涉及治疗性药物组合物领域。尤其是，本发明涉及医药制剂领域，该药物组合物含有一种与B和T淋巴细胞弱化子(BTLA)特异性结合的人源化抗体。
背景技术
正性和负性共刺激信号在B细胞和T细胞活性的调节中起决定性作用，而且已证实介导这些信号的分子是免疫调节剂的有效靶标。除T细胞受体(TCR)参与外，幼稚T细胞的最佳活化也需要正性共刺激，而负性共刺激则被认为是自身免疫耐受性的获得以及效应T细胞功能的终止所需要的。与抗原呈递细胞(APC)表面上的B7.1或B7.2相互作用时，原型T细胞共刺激分子CD28响应TCR参与而发出促进T细胞增殖和分化的信号，但是CD28同源物细胞毒性T淋巴细胞抗原-4(CTLA-4)介导T细胞增殖和效应子功能的抑制(Chambers等，Ann.Rev.Immunol.，19：565-594，2001；Egen等，Nature Immunol，3：611-618，2002)。已发现与B7家族同源的若干新分子(Abbas等，Nat.Med.，5：1345-6，1999；Coyle等，Nat.Immunol.，2：203-9，2001；Carreno等，Annu.Rev.Immunol.，20：29-53，2002；Liang等，Curr.Opin.Immunol.，14：384-90，2002)，并且刚开始阐明它们在T细胞活化中的作用。
B和T淋巴细胞弱化子(BTLA)是CD28家族的成员，该家族还包括CD28、ICOS、CTLA-4和PD-1。根据加入单抗后对提高T细胞增殖的功能作用，发现了该家族最初的成员CD28和ICOS具有免疫激活作用(Hutloff等，1999)。而BTLA、CTLA-4和PD-1等被描述为负性调节蛋白。若干体内研究证实了BTLA在淋巴细胞应答中的抑制作用。由Murphy与同事(Washington University St.Louis)制备的BTLA缺陷型小鼠在响应T依赖性抗原产生的IgG方面表现出3倍增加。另外，自BTLA -小鼠分离的T细胞和B细胞对分别使用CD3-和抗IgM进行抗原-受体刺激显示较大的增殖应答(Watanabe，2003)。在过量表达研究中，发现BTLA与B细胞受体复合物以及与T细胞受体缔合。与该研究结果一致，在BTLA缺陷型淋 巴细胞中，使用ConA(T细胞)或LPS(B细胞)进行抗原-受体非依赖性刺激不受影响，而且使用抗BTLA抗体也不能被调节。已经表明BTLA敲除小鼠随时间推移发生自发性自身免疫病，且寿命缩短(Oya，2008)。BTLA敲除小鼠显示在自身免疫性脑脊髓炎(EAE)和变应性气道炎症模型中疾病严重程度加剧，这两种模型均有赖于T细胞活性(Watanabe，2005；Deppong，2006)。
已经表明疱疹病毒进入介体(HVEM)是BTLA的配体(Scully等，2005)。HVEM是I型跨膜糖蛋白，属于TNF受体超家族的成员，带有4个胞外半胱氨酸富含区域(CDRs)，包含6个假性重复的半胱氨酸。BTLA及HVEM主要通过在细胞表面的动态表达来调节T细胞和APC的功能。BTLA与配体结合不仅抑制T细胞增殖，下调T细胞活化标志CD25，还可以抑制IFN-γ，IL-2，IL-4，和IL-10等的产生，但不能诱导细胞凋亡。HVEM与BTLA结合导致T细胞活化和增殖的下调(Sedy，2005)。这些研究结果表明，BTLA的表达或BTLA-HVEM结合情况与T细胞的活化与增值密切相关。
抗体可用作治疗药物。某些抗体在体内用作治疗药物时可引起不需要的抗体免疫原性。因为大多数单克隆抗体来源于啮齿动物，所以在人中重复使用导致产生针对治疗性抗体(例如，人抗小鼠抗体或HAMA)的免疫应答。这类免疫应答至少导致丧失治疗功效，而最高则导致潜在致死过敏反应。降低啮齿动物抗体的免疫原性的一种方法包括嵌合抗体的产生，其中将小鼠可变区(Fv)与人恒定区融合(Liu等(1987)Proc.Natl.Acad.Sci.USA 84：3439-43)。然而，用人可变区和小鼠恒定区的杂合体注射的小鼠发生针对人可变区的强抗体应答，这就表明这种嵌合抗体中的完整啮齿动物Fv区的保留仍可在患者中引起有害的免疫原性。
另外，将啮齿动物可变结构域的互补决定区(CDR)环移植到人构架上(即人源化)已被用于进一步将啮齿动物序列减至最低。Jones等(1986)Nature 321：522；Verhoeyen等(1988)Science 239：1534。然而，CDR环交换仍不能均匀产生具有与起始抗体相同的结合性质的抗体。在人源化抗体中，常常还需要构架残基(FR)(参与CDR环支持的残基)改变以保持抗原结合亲和力。Kabat等(1991)J.Immunol.147：1709。虽然已经报道了许多人源化抗体构建体中CDR移植和构架残基保持的使用，但是难以预测特定序列是否可产生具有所需结合性质以及间或具有生物学性质的抗体。参见例如Queen等，(1989)Proc.Natl.Acad.Sci.USA 86：10029；Gorman等， (1991)Proc.Natl.Acad.Sci.USA 88：4181；及Hodgson，(1991)Biotechnology(NY)，9：421-5。此外，大部分现有研究对动物轻链和重链可变序列使用不同人序列，致使这种研究的预测性成问题。已在使用已知抗体的序列，或者更通常使用具有已知X射线晶体结构的抗体例如抗体NEW和KOL的序列。参见例如Jones等，同上；Verhoeyen等，同上；以及Gorman等，同上。已经报道了少数人源化构建体的确切序列信息。
存在对用于治疗人病症(例如炎性病症、自身免疫病症和增殖性病症)的抗BTLA抗体、特别是抗BTLA单克隆抗体的需要。这种抗体可优选在人受试者中具有低免疫原性，允许重复给予而无不良免疫应答。
因此，本领域存在着对于高稳定性的蛋白质制剂的需求。
发明内容
本发明所述的药物组合物是一种含有与BTLA特异性结合的人源抗体的高稳定性药物组合物。特别地，本发明发现海藻糖与氯化钠的组合能明显提高药物组合物的稳定性。
本发明提供了一种药物组合物，包含：(1)缓冲液；和(2)抗BTLA抗体或其抗原结合片段。
在一些方案中，上述抗BTLA抗体或其抗原结合片段具有氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在一些方案中，所述药物组合物中抗BTLA抗体或其抗原结合片段的浓度约为1-200mg/mL，优选约为5-100mg/mL，优选约为10-50mg/mL；更优选地，上述抗BTLA抗体或其抗原结合片段浓度约为10mg/mL，15mg/mL，20mg/mL，25mg/mL，30mg/mL，35mg/mL，40mg/mL，45mg/mL或50mg/mL优选约为10mg/mL，20mg/mL，40mg/mL。
在一些方案中，上述缓冲液选自醋酸缓冲液、柠檬酸缓冲液和组氨酸缓冲液中一种或两种以上。
在一些方案中，上述缓冲液为组氨酸缓冲液，优选地，所述组氨酸缓冲液选自 组氨酸-盐酸盐缓冲液或组氨酸-醋酸盐缓冲液，优选组氨酸-盐酸盐缓冲液。
在一些方案中，上述组氨酸-盐酸盐缓冲液由组氨酸和组氨酸盐酸盐组成，优选L-组氨酸和L-组氨酸单盐酸盐。在一些方案中，组氨酸缓冲液由1-20mM的L-组氨酸和1-20mM的L-组氨酸单盐酸盐制成。在一些方案中，组氨酸缓冲液由摩尔比为1:1到1:4的组氨酸和组氨酸盐酸盐组成。在一些方案中，组氨酸缓冲液由摩尔比为1:1组氨酸和组氨酸盐酸盐组成。在一些方案中，组氨酸缓冲液由摩尔比为1:3的组氨酸和组氨酸盐酸盐组成。在一些方案中，组氨酸制剂为：由4.5mM的L-组氨酸和15.5mM的L-组氨酸单盐酸盐制成的pH为5.5的组氨酸缓冲剂。在一些方案中，组氨酸制剂为：由15mM的组氨酸和15mM的组氨酸盐酸盐制成的pH为6.0的组氨酸缓冲液。
在一些方案中，上述组氨酸缓冲液为组氨酸-醋酸缓冲液，优选地，两者的摩尔比为1:1到1.5:1，优选地，此类缓冲液的pH为5.5±0.3，优选约为5.5，优选地，这类缓冲液含有15-20mM的组氨酸和12-15mM的醋酸。
在一些方案中，上述缓冲液为醋酸缓冲液，优选地，所述醋酸缓冲液为醋酸-醋酸钠缓冲液或醋酸-醋酸钾缓冲液，优选醋酸-醋酸钠缓冲液。
在一些方案中，上述缓冲液为柠檬酸缓冲液，优选地，所述柠檬酸缓冲液为柠檬酸-柠檬酸钠缓冲液。
在一些方案中，上述缓冲液的浓度约为1-100mM，优选约为5-50mM，优选约为10-30mM，优选约为20-30mM；优选约为10-20mM，上述缓冲液浓度非限制性实施例约为5mM，10mM，15mM，20mM，25mM，30mM、40mM、45mM、50mM、55mM、60mM、65mM、70mM、75mM、80mM、85mM、90mM、95mM或100mM或这些范围内任意两个数值作为端点形成的范围，优选为10mM、20mM或30mM。
在一些方案中，上述缓冲液的pH约为5.0-6.5，优选约为5.0-6.0，优选约为5.5-6.5，优选约为5.0-5.5，优选约为5.5-6.0，优选约为6.0-6.5，上述缓冲液的pH非限制性实施例约为5.0，5.1，5.2，5.3，5.4，5.5，5.6，5.7，5.8，5.9，6.0，6.1，6.2，6.3，6.4，6.5，优选约为5.0，5.5或6.0。
在一些方案中，上述的药物组合物还包括稳定剂，所述稳定剂选自氯化钠、甘露醇、山梨醇、蔗糖、麦芽糖、木糖醇和海藻糖中的一种或两种以上。优选地，所述稳定剂为海藻糖和氯化钠的组合。
在一些方案中，上述稳定剂的浓度为约20mM～300mM，优选50mM～300mM，更优选120mM～250mM。
在一些方案中，上述稳定剂为浓度约50-200mM的氯化钠；或所述稳定剂为浓度约100-300mM的甘露醇；或所述稳定剂为浓度约100-300mM的山梨醇；或所述稳定剂为浓度约100-300mM的蔗糖；或所述稳定剂为浓度约100-300mM的海藻糖；或所述稳定剂为约30-100mM的氯化钠与约50-200mM的甘露醇的组合；所述稳定剂为约30-100mM的氯化钠与约50-200mM的山梨醇的组合；所述稳定剂为约30-100mM的氯化钠与约50-200mM的蔗糖的组合；所述稳定剂为约30-100mM的氯化钠与约50-200mM的海藻糖的组合。
在一些方案中，上述稳定剂为氯化钠。在一些方案中，上述稳定剂为浓度约50-200mM的氯化钠，上述氯化钠的浓度优选约为100-190mM，优选约为120-180mM，优选约为130-170mM，优选约为130-150mM，上述氯化钠浓度的非限制性实施例为约100mM，110mM，120mM，125mM，130mM，135mM，140mM，145mM，150mM，155mM，160mM，170mM，180mM，190mM，200mM，优选135mM或140mM。
在一些方案中，上述稳定剂为甘露醇。在一些方案中，上述稳定剂为浓度约100-300mM的甘露醇，上述甘露醇的浓度优选约为150-300mM，优选约为200-280mM，上述甘露醇浓度的非限制性实施例为约200mM，210mM，220mM，230mM，240mM，250mM，260mM，270mM，280mM，优选为240mM。
在一些方案中，上述稳定剂为山梨醇。在一些方案中，上述稳定剂为浓度约100-300mM的山梨醇，上述山梨醇的浓度优选约为150-300mM，优选约为200-280mM，上述山梨醇浓度的非限制性实施例为约200mM，210mM，220mM，230mM，240mM，250mM，260mM，270mM，280mM，优选为240mM。
在一些方案中，上述稳定剂为蔗糖。在一些方案中，上述稳定剂为浓度约100-300mM的蔗糖，上述蔗糖的浓度优选约为150-300mM，优选约为200-280mM，上述蔗糖浓度的非限制性实施例为约200mM，210mM，220mM，230mM，240mM，250mM，260mM，270mM，280mM，优选为220mM。
在一些方案中，上述稳定剂为海藻糖。在一些方案中，上述稳定剂为浓度约100-300mM的海藻糖，上述海藻糖的浓度优选约为150-300mM，优选约为200-280 mM，上述海藻糖浓度的非限制性实施例为约180mM，200mM，210mM，220mM，230mM，240mM，250mM，260mM，270mM，280mM，优选为220mM。
在一些方案中，上述稳定剂为氯化钠与甘露醇的组合。在一些方案中，上述稳定剂为约30-100mM的氯化钠与约50-200mM的甘露醇的组合，优选约40-80mM的氯化钠与约100-180mM的甘露醇的组合，优选约40-60mM的氯化钠与约120-160mM的甘露醇的组合，上述稳定剂的非限制性实施例约为54mM的氯化钠与约144mM的甘露醇的组合，约50mM的氯化钠与约140mM的甘露醇的组合。
在一些方案中，上述稳定剂为氯化钠与山梨醇的组合。在一些方案中，上述稳定剂为约30-100mM的氯化钠与约50-200mM的山梨醇的组合，优选约40-80mM的氯化钠与约100-180mM的山梨醇的组合，优选约40-60mM的氯化钠与约120-160mM的山梨醇的组合，上述稳定剂的非限制性实施例约为54mM的氯化钠与约144mM的山梨醇的组合，约40mM的氯化钠与约160mM的山梨醇的组合。
在一些方案中，上述稳定剂为氯化钠与蔗糖的组合。在一些方案中，上述稳定剂为约30-100mM的氯化钠与约50-200mM的蔗糖的组合，优选约40-80mM的氯化钠与约100-180mM的蔗糖的组合，优选约40-60mM的氯化钠与约120-160mM的蔗糖的组合，上述稳定剂的非限制性实施例约为54mM的氯化钠与约132mM的蔗糖的组合，约50mM的氯化钠与约150mM的蔗糖的组合。
在一些方案中，上述稳定剂为氯化钠与海藻糖的组合。在一些方案中，上述稳定剂为约30-100mM的氯化钠与约50-200mM的海藻糖的组合，优选约40-80mM的氯化钠与约100-180mM的海藻糖的组合，优选约40-60mM的氯化钠与约120-160mM的海藻糖的组合，上述稳定剂的非限制性实施例约为54mM的氯化钠与约132mM的海藻糖的组合，约50mM的氯化钠与约140mM的海藻糖的组合，约60mM的氯化钠与约120mM的海藻糖的组合，优选约54mM的氯化钠与约132mM的海藻糖的组合，或约50mM的氯化钠与约140mM的海藻糖的组合。
在一些方案中，上述药物组合物还包括表面活性剂，所述表面活性剂选自聚山梨醇酯80、聚山梨醇酯20或泊洛沙姆188。
在一些方案中，上述表面活性剂选自聚山梨醇酯80。
在一些方案中，上述表面活性剂选自聚山梨醇酯20。
在一些方案中，以w/v计算，上述表面活性剂浓度约为0.001％-0.1％，优选约 为0.01％-0.05％，优选约为0.02％-0.04％；作为非限制性实施例，上述表面活性剂的浓度约为0.02％，0.03％或0.04％，优选0.02％。
在一些方案中，上述抗BTLA抗体或其抗原结合片段选自鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
在一些方案中，上述抗BTLA抗体或其抗原结合片段具有氨基酸序列如SEQ ID NO:7所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区。
在一些方案中，上述抗BTLA抗体或其抗原结合片段具有氨基酸序列如SEQ ID NO:7所示的重链可变区，和氨基酸序列如SEQ ID NO:9所示的轻链可变区。
在一些方案中，上述抗BTLA抗体或其抗原结合片段具有氨基酸序列如SEQ ID NO:7所示的重链可变区，和氨基酸序列如SEQ ID NO:10所示的轻链可变区。
在一些方案中，上述抗BTLA抗体或其抗原结合片段具有如SEQ ID NO:11所示的重链氨基酸序列，和如SEQ ID NO:12所示的轻链氨基酸序列。
在一些方案中，上述药物组合物，其包含如下(1)-(8)任一项所示的组分，或由如下(1)-(8)任一项所示的组分组成；其中，抗BTLA抗体或其抗原结合片段如本发明任一实施方案所述：
(1)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM组氨酸缓冲液，pH约为5.0-6.0；(c)约30mM至约100mM的氯化钠与约50-200mM的海藻糖的组合；(d)以及约0.01％-0.05％的聚山梨醇酯80；或
(2)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM组氨酸缓冲液，pH约为5.0-6.0；(c)约30mM至约100mM的氯化钠与约50-200mM的甘露醇的组合；(d)以及约0.01％-0.05％的聚山梨醇酯80；或
(3)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM组氨酸缓冲液，pH约为5.0-6.0；(c)约30mM至约100mM的氯化钠与约50-200mM的蔗糖的组合；(d)以及约0.01％-0.05％的聚山梨醇酯80；或
(4)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM组氨酸缓冲液，pH约为5.0-6.0；(c)约30mM至约100mM的氯化 钠与约50-200mM的山梨醇的组合；(d)以及约0.01％-0.05％的聚山梨醇酯80；或
(5)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM组氨酸缓冲液，pH约为5.0-6.0；(c)约100mM至约300mM的海藻糖；(d)以及约0.01％-0.05％的聚山梨醇酯80；
(6)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM柠檬酸缓冲液，pH约为5.5-6.5；(c)约100mM至约300mM的海藻糖；(d)以及约0.01％-0.05％的聚山梨醇酯80。
(7)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM柠檬酸缓冲液，pH约为5.5-6.5；(c)约30mM至约100mM的氯化钠与约50-200mM的甘露醇的组合；(d)以及约0.01％-0.05％的聚山梨醇酯80。
(8)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM醋酸缓冲液，pH约为5.0-6.0；(c)约30mM至约100mM的氯化钠与约50-200mM的甘露醇的组合；(d)以及约0.01％-0.05％的聚山梨醇酯80。
在一些方案中，上述的药物组合物包含如下(9)-(13)任一项所示的组分，或由如下(9)-(13)任一项所示的组分组成；：
(9)(a)约20mg/mL的抗BTLA抗体或其抗原结合片段；(b)约20mM组氨酸缓冲液，pH约为6.0；(c)约54mM的氯化钠与约132mM的海藻糖的组合；(d)以及约0.02％的聚山梨醇酯80；或
(10)(a)约20mg/mL的抗BTLA抗体或其抗原结合片段；(b)约20mM组氨酸缓冲液，pH约为5.5；(c)约220mM的海藻糖的组合；(d)以及约0.02％的聚山梨醇酯80；或
(11)(a)约20mg/mL的抗BTLA抗体或其抗原结合片段；(b)约20mM组氨酸缓冲液，pH约为5.5；(c)约50mM的氯化钠与约140mM的甘露醇的组合；(d)以及约0.02％的聚山梨醇酯80；
(12)(a)约20mg/mL的抗BTLA抗体或其抗原结合片段；(b)约20mM组氨酸缓冲液，pH约为5.5；(c)约50mM的氯化钠与约140mM的海藻糖的组合；(d)以及约0.02％的聚山梨醇酯80；和
(13)(a)约20mg/mL的抗BTLA抗体或其抗原结合片段；(b)约20mM组氨酸缓冲液，pH约为5.0-6.0；(c)约50mM的氯化钠与约140mM的海藻糖的组合；(d)以及约0.02％的聚山梨醇酯80。
在一些方案中，所述药物组合物为液体制剂或冻干制剂。
在一些方案中，所述药物组合物为液体制剂，如水溶液制剂。
在一些方案中，上述液体制剂或冻干制剂于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。
在一些方案中，上述水溶液或冻干制剂于40℃稳定至少7天，至少14天或至少28天。
本发明还提供了上述药物组合物在制备治疗或预防BTLA介导的疾病的药物中的用途。
在一些方案中，上述疾病包括肿瘤、感染性疾病、炎性或自身免疫性疾病。所述的肿瘤包括黑色素瘤、乳腺癌、肾癌、***癌、结肠癌、肺癌、胰腺癌、骨癌、皮肤癌、头或颈癌、子宫癌、卵巢癌、直肠癌、肛区癌、胃癌、睾丸癌、食道癌、小肠癌、***、***癌、何杰金氏病、非何杰金氏淋巴瘤、食道癌、内分泌***癌、甲状腺癌、肾上腺爱、软组织癌、尿道癌、慢性或急性白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病、儿童期实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经***的赘生物、原发性中枢神经***淋巴瘤、肿瘤血管发生、脊髓轴肿瘤、脑干胶质瘤、垂体腺瘤、卡波西氏肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症、其包括石棉所诱发的那些，和所述癌症的组合。所述自身免疫性疾病包括器官特异性自身免疫病和***性自身免疫病；所述器官特异性自身免疫病包括慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症、急性特发性多神经炎等；所述***性自身免疫病包括***性红斑狼疮、类分湿关节炎、***性血管炎、硬皮病、天胞疮、皮肌炎、混合性***病、自身免疫性溶血性贫血、溃疡性结肠炎等。
附图说明
图1：人源化抗体hu 17与不同种属BTLA结合的对比实验。
图2：hu18对MC38-hHVEM细胞移植B-hBTLA小鼠肿瘤体积的影响。
图3：受试品对MC38-hHVEM细胞移植B-hBTLA小鼠体重的影响。
定义和说明
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。应理解本发明不限于具体的方法、试剂、化合物、组合物或生物***，当然可以对以上进行变化。还应理解本申请所用术语仅为了描述具体的实施方式，并不旨在进行限制。
除非该内容被另外明确说明，否则本说明书以及所附权利要求中所用的单数形式"一个"、"一种"和"该"包括复数指代。因此，例如，提及"一种多肽"包括了两种或更多种多肽等的组合。
术语"药物组合物"或“制剂”表示含有一种或多种本文所述化合物或其药学上可接受的盐或前体药物与其他组分的混合物，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。
术语“液体制剂”是指处于液体状态下的制剂，且不意图指称重悬浮的冻干制剂。本发明的液体制剂在储存时稳定，并且其稳定性不依赖于冻干(或其他状态改变方法，例如喷雾干燥)。
术语“水性液体制剂”是指使用水作为溶剂的液体制剂。在一些方案中，水性液体制剂是不需冻干、喷雾干燥和/或冷冻来维持稳定性(例如化学和/或物理稳定性和/或生物活性)的制剂。
术语“赋形剂”是指可以向制剂添加以提供所需特性(例如稠度、提高的稳定性)和/或调节渗透压的试剂。常用赋形剂的实例包括但不限于糖类、多元醇、氨基酸、表面活性剂和聚合物。
本申请所用的"约"在指代可测量数值(如量、持续时间等)时意在涵盖相对于具体数值±20％或±10％的变化，包括±5％、±1％和±0.1％，因为这些变化适于进行所公开的方法。
术语"缓冲液pH约为5.0-6.5"是指这样的试剂，通过其酸/碱共轭组分的作用使得包含该试剂的溶液能抵抗pH变化。本发明的制剂中使用的缓冲液可具有约5.0至约6.5范围内的pH、或约5.5至约6.5范围内的pH、或约5.0至约6.0范围内的pH。
在本文中，将pH控制在该范围内的“缓冲液”实例包括乙酸盐(例如乙酸钠)、琥珀酸盐(例如琥珀酸钠)、葡萄糖酸、组氨酸、组氨酸盐酸盐、甲硫氨酸、柠檬酸盐、磷酸盐、柠檬酸盐/磷酸盐、咪唑、醋酸、醋酸盐、枸橼酸盐、其组合和其他有机酸缓冲剂。
"组氨酸缓冲液"为包含组氨酸离子的缓冲液。组氨酸缓冲液的实例包括组氨酸和组氨酸的盐，如组氨酸盐酸盐、组氨酸乙酸盐、组氨酸磷酸盐和组氨酸硫酸盐等，如含有组氨酸与组氨酸盐酸盐的组氨酸缓冲液；本发明的组氨酸缓冲液也包括含有组氨酸和醋酸盐(如钠盐或钾盐)的组氨酸缓冲液。
“柠檬酸盐缓冲液”是包括柠檬酸根离子的缓冲液。柠檬酸盐缓冲液的实例包括柠檬酸-柠檬酸纳、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液为柠檬酸-柠檬酸纳缓冲液。
“醋酸盐缓冲液”是包括醋酸根离子的缓冲液。醋酸盐缓冲液的实例包括醋酸-醋酸纳、醋酸-醋酸钾、醋酸-醋酸钙、醋酸－醋酸镁等。优选的醋酸盐缓冲液为醋酸-醋酸纳缓冲液。
术语“稳定剂”表示药学上可接受的赋形剂，其在制造，储存和应用过程中保护活性药物成分和/或制剂免受化学和/或物理降解。稳定剂包括但不限于如以下定义的糖，氨基酸，盐，多元醇和他们的代谢产物，例如氯化钠、氯化钙、氯化镁、甘露醇、山梨醇、蔗糖、海藻糖、盐酸精氨酸、精氨酸、甘氨酸、丙氨酸(α-丙氨酸、β-丙氨酸)、甜菜碱、亮氨酸、赖氨酸、谷氨酸、天冬氨酸、脯氨酸、4-羟基脯氨酸、肌氨酸、γ-氨基丁酸(GABA)、奥品类(opines)、丙氨奥品、章鱼碱、甘氨奥品(strombine))和三甲胺的N-氧化物(TMAO)、人血清白蛋白(hsa)、牛血清白蛋白(bsa)、α-酪蛋白、球蛋白、α-乳白蛋白、LDH、溶菌酶、肌红蛋白、卵清蛋白和RNAaseA。部分稳定剂，如氯化钠、氯化钙、氯化镁、甘露醇、山梨醇、蔗糖等也可起到控制渗透压的作用。在本发明中具体地使用的稳定剂选自多元醇、盐、糖中的一种或两种以上。优选的盐为氯化钠，优选的糖为蔗糖和海藻糖，优选的多元醇为山梨醇和 甘露醇。优选的稳定剂为氯化钠、甘露醇、山梨醇、蔗糖、海藻糖、氯化钠-山梨醇、氯化钠-甘露醇、氯化钠-蔗糖和氯化钠-海藻糖，更优选为氯化钠-山梨醇、氯化钠-甘露醇、氯化钠-蔗糖和氯化钠-海藻糖，更优选为氯化钠-海藻糖。
术语“表面活性剂”一般包括保护蛋白质例如抗体免受空气/溶液界面诱导的应力、溶液/表面诱导的应力的影响以减少抗体的聚集或使制剂中颗粒物的形成最小化的试剂。示例性的表面活性剂包括但不限于非离子型表面活性剂例如聚氧乙烯脱水山梨醇脂肪酸酯(如聚山梨醇酯20和聚山梨醇酯80)、聚乙烯-聚丙烯共聚物、聚乙烯-聚丙烯二醇、聚氧乙烯-硬脂酸酯、聚氧乙烯烷基醚、例如聚氧乙烯单月桂基醚、烷基苯基聚氧乙烯醚(Triton-X)、聚氧乙烯-聚氧丙烯共聚物(泊洛沙姆，Pluronic)、十二烷基硫酸钠(SDS)。
术语"等渗"是指该制剂具有与人血液基本相同的渗透压。等渗制剂一般具有约250至350mOsm的渗透压。可使用蒸汽压或冰点下降式的渗透压计测量等渗性。
术语“稳定的”制剂是其中的抗体在制造过程期间和/或储存时基本上保持其物理稳定性和/或化学稳定性和/或生物活性的制剂。即使所含的抗体在经过一定时间储存之后未能保持其100％的化学结构或生物功能，医药制剂也可以是稳定的。在某些情况下，在经过一定时间储存之后，能维持约90％、约95％、约96％、约97％、约98％或约99％的抗体结构或功能，也可被认为是“稳定的”。用于测量蛋白质稳定性的各种分析技术在本技术领域中是可得的，并综述在《肽和蛋白质药物递送》(Peptide and Protein Drug Delivery)247-301，Vincent Lee主编，Marcel Dekker，Inc.，New York，N.Y.，Pubs.(1991))，和Jones，A.(1993)Adv.Drug Delivery Rev.10：29-90中(二者引入作为参考)。
制剂在一定温度下经过一定时间的储存之后，通过测定其中剩余的天然抗体的百分比(及其它方法)，可以测量其稳定性。除其它方法外，天然抗体的百分比可以通过尺寸排阻色谱法(例如尺寸排阻高效液相色谱法[SEC-HPLC])来测量，“天然的”指未聚集的和未降解的。在一些方案中，蛋白质的稳定性按照具有低百分比的降解(例如片段化)和/或聚集蛋白质的溶液中单体蛋白质的百分数来确定。在一些方案中，制剂可以在室温、约25-30℃或40℃下稳定储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个 月、至少18个月、至少24个月，或更长，最多不超过约6％、5％、4％、3％、2％、1％、0.5％，或0.1％聚集形式的抗体。
通过测定在离子交换期间在此抗体主馏分(“主要荷电形式”)较为酸性的馏分中迁移的抗体(“酸性形式”)的百分比(及其它方法)，可以测量稳定性，其中稳定性与酸性形式抗体的百分比成反比。除其它方法外，“酸化”抗体的百分比可以通过离子交换色谱法(例如阳离子交换高效液相色谱法[CEX-HPLC])来测量。在一些实施方式中，可接受程度的稳定性意为当制剂在一定温度下经过一定时间的储存之后，其中可检测出的酸性形式的抗体最多不超过约49％、45％、40％、35％、30％、25％、20％、15％、10％、5％、4％、3％、2％、1％、0.5％或0.1％。在测量稳定性之前储存的一定时间可以是至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，或更长。当评估稳定性时，容许储存医药制剂的一定温度可以是约-80℃至约45℃范围内的任何温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约2-8℃、约5℃、约25℃，或约40℃。
如果抗体在颜色和/或澄清度目测检查时或通过UV光散射或通过孔径排阻层析测量时基本上不显示出例如聚集、沉淀和/或变性的迹象，则所述抗体在该药物组合物中“保持其物理稳定性”。聚集是单个分子或复合物共价或非共价缔合以形成聚集体的过程。聚集可以进行到形成可见沉淀物的程度。
制剂的稳定性例如物理稳定性可以通过本技术领域中公知的方法来评估，包括测量样品的表观消光度(吸光度或光密度)。这样的消光测量与制剂的浊度相关。制剂的浊度部分地是溶解在溶液中的蛋白质的固有性质，并且通常通过比浊法来测量，并用比浊法浊度单位(NTU)来量度。
随着例如溶液中一种或多种组分的浓度(例如蛋白质和/或盐浓度)而变化的浊度水平也被称为制剂的“乳浊”或“乳浊外观”。浊度水平可以参照使用已知浊度的悬液产生的标准曲线来计算。用于测定药物组合物的浊度水平的参比标准品可以基于《欧洲药典》标准(《欧洲药典》(European Pharmacopoeia)，第四版，“欧洲药品质量委员会指令”(Directorate for the Quality of Medicine of the Council of Europe)(EDQM)，Strasbourg，France)。根据《欧洲药典》标准，澄清溶液被定义 为浊度低于或等于按照《欧洲药典》标准具有约3的参比悬液的浊度的溶液。比浊法的浊度测量可以检测在不存在缔合或非理想效应的情况下的瑞利散射，其通常随浓度线性变化。用于评估物理稳定性的其他方法在本技术领域中是公知的。
如果抗体在给定时间点的化学稳定性使得抗体被认为仍保持如下文中所定义的其生物活性，则所述抗体在药物组合物中“保持其化学稳定性”。可以通过例如检测或定量抗体的化学改变的形式来评估化学稳定性。化学改变可以包括尺寸改变(例如剪短)，其可以使用例如孔径排阻层析、SDS-PAGE和/或基质辅助的激光解吸电离/飞行时间质谱(MALDI/TOF MS)来评估。其他类型的化学改变包括电荷改变(例如作为脱酰胺或氧化的结果而发生)，其可以通过例如离子交换层析来评估。
如果药物组合物中的抗体对于其预期目的来说是生物活性的，则所述抗体在药物组合物中“保持其生物活性”。例如，如果制剂于例如5℃、25℃、45℃等温度下储存一定时间(例如1至12个月)之后，该制剂所含抗BTLA抗体与BTLA结合的亲和力为所述储存之前抗体结合亲和力的至少90％、95％或以上，则可认为本发明之制剂是稳定的。结合亲和力也可用例如ELISA或等离子共振技术测定。
在本发明的情形中，在药理学意义上，抗体的“治疗有效量”或“有效量”是指在抗体可以有效治疗的障碍的症状的预防或治疗或减轻方面有效的量。本发明中，药物的“治疗有效量”或“治疗有效剂量”是当单独使用或与另一种治疗剂组合使用时保护受试者免于疾病发作或促进疾病消退的任何量的药物，所述疾病消退通过疾病症状的严重性的降低，疾病无症状期的频率和持续时间的增加，或由疾病痛苦引起的损伤或失能的预防来证明。药物促进疾病消退的能力可以使用本领域技术人员已知的多种方法来评价，比如在临床试验期间的人受试者中，在预测人类功效的动物模型***中，或通过在体外测定法中测定所述药剂的活性。药物治疗有效量包括“预防有效量”，即当单独或如与其它治疗药物组合给与处于患病风险的受试者或患病复发的受试者时，抑制疾病的发展或复发的任何量的药物。
术语“受试者”或“患者”意图包括哺乳动物生物体。受试者/患者的实例包括人类和非人类哺乳动物，例如非人类灵长动物、狗、奶牛、马、猪、绵羊、山羊、猫、小鼠、兔、大鼠和转基因非人类动物。在本发明的特定实施方式中，受试者是人类。
术语“施用”、“给与”及“处理”是指采用本领域技术人员已知的各种方法或递送***中的任意一种将包含治疗剂的组合物引入受试者。抗PD-1抗体的给药 途径包括静脉内、肌内、皮下、腹膜、脊髓或其他胃肠外给药途径，比如注射或输注。“胃肠外给药”是指除了肠内或局部给药以外的通常通过注射的给药方式，包括但不限于静脉内、肌内、动脉内、鞘内、淋巴内、损伤内、囊内、框内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜内和胸骨内注射和输注以及经体内电穿孔。
抗BTLA抗体
本文所用的术语“抗体”应被理解为包括完整抗体分子及其抗原结合片段。本文所用的术语抗体的“抗原结合部分”或“抗原结合片段”(或简称为“抗体部分”或“抗体片段”)是指抗体中保持了与人BTLA(B和T淋巴细胞弱化子)或其表位特异性结合能力的一个或多个片段。
本文所用的术语“全长抗体”或“完整抗体分子”指包含四条肽链的免疫球蛋白分子，两条重(H)链(全长时约50-70kDa)和两条轻(L)链(全长时约25kDa)通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫***的各种细胞(例如，效应细胞)和经典补体***的第一组分(Clq))的结合。
当在本文中使用时，术语“CDR”是指抗体可变序列内的互补决定区。在重链和轻链的各个可变区中存在3个CDR，其对于各个重链和轻链可变区被命名为HCDR1、HCDR2和HCDR3或LCDR1、LCDR2和LCDR3。这些CDR的准确边界按照不同的***有不同的定义。
本发明的所述抗体的可变区CDR的精确氨基酸序列边界可使用许多公知的方案的任何方案来确定，包括基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard  conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(1999 Nucleic Acids Research,27,209-212)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本发明抗体的CDR可以由本领域的技术人员根据本领域的任何方案(例如不同的指派***或组合)确定边界。
本文所使用的“抗原结合片段”包括抗体的片段或其衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗原结合片段的实例包括但不限于Fab，Fab'，F(ab')2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗体的结合活性在摩尔浓度基础上表示时，结合片段或其衍生物通常保持亲代抗体抗原结合活性的至少10％。优选结合片段或其衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。
本发明所述的抗BTLA抗体或其抗原结合片段包括申请号为CN201810870514.0中描述的任意一个抗BTLA抗体，本文将其所公开的全部内容以引入的方式纳入本文。在一些方案中，在本发明的方法和组合物中使用的抗体的CDR序列包括来自于CN201810870514.0中描述的抗体hu18的CDR序列。在一些方案中，在本发明的方法和组合物中使用的抗体的CDR序列包括来自于CN201810870514.0中描述的抗体hu17的CDR序列。在一些方案中，在本发明的方法和组合物中使用的抗体的CDR序列包括来自于CN201810870514.0中描述的抗体hu19的CDR序列。
在本文实施例中所用的非限制性、示范性抗体选自CN201810870514.0中描述的hu17、hu18和hu19，均是与人BTLA特异性结合的人源化完全抗体，其中，抗体hu17、hu18和hu19均具有氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID  NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；优选地，抗体hu17具有分别氨基酸序列如SEQ ID NO:7所示的重链可变区和氨基酸序列如SEQ ID NO:8所示的轻链可变区；抗体hu18具有氨基酸序列分别如SEQ ID NO:7所示的重链可变区和氨基酸序列如SEQ ID NO:9所示的轻链可变区；抗体hu19具有氨基酸序列分别如SEQ ID NO:7所示的重链可变区和氨基酸序列如SEQ ID NO:10所示的轻链可变区；优选地，抗体hu18具有分别如SEQ ID NO:11所示的重链氨基酸序列和如SEQ ID NO:12所示的轻链氨基酸序列。其中，上述人源化抗体的CDR氨基酸序列由IMGT***定义。
医药制剂
本发明所述的药物组合物是一种含有与BTLA特异性结合的人源抗体的高稳定性药物组合物。特别地，本发明发现海藻糖与氯化钠的组合能明显提高药物组合物的稳定性。
本发明提供了一种药物组合物，包含：(1)缓冲液；和(2)抗BTLA抗体或其抗原结合片段。
本发明药物组合物中的抗体可以是鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体，可具有分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。优选地，本发明药物组合物中的抗体具有如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；或具有分别如SEQ ID NO:7所示的重链可变区和如SEQ ID NO:9所示的轻链可变区；或具有分别如SEQ ID NO:7所示的重链可变区和如SEQ ID NO:10所示的轻链可变区；更优选地，本发明药物组合物中的抗体具有分别如SEQ ID NO:11所示的重链氨基酸序列和如SEQ ID NO:12所示的轻链氨基酸序列。
本发明的药物组合物中，抗BTLA抗体或其抗原结合片段浓度约为1-200mg/mL，优选约为5-100mg/mL，优选约为10-50mg/mL，更优选15-25mg/mL。作为非限制性实施例；抗BTLA抗体或其抗原结合片段浓度约为10mg/mL，15mg/mL，20mg/mL，25mg/mL，30mg/mL，35mg/mL，40mg/mL，45mg/mL或50mg/mL；优选约为10mg/mL，20mg/mL或40mg/mL。
本发明药物组合物中的缓冲液可选自醋酸缓冲液、柠檬酸缓冲液和组氨酸缓冲液，用以为本发明的药物组合物提供5.0到6.5、优选5.0到6.0的pH。另一方面，用于本发明药物组合物中的缓冲液的pH可为5.0-6.5，优选为5.0-6.0。
本发明药物组合物中特别优选的缓冲液是组氨酸缓冲液。优选地，本发明使用的组氨酸缓冲液的pH为5.0-6.0，更优选为5.5±0.3，优选约为5.5。优选，所述组氨酸缓冲液包括组氨酸-盐酸盐缓冲液或组氨酸-醋酸盐缓冲液，优选组氨酸-盐酸盐缓冲液。更优选地，所述组氨酸-盐酸盐缓冲液由组氨酸和组氨酸盐酸盐组成，优选L-组氨酸和L-组氨酸单盐酸盐。在一些方案中，组氨酸缓冲液由1-20mM的L-组氨酸和1-20mM的L-组氨酸单盐酸盐制成。在一些方案中，组氨酸缓冲液由摩尔比为1:1到1:4的组氨酸和组氨酸盐酸盐组成。在一些方案中，组氨酸缓冲液由摩尔比为1:1组氨酸和组氨酸盐酸盐组成。在一些方案中，组氨酸缓冲液由摩尔比为1:3的组氨酸和组氨酸盐酸盐组成。在一些方案中，组氨酸制剂为：由4.5mM的L-组氨酸和15.5mM的L-组氨酸单盐酸盐制成的pH为5.5的组氨酸缓冲剂。在一些方案中，组氨酸制剂为：由15mM的组氨酸和15mM的组氨酸盐酸盐制成的pH为6.0的组氨酸缓冲液。
本发明药物组合物中的组氨酸缓冲液可为组氨酸-醋酸盐缓冲液，优选地，两者的摩尔比为1:1到1.5:1，优选地，此类缓冲液的pH为5.5±0.3，优选约为5.5，优选地，这类缓冲液含有15-20mM的组氨酸和12-15mM的醋酸。
因此，本发明的药物组合物可含有：pH为5.0-6.0的组氨酸-组氨酸盐酸盐缓冲液，其在药物组合物中的浓度为10-30mM；和15-25mg/mL的前文任一实施方案所述的抗BTLA抗体或其抗原结合片段，尤其是本文所述的hu17、hu18或hu19或其抗原结合片段。
在一些方案中，本发明的药物组合物还含有稳定剂。优选地，所述稳定剂选自氯化钠、甘露醇、山梨醇、蔗糖、麦芽糖、木糖醇和海藻糖中的一种或两种以上。优选地，药物组合物中的稳定剂中至少含有氯化钠，任选含有甘露醇、山梨醇、蔗糖和海藻糖中的一种或多种。例如，药物组合物中可含有氯化钠与甘露醇、氯化钠与山梨醇、氯化钠与蔗糖、或者氯化钠与海藻糖。含有时，药物组合物中稳定剂的浓度为约20mM-300mM，优选50mM-300mM，更优选120mM-250mM。在一些方案中，稳定剂为浓度约50-200mM的氯化钠；或所述稳定剂为浓度约100-300mM 的甘露醇；或所述稳定剂为浓度约100-300mM的山梨醇；或所述稳定剂为浓度约100-300mM的蔗糖；或所述稳定剂为浓度约100-300mM的海藻糖；或所述稳定剂为约30-100mM的氯化钠与约50-200mM的甘露醇的组合；所述稳定剂为约30-100mM的氯化钠与约50-200mM的山梨醇的组合；所述稳定剂为约30-100mM的氯化钠与约50-200mM的蔗糖的组合；所述稳定剂为约30-100mM的氯化钠与约50-200mM的海藻糖的组合。
因此，在一些实施方案中，本发明的药物组合物含有：pH为5.0-6.0的组氨酸-组氨酸盐酸盐缓冲液，其在药物组合物中的浓度为10-30mM；15-25mg/mL的前文任一实施方案所述的抗BTLA抗体或其抗原结合片段，尤其是本文所述的hu17、hu18或hu19或其抗原结合片段；以及20mM-300mM的稳定剂，优选地，所述稳定剂至少包括氯化钠，任选包括甘露醇、山梨醇、蔗糖和海藻糖中的一种，优选为50-150mM的氯化钠，或40-80mM的氯化钠与120-150mM的甘露醇，或40-80mM的氯化钠与120-150mM的山梨醇，或40-80mM的氯化钠与120-150mM的蔗糖，或40-80mM的氯化钠与120-150mM的海藻糖。在一些实施方案中，所述稳定剂为200-300mM的海藻糖。
在一些方案中，本发明的药物组合物还包括表面活性剂。优选的表面活性剂选自聚山梨醇酯80、聚山梨醇酯20和泊洛沙姆188。最优选的表面活性剂是聚山梨醇酯80。以w/v计，本发明药物组合物中表面活性剂的浓度约为0.001％-0.1％，优选约为0.01％-0.05％，优选约为0.02％-0.04％。作为非限制性实施例，本发明药物组合物中表面活性剂的浓度约为0.02％，0.03％或0.04％，优选0.02％。
因此，在一些实施方案中，本发明的药物组合物含有：pH为5.0-6.0的组氨酸-组氨酸盐酸盐缓冲液，其在药物组合物中的浓度为10-30mM；15-25mg/mL的前文任一实施方案所述的抗BTLA抗体或其抗原结合片段，尤其是本文所述的hu17、hu18或hu19或其抗原结合片段；20mM-300mM的稳定剂，优选地，所述稳定剂为海藻糖，或至少包括氯化钠，任选包括甘露醇、山梨醇、蔗糖和海藻糖中的一种，优选为200-300mM的海藻糖，或50-150mM的氯化钠，或40-80mM的氯化钠与120-150mM的甘露醇，或40-80mM的氯化钠与120-150mM的山梨醇，或40-80mM的氯化钠与120-150mM的蔗糖，或40-80mM的氯化钠与120-150mM的海藻糖；以及以w/v计0.02％-0.04％的聚山梨醇酯80。在特别优选的实施方案中，本发明的 药物组合物含有以下组分，或由以下组分组成：pH为5.5±0.3的组氨酸-组氨酸盐酸盐缓冲液，其浓度为20±5mM；40-80mM的氯化钠；120-150mM的海藻糖，以w/v计0.02-0.04％的聚山梨酯80；15-25mg/mL的抗体hu18或其抗原结合片段。
本发明的药物组合物可以是液体制剂，或者是冻干制剂。应理解，对于液体制剂，除本文所述的缓冲液、稳定剂、抗体或其抗原结合片段和表面活性剂外，还含有用来配制该药物组合物的水。
医药用途和方法
本发明还提供了用于治疗或预防BTLA介导的疾病的本发明任一实施方案所述的药物组合物，本发明任一实施方案所述的药物组合物在制备治疗或预防BTLA介导的疾病的药物中的用途，以及给予需要的个体或患者治疗有效量的本发明任一实施方案所述的药物组合物以治疗或预防BTLA介导的疾病的方法。
本发明中，BTLA介导的疾病指在BTLA参与了疾病的发生和发展的疾病，包括但不限于肿瘤、感染性疾病、和炎性或自身免疫性疾病。本发明中，适用于本发明药物组合物进行治疗和预防的肿瘤包括黑色素瘤、乳腺癌、肾癌、***癌、结肠癌、肺癌、胰腺癌、骨癌、皮肤癌、头或颈癌、子宫癌、卵巢癌、直肠癌、肛区癌、胃癌、睾丸癌、食道癌、小肠癌、***、***癌、何杰金氏病、非何杰金氏淋巴瘤、食道癌、内分泌***癌、甲状腺癌、肾上腺爱、软组织癌、尿道癌、慢性或急性白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病、儿童期实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经***的赘生物、原发性中枢神经***淋巴瘤、肿瘤血管发生、脊髓轴肿瘤、脑干胶质瘤、垂体腺瘤、卡波西氏肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症、其包括石棉所诱发的那些，和所述癌症的组合。适用于本发明药物组合物进行治疗和预防的自身免疫性疾病包括器官特异性自身免疫病和***性自身免疫病；所述器官特异性自身免疫病包括慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症、急性特发性多神经炎等；所述***性自身免疫病包括***性红斑狼疮、类分湿关节炎、***性血管炎、硬皮病、天胞疮、皮肌炎、混合性*** 病、自身免疫性溶血性贫血、溃疡性结肠炎等。
具体实施方式
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是阐述性的，并非意图限制本发明的范围。实施例中所用到的方法和材料，除非另有说明，否则为本领域的常规方法和材料。
实施例1：缓冲液体系和pH筛选实验
液体型药物组合物中，缓冲液体系和pH密切影响抗体的稳定性，每种具有独特理化性质的抗体都具有最适宜的缓冲液的种类和pH。本实施例旨在筛选一种最佳缓冲液体系和pH，使本发明公开的抗BTLA抗体具有最佳的稳定性以适宜临床应用。
本实施例以约10mg/mL、20mg/mL和40mg/mL浓度的抗体hu18进行。样品使用VIVAFlow200进行超滤浓缩换液，换液后样品处于相应的处方中，样品放置在密闭的离心管中进行缓冲液筛选。筛选了醋酸缓冲液、柠檬酸缓冲液和组氨酸缓冲液，pH从5.0到6.5(如表1所示)。将样品放置在40℃环境下，分别在第0周、第2周和第4周取出进行分析检测。蛋白降解的主要途径是聚集物、裂解产品和带电变体的形成。采用尺寸排阻色谱法(SEC-HPLC)测定天然形式(蛋白单体)与聚集形式所占的百分比，采用阳离子交换色谱法(CEX-HPLC)测定酸性与碱性形式抗体所占的百分比。以试验起始(0W)、放置两周(2W)和放置四周(4W)的SEC-HPLC单体含量和CEX-HPLC主峰含量，拟合直线并计算下降斜率(％/周)考察不同缓冲液体系和pH对抗体hu18抗体稳定性的影响，结果汇总见表2和表3所示。
表1：缓冲液体系和pH筛选实验中的处方信息
Figure PCTCN2021072660-appb-000001
Figure PCTCN2021072660-appb-000002
注：“-”表示未添加。
表2：缓冲液体系和pH筛选实验中SEC-HPLC单体含量降解速率
Figure PCTCN2021072660-appb-000003
Figure PCTCN2021072660-appb-000004
表3：缓冲液体系和pH筛选实验中CEX-HPLC电荷异质体含量降解速率
Figure PCTCN2021072660-appb-000005
由表2和表3所示，SEC-HPLC实验检测中，高温40℃放置4周后，单体含 量随蛋白浓度的增高下降速率增快，但是在pH 5.5～6.0组氨酸缓冲体系下不同蛋白浓度的单体含量下降速率均较低，平均下降速率≤0.27％/周；CEX-HPLC实验检测中，可以看出CEX-HPLC主峰含量降解速率与pH有一定的相关性，pH越高主峰含量降解速率越快，在pH 5.0～6.0的缓冲体系中，主峰含量降解速度相对较低，主峰含量降解速率与蛋白浓度的相关性不明显。
综合考虑产品规格以及产品的目标质量属性(聚体含量水平)，选择蛋白浓度20mg/ml，pH为5.5或6.0的组氨酸缓冲液进行辅料(稳定剂)的处方筛选。
实施例2：稳定剂筛选实验
为了进一步探究不同辅料对抗体稳定性的影响，我们选取氯化钠、甘露醇、山梨醇、蔗糖或海藻糖之一或其组合的制剂进行了比较测试。即将上述不同的辅料或其组合分别加入含约20mg/mL抗体hu18的20mM组氨酸缓冲液或者柠檬酸缓冲液中，具体处方信息如表4所示。各处方制剂分装后放置于40℃条件下，分别在第0周、第2周、第4周取出进行分析检测。通过分子排阻高效液相色谱法(SEC-HPLC)检测抗体hu18单体含量变化、弱阳离子高效液相色谱法(CEX-HPLC)检测抗体hu18电荷主峰含量。结果如表5所示。
根据稳定性考察结果，不同辅料的制剂处方样品在40℃高温条件下放置2周后，抗体均有较强的热稳定性。
综合各项数据分析，对比F13～F17与F18～F21发现，甘露醇、山梨醇、蔗糖和海藻糖与氯化钠的辅料组合优于甘露醇、山梨醇、蔗糖和海藻糖的单一辅料，表现在SEC-HPLC的单体含量与CEX-HPLC的主峰含量的降解速率更低。在含海藻糖辅料中，pH5.5的组氨酸缓冲体系条件下最为稳定，抗体单体纯度的下降速率低至0.11％/周，抗体主要电荷的下降速率仅为3.72％/周，明显低于pH6.0。
因此，选用pH5.5、海藻糖与氯化钠的辅料组合更有利于产品的稳定性。同时可以看到，对于SEC-HPLC的单体含量的降解，pH6.0的组氨酸缓冲体系(F17)明显优于pH6.0的柠檬酸缓冲体系(F23)。
表4：辅料筛选实验中的处方信息
Figure PCTCN2021072660-appb-000006
Figure PCTCN2021072660-appb-000007
注：“-”表示未添加。
表5：辅料筛选实验结果汇总
Figure PCTCN2021072660-appb-000008
Figure PCTCN2021072660-appb-000009
实施例3：表面活性剂筛选实验
液体制剂中添加表面活性剂常用于保护蛋白质例如抗体在储存过程中免受空气/溶液界面诱导的应力、溶液/表面诱导的应力的影响以减少抗体的聚集或使制剂中颗粒物的形成最小化的试剂，其利于抗体理化性质的稳定。在含有20mM组氨酸缓冲液(组氨酸与组氨酸盐酸盐的摩尔比为1:1，pH为6.0)和20mg/ml的抗体hu18的制剂中分别加入不同浓度的聚山梨酯20或聚山梨酯80，40℃放置4周后分析检测。结果如表6所示。
综合分析显示，表面活性剂筛选试验(F17、F24、F25和F26)结果显示添加不同浓度的聚山梨酯80或聚山梨酯20对SEC-HPLC的单体含量和CEX-HPLC的主峰含量影响不明显。
表6：表面活性剂筛选结果
Figure PCTCN2021072660-appb-000010
综上所述，我们通过对不同缓冲体系，不同pH条件、不同抗体浓度以及不同辅料和表面活性剂组成进行考察，探索研究重组人源化抗BTLA单克隆抗体抗体hu18的稳定性，并确定最佳液体制剂配方。抗体hu18选择组氨酸缓冲液来调节pH， 海藻糖和氯化钠来调节制剂渗透压，添加聚山梨酯80来增加制剂溶解性。
实施例4：制剂长期稳定性研究
含有治疗性抗体的液体药物制品通常需要储存在2-8℃条件下，所以制剂能在长期储存中保持高稳定性非常重要。根据上述的筛选结果，我们在前期26个处方基础上设计了27号处方进行制剂的长期稳定性研究。
选用4批次原液，配制如表7所示的27号处方制剂分别储存于透明小瓶中，放置2～8℃条件下6个月后，对各样品进行分析测试。通过以下参数评估稳定性：(a)目视外观；(b)光阻法检测不溶性微粒(OD405nm)；(c)pH；(d)CE-SDS(十二烷基硫酸钠毛细管电泳)法检测抗体的分子量；(d)SEC-HPLC测量抗体单体(质量标准：≥97.0％)、聚体(质量标准：≤3.0％)或片段的含量(质量标准：≤1.0％)；(e)CEX-HPLC测量抗体主要电荷(质量标准：≥70.0％)、酸性电荷(质量标准：≤30.0％)或碱性电荷含量(质量标准：≤15.0％)；(f)ELISA法检测抗体结合活性(质量标准：参比品的70％-130％)；(g)蛋白质含量(质量标准：18-22mg/ml)。
结果显示，4批次原液在27号制剂处方的外观、pH值、不溶性微粒、蛋白质含量、纯度(SEC-HPLC(分子排阻高效液相色谱法)、CEX-HPLC(弱阳离子交换高效液相色谱法)、R-CE-SDS(还原电泳法)、NR-CE-SDS(非还原电泳法)和生物学活性均无显著变化，具体见表8。结果表明，上述4批次原液在27号制剂处方保存在2-8℃条件下，0-24月储存过程具有非常好的稳定性。
表7：制剂长期稳定性研究的制剂处方
Figure PCTCN2021072660-appb-000011
注：组氨酸缓冲液由组氨酸与组氨酸盐酸盐的摩尔比为1:3配置。
表8：制剂处方的长期稳定性数据
Figure PCTCN2021072660-appb-000012
Figure PCTCN2021072660-appb-000013
Figure PCTCN2021072660-appb-000014
Figure PCTCN2021072660-appb-000015
Figure PCTCN2021072660-appb-000016
注：表格中NA表示在此时间点为非重点检项，在该取样点不做检测，NR表示未到该时间点，下同。
实施例5：制剂的加速稳定性研究
选用4批次原液，配制如表7所示的27号处方制剂分别储存于透明小瓶中，放置25±2℃，60±5％相对湿度(RH)条件下，0-12个月后，对各样品进行分析测试。
如表9所示，27号处方制剂对抗蛋白质降解具有更高稳定性，在25±2℃下测量的所得降解动力学参数满足室温环境储存长达12个月的要求。
表9：制剂处方的加速稳定性数据
Figure PCTCN2021072660-appb-000017
Figure PCTCN2021072660-appb-000018
Figure PCTCN2021072660-appb-000019
注：表格中NA表示在此时间点为非重点检项，在该取样点不做检测，NR表示未到该时间点。
实施例6：ELISA检测人源化抗体与hBTLA的结合
利用常规ELISA检测方法检测人源化抗体hu17，hu18和hu19与hBTLA结合特异性。用0.5μg/ml的hBTLA包被96孔酶标板，37摄氏度恒温孵育60分钟。然后弃去孔内溶液，用洗涤缓冲液洗3次，加入含有2％BSA的PBS溶液封闭60分 钟。用洗涤缓冲液洗3次后加入梯度稀释的抗体溶液(溶液中除抗体外的其它成分与处方编号27相同)，37摄氏度孵育60分钟后用洗涤缓冲液冲洗3次，然后加入1：10000倍稀释的HRP标记的小鼠抗人IgG4二抗，37摄氏度孵育1小时，经洗涤缓冲液冲洗三次后，加入100μl TMB底物溶液显色，室温反应30分钟后，以100μl 2M的盐酸溶液终止反应并在450nm处读出吸光度。
其EC 50值如下表10所示。结果显示，人源化抗体hu17，hu18和hu19可以与hBTLA特异性结合。
表10：人源化抗体组合hu17，hu18和hu19与hBTLA特异性结合数据
人源化抗体 hu17 hu18 hu19
EC 50(ng/mL) 6.3 5.4 6.6
实施例7：FACS检测人源化抗体与293F细胞上hBTLA的结合
采用基于细胞的流式细胞(FACS)测定人源化抗BTLA抗体hu17，hu18和hu19与细胞上表达的hBTLA结合能力。将表达hBTLA的293F细胞消化后离心重悬到FACS缓冲液中，按细胞量为2.5×10 4，体积为50ul加入到96孔圆底板孔中，加入50ul不同浓度的抗体稀释液(起始浓度10ug/ml，3倍滴定；稀释液中除抗体外的其它成分与处方编号27相同)混匀，室温孵育30min；用FACS缓冲液洗涤细胞两次后加入100ul山羊抗人IgG-PE抗体，避光孵育30min；用FACS buffer洗涤两次后进行FACS检测。将经洗涤的细胞重悬于含有碘化丙啶(PI)以及防止受体内化的0.02％叠氮化钠的4℃缓冲剂中后，通过流式细胞术分析。根据从FSC/SSC门排除PI阳性细胞，对活细胞进行门控，并测量其几何平均荧光。使用Prism TM软件内的S形剂量-响应模型进行分析数据。
各抗体的EC 50值如下表11所示。结果显示，人源化抗体hu17、hu18、hu19能与293F细胞上的hBTLA有效结合。
表11
  hu17 hu18 hu19
EC 50(ng/mL) 96.74 89.34 95.06
实施例8：FACS检测人源化抗体对阻断BTLA与HVEM结合的阻断作用
采用基于细胞的流式细胞(FACS)测定，检测人源化抗体hu17，hu18和hu19阻断hBTLA结合表达于293F细胞上hHVEM的能力。将hHVEM-293F稳定表达细胞消化后离心重悬到FACS缓冲液中，按细胞量为2.5×10 4个，体积为50ul加入到96孔圆底板孔中，加入预先生物素标记的hBTLA(1ug/ml)蛋白混合，4℃孵育15分钟；再加入50ul不同浓度的人源化抗体稀释液(起始5ug/ml，3倍滴定；稀释液中除抗体外的其它成分与处方编号27相同)混匀，室温孵育30min；用FACS缓冲液洗涤细胞两次后加入100ul山羊抗人IgG-PE抗体，避光孵育30min；用FACS缓冲液洗涤两次后进行FACS检测。将经洗涤的细胞重悬于含有碘化丙啶(PI)以及防止受体内化的0.02％叠氮化钠的4℃缓冲剂中后，通过流式细胞术分析。根据从FSC/SSC门排除PI阳性细胞，对活细胞进行门控，并测量其几何平均荧光。使用Prism TM软件内的S形剂量-响应模型进行分析数据。
各抗体的IC 50值如下表12所示。结果显示，人源化抗体hu17、hu18、hu19能有效阻断BTLA与细胞表面的HVEM结合。
表12
  hu17 hu18 hu19
IC 50(ng/mL) 142.7 172.2 161.5
实施例9：人源化抗BTLA抗体促进T细胞的激活
将稳定表达hPD-L1/hHVEM的CHO细胞铺到96孔板，每孔细胞量为5×10 4，37℃，7％CO 2培养过夜，去除细胞上清，每孔中加入40ul人源化抗BTLA抗体稀释液(起始浓度为60ug/ml，3倍浓度梯度稀释；稀释液中除抗体外的其它成分与处方编号27相同)，加入40ul可以持续表达hPD-1/hBTLA/NFAT-荧光素酶的Jurkat报告细胞，总细胞数为1×10 5细胞，37℃，7％CO 2培养6小时，加入荧光素酶试剂，酶标仪检测发光值。
各抗体的EC 50值如下表13所示。结果显示，人源化抗BTLA抗体hu17、hu18和hu19能有效促进T细胞活化。
表13
  hu17 hu18 hu19
EC 50(ng/mL) 256.7 277.3 290.4
实施例10：人源化抗BTLA抗体与hBTLA的亲和力
使用GE医疗生命科学公司的Biacore T200仪器进行检测实验，将Series S CM5芯片装载到仪器上，使用的***缓冲液为HBS-EP+(10mM HEPES,pH 7.4,150mM NaCl,3mM EDTA,0.05％表面活性剂P20)。进行BTLA-Fc抗原在芯片检测通道的偶联，用400mM EDC与100mM NHS的混合液以10μL/min注入420s活化芯片表面，将BTLA-Fc抗原稀释在10mM醋酸钠/醋酸(pH 5.5)缓冲液中至终浓度20μg/mL，并以10μL/min注入进行偶联，将1M乙醇胺-盐酸溶液(pH 8.5)以10μL/min注入420s进行封闭。
用Biacore***缓冲液将抗体进行2倍梯度稀释(稀释液中除抗体外的其它成分与处方编号27相同)，共6个浓度点。浓度梯度为24nM、12nM、6nM、3nM、1.5nM和0.75nM，其中24nM为重复测试。数据分析使用GE医疗生命科学公司的数据分析软件Biacore T200Evaluation Software版本号3.0。数据拟合使用模型为1:1Binding。拟合得到抗体与抗原间结合的动力学常数结合速率ka(1/Ms)，解离速率kd(1/s)，亲和力常数KD(M)，结果如表14所示。
表14：人源化抗体的亲和力数据
  ka(1/Ms) kd(1/s) KD(M)
hu17 6.24E+05 1.16E-04 1.86E-10
hu18 6.39E+05 8.67E-05 1.36E-10
实施例11：人源化抗体与不同种属BTLA结合动力学的表征
为了检测嵌合抗体与食蟹猴源和鼠源BTLA之间的交叉反应，使用Fortebio检定。简而言之，将人BTLA、食蟹猴BTLA或鼠BTLA偶联至已活化的CM5生物传感器芯片以实现大约100-200个响应单位(RU)，然后用IM乙醇胺封闭未反应 基团。以30次/分钟在SPR运行缓冲液中注射自0.12nM至90nM递增浓度的人源化抗体样品(样品中抗体为hu17，其它成分与处方编号27相同)，且藉由减去来自空白流动室的RU来计算在不同种源BTLA的结合响应。
结果如图1所示。hu17与不同种属BTLA结合对比结果显示：其不仅与人BTLA具有高亲和力，并且与食蟹猴源BTLA有相近的亲和力，但其与鼠BTLA基本不发生结合。
实施例12：人源化抗体(处方编号：27)对小鼠肿瘤生长的抑制作用
在小鼠结肠癌(MC38)细胞(ATCC)上通过电转Hxp-hHVEM质粒，构建MC38-hHVME细胞库，然后通过有限稀释法进行细胞亚克隆，通过流式筛选出单克隆，得到MC38-HVEM细胞；接着将MC38-hHVEM细胞以1×10 6个/0.1mL浓度接种于B-hBTLA人源化雌性小鼠的右侧皮下，待肿瘤生长到约118mm 3时按肿瘤体积随机分组，每组8只，共5组，分别为：G1 0.9％氯化钠注射液溶剂对照组、G2KLH(抗钥孔血蓝蛋白抗体)(10mg/kg)阴性对照组、G3hu18(1mg/kg)组、G4hu18(3mg/kg)组和G5hu18(10mg/kg)组。所有组给药途径均为腹腔注射，每周给药2次，连续给药7次，末次给药4天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，动物安乐死，剥取肿瘤称重、拍照，计算相对肿瘤抑制率(TGI％，TGI％＝(1-(T i-T 0)/(V i-V 0))×100％，T i治疗组给药结束时肿瘤体积，T 0治疗组开始给药时肿瘤体积，V i空白组给药结束时肿瘤体积，V 0空白组开始给药时肿瘤体积)。
各组受试品对MC38-hHVEM细胞移植B-hBTLA小鼠肿瘤体积的影响结果如表15和图2所示。首次给药后21天，KLH(10mg/kg)阴性对照组平均肿瘤体积为1560±256mm 3，其他给药组平均肿瘤体积分别为1073±224mm 3、747±268mm 3和868±211mm 3。各给药组与KLH阴性对照组相比，TGI％分别为33.7％、56.4％和48.0％，P值分别为0.175、0.046和0.056，表明受试药物hu18对肿瘤生长有一定抑制作用。
表15：hu18对MC38-hHVEM细胞移植B-hBTLA小鼠肿瘤体积的影响
Figure PCTCN2021072660-appb-000020
Figure PCTCN2021072660-appb-000021
注：a：平均数±标准误；b：给药组肿瘤体积与KLH阴性对照组肿瘤体积在给药21天后统计学比较，t-test。
各组受试品对MC38-hHVEM细胞移植B-hBTLA小鼠体重的影响结果如表16和图3所示。所有实验动物在给药期间活动和进食状态良好，各给药组动物体重均有一定程度的上升。实验期间未出现实验动物死亡情况。给药后21天，与KLH阴性对照组小鼠体重相比，各给药组小鼠体重无显著变化(P＞0.05)，表明实验动物对受试品耐受性良好。
表16：受试品对MC38-hHVEM细胞移植B-hBTLA小鼠体重的影响
Figure PCTCN2021072660-appb-000022
注：a：平均数±标准误；b：给药组体重与KLH阴性对照组体重在给药21天后统计学比较，t-test。
实施例13：人源化抗体hu18(处方编号：27)没有ADCC效应功能
当抗体结合至细胞表面靶蛋白质，然后连接至效应细胞上所表达的Fcγ受体(FcγR)时，启动ADCC。很清楚地记载了人类IgG1对FcγR具有比IgG4明显更高的结合亲和力，尤其是结合FcγR-I及FcγR-IIIA，该亲和力与IgG1激活ADCC的强度相关联。联想到ADCC，当抗体交联细胞表面靶点及C1q蛋白质，继之以补体复合物形成及靶细胞裂解的级联反应时，激活CDC。作为ADCC和CDC的代理，抗体结合至FcγR及C1q的检测就可充当ADCC及CDC的基本指标。因此，本发明利用biacore T200(GE)评估了单抗对主要FcγR结合的动力学亲和力。
GE的抗-His抗体固定在传感器晶片上。各种Fc受体，包括重组人FcγRIIIA(CD16a)V176，重组人FcγRIIA(CD32a)R167，重组人FcγRI(CD64)和重组人FcRn被捕获，然后注射一系列稀释的重组人抗-BTLA抗体(即hu18)，检测并分析相互作用的结合性质。hu18是IgG4亚型抗体，hu18-IgG1是IgG1亚型，其与hu18共享相同的Fab，并作为阳性对照。
结果如表17所示，相比于IgG1亚型的对照抗体，IgG4亚型的重组人抗-BTLA抗体与Fc受体的结合相对较弱，hu18抗体与IgG1亚型对照抗体相比，其与FcγRIIIA(CD16a)V176的相互作用弱400倍。这显示hu18具有较弱或无ADCC效应。如表17所示，hu18抗体不结合C1q，而hu18-IgG1抗体能够结合C1q。
表17：抗-BTLA抗体与Fc受体之间相互作用的结合亲和性
Figure PCTCN2021072660-appb-000023
表18：抗-BTLA抗体与人C1q之间相互作用的结合亲和力
Figure PCTCN2021072660-appb-000024

Claims (13)

  1. 一种药物组合物，包含：
    (1)缓冲液；和
    (2)抗BTLA抗体或其抗原结合片段，其中所述抗BTLA抗体或其抗原结合片段具有氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
  2. 如权利要求1所述的药物组合物，其中所述缓冲液选自醋酸缓冲液、柠檬酸缓冲液和组氨酸缓冲液；优选地，所述缓冲液为组氨酸缓冲液；更优选地，所述缓冲液为组氨酸-盐酸盐缓冲液，更优选为组氨酸-组氨酸盐酸盐缓冲液。
  3. 如权利要求2所述的药物组合物，其中所述缓冲液的浓度约为10～30mM；优选地，所述缓冲液的pH约为5.0-6.5，优选约为5.0-6.0。
  4. 如权利要求1-3任一项所述的药物组合物，其中所述药物组合物还包括稳定剂，所述稳定剂选自氯化钠、甘露醇、山梨醇、蔗糖、麦芽糖、木糖醇和海藻糖中的一种或两种以上；优选地，所述稳定剂为海藻糖和氯化钠的组合。
  5. 如权利要求4所述的药物组合物，其中所述稳定剂为浓度约50-200mM的氯化钠；或所述稳定剂为浓度约100-300mM的甘露醇；或所述稳定剂为浓度约100-300mM的山梨醇；或所述稳定剂为浓度约100-300mM的蔗糖；或所述稳定剂为浓度约100-300mM的海藻糖；或所述稳定剂为约30-100mM的氯化钠与约50-200mM的甘露醇的组合；或所述稳定剂为约30-100mM的氯化钠与约50-200mM的山梨醇的组合；或所述稳定剂为约30-100mM的氯化钠与约50-200mM的蔗糖的组合；或所述稳定剂为约30-100mM的氯化钠与约50-200mM的海藻糖的组合；优选地，所述稳定剂为约30-100mM的氯化钠与约50-200mM的海藻糖的组合。
  6. 如权利要求1-5任一项所述的药物组合物，其中所述药物组合物还包括表面活性剂，所述表面活性剂选自聚山梨醇酯80、聚山梨醇酯20或泊洛沙姆188，优选地，所述表面活性剂为聚山梨醇酯80。
  7. 如权利要求6所述的药物组合物，其中所述表面活性剂浓度约为0.01％-0.1％；优选地，所述表面活性剂浓度约为0.01％-0.05％。
  8. 如权利要求1-7任一项所述的药物组合物，其中所述抗BTLA抗体具有如氨基酸序列SEQ ID NO:7所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区；或具有氨基酸序列如SEQ ID NO:7所示的重链可变区，和氨基酸序列如SEQ ID NO:9所示的轻链可变区；或具有氨基酸序列如SEQ ID NO:7所示的重链可变区，和氨基酸序列如SEQ ID NO:10所示的轻链可变区。
  9. 如权利要求1-7任一项所述的药物组合物，其中所述抗BTLA抗体具有如SEQ ID NO:11所示的重链氨基酸序列和如SEQ ID NO:12所示的轻链氨基酸序列。
  10. 如权利要求1-9任一项所述的药物组合物，其中所述抗BTLA抗体或其抗原结合片段浓度约为5-100mg/mL，优选约为10-50mg/mL，更优选15-25mg/mL。
  11. 如权利要求1-9任一项所述的药物组合物，其包含如下(1)-(13)任一项所示的组分，或由如下(1)-(13)任一项所示的组分组成：
    (1)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM组氨酸缓冲液，pH约为5.0-6.0；(c)约30mM至约100mM的氯化钠与约50-200mM的海藻糖的组合；(d)以及约0.01％-0.05％的聚山梨醇酯80；
    (2)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM组氨酸缓冲液，pH约为5.0-6.0；(c)约30mM至约100mM的氯化钠与约50-200mM的甘露醇的组合；(d)以及约0.01％-0.05％的聚山梨醇酯80；
    (3)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM组氨酸缓冲液，pH约为5.0-6.0；(c)约30mM至约100mM的氯化钠与约50-200mM的蔗糖的组合；(d)以及约0.01％-0.05％的聚山梨醇酯80；
    (4)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM组氨酸缓冲液，pH约为5.0-6.0；(c)约30mM至约100mM的氯化钠与约50-200mM的山梨醇的组合；(d)以及约0.01％-0.05％的聚山梨醇酯80；
    (5)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM组氨酸缓冲液，pH约为5.0-6.0；(c)约100mM至约300mM的海藻糖；(d)以及约0.01％-0.05％的聚山梨醇酯80；
    (6)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM柠檬酸缓冲液，pH约为5.5-6.5；(c)约100mM至约300mM的海藻糖；(d)以及约0.01％-0.05％的聚山梨醇酯80；
    (7)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM柠檬酸缓冲液，pH约为5.5-6.5；(c)约30mM至约100mM的氯化钠与约50-200mM的甘露醇的组合；(d)以及约0.01％-0.05％的聚山梨醇酯80；
    (8)(a)约10mg/mL-50mg/mL的抗BTLA抗体或其抗原结合片段；(b)约10-30mM醋酸缓冲液，pH约为5.0-6.0；(c)约30mM至约100mM的氯化钠与约50-200mM的甘露醇的组合；(d)以及约0.01％-0.05％的聚山梨醇酯80；
    (9)(a)约20mg/mL的抗BTLA抗体或其抗原结合片段；(b)约20mM组氨酸缓冲液，pH约为6.0；(c)约54mM的氯化钠与约132mM的海藻糖的组合；(d)以及约0.02％的聚山梨醇酯80；
    (10)(a)约20mg/mL的抗BTLA抗体或其抗原结合片段；(b)约20mM组氨酸缓冲液，pH约为5.5；(c)约220mM的海藻糖的组合；(d)以及约0.02％的聚山梨醇酯80；
    (11)(a)约20mg/mL的抗BTLA抗体或其抗原结合片段；(b)约20mM组氨酸缓冲液，pH约为5.5；(c)约50mM的氯化钠与约140mM的甘露醇的组合；(d)以及约0.02％的聚山梨醇酯80；
    (12)(a)约20mg/mL的抗BTLA抗体或其抗原结合片段；(b)约20mM组氨酸缓冲液，pH约为5.5；(c)约50mM的氯化钠与约140mM的海藻糖的组合；(d)以及约0.02％的聚山梨醇酯80；和
    (13)(a)约20mg/mL的抗BTLA抗体或其抗原结合片段；(b)约20mM组氨酸缓冲液，pH约为5.0-6.0；(c)约50mM的氯化钠与约140mM的海藻糖的组合；(d)以及约0.02％的聚山梨醇酯80。
  12. 如权利要求1所述的药物组合物，其特征在于，所述药物组合物含有以下组分，或由以下组分组成：pH为5.5±0.3的组氨酸-组氨酸盐酸盐缓冲液，其浓度为20±5mM；40-80mM的氯化钠；120-150mM的海藻糖；以w/v计0.02-0.04％的聚山梨酯80；15-25mg/mL的抗BTLA抗体或其抗原结合片段，其中，所述抗BTLA抗体具有如SEQ ID NO:11所示的重链氨基酸序列和如SEQ ID NO:12所示的轻链氨基酸序列。
  13. 如权利要求1-12任一项所述的药物组合物在制备治疗或预防BTLA介导的疾病的药物中的用途；优选地，所述疾病为肿瘤、感染性疾病或自身免疫性疾病； 优选地，所述肿瘤包括黑色素瘤、乳腺癌、肾癌、***癌、结肠癌、肺癌、胰腺癌、骨癌、皮肤癌、头或颈癌、子宫癌、卵巢癌、直肠癌、肛区癌、胃癌、睾丸癌、食道癌、小肠癌、***、***癌、何杰金氏病、非何杰金氏淋巴瘤、食道癌、内分泌***癌、甲状腺癌、肾上腺爱、软组织癌、尿道癌、慢性或急性白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病、儿童期实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经***的赘生物、原发性中枢神经***淋巴瘤、肿瘤血管发生、脊髓轴肿瘤、脑干胶质瘤、垂体腺瘤、卡波西氏肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症；所述自身免疫性疾病包括器官特异性自身免疫病和***性自身免疫病，其中，所述器官特异性自身免疫病包括慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症和急性特发性多神经炎，所述***性自身免疫病包括***性红斑狼疮、类分湿关节炎、***性血管炎、硬皮病、天胞疮、皮肌炎、混合性***病、自身免疫性溶血性贫血和溃疡性结肠炎。
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