WO2023134771A1 - Pharmaceutical composition of anti-ctla-4 antibody and use thereof - Google Patents

Pharmaceutical composition of anti-ctla-4 antibody and use thereof Download PDF

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WO2023134771A1
WO2023134771A1 PCT/CN2023/072509 CN2023072509W WO2023134771A1 WO 2023134771 A1 WO2023134771 A1 WO 2023134771A1 CN 2023072509 W CN2023072509 W CN 2023072509W WO 2023134771 A1 WO2023134771 A1 WO 2023134771A1
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seq
antibody
amino acid
ctla
acid sequence
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PCT/CN2023/072509
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French (fr)
Chinese (zh)
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刘沛想
刘洪川
李宝贤
刘辉
姚盛
冯辉
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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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Publication of WO2023134771A1 publication Critical patent/WO2023134771A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—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

Definitions

  • the invention relates to the field of therapeutic pharmaceutical compositions, in particular to anti-CTLA-4 antibody pharmaceutical compositions and uses thereof.
  • Cytotoxic T lymphocyte-associated protein 4 (CTLA4 or CTLA-4, cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a transmembrane protein encoded by the CTLA-4 gene. Located on human chromosome 2q33.
  • CTLA-4 is a member of the immunoglobulin superfamily and consists of an extracellular V domain, a transmembrane domain and a cytoplasmic domain.
  • CTLA-4 has homology with the co-stimulatory molecule receptor CD28 on the surface of T cells, and the two compete for the binding of its ligands B7-1 (CD80) and B7-2 (CD86), which are mainly expressed in antigen presentation cell surface.
  • CTLA-4 binds CD80 and CD86 with a higher affinity than CD28, thus competing and blocking CD28-mediated activation.
  • CTLA-4 is normally expressed on the surface of regulatory T cells (Treg) and conventional T cells in the activated state. After it binds to B7 molecules, it inhibits the activation of T cells, participates in the negative regulation of the immune response, acts as an immune checkpoint and down-regulates the immune response, so CTLA-4 plays a very important role in immune regulation.
  • Treg regulatory T cells
  • T cells require the stimulation of two signals, the first signal comes from the T cell receptor (TCR) that specifically binds the antigen peptide-MHC complex on the surface of the antigen presenting cell (APC), and the second signaling pathway requires co-stimulation
  • TCR T cell receptor
  • APC antigen presenting cell
  • CD28 T cell receptor
  • B7-1/B7-2 CD80/CD86
  • CTLA-4 down-regulates the function of T cells through the following pathways: First, CTLA-4 can competitively block the co-expression of CD28 and CD80/86 through its high affinity with CD80/CD86; Stimulate the transmission of signals, thereby inhibiting the proliferation of T cells and reducing the secretion of IL-2. Second, CTLA-4 can reduce the expression level of CD80/CD86 on antigen-presenting cells (APCs) or remove CD80/CD86 molecules from the surface of antigen-presenting cells (APCs) through trans-endocytosis, thereby reducing CD28 involved in T cell activation.
  • APCs antigen-presenting cells
  • APCs antigen-presenting cells
  • CTLA-4 can inhibit TCR signaling by mediating dendritic cells binding to CD80/CD86 and inducing the expression of tryptophan-degrading enzyme IDO.
  • CTLA-4 can also induce the production of regulatory cytokines by recruiting inhibitory molecules to the immune synapse, thereby inhibiting the transmission of APC and TCR signals.
  • CTLA-4 Blockade of CTLA-4 has been demonstrated in many studies to induce tumor regression.
  • the anti-CTLA-4 antibody can effectively and specifically inhibit cellular and humoral immune responses in vivo and in vitro, and has a significant therapeutic effect on transplant rejection and various autoimmune diseases, with low toxic and side effects.
  • CTLA-4 monoclonal antibody drugs Ipilimumab (Bristol-Myers Squibb) and Tremelimumab (AstraZeneca) are currently used for some cancer treatments and are being tested for other anti-cancer indications, there is still a need to include activities in all aspects compared to Novel anti-CTLA-4 antibodies with known antibody improvements.
  • antibody drugs have a large molecular weight and complex structure, and are prone to degradation, polymerization, or undesired chemical modification and become unstable.
  • the pharmaceutical composition of the present invention is a highly stable pharmaceutical composition containing an antibody specifically binding to CTLA-4 or an antigen-binding fragment thereof.
  • the antibody preparations developed by the present invention can be used for intravenous injection, and each component interacts and cooperates synergistically, which is an anti-CTLA-4 antibody or Its antigen-binding fragment provides a storage environment suitable for long-term storage, which can avoid antibody degradation, aggregation or precipitation caused by preparations during long-term storage or transportation.
  • Antibodies can be stored in this environment for a long time, and antibody activity and purity can be guaranteed during storage. Maintaining stability ensures the efficacy of biological drugs.
  • the present invention provides a pharmaceutical composition, comprising: (1) a buffer; (2) an anti-CTLA-4 antibody or an antigen-binding fragment thereof.
  • the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 comprised by the anti-CTLA-4 antibody or antigen-binding fragment thereof are respectively:
  • LCDR1 RASQNVGTYVA (SEQ ID NO: 1);
  • LCDR2 STSYRYS (SEQ ID NO: 2);
  • LCDR3 HQYDTYPLT (SEQ ID NO: 3);
  • HCDR1 SGYYWN (SEQ ID NO: 4);
  • HCDR2 YIGYDGSNX1YNPSLKX2, where X1 is N or Y, X2 is S or N;
  • HCDR3 X3YYSGYFDS, X3 is D or N.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof described above comprises:
  • amino acid sequence is LCDR1 shown in SEQ ID NO:1;
  • amino acid sequence is LCDR2 shown in SEQ ID NO:2;
  • amino acid sequence is LCDR3 shown in SEQ ID NO:3;
  • amino acid sequence is HCDR1 as shown in SEQ ID NO:4;
  • amino acid sequence is HCDR2 as shown in SEQ ID NO:5 or 7 or 9;
  • amino acid sequence is HCDR3 as shown in SEQ ID NO:6 or 8.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof described above comprises:
  • Amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, and amino acid sequences such as SEQ ID NO:4, SEQ ID NO:5 and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 6; or
  • Amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and amino acid sequences such as SEQ ID NO:4, SEQ ID NO:9 and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:6.
  • the above-mentioned anti-CTLA-4 antibody or antigen-binding fragment thereof is selected from murine antibody or antigen-binding fragment thereof, chimeric antibody or antigen-binding fragment thereof, humanized antibody or antigen-binding fragment thereof, preferably humanized Antibodies or antigen-binding fragments thereof.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof described above comprises:
  • the anti-CTLA-4 antibody described above comprises:
  • Amino acid sequence such as the light chain amino acid sequence shown in SEQ ID NO:22, and the amino acid sequence such as the heavy chain amino acid sequence shown in SEQ ID NO:23.
  • the concentration of the anti-CTLA-4 antibody or its antigen-binding fragment in the above pharmaceutical composition is about 1-100 mg/mL, preferably about 1-50 mg/mL, more preferably about 5-30 mg/mL; more preferably Preferably, the above-mentioned anti-CTLA-4 antibody or its antigen-binding fragment concentration is about 1 mg/mL, 5 mg/mL, 7 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg /mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL or any two values in these ranges as the range formed by the endpoint, preferably About 9mg/mL, 10mg/mL, 11mg/mL or 15mg/mL.
  • the pH of the above-mentioned pharmaceutical composition is about 4.5-6.5, preferably about 5.0-6.0, more preferably about 5.3-5.7, the non-limiting examples of the pH of the above-mentioned pharmaceutical composition are about 4.5, 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 or any two of these ranges as endpoints, preferably about 5.4, 5.5 or 5.6 .
  • the osmolarity of the above pharmaceutical composition is about 250-350 mOsm/kg.
  • the buffer is selected from one or more of acetate buffer, citrate buffer, phosphate buffer and histidine buffer; preferably, the buffer is acetate buffer.
  • the above-mentioned buffer is an acetate buffer, preferably, the acetate buffer is an acetate-sodium acetate buffer or an acetate-potassium acetate buffer, preferably an acetate-sodium acetate buffer.
  • the acetate buffer is an acetate-sodium acetate buffer.
  • the acetic acid-sodium acetate buffer is made of about 1-20 mM acetic acid and about 1-20 mM sodium acetate.
  • the acetic acid-sodium acetate buffer is made of acetic acid and sodium acetate in a molar ratio of about 1:2 to about 1:3.
  • the acetic acid-sodium acetate buffer is made of acetic acid and sodium acetate in a molar ratio of about 1:5 to about 1:8.
  • the acetic acid-sodium acetate buffer is: a pH of about 6.5 mM acetic acid and about 13.5 mM sodium acetate 5.0 in acetate buffer.
  • the acetic acid-sodium acetate buffer is: an acetate buffer with a pH of about 5.0 to 6.0 made from about 2 to 4 mM acetic acid and about 16 to 18 mM sodium acetate; preferably about 3 mM acetic acid and about 17 mM sodium acetate to make an acetate buffer with a pH of about 5.5.
  • the above-mentioned histidine buffer is selected from histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, preferably histidine-histidine hydrochloride buffer.
  • the above-mentioned histidine-histidine hydrochloride buffer is made of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride.
  • the histidine buffer is made of about 1-20 mM L-histidine and about 1-20 mM L-histidine monohydrochloride.
  • the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of about 1:1 to about 1:4. In some protocols, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of about 1:1. In some protocols, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of about 1:3. In some aspects, the histidine formulation is a histidine buffer at a pH of about 5.5 made from about 4.5 mM L-histidine and about 15.5 mM L-histidine monohydrochloride. In some aspects, the histidine formulation is a histidine buffer at a pH of about 6.0 made from about 10 mM histidine and about 10 mM histidine hydrochloride.
  • the above-mentioned buffer is a citric acid buffer, preferably, the citric acid buffer is a citric acid-sodium citrate buffer.
  • the citrate buffer is made from about 1-20 mM citric acid and about 1-20 mM sodium citrate.
  • the citrate buffer is made of citric acid and sodium citrate in a molar ratio of about 1:1 to 1:4.
  • the citrate buffer is: a citrate buffer having a pH of about 6.5 made from about 5.0 mM citric acid and about 15.0 mM sodium citrate.
  • the citrate buffer is: a citrate buffer having a pH of about 6.0 made from about 10 mM citric acid and about 10 mM sodium citrate.
  • the above-mentioned buffer is a phosphate buffer, preferably, the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.
  • the phosphate buffer is made from about 1-20 mM disodium phosphate and about 1-20 mM sodium phosphate.
  • the phosphate buffer is made of disodium hydrogen phosphate and monobasic sodium phosphate in a molar ratio of about 1:1 to 1:4.
  • the phosphate buffer is: a phosphate buffer having a pH of about 7.0 made from about 10 mM disodium hydrogen phosphate and about 10 mM monobasic sodium phosphate.
  • the concentration of the above-mentioned buffer solution is about 5-100 mM, preferably about 10-50 mM, preferably about 10-30 mM; preferably about 15-25 mM, and the non-limiting example of the above-mentioned buffer solution concentration is about 10 mM, 15mM, 20mM, 25mM, 30mM, 40mM, 45mM, 50mM or any two values within these ranges as the range formed by the endpoints, preferably about 15mM, 20mM or 25mM.
  • the pH of the above-mentioned buffer is about 4.5-6.5, preferably about 5.0-6.0, more preferably about 5.3-5.7, and the non-limiting examples of the pH of the above-mentioned buffer are 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 or any two values in these ranges as the range formed by the endpoints, preferably about 5.4, 5.5 or 5.6.
  • the above-mentioned pharmaceutical composition also includes a stabilizer, and the stabilizer is selected from one or more of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose and trehalose species; preferably, the above-mentioned arginine salt is arginine hydrochloride.
  • the concentration of the above-mentioned stabilizer is about 10-400mM, preferably about 100-300mM, preferably about 130-280mM, preferably about 200-260mM, and the non-limiting example of the above-mentioned stabilizer concentration is about 130mM, 135mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 228mM, 230mM, 240mM, 250mM, 260mM or any two values in these ranges as the range formed by the endpoint, preferably about 210mM, 220mM, 228mM or 230mM.
  • the above-mentioned stabilizer is mannitol with a concentration of about 130-280mM; or the above-mentioned stabilizer is sucrose with a concentration of about 130-280mM; or the above-mentioned stabilizer is sodium chloride with a concentration of about 20-80mM A combination of 170mM mannitol; or the above-mentioned stabilizer is a combination of sodium chloride at a concentration of about 20-80mM and sucrose at a concentration of about 110-170mM; in some embodiments, the above-mentioned stabilizer is mannitol at a concentration of about 200-260mM; Or the above-mentioned stabilizer is sucrose with a concentration of about 200-260mM; or the above-mentioned stabilizer is a combination of sodium chloride with a concentration of about 30-70mM and mannitol with a concentration of about 120-160mM; or the above-mentioned stabilizer is about 30-70
  • the aforementioned stabilizer is mannitol.
  • the above-mentioned stabilizer is mannitol at a concentration of about 100-300 mM
  • the concentration of the above-mentioned mannitol is preferably about 130-280 mM, preferably about 200-260 mM
  • the non-limiting example of the above-mentioned mannitol concentration is about 200 mM , 210mM, 220mM, 230mM, 240mM, 250mM, 260mM or any two values within these ranges as the range formed by the endpoints, preferably about 240mM.
  • the aforementioned stabilizer is sucrose.
  • the above-mentioned stabilizer is sucrose with a concentration of about 100-300mM
  • the concentration of the above-mentioned sucrose is preferably about 130-280mM, preferably about 200-260mM
  • the non-limiting examples of the above-mentioned sucrose concentration are about 200mM, 210mM, 220mM, 228mM, 230mM, 240mM, 250mM or any two values in these ranges as the range formed by the endpoint, preferably about 220mM or about 228mM.
  • the aforementioned stabilizer is a combination of sodium chloride and mannitol.
  • the aforementioned stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM mannitol, preferably about 20-80 mM sodium chloride and about 110-170 mM mannitol, preferably about A combination of 30-70 mM sodium chloride and about 120-160 mM mannitol, a non-limiting example of the above stabilizer is a combination of about 50 mM sodium chloride and about 140 mM mannitol, or about 50 mM sodium chloride Combination with about 150mM mannitol.
  • the aforementioned stabilizer is a combination of sodium chloride and sucrose.
  • the above-mentioned stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose, preferably about 20-80 mM sodium chloride and about 110-170 mM sucrose, preferably about 30-200 mM
  • the combination of sodium chloride of 70mM and the sucrose of about 120 ⁇ 160mM, the non-limiting example of above-mentioned stabilizer is the combination of the sodium chloride of about 50mM and the sucrose of about 140mM, or the sodium chloride of about 50mM and the sucrose of about 160mM Combination of sucrose.
  • the above pharmaceutical composition further includes a surfactant, and the surfactant is selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188.
  • the aforementioned surfactant is selected from polysorbate 80.
  • the aforementioned surfactant is selected from polysorbate 20.
  • the above-mentioned surfactant concentration is about 0.001%-0.1%, preferably about 0.01%-0.1%, preferably about 0.01%-0.05%; as a non-limiting example, the above-mentioned The concentration of the surfactant is about 0.01%, 0.02%, 0.03%, 0.04%, 0.08%, or any two values within these ranges as endpoints forming a range, preferably about 0.02%.
  • the pharmaceutical composition comprises the components shown in any one of (1) to (13) below, or consists of the components shown in any one of (1) to (13):
  • an anti-CTLA-4 antibody of about 10 mg/mL said anti-CTLA-4 antibody comprising a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain as shown in SEQ ID NO: 17 the amino acid sequence;
  • an anti-CTLA-4 antibody of about 10 mg/mL said anti-CTLA-4 antibody comprising the light chain amino acid sequence shown in SEQ ID NO: 16 and the heavy chain shown in SEQ ID NO: 17 the amino acid sequence; (b) about 20 mM acetate buffer, pH about 5.5; (c) about 220 mM sucrose; and (d) about 0.02% (w/v) polysorbate 80; or
  • an anti-CTLA-4 antibody of about 10 mg/mL said anti-CTLA-4 antibody comprising a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain as shown in SEQ ID NO: 17 Amino acid sequence; (b) about 20 mM acetate buffer, pH about 5.5; (c) about 228 mM sucrose; and (d) about 0.02% (w/v) polysorbate 80; or
  • an anti-CTLA-4 antibody of about 10 mg/mL said anti-CTLA-4 antibody comprising a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain as shown in SEQ ID NO: 17 Amino acid sequence;
  • an anti-CTLA-4 antibody of about 10 mg/mL said anti-CTLA-4 antibody comprising a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain as shown in SEQ ID NO: 17 Amino acid sequence;
  • the pharmaceutical composition described in any of the aspects herein is a liquid formulation or a lyophilized formulation.
  • the pharmaceutical composition described above is a liquid formulation.
  • the above 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.
  • liquid formulation or lyophilized formulation described above is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.
  • the present invention also provides an injection, which contains the pharmaceutical composition described in any scheme herein and sodium chloride solution or glucose solution; preferably, the concentration of the sodium chloride solution is about 0.85-0.9% (w /v); Preferably, the concentration of the glucose solution is about 5-25% (w/v), more preferably about 5-10% (w/v); Preferably, in the injection, the anti-CTLA
  • the concentration of the -4 antibody is about 0.05-10.5 mg/mL, more preferably about 0.1-5 mg/mL or about 1-5 mg/mL; the pH of the injection is about 4.5-6.5, preferably about 5.0-6.0, more preferably Preferably it is about 5.3 to 5.7.
  • the above-mentioned pharmaceutical composition or injection is administered through intravenous injection.
  • the present invention also provides the use of the pharmaceutical composition or injection described in any of the schemes herein in the preparation of medicines for treating and/or preventing CTLA-4-mediated diseases or disorders.
  • the present invention also provides the pharmaceutical composition or injection described in any one of the schemes herein, which is used for treating and/or preventing CTLA-4-mediated diseases or conditions.
  • the present invention also provides a method for treating and/or preventing CTLA-4-mediated diseases or conditions, which comprises administering the pharmaceutical composition or injection as described in any one of the regimens herein to a subject in need.
  • the aforementioned disease or condition is cancer.
  • Figure 1 The results of the affinity test between the anti-CTLA-4 pharmaceutical composition and the recombinant human CTLA-4 protein.
  • Figure 2 Test results of the inhibitory effect of the anti-CTLA-4 pharmaceutical composition on the growth of EMT6 tumor transplanted in hCTLA4 humanized mice.
  • Figure 3 Detection of the binding of humanized anti-CTLA-4 antibody to huCTLA-4 by ELISA.
  • Figure 4 ELISA method to detect the ability of humanized anti-CTLA-4 antibody to block the binding of huCTLA-4 to CD80.
  • Figure 5 Detection of biological activity of human anti-CTLA-4ylation antibody by luciferase method.
  • Figure 6 ADCC activity of humanized anti-CTLA-4 antibodies.
  • Figure 7 CDC activity of humanized anti-CTLA-4 antibodies.
  • Figure 8 Inhibition of tumor growth in mice by humanized anti-CTLA-4 antibodies.
  • Figure 9 Fortebio binding assay identifies antigenic epitopes.
  • Figure 10 Inhibitory effect of huJS007-47 on the growth of MC38 tumors transplanted in hCTLA4 humanized mice.
  • Figure 11 Inhibitory effect of huJS007-47 on the growth of H22 tumor transplanted in hCTLA4 humanized mice.
  • pharmaceutical composition means a mixture comprising one or more of the antibodies described herein and other components, such as physiologically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
  • liquid formulation refers to a formulation in a liquid state and is not intended to refer to a resuspended lyophilized formulation.
  • the liquid formulations of the present invention are stable on storage and are not dependent on lyophilization (or other state changing methods such as spray drying) for their stability.
  • aqueous liquid formulation refers to a liquid formulation using water as a solvent.
  • aqueous liquid formulations are formulations that do not require lyophilization, spray drying, and/or freezing to maintain stability (eg, chemical and/or physical stability and/or biological activity).
  • excipient refers to an agent that can be added to a formulation to provide desired properties (eg, consistency, increased stability) and/or to adjust osmotic pressure.
  • excipients include, but are not limited to, carbohydrates, polyols, amino acids, surfactants, and polymers.
  • buffer pH of about 4.5 to 6.5 refers to an agent that renders a solution containing the agent resistant to pH changes through the action of its acid/base conjugate component.
  • Buffers used in formulations of the 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 “buffers” for controlling the pH within this range include acetic acid, acetate salts (such as sodium acetate), succinic acid, succinates (such as sodium succinate), gluconic acid, histidine, histidine Amine salts (e.g., histidine hydrochloride), methionine, citric acid (citric acid), citrate (citrate), phosphate, citrate/phosphate, imidazole, combinations thereof and other organic acid buffers.
  • a “histidine buffer” is a buffer comprising histidine ions.
  • histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate, etc., such as histidine containing A histidine buffer of acid and histidine hydrochloride; the histidine buffer of the present invention also includes a histidine buffer containing histidine and acetate (eg sodium or potassium salt).
  • citrate buffer also known as “citrate buffer” is a buffer comprising citrate ions.
  • citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like.
  • a preferred citrate buffer is 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.
  • a preferred acetate buffer is acetic acid-sodium acetate buffer.
  • succinate buffer is a buffer that includes succinate ions.
  • succinate buffers include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate, succinate-magnesium succinate, and the like.
  • a preferred succinate buffer is succinic acid-sodium succinate buffer.
  • Phosphate buffer is a buffer that includes phosphate ions.
  • examples of the phosphate buffer include sodium dihydrogenphosphate-disodium hydrogenphosphate, potassium dihydrogenphosphate-dipotassium hydrogenphosphate, and the like.
  • a preferred phosphate buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.
  • stabilizer denotes a pharmaceutically acceptable excipient which 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 or Salts (such as arginine hydrochloride), glycine, alanine (alpha-alanine, beta-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline N-oxidation of acid, 4-hydroxyproline, sarcosine, gamma-aminobutyric acid (GABA), opines, alanine, octopine, glycine, and trimethylamine (TMAO), human serum albumin (hsa), bovine serum albumin (BSA),
  • 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 more of polyols, amino acids, salts, and sugars.
  • Preferred salts are sodium chloride
  • preferred sugars are sucrose and trehalose
  • preferred polyols are sorbitol and mannitol.
  • Preferred amino acids are arginine, glycine, proline
  • amino acids can exist in their D- and/or L-forms, but are typically L-forms
  • amino acids can Any suitable salt is present, for example a hydrochloride such as arginine hydrochloride.
  • Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose , Sodium chloride-trehalose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose.
  • surfactant generally includes agents that protect proteins, such as antibodies, from air/solution interface-induced stress, solution/surface-induced stress, to reduce aggregation of antibodies, or to 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 ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene- Polyoxypropylene copolymer (poloxamer, Pluronic), sodium dodecyl sulfate (SDS).
  • nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-poly
  • the terms “the concentration of polysorbate 20" and “the concentration of polysorbate 80” all refer to the mass volume concentration (w/v), such as “about 0.02% polysorbate 80 "0.02%” means "100mL liquid contains 0.02g polysorbate 80".
  • isotonic means that the formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using vapor pressure or freezing point depression osmometers.
  • stable formulation is one in which the antibody substantially retains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage. Pharmaceutical formulations may be stable even if the contained antibodies fail to retain 100% of their chemical structure or biological function after storage over a period of time. In certain instances, maintaining about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the structure or function of an antibody after storage over a period of time may also be considered a " stable”.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery 247-301, edited by Vincent Lee, Marcel Dekker, Inc ., New York, N.Y., Pubs. (1991), and Jones, A. (1993) Adv. Drug Delivery Rev. 10:29-90 (both incorporated by reference).
  • Stability can be measured by determining the percentage of natural antibody remaining (among other methods) in a formulation after storage at a temperature for a period of time.
  • the percentage of native antibodies can be measured by size exclusion chromatography (eg, size exclusion high performance liquid chromatography [SEC-HPLC]), "native" meaning unaggregated and undegraded, among other methods.
  • SEC-HPLC size exclusion high performance liquid chromatography
  • the stability of a protein is determined as the percentage of monomeric protein in solution with a low percentage of degraded (eg, fragmented) and/or aggregated protein.
  • the formulation is stable 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 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, up to about 6%, 5%, 4%, 3%, 2%, 1%, 0.5% , or 0.1% aggregated form of the antibody.
  • Stability can be measured by determining (among other methods) the percentage of antibody (“acidic form") that migrates in the more acidic fraction of the antibody's main fraction ("primary charged form") during ion exchange, where stability is related to The percentage of acidic form of antibody is inversely proportional.
  • the percentage of "acidified” antibodies can be measured by ion exchange chromatography (eg, cation exchange high performance liquid chromatography [CEX-HPLC]), among other methods.
  • an acceptable degree of stability means that no more than about 49%, 45%, 40%, 35% or more of the acidic form of the antibody can be detected in the formulation after storage at a certain temperature for a certain period of time.
  • the certain period of storage prior to measuring stability may 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.
  • certain temperatures that allow storage of pharmaceutical formulations 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, about 0°C , about 2-8°C, about 5°C, about 25°C, or about 40°C.
  • Antibodies are present in the pharmaceutical combination if they show substantially no signs of, e. "maintain its physical stability" in the substance. Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation may proceed to the extent that a visible precipitate is formed.
  • Stability of formulations can be assessed by methods known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements are related to the turbidity of the formulation.
  • the turbidity of a formulation is, in part, an intrinsic property of proteins dissolved in solution, and is usually measured by nephelometric methods and is measured in nephelometric turbidity units (NTU).
  • the level of turbidity as a function of, for example, the concentration of one or more components in a solution (eg, protein and/or salt concentration) is also referred to as the "opacity" or "opacity appearance" of a formulation.
  • Turbidity levels can be calculated by reference to a standard curve generated using suspensions of known turbidity. Reference standards for the determination of turbidity levels in pharmaceutical compositions can be based on the standards of the European Pharmacopoeia (European Pharmacopoeia, Fourth Edition, "European Pharmacopoeia").
  • a clear solution is defined as having a turbidity lower than or equal to that according to the European Pharmacopoeia Standard A solution that has a turbidity of a reference suspension of about 3.
  • Nephelometric turbidity measurements detect Rayleigh scattering in the absence of association or non-ideal effects, which typically vary linearly with concentration. Used in Other methods of assessing physical stability are known in the art.
  • Chemical stability can be assessed by, for example, detecting or quantifying chemically altered forms of the antibody.
  • Chemical alterations can include size alterations (e.g., clipping), 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).
  • size alterations e.g., clipping
  • MALDI/TOF MS matrix-assisted laser desorption ionization/time-of-flight mass spectrometry
  • charge alterations such as occur as a result of deamidation or oxidation
  • An antibody "retains its biological activity" in a pharmaceutical composition if the antibody in the pharmaceutical composition is biologically active for its intended purpose. For example, if the preparation is stored for a certain period of time (for example, 1 to 12 months) at a temperature such as 5°C, 25°C, 45°C, etc., the binding affinity of the anti-CTLA-4 antibody contained in the preparation to CTLA-4 is equal to that of the storage A formulation of the invention is considered stable if it has at least 90%, 95% or more of the previous antibody binding affinity. Binding affinity can also be determined using, for example, ELISA or plasmon resonance techniques.
  • a “therapeutically effective amount” or “effective amount” of an antibody in a pharmacological sense refers to an amount effective in the prevention or treatment or alleviation of symptoms of a disorder that the antibody can effectively treat.
  • a “therapeutically effective amount” or “therapeutically effective dose” of a drug is any amount of a drug that protects a subject from disease onset or promotes disease regression when used alone or in combination with another therapeutic agent, so Such regression of disease is evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of impairment or disability resulting from disease affliction.
  • a therapeutically effective amount of a drug includes a "prophylactically effective amount", i.e. any amount that inhibits the development or recurrence of disease when administered alone or as in combination with other therapeutic agents to a subject at risk of disease or to a subject with relapsed disease Drug.
  • 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.
  • administering refers to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those skilled in the art.
  • Routes of administration of anti-CTLA-4 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion.
  • Parenteral administration means administration other than enteral or topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intrasaccular , Intratracheal, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, intradural, and intrasternal injections and infusions and in vivo electroporation.
  • antibody as used herein should be understood to include whole antibody molecules and antigen-binding fragments thereof.
  • antigen-binding portion or “antigen-binding fragment” of an antibody (or simply referred to as “antibody portion” or “antibody fragment”) as used herein refers to an antibody that retains the ability to specifically bind to human CTLA-4 or its epitope. One or more fragments of . Accordingly, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric antibodies and camelized single domain antibodies.
  • isolated antibody refers to the purified state of a binding compound, and in this context means that the molecule is substantially free of other biomolecules such as nucleic acids, proteins, lipids, sugars or other substances such as cell debris and growth media .
  • isolated does not imply the complete absence of such substances or the absence of water, buffers or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compounds described herein.
  • the term "monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes.
  • the modifier "monoclonal” characterizes an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.
  • murine antibody or “hybridoma antibody” in this disclosure refers to an anti-human CTLA-4 monoclonal antibody prepared according to the knowledge and skill in the art. In preparation, test subjects are injected with the CTLA-4 antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.
  • chimeric antibody is an antibody having the variable domains of a first antibody and the constant domains of a second antibody. Individuals in which the primary and secondary antibodies are from different species. Typically, the variable domains are derived from an antibody of a rodent, etc. (the "parent antibody”), while the constant domain sequences are derived from a human antibody such that the resulting chimeric antibody induces greater The likelihood of an adverse immune response is low.
  • humanized antibody refers to forms of antibodies that contain sequences from both human and non-human (eg, mouse, rat) antibodies.
  • a humanized antibody will comprise substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin.
  • Framework (FR) regions are the framework regions of human immunoglobulin sequences.
  • a humanized antibody optionally can comprise at least a portion of a human immunoglobulin constant region (Fc).
  • full-length antibody or "intact antibody molecule” refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70 kDa in full length) and two light (L) chains (full-length 25 kDa) are interconnected by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into complementarity determining regions (CDRs) of high variability separated by more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs 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 amino-terminus to carboxy-terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin 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 regions within the variable sequences of an antibody. There are three CDRs in each variable region of the heavy and light chains, which are named HCDR1, HCDR2, and HCDR3 or LCDR1, LCDR2, and LCDR3 for each variable region of the heavy and light chains. The exact boundaries of these CDRs are defined differently according to different systems.
  • variable region CDRs of the antibodies of the invention can be determined using any of a number of 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, typically comprising at least a fragment of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody that retains at least some of the parent antibody's 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 scFv; specific antibody. Binding fragments or derivatives thereof generally retain at least 10% of the antigen-binding activity of the parent antibody when the binding activity of the antibody is expressed on a molar basis.
  • 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.
  • antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not appreciably alter their biological activity (referred to as “conservative variants” or “functionally conservative variants” of the antibody).
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof described in the present invention includes any anti-CTLA-4 antibody or antigen-binding fragment thereof described in patent applications with application numbers CN202010708105.8 and PCT/CN2021/107707, which will be applied herein The entire contents disclosed in the patent applications CN 202010708105.8 and PCT/CN2021/107707 are incorporated herein by reference.
  • the CDR sequences of the antibodies used in the methods and compositions of the invention include humanized anti-CTLA-4 antibodies huJS007-47, huJS007-48, huJS007-79 and huJS007 from CN202010708105.8 -106, the amino acid sequence of which is shown in Table A.
  • Table A Amino acid sequences of parts of four humanized anti-CTLA-4 antibodies (KABAT protocol)
  • the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprise, respectively:
  • LCDR1 RASQNVGTYVA (SEQ ID NO: 1);
  • LCDR2 STSYRYS (SEQ ID NO: 2);
  • LCDR3 HQYDTYPLT (SEQ ID NO: 3);
  • HCDR1 SGYYWN (SEQ ID NO: 4);
  • HCDR2 YIGYDGSNX1YNPSLKX2, where X1 is N or Y, X2 is S or N;
  • HCDR3 X3YYSGYFDS, X3 is D or N.
  • the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention comprise:
  • amino acid sequence is LCDR1 shown in SEQ ID NO:1;
  • amino acid sequence is LCDR2 shown in SEQ ID NO:2;
  • amino acid sequence is LCDR3 shown in SEQ ID NO:3;
  • amino acid sequence is HCDR1 as shown in SEQ ID NO:4;
  • amino acid sequence is HCDR2 as shown in SEQ ID NO:5 or 7 or 9;
  • amino acid sequence is HCDR3 as shown in SEQ ID NO:6 or 8.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises an amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively LCDR1, LCDR2 and LCDR3, and amino acid sequences such as HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises an amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively LCDR1, LCDR2 and LCDR3, and amino acid sequences such as HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively LCDR1, LCDR2 and LCDR3, and amino acid sequences such as SEQ ID NO:4, SEQ ID NO:9 and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:6.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention is selected from murine antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, humanized antibodies or an antigen-binding fragment thereof, preferably a humanized antibody or an antigen-binding fragment thereof.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises a light chain variable region as set forth in SEQ ID NO: 10, and a light chain variable region as set forth in SEQ ID NO: 11 The heavy chain variable region shown.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises a light chain variable region as set forth in SEQ ID NO: 12, and a light chain variable region as set forth in SEQ ID NO: 11 The heavy chain variable region shown.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises a light chain variable region as set forth in SEQ ID NO: 13, and a light chain variable region as set forth in SEQ ID NO: 14 The heavy chain variable region shown.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises a light chain variable region as set forth in SEQ ID NO: 13, and a light chain variable region as set forth in SEQ ID NO: 15 The heavy chain variable region shown.
  • the anti-CTLA-4 antibody used in the methods and compositions of the invention comprises a light chain amino acid sequence as set forth in SEQ ID NO: 16, and a heavy chain amino acid sequence as set forth in SEQ ID NO: 17 .
  • the anti-CTLA-4 antibody used in the methods and compositions of the invention comprises a light chain amino acid sequence as set forth in SEQ ID NO: 18, and a heavy chain amino acid sequence as set forth in SEQ ID NO: 19 .
  • the anti-CTLA-4 antibody used in the methods and compositions of the invention comprises a light chain amino acid sequence as set forth in SEQ ID NO:20, and a heavy chain amino acid sequence as set forth in SEQ ID NO:21 .
  • the anti-CTLA-4 antibody used in the methods and compositions of the invention comprises a light chain amino acid sequence as set forth in SEQ ID NO:22, and a heavy chain amino acid sequence as set forth in SEQ ID NO:23 .
  • the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention are humanized or chimeric antibodies and may include human constant regions.
  • the constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions; preferably, an anti-CTLA-4 antibody or antigen-binding fragment thereof suitable for use in the methods and compositions described herein Comprising the heavy chain constant region of the human IgGl or IgG4 isotype.
  • the present invention provides a method for preparing an anti-CTLA-4 antibody or antigen-binding fragment thereof as described herein, the method comprising performing the expression of the antibody or antigen-binding fragment thereof in a The antibody or antigen-binding fragment thereof is expressed in a host cell described herein under conditions and the expressed antibody or antigen-binding fragment thereof is recovered from the host cell.
  • the invention provides mammalian host cells for expressing the recombinant antibodies of the invention, including the many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many others cell line. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.
  • ATCC American Type Culture Collection
  • the present invention provides a method for preparing an anti-CTLA-4 antibody, wherein the method comprises, when introducing the expression vector into a mammalian host cell, culturing the host cell for a sufficient period of time to allow the antibody to express in the host cell
  • the antibody is produced by expressing it in the cell, or more preferably secreting the antibody into the medium in which the host cell is grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • non-fucosylated antibodies are advantageous because they generally have more potent potency than their fucosylated counterparts in vitro and in vivo and are less likely to be immunogenic , because their sugar structure is a normal component of natural human serum IgG.
  • the present invention provides a pharmaceutical composition, comprising: (1) a buffer; (2) an anti-CTLA-4 antibody or an antigen-binding fragment thereof.
  • anti-CTLA-4 antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention is as described in any embodiment of the "anti-CTLA-4 antibody” section of this application.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequence are respectively shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 HCDR1, HCDR2 and HCDR3 are indicated.
  • the above-mentioned anti-CTLA-4 antibody or antigen-binding fragment thereof is selected from murine antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, preferably humanized antibodies or its antigen-binding fragment.
  • the above-mentioned anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a light chain variable region as shown in SEQ ID NO:10, and a heavy chain variable region as shown in SEQ ID NO:11. More preferably, the above-mentioned anti-CTLA-4 antibody comprises the light chain amino acid sequence shown in SEQ ID NO:16, and the heavy chain amino acid sequence shown in SEQ ID NO:17.
  • the concentration of the anti-CTLA-4 antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention is about 1-100 mg/mL, preferably about 1-50 mg/mL, more preferably about 5-30 mg/mL.
  • the pH of the pharmaceutical composition of the present invention is about 4.5-6.5, preferably about 5.0-6.0, more preferably about 5.3-5.7.
  • the buffer in the pharmaceutical composition of the present invention is selected from one or more of acetate buffer, citrate buffer, phosphate buffer and histidine buffer; preferably, the buffer is acetate buffer liquid.
  • the acetic acid buffer is acetic acid-sodium acetate buffer or acetic acid-potassium acetate buffer, preferably acetic acid-sodium acetate buffer.
  • the concentration of the buffer is about 5-100 mM, preferably about 10-50 mM, preferably about 10-30 mM; preferably about 15-25 mM.
  • the pH of the buffer is about 4.5-6.5, preferably about 5.0-6.0, preferably about 5.3-5.7.
  • the pharmaceutical composition of the present invention may contain: an acetate buffer solution with a pH of about 5.0-6.0, and its concentration in the pharmaceutical composition is about 10-30 mM; Anti-CTLA-4 antibodies or antigen-binding fragments thereof, especially the humanized antibody huJS007-47 or antigen-binding fragments thereof described herein.
  • the pharmaceutical composition of the present invention also includes a stabilizer, and the stabilizer is selected from one of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose and trehalose or more; preferably, the above-mentioned arginine salt is arginine hydrochloride.
  • the above stabilizer has a concentration of about 10-400 mM, preferably about 100-300 mM, preferably about 130-280 mM, preferably about 200-260 mM.
  • the aforementioned stabilizer is mannitol with a concentration of about 130-280mM; or the aforementioned stabilizer is sucrose with a concentration of about 130-280mM; or the aforementioned stabilizer is sodium chloride with a concentration of about 20-80mM and A combination of mannitol; or the above-mentioned stabilizer is a combination of sodium chloride at a concentration of about 20-80mM and sucrose at a concentration of about 110-170mM; in some embodiments, the above-mentioned stabilizer is mannitol at a concentration of about 200-260mM; or the above-mentioned The stabilizer is the concentration about 200-260mM sucrose; or the above-mentioned stabilizer is a combination of sodium chloride with a concentration of about 30-70mM and mannitol with a concentration of about 120-160mM; or the above-mentioned stabilizer is a combination of sodium chloride with a concentration of about 30-70mM
  • the pharmaceutical composition of the present invention may contain: an acetate buffer solution with a pH of about 5.0-6.0, and its concentration in the pharmaceutical composition is about 10-30 mM; anti-CTLA-4 antibody or antigen-binding fragment thereof, especially the humanized antibody huJS007-47 or antigen-binding fragment thereof described herein; and a stabilizer of about 130-280 mM, preferably, the stabilizer is selected from sperm One or more of amino acid, arginine salt, sodium chloride, mannitol, sorbitol, sucrose and trehalose, preferably, the above-mentioned arginine salt is arginine hydrochloride.
  • the above-mentioned stabilizer is sucrose with a concentration of about 200-260mM; or the above-mentioned stabilizer is a combination of sodium chloride with a concentration of about 30-70mM and mannitol with a concentration of about 120-160mM; A combination of 70 mM sodium chloride and sucrose at a concentration of about 120-160 mM.
  • the above pharmaceutical composition further includes a surfactant, and the surfactant is selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188.
  • the above-mentioned surfactant concentration is about 0.001%-0.1%, preferably about 0.01%-0.1%, preferably about 0.01%-0.05%, calculated by w/v.
  • the pharmaceutical composition of the present invention may contain: an acetate buffer solution with a pH of about 5.0-6.0, and its concentration in the pharmaceutical composition is about 10-30 mM; anti-CTLA-4 antibody or antigen-binding fragment thereof, especially the humanized antibody huJS007-47 or antigen-binding fragment thereof described herein; and a stabilizer of about 130-280 mM, preferably, the stabilizer is selected from sperm One or more of amino acid, arginine salt, sodium chloride, mannitol, sorbitol, sucrose and trehalose, preferably, the above-mentioned arginine salt is arginine hydrochloride.
  • the above-mentioned stabilizer is sucrose with a concentration of about 200-260mM; or the above-mentioned stabilizer is a combination of sodium chloride with a concentration of about 30-70mM and mannitol with a concentration of about 120-160mM; 70 mM sodium chloride in combination with sucrose at a concentration of about 120-160 mM; and polysorbate 80 at about 0.01%-0.1% w/v.
  • the pharmaceutical composition of the present invention is a liquid formulation or a freeze-dried formulation.
  • the present invention also provides an injection, which contains the pharmaceutical composition described in any scheme herein and sodium chloride solution or glucose solution; preferably, the concentration of the sodium chloride solution is about 0.85-0.9% (w /v); Preferably, the concentration of the glucose solution is about 5-25% (w/v), more preferably about 5-10% (w/v); Preferably, in the injection, the anti-CTLA -4 antibody concentration is about 0.05-10.5mg/mL, more preferably It is selected as about 0.1-5 mg/mL or about 1-5 mg/mL; the pH of the injection is about 4.5-6.5, preferably about 5.0-6.0, more preferably about 5.3-5.7.
  • the above-mentioned pharmaceutical composition or injection is administered through intravenous injection.
  • the present invention also provides the use of the pharmaceutical composition or injection described in any of the schemes herein in the preparation of medicines for treating and/or preventing CTLA-4-mediated diseases or disorders.
  • the present invention also provides the pharmaceutical composition or injection described in any one of the schemes herein, which is used for treating and/or preventing CTLA-4-mediated diseases or conditions.
  • the present invention also provides a method for treating and/or preventing CTLA-4-mediated diseases or conditions, which comprises administering the pharmaceutical composition or injection as described in any one of the regimens herein to a subject in need.
  • CTLA-4-mediated diseases refer to diseases in which CTLA-4 is involved in the occurrence and development of diseases, including but not limited to cancer.
  • Cancer and “cancerous” refer to or describe the physiological condition in mammals that is often characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal carcinoma, hepatocellular carcinoma, carcinoma of the stomach or gastric cancer ( Including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, Salivary gland cancer, renal cancer or renal carcinoma, nasopharyngeal carcinoma, esophageal squamous cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, and B-cell lymphoma (including Low-grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade
  • the appearance is checked by visual inspection. Make sure that the light intensity of the clarity detector is kept between 1000lx and 1500lx. Hold the sample at eye level and shake or invert gently to avoid air bubbles. Visual inspection was performed in front of black and white backgrounds, respectively. The results were recorded in terms of color, opalescence and visible foreign matter.
  • Protein concentration was detected using a UV spectrophotometer (Thermofisher, BIO MATE 3S). The percent extinction coefficient (E1%) was set at 1.5367 (mg/ml)-1 cm-1.
  • BIO MATE 3S instrument wash the cuvette with ultrapure water three times, then add 150 ⁇ L of ultrapure water to the cuvette, click to measure, and use ultrapure water for blank calibration. The absorbance of the blank sample does not exceed ⁇ 0.003.
  • Use ultrapure water to dilute to 0.3mg/ml ⁇ 0.7mg/ml, the final volume of each part should not be less than 900 ⁇ L, and the single dilution factor should not exceed 10 times.
  • CE-SDS electrophoresis uses a high-voltage direct current electric field as the driving force and a capillary as the separation channel.
  • the pre-filled gel will form a molecular sieve in the capillary
  • sodium lauryl sulfate can eliminate the charge effect of different protein molecules
  • the reducing agent ⁇ -mercaptoethanol can cut the disulfide bond in the sample, and samples with different molecular sizes are in the capillary.
  • Different speeds of movement allow separation. Dilute the sample to 1 mg/mL with the sample buffer (SDS-MW Sample Buffer); take 95 ⁇ L of the sample buffer (SDS-MW Sample Buffer), add 5 ⁇ L of ⁇ -mercaptoethanol, vortex and mix well as a blank control.
  • the non-reducing CE-SDS electrophoresis method for purity detection of antibody preparations uses a high-voltage direct current electric field as the driving force and a capillary as the separation channel.
  • the pre-filled gel will form a molecular sieve in the capillary, and the sample is treated with sodium dodecyl sulfate to eliminate the charge effect of different protein molecules, so that samples with different molecular sizes and different moving speeds in the capillary can be separated.
  • Adding an alkylating agent to the test solution can effectively reduce the diffusion of the components, make the obtained peak shape sharp, and the separation efficiency is high, and it can ensure that the sample remains in a non-reducing state.
  • the huJS007-47 antibody was diluted to 80 ⁇ g/ml (4 ⁇ analysis concentration) with huJS007-47 assay buffer (RPMI 1640 Medium+1% FBS), 1.8-fold serial dilution, 10 concentrations.
  • the concentration of CTLA-4 Fc Protein was diluted to 8 ⁇ g/ml (4 ⁇ analytical concentration) for use.
  • the GS-J1/CD28 cell density was adjusted to 2.0 ⁇ 10 6 cells/ml, and PHA was added to make the concentration 20 ⁇ g/ml (4 ⁇ analysis concentration) for later use.
  • the density of GS-C1/CD80 cells was adjusted to 1.0 ⁇ 10 6 cells/ml for later use.
  • Binding ELISA (Binding ELISA) experiment: use a microplate reader (Thermo Scientific, Multiskan Go), coat the plate with a fixed concentration of His-tagged CTLA4 antigen (1.0 ⁇ g/mL), block with 2% BSA, and add a serially diluted antibody (1 ⁇ g /ml starting, 2.5 times serial dilution, a total of 12 concentrations), Anti-Human IgG (Fc Specific)-Peroxidase antibody produced in goat (Sigma, A0170) was diluted 5000 as the detection antibody for detection, and then 0.1mg/ml TMB The color was developed, and finally the reaction was terminated with 2M HCl, and the plate was read at 450nm/620nm. EC50s were fitted using a four parameter logarithmic regression (4PL) model.
  • 4PL logarithmic regression
  • the detection of sub-visible particles is carried out by a particle detector (MFI5100). Since the sample is a high-concentration sample, it needs to be diluted to a certain extent before loading the sample. After dilution, mix gently and fully to avoid air bubbles, then use a pyrogen-free pipette tip to sample 1.3mL into the sample plate in the ultra-clean bench, and use a clean tin foil paper on the sample plate The coverage is tight. After moving from the ultra-clean bench to the corresponding working plate of the instrument, input the sample loading position into the instrument, run the detection sequence, and when the sample flows through the flow cell, the sub-visible particles in it are photographed and counted by the micro camera.
  • MFI5100 particle detector
  • Embodiment 1 buffer system and stabilizer preliminary screening experiment
  • the buffer system closely affects the stability of antibodies, and each antibody with unique physical and chemical properties has the most suitable type of buffer.
  • This example aims to preliminarily screen out the best buffer system and stabilizer, so that the anti-CTLA-4 antibody disclosed in the present invention has the best stability and is suitable for clinical application.
  • This example was performed with antibody huJS007-47.
  • the sample was concentrated by UF/DF ultrafiltration using Millipore Pellicon3 88cm2 membrane to a concentration of about 21mg/mL, then the sample was dialyzed into the corresponding formula shown in Table 3, and the final concentration was adjusted to about 20mg/mL, and then the corresponding concentration was added Polysorbate 80. Aseptically fill it into 2R vials, 2.0mL/bottle, in a clean bench, and carry out stability sampling and testing.
  • Table 3 The first round of formulation screening - the formulation information in the preliminary screening experiment of buffer system and stabilizer
  • Table 4 The first round of formulation screening - protein content data
  • Table 7 The first round of prescription screening—R-CE-SDS data
  • Table 8 The first round of prescription screening—NR-CE-SDS data
  • HHL refers to antibody fragments with two heavy chains and one light chain.
  • acetate buffer (FS1-3) and citrate buffer (FS1-5) performed relatively better. Therefore, 20Mm acetate buffer (pH5.5) and 20Mm citrate buffer (pH6.0) were selected as the buffer system to enter the next round of screening experiments.
  • Embodiment 2 stabilizer and surfactant screening experiment
  • This example was performed with antibody huJS007-47.
  • the sample was concentrated by UF/DF ultrafiltration using Millipore Pellicon3 0.11m2 membrane to a concentration of about 10mg/mL, and then the sample was dialyzed into the corresponding formula shown in Table 13, and the final concentration was adjusted to about 10mg/mL, and then the corresponding concentration of polysorbate 80.
  • Table 14 The second round of formulation screening - protein content data
  • Table 17 The second round of prescription screening—R-CE-SDS data
  • Embodiment 3 Influencing factor experiment
  • the prescriptions showed that the four prescriptions FS2-3, FS2-4, FS2-7 and FS2-8 were more stable, and the four candidate prescriptions were selected for the experiment of influencing factors.
  • the antibody huJS007- 47 carried out. See Table 23 for specific plans.
  • Embodiment 4 Compatibility stability experiment
  • Prescription FS2-4 was selected to investigate the stability of 0.9% Sodium Chloride Injection and 5% Glucose Injection after dilution in two media. This embodiment was carried out with antibody huJS007-47. Dilute to different concentrations of the corresponding medium according to the following scheme (Table 25), and place in a 25°C incubator for 24 hours before testing.
  • sample number "FS2-4-Glu” means diluted with 5% glucose injection
  • sample number "FS2-4-NaCl” means diluted with 0.9% sodium chloride injection.
  • the target pH range was controlled at 5.0-6.0, and the osmotic pressure range was 250-350mOsm/kg, and the optimal preparation was selected.
  • the gradient dilution concentration of CTLA-4 antigen protein is 24nM, 12nM, 6nM, 3nM, 1.5nM, 0.75nM, wherein 24nM is repeated.
  • Biacore T200 Evaluation Software 3.0 the affinity KD value was obtained by fitting.
  • the experimental results are shown in Table 27 and Figure 1.
  • the anti-CTLA-4 pharmaceutical composition has a high affinity with human CTLA-4 protein, and the K D value is 2.06E-10M.
  • Table 27 Affinity result table of anti-CTLA-4 pharmaceutical composition and recombinant human CTLA-4 protein
  • Example 6 Inhibitory Effect of Anti-CTLA-4 Pharmaceutical Composition on EMT6 Tumor Growth Transplanted in hCTLA4 Humanized Mice
  • Mouse breast cancer EMT6 cells (0.5 ⁇ 10 6 ) (ATCC CRL-2755) were inoculated subcutaneously on the right back of hCTLA-4 humanized mice (Shanghai Southern Model Biotechnology Co., Ltd.). When the tumor size was about 99 mm, the mice were randomly divided into 5 groups with 6 mice in each group.
  • Anti-KLH hIgG1, 1, 3 or 10 mg/kg anti-CTLA-4 pharmaceutical composition prepared according to prescription FS2-4, antibody is antibody huJS007-47) of intraperitoneal injection of 10 mg/kg, administration twice a week times, a total of 5 times.
  • the experimental results are shown in Figure 2.
  • the 10 mg/kg anti-CTLA-4 pharmaceutical composition significantly inhibited tumor growth, and the TGI value was 96.4% (p ⁇ 0.05) .
  • the anti-CTLA-4 pharmaceutical composition was well tolerated by the animals, and the mice gradually gained weight during the study period.
  • Example 7 Biological activity of pharmaceutical compositions of anti-CTLA-4 antibodies
  • humanized anti-CTLA-4 antibody The binding of humanized anti-CTLA-4 antibody to huCTLA-4, the ability to block the binding of huCTLA-4 to CD80/CD86, and the biological activity of antagonizing huCTLA-4 were detected.
  • the humanized anti-CTLA-4 antibody was prepared according to the formula of prescription FS2-4, and the experimental methods and results are as follows.
  • the humanized anti-CTLA-4 antibody has good binding to huCTLA-4, which is comparable to or better than the control antibody Ipilimumab.
  • the humanized anti-CTLA-4 antibody has a good ability to block the binding of huCTLA-4 to CD80, which is comparable to or better than the control antibody Ipilimumab.
  • Humanized anti-CTLA-4 antibodies huJS007-46, 47, 48, 49, 55, 56, 73, 79, 82, 88, 100 and 106 were selected for biological Activity analysis.
  • the Jurkat cells expressing huCTLA-4 were plated at 6 ⁇ 104 cells per well, and 3 ⁇ 104 Raji APC cells and different concentrations of humanized anti-CTLA-4 antibodies or control antibodies (Ipilimumab, Junshi Self-made, see patents WO2001014424A2 and CN1371416A), after incubation for 6 hours, T cell activation activity was measured by luciferase method.
  • Example 8 ADCC activity of humanized anti-CTLA-4 antibodies
  • 293T-CTLA4 cells were labeled with CFSE, and peripheral blood mononuclear cells (PBMC2144896) and different concentrations of humanized anti-CTLA-4 antibodies or control antibodies (Ipilimumab , made by Junshi, see patents WO2001014424A2 and CN1371416A), incubated overnight. Cells were stained with propidium iodide (PI) and analyzed by flow cytometry. ADCC killing (ADCC Killing,%) was expressed as the percentage of dead target cells (PI and CFSE positive) in total target cells (CFSE positive).
  • the humanized anti-CTLA-4 antibody was prepared according to the formula of prescription FS2-4.
  • 293T-CTLA4 cells were activated at 37°C for 15 minutes with 12 humanized anti-CTLA-4 antibodies or control antibodies (Ipilimumab, made by Junshi, see patents WO2001014424A2 and CN1371416A) at different concentrations (0.8-100 ⁇ g/mL), in which this Example Humanized anti-CTLA-4 antibody was prepared according to the formula of prescription FS2-4, and then human serum complement with different dilution gradients (1:5, 1:10, 1:20) was added and incubated for 1 hour. After culturing, cells were stained with propidium iodide (PI) and analyzed with a BD FACSCalibur flow cytometer. CDC Killing (CDC Killing,%) was expressed as the percentage of PI-positive target cells in the total target cells. The results are shown in Figure 7, showing that 12 humanized anti-CTLA-4 antibodies had no or negligible CDC activity.
  • PI propidium iodide
  • Example 10 Inhibition of tumor growth in mice by anti-CTLA-4 antibody pharmaceutical composition
  • mice Take 50 6-8-week-old female B-hCTLA4 humanized mice (Biocytogen), and use 1 ⁇ 106 /0.1mL mouse colon cancer cell MC38 WT cells (Shanghai Sunran Biotechnology Co., Ltd.) The concentration was inoculated subcutaneously on the right side of the mouse, and when the tumor grew to about 138mm3 , the mice were randomly divided into groups according to the tumor volume, with 6 mice in each group, a total of 6 groups, respectively:
  • the humanized anti-CTLA-4 antibody was prepared according to the formula of prescription FS2-4.
  • the administration route of all groups was intraperitoneal injection, the administration dose was 0.3mg/kg, and the administration concentration was 0.03mg/ml.
  • the administration was given twice a week, 5 consecutive administrations, and the experiment was ended 3 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded.
  • Ti the average tumor volume of the treatment group and the positive control group on the i-th day of administration
  • T0 the tumor volume of the treatment group and the positive control group on the 0th day of administration
  • Vi the average tumor volume of the negative control group on day i of administration
  • V0 the average tumor volume of the negative control group on day 0 of administration.
  • the average tumor volume of the KLH IgG1 negative control group was 975 ⁇ 115mm 3
  • the average tumor volume of the Ipilimumab (made by Junshi, see patents WO2001014424A2 and CN1371416A) positive control group was 975 ⁇ 115mm 3 .
  • the relative tumor inhibition rate was 18.1%; the average tumor volume of huJS007-47 treatment group, huJS007-48 treatment group, huJS007-79 treatment group and huJS007-106 treatment group were 229 ⁇ 85mm 3 , 313 ⁇ 197mm 3 , 550 ⁇ 229mm 3 and 472 ⁇ 125mm 3 , compared with KLH IgG1, the relative tumor inhibition rates were 89.2%, 79.1%, 50.9% and 60.1%, respectively, indicating that the above-mentioned humanized anti-CTLA-4
  • the antibody can inhibit the growth of B-hCTLA4 humanized mouse MC38-WT cell subcutaneously transplanted tumor in vivo, and it is significantly better than the control antibody Ipilimumab.
  • Protein A probe (Fortebio) to first capture the full-length antibody, namely 2.7 ⁇ g/mL humanized anti-CTLA-4 antibody huJS007-47 and 2 ⁇ g/mL control antibody (Ipilimumab, made by Junshi, see patents WO2001014424A2 and CN1371416A). Then the probe was immersed in 55nM human CTLA (huCTLA) antigen solution to make the full-length antibody bind to the antigen. Finally, the probes were immersed in 600nM Fab solution, including humanized Fab huJS00-47 and control Fab (Ipilimumab), to detect whether the antigen combined with the Fab.
  • huCTLA-4 can continue to bind to the control Fab (Ipilimumab). 4 different epitopes.
  • Example 12 Effect of huJS007-47 pharmaceutical composition on hCTLA4 humanized mice transplanted with MC38
  • mice 6-8 weeks old female hCTLA4 humanized mice (Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were subcutaneously inoculated with 1 ⁇ 10 6 mouse colon cancer MC38 cells (Shanghai Sunran Biotechnology Co., Ltd. ) (0.1ml/only (cell-containing medium RMPI1640 (Gibco))).
  • MC38 cells Cell-containing medium RMPI1640 (Gibco)
  • Anti-KLH hIgG1 negative control group 1mg/kg
  • Ipilimumab positive control group 1mg/kg
  • huJS007-47 treatment group 0.1mg/kg
  • huJS007-47 treatment group 0.3mg/kg
  • huJS007-47 treatment group 1mg/kg
  • the humanized anti-CTLA-4 antibody was prepared according to the formula of prescription FS2-4.
  • the administration was administered on the day of grouping, and the administration route of all groups was intraperitoneal injection, administered twice a week, for 6 consecutive administrations, and the experiment ended 3 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded.
  • TGI% tumor inhibition rate
  • Ti the average tumor volume of the treatment group on day i of administration
  • T0 the average tumor volume of the treatment group on day 0 of administration
  • Vi the average tumor volume of the negative control group on day i of administration
  • V0 negative The average tumor volume of the control group on the 0th day of administration
  • the average tumor volume of the anti-KLH hIgG1 negative control group at a dose of 1 mg/kg was 1116 ⁇ 106 mm 3 on day 21 after the start of administration.
  • Ipilimumab (made by Junshi, refer to patents WO2001014424A2 and CN1371416A) in the positive control group had an average tumor volume of 255 ⁇ 88 mm 3 and a TGI% of 86.36% at a dose of 1 mg/kg.
  • huJS007-47 at doses of 0.1, 0.3 and 1 mg/kg, the average tumor volumes were 736 ⁇ 203mm 3 , 47 ⁇ 12mm 3 , 33 ⁇ 15mm 3 , and TGI% were 38.11%, 107.22% and 108.63%, respectively. It was shown that huJS007-47 significantly inhibited the growth of MC38 tumor volume in hCTLA4 humanized mice at doses of 0.1, 0.3 and 1 mg/kg, and showed a good dose effect, and at the same dose (1 mg/kg), huJS007 The anti-tumor effect of -47 was significantly better than that of Ipilimumab.
  • Example 13 Inhibitory Effect of huJS007-47 Pharmaceutical Composition on H22 Tumor Growth Transplanted in hCTLA4 Humanized Mice
  • mice Take 6-8 week-old female hCTLA4 humanized mice (Shanghai Ncapturing Model Biotechnology Co., Ltd.), and subcutaneously inoculate 1 ⁇ 106 mouse liver cancer cell H22 cells (Shanghai Nuobai Biotechnology Co., Ltd., Cat. No. C01-FV) (0.1 ml/one (cell-containing medium RMPI1640 (Gibco))).
  • the average tumor volume was about 119 mm
  • 35 animals were selected and randomly divided into 5 animals according to the tumor volume. groups, with 7 animals in each group. respectively
  • Anti-KLH hIgG1 negative control group 0.3mg/kg
  • Ipilimumab positive control group 0.1mg/kg
  • huJS007-47 treatment group 0.03mg/kg
  • huJS007-47 treatment group 0.1mg/kg
  • huJS007-47 treatment group 0.3mg/kg
  • the humanized anti-CTLA-4 antibody was prepared according to the formula of prescription FS2-4.
  • the administration was administered on the day of grouping, and the administration route of all groups was intraperitoneal injection, administered twice a week, for 6 consecutive administrations, and the experiment ended 3 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded.
  • the mice were euthanized, and the tumor inhibition rate TGI% was calculated in the same way as in Example 15.
  • the average tumor volume of the anti-KLH hIgG1 negative control group at a dose of 0.3 mg/kg was 2304 ⁇ 402 mm 3 .
  • Ipilimumab (made by Junshi, see patents WO2001014424A2 and CN1371416A) in the positive control group at a dose of 0.1 mg/kg had an average tumor volume of 837 ⁇ 397mm 3 and a TGI% of 67.14%.
  • huJS007-47 significantly inhibited the growth of hCTLA4 humanized mouse transplanted H22 tumor volume at the dose of 0.1 and 0.3 mg/kg, and showed a good dose effect, and at the same dose (0.1 mg/kg), huJS007
  • the anti-tumor effect of -47 was significantly better than that of Ipilimumab.

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Abstract

Provided in the present invention are a pharmaceutical composition of an anti-CTLA-4 antibody, an injection agent, and the use thereof. The pharmaceutical composition or injection agent contains a buffer solution and an anti-CTLA-4 antibody or an antigen binding fragment thereof, wherein the concentration of the anti-CTLA-4 antibody or the antigen binding fragment thereof is approximately 1-100 mg/mL. According to the present invention, by means of selecting an appropriate buffer system and pH and optimizing a stabilizer and a surfactant, the developed antibody preparation can be used for intravenous injection.

Description

抗CTLA-4抗体药物组合物及其用途Anti-CTLA-4 antibody pharmaceutical composition and use thereof 技术领域technical field
本发明涉及治疗性药物组合物领域，具体涉及抗CTLA-4抗体药物组合物及其用途。The invention relates to the field of therapeutic pharmaceutical compositions, in particular to anti-CTLA-4 antibody pharmaceutical compositions and uses thereof.
背景技术Background technique
细胞毒性T淋巴细胞相关蛋白4(CTLA4或CTLA-4，cytotoxic T-lymphocyte-associated protein 4)，也称为CD152(cluster of differentiation 152)，是由CTLA-4基因编码的跨膜蛋白，该基因位于人类第2号染色体2q33。CTLA-4是免疫球蛋白超家族的一员，由胞外V区、跨膜区和胞浆区组成。CTLA-4与T细胞表面的协同刺激分子受体CD28具有同源性，两者竞争结合其配体B7-1(CD80)和B7-2(CD86)，这类配体主要表达于抗原递呈细胞表面。与CD28相比，CTLA-4与CD80和CD86的结合亲和力更高，因此能够竞争和阻断CD28介导的激活作用。CTLA-4通常表达在调节性T细胞(Treg)和活化状态的常规T细胞表面上。其在与B7类分子结合后，抑制T细胞的激活，参与免疫反应的负调控，充当免疫检查点并下调免疫应答，所以CTLA-4在免疫调节中起到非常重要的作用。Cytotoxic T lymphocyte-associated protein 4 (CTLA4 or CTLA-4, cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a transmembrane protein encoded by the CTLA-4 gene. Located on human chromosome 2q33. CTLA-4 is a member of the immunoglobulin superfamily and consists of an extracellular V domain, a transmembrane domain and a cytoplasmic domain. CTLA-4 has homology with the co-stimulatory molecule receptor CD28 on the surface of T cells, and the two compete for the binding of its ligands B7-1 (CD80) and B7-2 (CD86), which are mainly expressed in antigen presentation cell surface. CTLA-4 binds CD80 and CD86 with a higher affinity than CD28, thus competing and blocking CD28-mediated activation. CTLA-4 is normally expressed on the surface of regulatory T cells (Treg) and conventional T cells in the activated state. After it binds to B7 molecules, it inhibits the activation of T cells, participates in the negative regulation of the immune response, acts as an immune checkpoint and down-regulates the immune response, so CTLA-4 plays a very important role in immune regulation.
T细胞的激活需要两个信号的刺激，第一个信号来自T细胞受体(TCR)特异性结合抗原递呈细胞(APC)表面的抗原肽-MHC复合物，第二个信号通路需要共刺激分子(如CD28)的参与，当CD28与B7-1/B7-2(CD80/CD86)结合后可以进一步活化T细胞，促进其成熟和增殖。目前的研究表明，在免疫应答过程中，CTLA-4通过下列途径下调T细胞的功能：其一，CTLA-4可以通过其与CD80/CD86的高亲和力，竞争性阻断CD28与CD80/86共刺激信号的传递，进而抑制T细胞的增殖，减少IL-2的分泌。其二，CTLA-4可以通过降低CD80/CD86在抗原递呈细胞(APC)上的表达水平或者通过反式内吞作用将CD80/CD86分子从抗原递呈细胞(APC)表面移除，从而减少了CD28参与的T细胞激活。其三，CTLA-4可以通过介导树突状细胞结合CD80/CD86并诱导色氨酸降解酶IDO的表达，抑制TCR信号。此外，CTLA-4还可以通过招募抑制性分子结合到免疫突触，诱导产生调节性细胞因子，从而抑制APC和TCR信号的传递。 The activation of T cells requires the stimulation of two signals, the first signal comes from the T cell receptor (TCR) that specifically binds the antigen peptide-MHC complex on the surface of the antigen presenting cell (APC), and the second signaling pathway requires co-stimulation The participation of molecules (such as CD28), when CD28 combines with B7-1/B7-2 (CD80/CD86), can further activate T cells and promote their maturation and proliferation. Current studies have shown that during the immune response, CTLA-4 down-regulates the function of T cells through the following pathways: First, CTLA-4 can competitively block the co-expression of CD28 and CD80/86 through its high affinity with CD80/CD86; Stimulate the transmission of signals, thereby inhibiting the proliferation of T cells and reducing the secretion of IL-2. Second, CTLA-4 can reduce the expression level of CD80/CD86 on antigen-presenting cells (APCs) or remove CD80/CD86 molecules from the surface of antigen-presenting cells (APCs) through trans-endocytosis, thereby reducing CD28 involved in T cell activation. Third, CTLA-4 can inhibit TCR signaling by mediating dendritic cells binding to CD80/CD86 and inducing the expression of tryptophan-degrading enzyme IDO. In addition, CTLA-4 can also induce the production of regulatory cytokines by recruiting inhibitory molecules to the immune synapse, thereby inhibiting the transmission of APC and TCR signals.
已在许多研究中证明CTLA-4的阻断可以诱导肿瘤消退。抗CTLA-4抗体能够在体内外有效、特异地抑制细胞和体液免疫应答，对移植排斥反应及各种自身免疫性疾病有显著的治疗作用，毒副作用低。Blockade of CTLA-4 has been demonstrated in many studies to induce tumor regression. The anti-CTLA-4 antibody can effectively and specifically inhibit cellular and humoral immune responses in vivo and in vitro, and has a significant therapeutic effect on transplant rejection and various autoimmune diseases, with low toxic and side effects.
尽管目前已有CTLA-4的单抗药物Ipilimumab(百时美施贵宝)和Tremelimumab(阿斯利康)用于一些癌症治疗并且正在测试其他抗癌适应症，但是仍需要在各方面包括活性相比于已知抗体改善的新型抗CTLA-4抗体。Although CTLA-4 monoclonal antibody drugs Ipilimumab (Bristol-Myers Squibb) and Tremelimumab (AstraZeneca) are currently used for some cancer treatments and are being tested for other anti-cancer indications, there is still a need to include activities in all aspects compared to Novel anti-CTLA-4 antibodies with known antibody improvements.
但是，抗体药物其分子量大，结构复杂，容易降解、聚合或发生不希望发生的化学修饰等而变得不稳定。为了使抗体适合于生产和给药，并且在储存和随后使用过程中能保持稳定性，及发挥更好的效果，抗体药物的稳定制剂研究显得尤为重要。However, antibody drugs have a large molecular weight and complex structure, and are prone to degradation, polymerization, or undesired chemical modification and become unstable. In order to make antibodies suitable for production and administration, maintain stability during storage and subsequent use, and exert better effects, research on stable formulations of antibody drugs is particularly important.
发明内容Contents of the invention
本发明所述的药物组合物是一种含有与CTLA-4特异性结合的抗体或其抗原结合片段的高稳定性药物组合物。特别地，本发明通过选择适当的缓冲体系和pH，优化稳定剂和表面活性剂，开发得到的抗体制剂可用于静脉注射，且各成分之间相互作用、协同配合，为抗CTLA-4抗体或其抗原结合片段提供了适宜长期保存的存储环境，能够避免制剂在长期保存或运输过程中导致的抗体降解、聚集或沉淀，抗体在该环境中可以长期存储，存储期间能够保证抗体活性和纯度均保持稳定，保证了生物药物的药效。The pharmaceutical composition of the present invention is a highly stable pharmaceutical composition containing an antibody specifically binding to CTLA-4 or an antigen-binding fragment thereof. In particular, by selecting an appropriate buffer system and pH, and optimizing stabilizers and surfactants, the antibody preparations developed by the present invention can be used for intravenous injection, and each component interacts and cooperates synergistically, which is an anti-CTLA-4 antibody or Its antigen-binding fragment provides a storage environment suitable for long-term storage, which can avoid antibody degradation, aggregation or precipitation caused by preparations during long-term storage or transportation. Antibodies can be stored in this environment for a long time, and antibody activity and purity can be guaranteed during storage. Maintaining stability ensures the efficacy of biological drugs.
本发明提供了一种药物组合物，包含：(1)缓冲液；(2)抗CTLA-4抗体或其抗原结合片段。The present invention provides a pharmaceutical composition, comprising: (1) a buffer; (2) an anti-CTLA-4 antibody or an antigen-binding fragment thereof.
在一些方案中，上述抗CTLA-4抗体或其抗原结合片段包含的LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3分别为：In some aspects, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 comprised by the anti-CTLA-4 antibody or antigen-binding fragment thereof are respectively:
LCDR1：RASQNVGTYVA(SEQ ID NO:1)；LCDR1: RASQNVGTYVA (SEQ ID NO: 1);
LCDR2：STSYRYS(SEQ ID NO:2)；LCDR2: STSYRYS (SEQ ID NO: 2);
LCDR3：HQYDTYPLT(SEQ ID NO:3)；LCDR3: HQYDTYPLT (SEQ ID NO: 3);
HCDR1：SGYYWN(SEQ ID NO:4)；HCDR1: SGYYWN (SEQ ID NO: 4);
HCDR2：YIGYDGSNX1YNPSLKX2，其中，X1为N或Y，X2为S或N；HCDR2: YIGYDGSNX1YNPSLKX2, where X1 is N or Y, X2 is S or N;
HCDR3：X3YYSGYFDS，X3为D或N。HCDR3: X3YYSGYFDS, X3 is D or N.
在一些方案中，上述抗CTLA-4抗体或其抗原结合片段包含： In some aspects, the anti-CTLA-4 antibody or antigen-binding fragment thereof described above comprises:
氨基酸序列如SEQ ID NO:1所示的LCDR1；The amino acid sequence is LCDR1 shown in SEQ ID NO:1;
氨基酸序列如SEQ ID NO:2所示的LCDR2；The amino acid sequence is LCDR2 shown in SEQ ID NO:2;
氨基酸序列如SEQ ID NO:3所示的LCDR3；The amino acid sequence is LCDR3 shown in SEQ ID NO:3;
氨基酸序列如SEQ ID NO:4所示的HCDR1；The amino acid sequence is HCDR1 as shown in SEQ ID NO:4;
氨基酸序列如SEQ ID NO:5或7或9所示的HCDR2；The amino acid sequence is HCDR2 as shown in SEQ ID NO:5 or 7 or 9;
氨基酸序列如SEQ ID NO:6或8所示的HCDR3。The amino acid sequence is HCDR3 as shown in SEQ ID NO:6 or 8.
在一些方案中，上述抗CTLA-4抗体或其抗原结合片段包含：In some aspects, the anti-CTLA-4 antibody or antigen-binding fragment thereof described above comprises:
(1)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的LCDR1、LCDR2和LCDR3，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HCDR1、HCDR2和HCDR3；或(1) Amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, and amino acid sequences such as SEQ ID NO:4, SEQ ID NO:5 and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 6; or
(2)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:7和SEQ ID NO:8所示的HCDR1、HCDR2和HCDR3；或(2) Amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, and amino acid sequences such as SEQ ID NO:4, SEQ ID NO:7 and HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO:8; or
(3)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:9和SEQ ID NO:6所示的HCDR1、HCDR2和HCDR3。(3) Amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and amino acid sequences such as SEQ ID NO:4, SEQ ID NO:9 and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:6.
在一些方案中，上述抗CTLA-4抗体或其抗原结合片段选自鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、人源化抗体或其抗原结合片段，优选为人源化抗体或其抗原结合片段。In some embodiments, the above-mentioned anti-CTLA-4 antibody or antigen-binding fragment thereof is selected from murine antibody or antigen-binding fragment thereof, chimeric antibody or antigen-binding fragment thereof, humanized antibody or antigen-binding fragment thereof, preferably humanized Antibodies or antigen-binding fragments thereof.
在一些方案中，上述抗CTLA-4抗体或其抗原结合片段包含：In some aspects, the anti-CTLA-4 antibody or antigen-binding fragment thereof described above comprises:
(1)氨基酸序列如SEQ ID NO:10所示的轻链可变区，和氨基酸序列如SEQ ID NO:11所示的重链可变区；或(1) a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 10, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 11; or
(2)氨基酸序列如SEQ ID NO:12所示的轻链可变区，和氨基酸序列如SEQ ID NO:11所示的重链可变区；或(2) a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 12, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 11; or
(3)氨基酸序列如SEQ ID NO:13所示的轻链可变区，和氨基酸序列如SEQ ID NO:14所示的重链可变区；或(3) a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 13, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 14; or
(4)氨基酸序列如SEQ ID NO:13所示的轻链可变区，和氨基酸序列如SEQ ID NO:15所示的重链可变区。(4) The light chain variable region with amino acid sequence as shown in SEQ ID NO:13, and the heavy chain variable region with amino acid sequence as shown in SEQ ID NO:15.
在一些方案中，上述抗CTLA-4抗体包含： In some aspects, the anti-CTLA-4 antibody described above comprises:
(1)氨基酸序列如SEQ ID NO:16所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO:17所示的重链氨基酸序列。(1) the amino acid sequence of the light chain amino acid sequence shown in SEQ ID NO: 16, and the heavy chain amino acid sequence of the amino acid sequence shown in SEQ ID NO: 17.
(2)氨基酸序列如SEQ ID NO:18所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO:19所示的重链氨基酸序列。(2) The amino acid sequence of the light chain as shown in SEQ ID NO:18, and the amino acid sequence of the heavy chain as shown in SEQ ID NO:19.
(3)氨基酸序列如SEQ ID NO:20所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO:21所示的重链氨基酸序列。(3) The amino acid sequence of the light chain amino acid sequence shown in SEQ ID NO: 20, and the heavy chain amino acid sequence of the amino acid sequence shown in SEQ ID NO: 21.
(4)氨基酸序列如SEQ ID NO:22所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO:23所示的重链氨基酸序列。(4) Amino acid sequence such as the light chain amino acid sequence shown in SEQ ID NO:22, and the amino acid sequence such as the heavy chain amino acid sequence shown in SEQ ID NO:23.
在一些方案中，上述药物组合物中抗CTLA-4抗体或其抗原结合片段的浓度为约1～100mg/mL，优选为约1～50mg/mL，更优选为约5～30mg/mL；更优选地，上述抗CTLA-4抗体或其抗原结合片段浓度为约1mg/mL，5mg/mL，7mg/mL，9mg/mL，10mg/mL，11mg/mL，15mg/mL，20mg/mL，25mg/mL，30mg/mL，40mg/mL，50mg/mL，60mg/mL，70mg/mL，80mg/mL，90mg/mL，100mg/mL或这些范围内任意两个数值作为端点形成的范围，优选为约9mg/mL，10mg/mL，11mg/mL或15mg/mL。In some schemes, the concentration of the anti-CTLA-4 antibody or its antigen-binding fragment in the above pharmaceutical composition is about 1-100 mg/mL, preferably about 1-50 mg/mL, more preferably about 5-30 mg/mL; more preferably Preferably, the above-mentioned anti-CTLA-4 antibody or its antigen-binding fragment concentration is about 1 mg/mL, 5 mg/mL, 7 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg /mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL or any two values in these ranges as the range formed by the endpoint, preferably About 9mg/mL, 10mg/mL, 11mg/mL or 15mg/mL.
在一些方案中，上述药物组合物的pH为约4.5～6.5，优选为约5.0～6.0，更优选为约5.3～5.7，上述药物组合物的pH非限制性实施例为约4.5，5.0，5.1，5.2，5.3，5.4，5.5，5.6，5.7，5.8，5.9，6.0，6.1，6.2，6.3，6.4，6.5或这些范围内任意两个数值作为端点形成的范围，优选为约5.4，5.5或5.6。In some schemes, the pH of the above-mentioned pharmaceutical composition is about 4.5-6.5, preferably about 5.0-6.0, more preferably about 5.3-5.7, the non-limiting examples of the pH of the above-mentioned pharmaceutical composition are about 4.5, 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 or any two of these ranges as endpoints, preferably about 5.4, 5.5 or 5.6 .
在一些方案中，上述药物组合物的渗透压为约250～350mOsm/kg。In some aspects, the osmolarity of the above pharmaceutical composition is about 250-350 mOsm/kg.
在一些方案中，上述缓冲液选自醋酸缓冲液、柠檬酸缓冲液、磷酸盐缓冲液和组氨酸缓冲液中的一种或多种；优选地，所述缓冲液为醋酸缓冲液。In some schemes, the buffer is selected from one or more of acetate buffer, citrate buffer, phosphate buffer and histidine buffer; preferably, the buffer is acetate buffer.
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，优选为醋酸-醋酸钠缓冲液。In some schemes, the above-mentioned buffer is an acetate buffer, preferably, the acetate buffer is an acetate-sodium acetate buffer or an acetate-potassium acetate buffer, preferably an acetate-sodium acetate buffer.
在一些方案中，上述醋酸缓冲液为醋酸-醋酸钠缓冲液。在一些方案中，醋酸-醋酸钠缓冲液由约1～20mM的醋酸和约1～20mM的醋酸钠制成。在一些方案中，醋酸-醋酸钠缓冲液由摩尔比为约1:2到约1:3的醋酸和醋酸钠制成。在一些方案中，醋酸-醋酸钠缓冲液由摩尔比为约1:5到约1:8的醋酸和醋酸钠制成。在一些方案中，醋酸-醋酸钠缓冲液为：由约6.5mM的醋酸和约13.5mM的醋酸钠制成的pH为约 5.0的醋酸缓冲液。在一些方案中，醋酸-醋酸钠缓冲液为：由约2～4mM的醋酸和约16～18mM的醋酸钠制成的pH为约5.0～6.0的醋酸缓冲液；优选为由约3mM的醋酸和约17mM的醋酸钠制成的pH为约5.5的醋酸缓冲液。In some embodiments, the acetate buffer is an acetate-sodium acetate buffer. In some protocols, the acetic acid-sodium acetate buffer is made of about 1-20 mM acetic acid and about 1-20 mM sodium acetate. In some aspects, the acetic acid-sodium acetate buffer is made of acetic acid and sodium acetate in a molar ratio of about 1:2 to about 1:3. In some aspects, the acetic acid-sodium acetate buffer is made of acetic acid and sodium acetate in a molar ratio of about 1:5 to about 1:8. In some protocols, the acetic acid-sodium acetate buffer is: a pH of about 6.5 mM acetic acid and about 13.5 mM sodium acetate 5.0 in acetate buffer. In some protocols, the acetic acid-sodium acetate buffer is: an acetate buffer with a pH of about 5.0 to 6.0 made from about 2 to 4 mM acetic acid and about 16 to 18 mM sodium acetate; preferably about 3 mM acetic acid and about 17 mM sodium acetate to make an acetate buffer with a pH of about 5.5.
在一些方案中，上述组氨酸缓冲液选自组氨酸-组氨酸盐酸盐缓冲液或组氨酸-组氨酸醋酸盐缓冲液，优选组氨酸-组氨酸盐酸盐缓冲液。在一些方案中，上述组氨酸-组氨酸盐酸盐缓冲液由组氨酸和组氨酸盐酸盐制成，优选L-组氨酸和L-组氨酸单盐酸盐。在一些方案中，组氨酸缓冲液由约1～20mM的L-组氨酸和约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约1:1到约1:4的组氨酸和组氨酸盐酸盐制成。在一些方案中，组氨酸缓冲液由摩尔比为约1:1组氨酸和组氨酸盐酸盐制成。在一些方案中，组氨酸缓冲液由摩尔比为约1:3的组氨酸和组氨酸盐酸盐制成。在一些方案中，组氨酸制剂为：由约4.5mM的L-组氨酸和约15.5mM的L-组氨酸单盐酸盐制成的pH为约5.5的组氨酸缓冲剂。在一些方案中，组氨酸制剂为：由约10mM的组氨酸和约10mM的组氨酸盐酸盐制成的pH为约6.0的组氨酸缓冲液。In some schemes, the above-mentioned histidine buffer is selected from histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, preferably histidine-histidine hydrochloride buffer. In some aspects, the above-mentioned histidine-histidine hydrochloride buffer is made of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some protocols, the histidine buffer is made of about 1-20 mM L-histidine and about 1-20 mM L-histidine monohydrochloride. In some aspects, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of about 1:1 to about 1:4. In some protocols, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of about 1:1. In some protocols, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of about 1:3. In some aspects, the histidine formulation is a histidine buffer at a pH of about 5.5 made from about 4.5 mM L-histidine and about 15.5 mM L-histidine monohydrochloride. In some aspects, the histidine formulation is a histidine buffer at a pH of about 6.0 made from about 10 mM histidine and about 10 mM histidine hydrochloride.
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，柠檬酸缓冲液由约1～20mM的柠檬酸和约1～20mM的柠檬酸钠制成。在一些方案中，柠檬酸缓冲液由摩尔比为约1:1到1:4的柠檬酸和柠檬酸钠制成。在一些方案中，柠檬酸缓冲液为：由约5.0mM的柠檬酸和约15.0mM的柠檬酸钠制成的pH为约6.5的柠檬酸缓冲液。在一些方案中，柠檬酸缓冲液为：由约10mM的柠檬酸和约10mM的柠檬酸钠制成的pH为约6.0的柠檬酸缓冲液。In some solutions, the above-mentioned buffer is a citric acid buffer, preferably, the citric acid buffer is a citric acid-sodium citrate buffer. In some protocols, the citrate buffer is made from about 1-20 mM citric acid and about 1-20 mM sodium citrate. In some aspects, the citrate buffer is made of citric acid and sodium citrate in a molar ratio of about 1:1 to 1:4. In some aspects, the citrate buffer is: a citrate buffer having a pH of about 6.5 made from about 5.0 mM citric acid and about 15.0 mM sodium citrate. In some aspects, the citrate buffer is: a citrate buffer having a pH of about 6.0 made from about 10 mM citric acid and about 10 mM sodium citrate.
在一些方案中，上述缓冲液为磷酸盐缓冲液，优选地，所述磷酸盐缓冲液为磷酸氢二钠-磷酸二氢钠缓冲液。在一些方案中，磷酸盐缓冲液由约1～20mM的磷酸氢二钠和约1～20mM的磷酸二氢钠制成。在一些方案中，磷酸盐缓冲液由摩尔比为约1:1到1:4的磷酸氢二钠和磷酸二氢钠制成。在一些方案中，磷酸盐缓冲液为：由约10mM的磷酸氢二钠和约10mM的磷酸二氢钠制成的pH为约7.0的磷酸盐缓冲液。In some schemes, the above-mentioned buffer is a phosphate buffer, preferably, the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. In some protocols, the phosphate buffer is made from about 1-20 mM disodium phosphate and about 1-20 mM sodium phosphate. In some protocols, the phosphate buffer is made of disodium hydrogen phosphate and monobasic sodium phosphate in a molar ratio of about 1:1 to 1:4. In some aspects, the phosphate buffer is: a phosphate buffer having a pH of about 7.0 made from about 10 mM disodium hydrogen phosphate and about 10 mM monobasic sodium phosphate.
在一些方案中，上述缓冲液的浓度为约5～100mM，优选为约10～50mM，优选为约10～30mM；优选为约15～25mM，上述缓冲液浓度非限制性实施例为约10mM， 15mM，20mM，25mM，30mM，40mM，45mM，50mM或这些范围内任意两个数值作为端点形成的范围，优选为约15mM，20mM或25mM。In some schemes, the concentration of the above-mentioned buffer solution is about 5-100 mM, preferably about 10-50 mM, preferably about 10-30 mM; preferably about 15-25 mM, and the non-limiting example of the above-mentioned buffer solution concentration is about 10 mM, 15mM, 20mM, 25mM, 30mM, 40mM, 45mM, 50mM or any two values within these ranges as the range formed by the endpoints, preferably about 15mM, 20mM or 25mM.
在一些方案中，上述缓冲液的pH为约4.5～6.5，优选为约5.0～6.0，更优选为约5.3～5.7，上述缓冲液的pH非限制性实施例为约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或这些范围内任意两个数值作为端点形成的范围，优选为约5.4，5.5或5.6。在一些方案中，上述的药物组合物还包括稳定剂，所述稳定剂选自精氨酸、精氨酸盐、氯化钠、甘露醇、山梨醇、蔗糖和海藻糖中的一种或多种；优选地，上述精氨酸盐为盐酸精氨酸。在一些方案中，上述稳定剂的浓度为约10～400mM，优选为约100～300mM，优选为约130～280mM，优选为约200～260mM，上述稳定剂浓度非限制性实施例为约130mM，135mM，140mM，150mM，160mM，170mM，180mM，190mM，200mM，210mM，220mM，228mM，230mM，240mM，250mM，260mM或这些范围内任意两个数值作为端点形成的范围，优选为约210mM，220mM，228mM或230mM。In some schemes, the pH of the above-mentioned buffer is about 4.5-6.5, preferably about 5.0-6.0, more preferably about 5.3-5.7, and the non-limiting examples of the pH of the above-mentioned buffer are 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 or any two values in these ranges as the range formed by the endpoints, preferably about 5.4, 5.5 or 5.6. In some schemes, the above-mentioned pharmaceutical composition also includes a stabilizer, and the stabilizer is selected from one or more of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose and trehalose species; preferably, the above-mentioned arginine salt is arginine hydrochloride. In some schemes, the concentration of the above-mentioned stabilizer is about 10-400mM, preferably about 100-300mM, preferably about 130-280mM, preferably about 200-260mM, and the non-limiting example of the above-mentioned stabilizer concentration is about 130mM, 135mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 228mM, 230mM, 240mM, 250mM, 260mM or any two values in these ranges as the range formed by the endpoint, preferably about 210mM, 220mM, 228mM or 230mM.
在一些方案中，上述稳定剂为浓度约130～280mM的甘露醇；或上述稳定剂为浓度约130～280mM的蔗糖；或上述稳定剂为浓度约20～80mM的氯化钠与浓度约110-170mM的甘露醇的组合；或上述稳定剂为浓度约20～80mM的氯化钠与浓度约110-170mM的蔗糖的组合；在一些方案中，上述稳定剂为浓度约200～260mM的甘露醇；或上述稳定剂为浓度约200～260mM的蔗糖；或上述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的甘露醇的组合；或上述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的蔗糖的组合。In some schemes, the above-mentioned stabilizer is mannitol with a concentration of about 130-280mM; or the above-mentioned stabilizer is sucrose with a concentration of about 130-280mM; or the above-mentioned stabilizer is sodium chloride with a concentration of about 20-80mM A combination of 170mM mannitol; or the above-mentioned stabilizer is a combination of sodium chloride at a concentration of about 20-80mM and sucrose at a concentration of about 110-170mM; in some embodiments, the above-mentioned stabilizer is mannitol at a concentration of about 200-260mM; Or the above-mentioned stabilizer is sucrose with a concentration of about 200-260mM; or the above-mentioned stabilizer is a combination of sodium chloride with a concentration of about 30-70mM and mannitol with a concentration of about 120-160mM; or the above-mentioned stabilizer is about 30-70mM Sodium chloride in combination with sucrose at a concentration of about 120-160 mM.
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为约130～280mM，优选为约200～260mM，上述甘露醇浓度的非限制性实施例为约200mM，210mM，220mM，230mM，240mM，250mM，260mM或这些范围内任意两个数值作为端点形成的范围，优选为约240mM。In some aspects, the aforementioned stabilizer is mannitol. In some schemes, the above-mentioned stabilizer is mannitol at a concentration of about 100-300 mM, the concentration of the above-mentioned mannitol is preferably about 130-280 mM, preferably about 200-260 mM, and the non-limiting example of the above-mentioned mannitol concentration is about 200 mM , 210mM, 220mM, 230mM, 240mM, 250mM, 260mM or any two values within these ranges as the range formed by the endpoints, preferably about 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为约130～280mM，优选为约200～260mM，上述蔗糖浓度的非限制性实施例为约200mM，210mM，220mM，228mM，230mM，240mM，250mM或这些范围内任意两个数值作为端点形成的范围，优选为约 220mM或约228mM。In some aspects, the aforementioned stabilizer is sucrose. In some schemes, the above-mentioned stabilizer is sucrose with a concentration of about 100-300mM, the concentration of the above-mentioned sucrose is preferably about 130-280mM, preferably about 200-260mM, and the non-limiting examples of the above-mentioned sucrose concentration are about 200mM, 210mM, 220mM, 228mM, 230mM, 240mM, 250mM or any two values in these ranges as the range formed by the endpoint, preferably about 220mM or about 228mM.
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30～200mM的氯化钠与约30～200mM的甘露醇的组合，优选约20～80mM的氯化钠与约110～170mM的甘露醇的组合，优选约30～70mM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为约50mM的氯化钠与约140mM的甘露醇的组合，或约50mM的氯化钠与约150mM的甘露醇的组合。In some aspects, the aforementioned stabilizer is a combination of sodium chloride and mannitol. In some schemes, the aforementioned stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM mannitol, preferably about 20-80 mM sodium chloride and about 110-170 mM mannitol, preferably about A combination of 30-70 mM sodium chloride and about 120-160 mM mannitol, a non-limiting example of the above stabilizer is a combination of about 50 mM sodium chloride and about 140 mM mannitol, or about 50 mM sodium chloride Combination with about 150mM mannitol.
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30～200mM的氯化钠与约30～200mM的蔗糖的组合，优选约20～80mM的氯化钠与约110～170mM的蔗糖的组合，优选约30～70mM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为约50mM的氯化钠与约140mM的蔗糖的组合，或约50mM的氯化钠与约160mM的蔗糖的组合。In some aspects, the aforementioned stabilizer is a combination of sodium chloride and sucrose. In some schemes, the above-mentioned stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose, preferably about 20-80 mM sodium chloride and about 110-170 mM sucrose, preferably about 30-200 mM The combination of sodium chloride of 70mM and the sucrose of about 120～160mM, the non-limiting example of above-mentioned stabilizer is the combination of the sodium chloride of about 50mM and the sucrose of about 140mM, or the sodium chloride of about 50mM and the sucrose of about 160mM Combination of sucrose.
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和泊洛沙姆188中的一种或多种。In some embodiments, the above pharmaceutical composition further includes a surfactant, and the surfactant is selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188.
在一些方案中，上述表面活性剂选自聚山梨醇酯80。In some aspects, the aforementioned surfactant is selected from polysorbate 80.
在一些方案中，上述表面活性剂选自聚山梨醇酯20。In some aspects, the aforementioned surfactant is selected from polysorbate 20.
在一些方案中，以w/v计算，上述表面活性剂浓度为约0.001％～0.1％，优选为约0.01％～0.1％，优选为约0.01％～0.05％；作为非限制性实施例，上述表面活性剂的浓度为约0.01％，0.02％，0.03％，0.04％，0.08％或这些范围内任意两个数值作为端点形成的范围，优选为约0.02％。In some schemes, calculated by w/v, the above-mentioned surfactant concentration is about 0.001%-0.1%, preferably about 0.01%-0.1%, preferably about 0.01%-0.05%; as a non-limiting example, the above-mentioned The concentration of the surfactant is about 0.01%, 0.02%, 0.03%, 0.04%, 0.08%, or any two values within these ranges as endpoints forming a range, preferably about 0.02%.
在一些方案中，药物组合物包含如下(1)～(13)中任一项所示的组分，或分别由(1)～(13)任一项所示的组分组成：In some schemes, the pharmaceutical composition comprises the components shown in any one of (1) to (13) below, or consists of the components shown in any one of (1) to (13):
(1)(a)约1～50mg/mL的上述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)130～280mM的甘露醇；以及(d)约0.01％～0.1％(w/v)的聚山梨醇酯80；或(1) (a) about 1 to 50 mg/mL of the above-mentioned anti-CTLA-4 antibody or an antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer, pH about 5.0 to 6.0; (c) 130 to 280 mM Mannitol; and (d) about 0.01% to 0.1% (w/v) polysorbate 80; or
(2)(a)约1～50mg/mL的上述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)130～280mM的蔗糖；以及(d)约0.01％～0.1％(w/v)的聚山梨醇酯80；或(2) (a) about 1 to 50 mg/mL of the above-mentioned anti-CTLA-4 antibody or an antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer solution with a pH of about 5.0 to 6.0; (c) 130 to 280 mM of sucrose; and (d) about 0.01% to 0.1% (w/v) polysorbate 80; or
(3)(a)约1～50mg/mL的上述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)稳定剂，所述稳定剂为浓度约20～80mM 的氯化钠与浓度约110-170mM的甘露醇的组合；以及(d)约0.01％～0.1％(w/v)的聚山梨醇酯80；或(3) (a) about 1 to 50 mg/mL of the above-mentioned anti-CTLA-4 antibody or an antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer solution with a pH of about 5.0 to 6.0; (c) a stabilizer, the The above stabilizer has a concentration of about 20-80mM A combination of sodium chloride and mannitol at a concentration of about 110-170 mM; and (d) polysorbate 80 at about 0.01% to 0.1% (w/v); or
(4)(a)约1～50mg/mL的上述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)稳定剂，所述稳定剂为浓度约20～80mM的氯化钠与浓度约110-170mM的蔗糖的组合；以及(d)约0.01％～0.1％(w/v)的聚山梨醇酯80；或(4) (a) about 1 to 50 mg/mL of the above-mentioned anti-CTLA-4 antibody or an antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer solution with a pH of about 5.0 to 6.0; (c) a stabilizer, the The stabilizer is a combination of sodium chloride at a concentration of about 20-80 mM and sucrose at a concentration of about 110-170 mM; and (d) polysorbate 80 at about 0.01%-0.1% (w/v); or
(5)(a)约5～30mg/mL的上述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)200～260mM的甘露醇；以及(d)约0.01％～0.05％(w/v)的聚山梨醇酯80；或(5) (a) about 5 to 30 mg/mL of the above-mentioned anti-CTLA-4 antibody or an antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer solution with a pH of about 5.0 to 6.0; (c) 200 to 260 mM of Mannitol; and (d) about 0.01% to 0.05% (w/v) polysorbate 80; or
(6)(a)约5～30mg/mL的上述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)200～260mM的蔗糖；以及(d)约0.01％～0.05％(w/v)的聚山梨醇酯80；或(6) (a) about 5 to 30 mg/mL of the above-mentioned anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer solution with a pH of about 5.0 to 6.0; (c) 200 to 260 mM of sucrose; and (d) about 0.01% to 0.05% (w/v) polysorbate 80; or
(7)(a)约5～30mg/mL的上述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)稳定剂，所述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的甘露醇的组合；以及(d)约0.01％～0.05％(w/v)的聚山梨醇酯80；或(7) (a) about 5-30 mg/mL of the above-mentioned anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer solution with a pH of about 5.0-6.0; (c) a stabilizer, the The stabilizer is a combination of sodium chloride at a concentration of about 30-70mM and mannitol at a concentration of about 120-160mM; and (d) polysorbate 80 at about 0.01%-0.05% (w/v); or
(8)(a)约5～30mg/mL的上述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)稳定剂，所述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的蔗糖的组合；以及(d)约0.01％～0.05％(w/v)的聚山梨醇酯80；或(8) (a) about 5 to 30 mg/mL of the above-mentioned anti-CTLA-4 antibody or an antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer solution with a pH of about 5.0 to 6.0; (c) a stabilizer, the The stabilizer is a combination of sodium chloride at a concentration of about 30-70 mM and sucrose at a concentration of about 120-160 mM; and (d) polysorbate 80 at about 0.01%-0.05% (w/v); or
(9)(a)约10mg/mL的抗CTLA-4抗体，所述抗CTLA-4抗体包含如SEQ ID NO:16所示的轻链氨基酸序列和如SEQ ID NO:17所示的重链氨基酸序列；(b)约20mM醋酸缓冲液，pH为约5.5；(c)约240mM的甘露醇；以及(d)约0.02％(w/v)的聚山梨醇酯80；或(9) (a) an anti-CTLA-4 antibody of about 10 mg/mL, said anti-CTLA-4 antibody comprising a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain as shown in SEQ ID NO: 17 the amino acid sequence; (b) about 20 mM acetate buffer, pH about 5.5; (c) about 240 mM mannitol; and (d) about 0.02% (w/v) polysorbate 80; or
(10)(a)约10mg/mL的抗CTLA-4抗体，所述抗CTLA-4抗体包含如SEQ ID NO:16所示的轻链氨基酸序列和如SEQ ID NO:17所示的重链氨基酸序列；(b)约20mM醋酸缓冲液，pH为约5.5；(c)约220mM的蔗糖；以及(d)约0.02％(w/v)的聚山梨醇酯80；或 (10) (a) an anti-CTLA-4 antibody of about 10 mg/mL, said anti-CTLA-4 antibody comprising the light chain amino acid sequence shown in SEQ ID NO: 16 and the heavy chain shown in SEQ ID NO: 17 the amino acid sequence; (b) about 20 mM acetate buffer, pH about 5.5; (c) about 220 mM sucrose; and (d) about 0.02% (w/v) polysorbate 80; or
(11)(a)约10mg/mL的抗CTLA-4抗体，所述抗CTLA-4抗体包含如SEQ ID NO:16所示的轻链氨基酸序列和如SEQ ID NO:17所示的重链氨基酸序列；(b)约20mM醋酸缓冲液，pH为约5.5；(c)约228mM的蔗糖；以及(d)约0.02％(w/v)的聚山梨醇酯80；或(11) (a) an anti-CTLA-4 antibody of about 10 mg/mL, said anti-CTLA-4 antibody comprising a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain as shown in SEQ ID NO: 17 Amino acid sequence; (b) about 20 mM acetate buffer, pH about 5.5; (c) about 228 mM sucrose; and (d) about 0.02% (w/v) polysorbate 80; or
(12)(a)约10mg/mL的抗CTLA-4抗体，所述抗CTLA-4抗体包含如SEQ ID NO:16所示的轻链氨基酸序列和如SEQ ID NO:17所示的重链氨基酸序列；(b)约20mM醋酸缓冲液，pH为约5.5；(c)稳定剂，所述稳定剂为浓度约50mM的氯化钠与浓度约140mM的甘露醇的组合；以及(d)约0.02％(w/v)的聚山梨醇酯80；或(12) (a) an anti-CTLA-4 antibody of about 10 mg/mL, said anti-CTLA-4 antibody comprising a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain as shown in SEQ ID NO: 17 Amino acid sequence; (b) about 20 mM acetate buffer, pH is about 5.5; (c) stabilizer, the combination of the sodium chloride of concentration about 50 mM and the mannitol of concentration about 140 mM; And (d) about 0.02% (w/v) polysorbate 80; or
(13)(a)约10mg/mL的抗CTLA-4抗体，所述抗CTLA-4抗体包含如SEQ ID NO:16所示的轻链氨基酸序列和如SEQ ID NO:17所示的重链氨基酸序列；(b)约20mM醋酸缓冲液，pH为约5.5；(c)稳定剂，所述稳定剂为浓度约50mM的氯化钠与浓度约140mM的蔗糖的组合；以及(d)约0.02％(w/v)的聚山梨醇酯80。(13) (a) an anti-CTLA-4 antibody of about 10 mg/mL, said anti-CTLA-4 antibody comprising a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain as shown in SEQ ID NO: 17 Amino acid sequence; (b) about 20 mM acetate buffer at a pH of about 5.5; (c) a stabilizer comprising a combination of sodium chloride at a concentration of about 50 mM and sucrose at a concentration of about 140 mM; and (d) about 0.02 % (w/v) polysorbate 80.
在一些方案中，本文任一项方案中所述的药物组合物为液体制剂或冻干制剂。In some aspects, the pharmaceutical composition described in any of the aspects herein is a liquid formulation or a lyophilized formulation.
在一些方案中，上述药物组合物为液体制剂。In some aspects, the pharmaceutical composition described above is a liquid formulation.
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。In some aspects, the above 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.
在一些方案中，上述液体制剂或冻干制剂于40℃稳定至少7天，至少14天或至少28天。In some aspects, the liquid formulation or lyophilized formulation described above is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.
本发明还提供了一种注射剂，其含有本文任一项方案中所述的药物组合物与氯化钠溶液或葡萄糖溶液；优选地，所述氯化钠溶液浓度为约0.85～0.9％(w/v)；优选地，所述葡萄糖溶液浓度为约5～25％(w/v)，更优选为约5～10％(w/v)；优选地，所述注射剂中，所述抗CTLA-4抗体的浓度为约0.05～10.5mg/mL，更优选为约0.1～5mg/mL或约1～5mg/mL；所述注射剂的pH为约4.5～6.5，优选为约5.0～6.0，更优选为约5.3～5.7。The present invention also provides an injection, which contains the pharmaceutical composition described in any scheme herein and sodium chloride solution or glucose solution; preferably, the concentration of the sodium chloride solution is about 0.85-0.9% (w /v); Preferably, the concentration of the glucose solution is about 5-25% (w/v), more preferably about 5-10% (w/v); Preferably, in the injection, the anti-CTLA The concentration of the -4 antibody is about 0.05-10.5 mg/mL, more preferably about 0.1-5 mg/mL or about 1-5 mg/mL; the pH of the injection is about 4.5-6.5, preferably about 5.0-6.0, more preferably Preferably it is about 5.3 to 5.7.
在一些方案中，上述药物组合物或注射剂，其经静脉注射施用。In some aspects, the above-mentioned pharmaceutical composition or injection is administered through intravenous injection.
本发明还提供了本文任一项方案中所述的药物组合物或注射剂在制备用于治疗和/或预防CTLA-4介导的疾病或病症的药物中的用途。 The present invention also provides the use of the pharmaceutical composition or injection described in any of the schemes herein in the preparation of medicines for treating and/or preventing CTLA-4-mediated diseases or disorders.
本发明还提供了本文任一项方案中所述的药物组合物或注射剂，其用于治疗和/或预防CTLA-4介导的疾病或病症。The present invention also provides the pharmaceutical composition or injection described in any one of the schemes herein, which is used for treating and/or preventing CTLA-4-mediated diseases or conditions.
本发明还提供了一种治疗和/或预防CTLA-4介导的疾病或病症的方法，其包括向有需要的受试者施用如本文任一项方案中所述的药物组合物或注射剂。The present invention also provides a method for treating and/or preventing CTLA-4-mediated diseases or conditions, which comprises administering the pharmaceutical composition or injection as described in any one of the regimens herein to a subject in need.
在一些方案中，上述疾病或病症为癌症。In some aspects, the aforementioned disease or condition is cancer.
附图说明Description of drawings
图1：抗CTLA-4药物组合物与重组人CTLA-4蛋白的亲和力测试结果。Figure 1: The results of the affinity test between the anti-CTLA-4 pharmaceutical composition and the recombinant human CTLA-4 protein.
图2：抗CTLA-4药物组合物对hCTLA4人源化小鼠移植EMT6肿瘤生长的抑制作用测试结果。Figure 2: Test results of the inhibitory effect of the anti-CTLA-4 pharmaceutical composition on the growth of EMT6 tumor transplanted in hCTLA4 humanized mice.
图3：ELISA法检测人源化抗CTLA-4抗体与huCTLA-4的结合。Figure 3: Detection of the binding of humanized anti-CTLA-4 antibody to huCTLA-4 by ELISA.
图4：ELISA法检测人源化抗CTLA-4抗体阻断huCTLA-4与CD80结合的能力。Figure 4: ELISA method to detect the ability of humanized anti-CTLA-4 antibody to block the binding of huCTLA-4 to CD80.
图5：荧光素酶法检测人源抗CTLA-4化抗体的生物学活性。Figure 5: Detection of biological activity of human anti-CTLA-4ylation antibody by luciferase method.
图6：人源化抗CTLA-4抗体的ADCC活性。Figure 6: ADCC activity of humanized anti-CTLA-4 antibodies.
图7：人源化抗CTLA-4抗体的CDC活性。Figure 7: CDC activity of humanized anti-CTLA-4 antibodies.
图8：人源化抗CTLA-4抗体对小鼠肿瘤生长的抑制。Figure 8: Inhibition of tumor growth in mice by humanized anti-CTLA-4 antibodies.
图9：Fortebio结合实验鉴定抗原表位。Figure 9: Fortebio binding assay identifies antigenic epitopes.
图10：huJS007-47对hCTLA4人源化小鼠移植MC38肿瘤生长的抑制作用。Figure 10: Inhibitory effect of huJS007-47 on the growth of MC38 tumors transplanted in hCTLA4 humanized mice.
图11：huJS007-47对hCTLA4人源化小鼠移植H22肿瘤生长的抑制作用。Figure 11: Inhibitory effect of huJS007-47 on the growth of H22 tumor transplanted in hCTLA4 humanized mice.
具体实施方式Detailed ways
定义和说明Definition and Description
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。本文所引用的所有参考文献，包括专利、专利申请、论文、教科书诸如此类，以及其中所引用的参考文献，就其尚未被引用的程 度而言，在此其全文通过引用并入。如果所并入的文献和类似材料中的一个或多个与本申请不同或矛盾，包括但不限于所定义的术语、术语用法、所描述的技术诸如此类，则以本申请为准。For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. It is to be understood that this invention is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It should also be understood that the terminology used in this application is for the purpose of describing specific embodiments only and is not intended to be limiting. All references cited herein, including patents, patent applications, treatises, textbooks, and the like, and references cited therein, are for the purposes in which they have not been cited To the extent that it is hereby incorporated by reference in its entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, and the like, this application controls.
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。As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes combinations of two or more polypeptides, and the like.
术语“药物组合物”或“制剂”表示含有一种或多种本文所述抗体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。The term "pharmaceutical composition" or "formulation" means a mixture comprising one or more of the antibodies described herein and other components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
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)。The term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a resuspended lyophilized formulation. The liquid formulations of the present invention are stable on storage and are not dependent on lyophilization (or other state changing methods such as spray drying) for their stability.
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。The term "aqueous liquid formulation" refers to a liquid formulation using water as a solvent. In some aspects, aqueous liquid formulations are formulations that do not require lyophilization, spray drying, and/or freezing to maintain stability (eg, chemical and/or physical stability and/or biological activity).
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。The term "excipient" refers to an agent that can be added to a formulation to provide desired properties (eg, consistency, increased stability) and/or to adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, carbohydrates, polyols, amino acids, surfactants, and polymers.
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。As used herein, "about" when referring to a measurable value (such as amount, duration, etc.) is intended to cover a variation of ±20% or ±10% relative to the specified value, including ±5%, ±1% and ±0.1 %, as these variations are suitable for carrying out the disclosed methods.
术语“缓冲液pH为约4.5～6.5”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。The term "buffer pH of about 4.5 to 6.5" refers to an agent that renders a solution containing the agent resistant to pH changes through the action of its acid/base conjugate component. Buffers used in formulations of the 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.
在本文中，将pH控制在该范围内的“缓冲液”实例包括醋酸、醋酸盐(例如醋酸钠)、琥珀酸、琥珀酸盐(例如琥珀酸钠)、葡萄糖酸、组氨酸、组胺酸盐(例如组氨酸盐酸盐)、甲硫氨酸、柠檬酸(枸橼酸)、柠檬酸盐(枸橼酸盐)、磷酸盐、柠檬酸盐/磷酸盐、咪唑、其组合和其他有机酸缓冲剂。 Herein, examples of "buffers" for controlling the pH within this range include acetic acid, acetate salts (such as sodium acetate), succinic acid, succinates (such as sodium succinate), gluconic acid, histidine, histidine Amine salts (e.g., histidine hydrochloride), methionine, citric acid (citric acid), citrate (citrate), phosphate, citrate/phosphate, imidazole, combinations thereof and other organic acid buffers.
“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。A "histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate, etc., such as histidine containing A histidine buffer of acid and histidine hydrochloride; the histidine buffer of the present invention also includes a histidine buffer containing histidine and acetate (eg sodium or potassium salt).
“柠檬酸缓冲液”，又称“枸橼酸缓冲液”，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-柠檬酸钠、柠檬酸-柠檬酸钾、柠檬酸-柠檬酸钙、柠檬酸-柠檬酸镁等。优选的柠檬酸盐缓冲液为柠檬酸-柠檬酸钠缓冲液。"Citrate buffer", also known as "citrate buffer", is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate buffer.
“醋酸缓冲液”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-醋酸钠、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-醋酸钠缓冲液。"Acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like. A preferred acetate buffer is acetic acid-sodium acetate buffer.
“琥珀酸缓冲液”是包括琥珀酸根离子的缓冲液。琥珀酸缓冲液的实例包括琥珀酸-琥珀酸钠、琥珀酸-琥珀酸钾、琥珀酸-琥珀酸钙、琥珀酸-琥珀酸镁等。优选的琥珀酸盐缓冲液为琥珀酸-琥珀酸钠缓冲液。A "succinate buffer" is a buffer that includes succinate ions. Examples of succinate buffers include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate, succinate-magnesium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate buffer.
“磷酸盐缓冲液”是包括磷酸根离子的缓冲液。磷酸盐缓冲液的实例包括磷酸二氢钠-磷酸氢二钠、磷酸二氢钾-磷酸氢二钾等。优选的磷酸盐缓冲液为磷酸二氢钠-磷酸氢二钠缓冲液。"Phosphate buffer" is a buffer that includes phosphate ions. Examples of the phosphate buffer include sodium dihydrogenphosphate-disodium hydrogenphosphate, potassium dihydrogenphosphate-dipotassium hydrogenphosphate, and the like. A preferred phosphate buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.
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、精氨酸或其盐(如盐酸精氨酸)、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RNAase A。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、氨基酸、盐、糖中的一种或一种以上。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。优选的氨基酸为精氨酸、甘氨酸、脯氨酸，氨基酸可以以其D-和/或L-型存在，但典型是L-型，氨基酸可以 任何合适的盐存在，例如盐酸盐，如盐酸精氨酸。优选的稳定剂为氯化钠、甘露醇、山梨醇、蔗糖、海藻糖、盐酸精氨酸、甘氨酸、脯氨酸、氯化钠-山梨醇、氯化钠-甘露醇、氯化钠-蔗糖、氯化钠-海藻糖、盐酸精氨酸-甘露醇、盐酸精氨酸-蔗糖。The term "stabilizer" denotes a pharmaceutically acceptable excipient which 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 or Salts (such as arginine hydrochloride), glycine, alanine (alpha-alanine, beta-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline N-oxidation of acid, 4-hydroxyproline, sarcosine, gamma-aminobutyric acid (GABA), opines, alanine, octopine, glycine, and trimethylamine (TMAO), human serum albumin (hsa), bovine serum albumin (BSA), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin and RNAase A. 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 more of polyols, amino acids, salts, and sugars. Preferred salts are sodium chloride, preferred sugars are sucrose and trehalose, preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine, glycine, proline, amino acids can exist in their D- and/or L-forms, but are typically L-forms, amino acids can Any suitable salt is present, for example a hydrochloride such as arginine hydrochloride. Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose , Sodium chloride-trehalose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose.
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)。本文中，如无特别说明，术语“聚山梨醇酯20的浓度”和“聚山梨醇酯80的浓度”均是指质量体积浓度(w/v)，如“约0.02％聚山梨醇酯80”中“0.02％”即指“100mL液体中含有0.02g的聚山梨醇酯80”。The term "surfactant" generally includes agents that protect proteins, such as antibodies, from air/solution interface-induced stress, solution/surface-induced stress, to reduce aggregation of antibodies, or to 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 ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene- Polyoxypropylene copolymer (poloxamer, Pluronic), sodium dodecyl sulfate (SDS). Herein, unless otherwise specified, the terms "the concentration of polysorbate 20" and "the concentration of polysorbate 80" all refer to the mass volume concentration (w/v), such as "about 0.02% polysorbate 80 "0.02%" means "100mL liquid contains 0.02g polysorbate 80".
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。The term "isotonic" means that the formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using vapor pressure or freezing point depression osmometers.
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)。The term "stable" formulation is one in which the antibody substantially retains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage. Pharmaceutical formulations may be stable even if the contained antibodies fail to retain 100% of their chemical structure or biological function after storage over a period of time. In certain instances, maintaining about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the structure or function of an antibody after storage over a period of time may also be considered a " stable". Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery 247-301, edited by Vincent Lee, Marcel Dekker, Inc ., New York, N.Y., Pubs. (1991), and Jones, A. (1993) Adv. Drug Delivery Rev. 10:29-90 (both incorporated by reference).
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。Stability can be measured by determining the percentage of natural antibody remaining (among other methods) in a formulation after storage at a temperature for a period of time. The percentage of native antibodies can be measured by size exclusion chromatography (eg, size exclusion high performance liquid chromatography [SEC-HPLC]), "native" meaning unaggregated and undegraded, among other methods. In some protocols, the stability of a protein is determined as the percentage of monomeric protein in solution with a low percentage of degraded (eg, fragmented) and/or aggregated protein. In some protocols, the formulation is stable 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 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, up to about 6%, 5%, 4%, 3%, 2%, 1%, 0.5% , or 0.1% aggregated form of the antibody.
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℃。Stability can be measured by determining (among other methods) the percentage of antibody ("acidic form") that migrates in the more acidic fraction of the antibody's main fraction ("primary charged form") during ion exchange, where stability is related to The percentage of acidic form of antibody is inversely proportional. The percentage of "acidified" antibodies can be measured by ion exchange chromatography (eg, cation exchange high performance liquid chromatography [CEX-HPLC]), among other methods. In some embodiments, an acceptable degree of stability means that no more than about 49%, 45%, 40%, 35% or more of the acidic form of the antibody can be detected in the formulation after storage at a certain temperature for a certain period of time. %, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%. The certain period of storage prior to measuring stability may 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. When assessing stability, certain temperatures that allow storage of pharmaceutical formulations 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, about 0°C , about 2-8°C, about 5°C, about 25°C, or about 40°C.
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。Antibodies are present in the pharmaceutical combination if they show substantially no signs of, e. "maintain its physical stability" in the substance. Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation may proceed to the extent that a visible precipitate is formed.
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。Stability of formulations, such as physical stability, can be assessed by methods known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements are related to the turbidity of the formulation. The turbidity of a formulation is, in part, an intrinsic property of proteins dissolved in solution, and is usually measured by nephelometric methods and is measured in nephelometric turbidity units (NTU).
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。The level of turbidity as a function of, for example, the concentration of one or more components in a solution (eg, protein and/or salt concentration) is also referred to as the "opacity" or "opacity appearance" of a formulation. Turbidity levels can be calculated by reference to a standard curve generated using suspensions of known turbidity. Reference standards for the determination of turbidity levels in pharmaceutical compositions can be based on the standards of the European Pharmacopoeia (European Pharmacopoeia, Fourth Edition, "European Pharmacopoeia"). According to the European Pharmacopoeia, a clear solution is defined as having a turbidity lower than or equal to that according to the European Pharmacopoeia Standard A solution that has a turbidity of a reference suspension of about 3. Nephelometric turbidity measurements detect Rayleigh scattering in the absence of association or non-ideal effects, which typically vary linearly with concentration. Used in Other methods of assessing physical stability are known in the art.
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。An antibody "retains its chemical stability" in a pharmaceutical composition if its chemical stability at a given point in time is such that the antibody is considered to still retain its biological activity as defined hereinafter. Chemical stability can be assessed by, for example, detecting or quantifying chemically altered forms of the antibody. Chemical alterations can include size alterations (e.g., clipping), 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 alterations include charge alterations (such as occur as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.
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CTLA-4抗体与CTLA-4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。An antibody "retains its biological activity" in a pharmaceutical composition if the antibody in the pharmaceutical composition is biologically active for its intended purpose. For example, if the preparation is stored for a certain period of time (for example, 1 to 12 months) at a temperature such as 5°C, 25°C, 45°C, etc., the binding affinity of the anti-CTLA-4 antibody contained in the preparation to CTLA-4 is equal to that of the storage A formulation of the invention is considered stable if it has at least 90%, 95% or more of the previous antibody binding affinity. Binding affinity can also be determined using, for example, ELISA or plasmon resonance techniques.
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。In the context of the present invention, a "therapeutically effective amount" or "effective amount" of an antibody in a pharmacological sense refers to an amount effective in the prevention or treatment or alleviation of symptoms of a disorder that the antibody can effectively treat. In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" of a drug is any amount of a drug that protects a subject from disease onset or promotes disease regression when used alone or in combination with another therapeutic agent, so Such regression of disease is evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of impairment or disability resulting from disease affliction. The ability of a drug to promote disease regression can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predictive of human efficacy, or by in vitro assays The activity of the agent is determined. A therapeutically effective amount of a drug includes a "prophylactically effective amount", i.e. any amount that inhibits the development or recurrence of disease when administered alone or as in combination with other therapeutic agents to a subject at risk of disease or to a subject with relapsed disease Drug.
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。The term "subject" or "patient" is intended to include mammalian organisms. Examples of 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. In a particular embodiment of the invention, the subject is a human.
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CTLA-4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。The terms "administering", "administering" and "treating" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those skilled in the art. Routes of administration of anti-CTLA-4 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion. "Parenteral administration" means administration other than enteral or topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intrasaccular , Intratracheal, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, intradural, and intrasternal injections and infusions and in vivo electroporation.
抗CTLA-4抗体anti-CTLA-4 antibody
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CTLA-4或其表位特异性结合能力的一个或多个片段。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、全人抗体、嵌合抗体和骆驼源化单结构域抗体。The term "antibody" as used herein should be understood to include whole antibody molecules and antigen-binding fragments thereof. The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply referred to as "antibody portion" or "antibody fragment") as used herein refers to an antibody that retains the ability to specifically bind to human CTLA-4 or its epitope. One or more fragments of . Accordingly, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric antibodies and camelized single domain antibodies.
术语“分离的抗体”是指结合化合物的纯化状态，且在这种情况下意指该分子基本不含其它生物分子，例如核酸、蛋白质、脂质、糖或其它物质例如细胞碎片和生长培养基。术语“分离(的)”并非意指完全不存在这类物质或不存在水、缓冲液或盐，除非它们以明显干扰本文所述结合化合物的实验或治疗应用的量存在。The term "isolated antibody" refers to the purified state of a binding compound, and in this context means that the molecule is substantially free of other biomolecules such as nucleic acids, proteins, lipids, sugars or other substances such as cell debris and growth media . The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compounds described herein.
术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" characterizes an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.
术语“鼠源抗体”或“杂交瘤抗体”在本公开中为根据本领域知识和技能制备的抗人CTLA-4的单克隆抗体。制备时用CTLA-4抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。The term "murine antibody" or "hybridoma antibody" in this disclosure refers to an anti-human CTLA-4 monoclonal antibody prepared according to the knowledge and skill in the art. In preparation, test subjects are injected with the CTLA-4 antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.
术语“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗 体，其中第一抗体和第二抗体来自不同物种。通常，可变结构域获自啮齿动物等的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体在人受试者中诱导不良免疫应答的可能性较低。The term "chimeric antibody" is an antibody having the variable domains of a first antibody and the constant domains of a second antibody. Individuals in which the primary and secondary antibodies are from different species. Typically, the variable domains are derived from an antibody of a rodent, etc. (the "parent antibody"), while the constant domain sequences are derived from a human antibody such that the resulting chimeric antibody induces greater The likelihood of an adverse immune response is low.
术语“人源化抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。The term "humanized antibody" refers to forms of antibodies that contain sequences from both human and non-human (eg, mouse, rat) antibodies. In general, a humanized antibody will comprise substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin. Framework (FR) regions are the framework regions of human immunoglobulin sequences. A humanized antibody optionally can comprise at least a portion of a human immunoglobulin constant region (Fc).
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。The term "full-length antibody" or "intact antibody molecule" refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70 kDa in full length) and two light (L) chains (full-length 25 kDa) are interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into complementarity determining regions (CDRs) of high variability separated by more conserved regions called framework regions (FRs). 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 amino-terminus to carboxy-terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin 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.
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。The term "CDR" refers to the complementarity determining regions within the variable sequences of an antibody. There are three CDRs in each variable region of the heavy and light chains, which are named HCDR1, HCDR2, and HCDR3 or LCDR1, LCDR2, and LCDR3 for each variable region of the heavy and light chains. The exact boundaries of these CDRs are defined differently according to different systems.
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。The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997) Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, USDepartment of Health and Human Services, National Institutes of Health (1987), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and neighbor propagation based on the use of a large number of crystal structures North CDR definition for affinity propagation clustering. The CDRs of the antibodies of the present invention can be bounded by those skilled in the art according to any scheme in the art (eg different assignment systems or combinations).
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scFv；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”)。As used herein, "antigen-binding fragment" includes fragments of antibodies or derivatives thereof, typically comprising at least a fragment of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody that retains at least some of the parent antibody's binding specificity. Examples of 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 scFv; specific antibody. Binding fragments or derivatives thereof generally retain at least 10% of the antigen-binding activity of the parent antibody when the binding activity of the antibody is expressed on a molar basis. Preferably, 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 contemplated that antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not appreciably alter their biological activity (referred to as "conservative variants" or "functionally conservative variants" of the antibody).
本发明所述的抗CTLA-4抗体或其抗原结合片段包括申请号为CN202010708105.8和PCT/CN2021/107707的专利申请中描述的任意一个抗CTLA-4抗体或其抗原结合片段，本文将申请号为CN 202010708105.8和PCT/CN2021/107707的专利申请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CN202010708105.8中描述的人源化抗CTLA-4抗体huJS007-47、huJS007-48、huJS007-79和huJS007-106，其氨基酸序列如表A所示。The anti-CTLA-4 antibody or antigen-binding fragment thereof described in the present invention includes any anti-CTLA-4 antibody or antigen-binding fragment thereof described in patent applications with application numbers CN202010708105.8 and PCT/CN2021/107707, which will be applied herein The entire contents disclosed in the patent applications CN 202010708105.8 and PCT/CN2021/107707 are incorporated herein by reference. In some aspects, the CDR sequences of the antibodies used in the methods and compositions of the invention include humanized anti-CTLA-4 antibodies huJS007-47, huJS007-48, huJS007-79 and huJS007 from CN202010708105.8 -106, the amino acid sequence of which is shown in Table A.
表A：4种人源化抗CTLA-4抗体的各部分的氨基酸序列(KABAT方案)

Table A: Amino acid sequences of parts of four humanized anti-CTLA-4 antibodies (KABAT protocol)

在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体或其抗原结合片段包含的LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3分别为：In some aspects, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprise, respectively:
LCDR1：RASQNVGTYVA(SEQ ID NO:1)；LCDR1: RASQNVGTYVA (SEQ ID NO: 1);
LCDR2：STSYRYS(SEQ ID NO:2)；LCDR2: STSYRYS (SEQ ID NO: 2);
LCDR3：HQYDTYPLT(SEQ ID NO:3)；LCDR3: HQYDTYPLT (SEQ ID NO: 3);
HCDR1：SGYYWN(SEQ ID NO:4)；HCDR1: SGYYWN (SEQ ID NO: 4);
HCDR2：YIGYDGSNX1YNPSLKX2，其中，X1为N或Y，X2为S或N；HCDR2: YIGYDGSNX1YNPSLKX2, where X1 is N or Y, X2 is S or N;
HCDR3：X3YYSGYFDS，X3为D或N。HCDR3: X3YYSGYFDS, X3 is D or N.
在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体或其抗原结合片段包含：In some aspects, the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention comprise:
氨基酸序列如SEQ ID NO:1所示的LCDR1；The amino acid sequence is LCDR1 shown in SEQ ID NO:1;
氨基酸序列如SEQ ID NO:2所示的LCDR2；The amino acid sequence is LCDR2 shown in SEQ ID NO:2;
氨基酸序列如SEQ ID NO:3所示的LCDR3；The amino acid sequence is LCDR3 shown in SEQ ID NO:3;
氨基酸序列如SEQ ID NO:4所示的HCDR1；The amino acid sequence is HCDR1 as shown in SEQ ID NO:4;
氨基酸序列如SEQ ID NO:5或7或9所示的HCDR2；The amino acid sequence is HCDR2 as shown in SEQ ID NO:5 or 7 or 9;
氨基酸序列如SEQ ID NO:6或8所示的HCDR3。The amino acid sequence is HCDR3 as shown in SEQ ID NO:6 or 8.
在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体或其抗原结合片段包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的LCDR1、LCDR2和LCDR3，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HCDR1、HCDR2和HCDR3。In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises an amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively LCDR1, LCDR2 and LCDR3, and amino acid sequences such as HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体或其抗原结合片段包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:7和SEQ ID NO:8所示的HCDR1、HCDR2和HCDR3。In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises an amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively LCDR1, LCDR2 and LCDR3, and amino acid sequences such as HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体或其抗原结合片段包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:9和 SEQ ID NO:6所示的HCDR1、HCDR2和HCDR3。In some aspects, the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively LCDR1, LCDR2 and LCDR3, and amino acid sequences such as SEQ ID NO:4, SEQ ID NO:9 and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:6.
在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体或其抗原结合片段选自鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、人源化抗体或其抗原结合片段，优选为人源化抗体或其抗原结合片段。In some aspects, the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention is selected from murine antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, humanized antibodies or an antigen-binding fragment thereof, preferably a humanized antibody or an antigen-binding fragment thereof.
在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体或其抗原结合片段包含如SEQ ID NO:10所示的轻链可变区，和如SEQ ID NO:11所示的重链可变区。In some aspects, the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises a light chain variable region as set forth in SEQ ID NO: 10, and a light chain variable region as set forth in SEQ ID NO: 11 The heavy chain variable region shown.
在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体或其抗原结合片段包含如SEQ ID NO:12所示的轻链可变区，和如SEQ ID NO:11所示的重链可变区。In some aspects, the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises a light chain variable region as set forth in SEQ ID NO: 12, and a light chain variable region as set forth in SEQ ID NO: 11 The heavy chain variable region shown.
在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体或其抗原结合片段包含如SEQ ID NO:13所示的轻链可变区，和如SEQ ID NO:14所示的重链可变区。In some aspects, the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises a light chain variable region as set forth in SEQ ID NO: 13, and a light chain variable region as set forth in SEQ ID NO: 14 The heavy chain variable region shown.
在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体或其抗原结合片段包含如SEQ ID NO:13所示的轻链可变区，和如SEQ ID NO:15所示的重链可变区。In some aspects, the anti-CTLA-4 antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises a light chain variable region as set forth in SEQ ID NO: 13, and a light chain variable region as set forth in SEQ ID NO: 15 The heavy chain variable region shown.
在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体包含如SEQ ID NO:16所示的轻链氨基酸序列，和如SEQ ID NO:17所示的重链氨基酸序列。In some aspects, the anti-CTLA-4 antibody used in the methods and compositions of the invention comprises a light chain amino acid sequence as set forth in SEQ ID NO: 16, and a heavy chain amino acid sequence as set forth in SEQ ID NO: 17 .
在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体包含如SEQ ID NO:18所示的轻链氨基酸序列，和如SEQ ID NO:19所示的重链氨基酸序列。In some aspects, the anti-CTLA-4 antibody used in the methods and compositions of the invention comprises a light chain amino acid sequence as set forth in SEQ ID NO: 18, and a heavy chain amino acid sequence as set forth in SEQ ID NO: 19 .
在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体包含如SEQ ID NO:20所示的轻链氨基酸序列，和如SEQ ID NO:21所示的重链氨基酸序列。In some aspects, the anti-CTLA-4 antibody used in the methods and compositions of the invention comprises a light chain amino acid sequence as set forth in SEQ ID NO:20, and a heavy chain amino acid sequence as set forth in SEQ ID NO:21 .
在一些方案中，在本发明的方法和组合物中使用的抗CTLA-4抗体包含如SEQ ID NO:22所示的轻链氨基酸序列，和如SEQ ID NO:23所示的重链氨基酸序列。In some aspects, the anti-CTLA-4 antibody used in the methods and compositions of the invention comprises a light chain amino acid sequence as set forth in SEQ ID NO:22, and a heavy chain amino acid sequence as set forth in SEQ ID NO:23 .
在一些实施方式中，在本发明的方法和组合物中使用的抗CTLA-4抗体或其抗原结合片段为人源化抗体或嵌合抗体，且可包括人恒定区。在一些实施方式中，恒定区是选自人IgG1、IgG2、IgG3及IgG4恒定区组成的组；优选地，适用于本发明所述的方法和组合物的抗CTLA-4抗体或其抗原结合片段包含人IgG1或IgG4同种型的重链恒定区。 In some embodiments, the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention are humanized or chimeric antibodies and may include human constant regions. In some embodiments, the constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions; preferably, an anti-CTLA-4 antibody or antigen-binding fragment thereof suitable for use in the methods and compositions described herein Comprising the heavy chain constant region of the human IgGl or IgG4 isotype.
在一些实施方式中，本发明提供了一种用于制备如本文所述的抗CTLA-4抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下在本文所述的宿主细胞中表达所述抗体或其抗原结合片段，并从所述宿主细胞回收所表达的抗体或其抗原结合片段。In some embodiments, the present invention provides a method for preparing an anti-CTLA-4 antibody or antigen-binding fragment thereof as described herein, the method comprising performing the expression of the antibody or antigen-binding fragment thereof in a The antibody or antigen-binding fragment thereof is expressed in a host cell described herein under conditions and the expressed antibody or antigen-binding fragment thereof is recovered from the host cell.
本发明提供用于表达本发明的重组抗体的哺乳动物宿主细胞，包括可获自美国典型培养物保藏中心(ATCC)的许多永生化细胞系。这些尤其包括中国仓鼠卵巢(CHO)细胞、NS0、SP2/0细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞、A549细胞、293T细胞和许多其它细胞系。哺乳动物宿主细胞包括人、小鼠、大鼠、狗、猴、猪、山羊、牛、马和仓鼠细胞。通过测定哪种细胞系具有高表达水平来选择特别优选的细胞系。The invention provides mammalian host cells for expressing the recombinant antibodies of the invention, including the many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many others cell line. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.
在一个实施方式中，本发明提供制备抗CTLA-4抗体的方法，其中所述方法包括，将表达载体导入哺乳动物宿主细胞中时，通过将宿主细胞培养足够的一段时间，以允许抗体在宿主细胞中表达，或者更优选抗体分泌到宿主细胞生长的培养基中,来产生抗体。可采用标准蛋白质纯化方法从培养基中回收抗体。In one embodiment, the present invention provides a method for preparing an anti-CTLA-4 antibody, wherein the method comprises, when introducing the expression vector into a mammalian host cell, culturing the host cell for a sufficient period of time to allow the antibody to express in the host cell The antibody is produced by expressing it in the cell, or more preferably secreting the antibody into the medium in which the host cell is grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
很可能由不同细胞系表达或在转基因动物中表达的抗体彼此具有不同的糖基化。然而，由本文提供的核酸分子编码的或包含本文提供的氨基酸序列的所有抗体是本发明的组成部分，而不论抗体的糖基化如何。同样，在某些实施方式中，非岩藻糖基化抗体是有利的，因为它们通常在体外和体内具有比其岩藻糖基化对应物更强力的功效，并且不可能是免疫原性的，因为它们的糖结构是天然人血清IgG的正常组分。It is likely that antibodies expressed by different cell lines or expressed in transgenic animals have different glycosylation from each other. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the invention, regardless of the glycosylation of the antibody. Also, in certain embodiments, non-fucosylated antibodies are advantageous because they generally have more potent potency than their fucosylated counterparts in vitro and in vivo and are less likely to be immunogenic , because their sugar structure is a normal component of natural human serum IgG.
医药制剂pharmaceutical preparations
本发明提供了一种药物组合物，包含：(1)缓冲液；(2)抗CTLA-4抗体或其抗原结合片段。The present invention provides a pharmaceutical composition, comprising: (1) a buffer; (2) an anti-CTLA-4 antibody or an antigen-binding fragment thereof.
本发明所述药物组合物中的抗CTLA-4抗体或其抗原结合片段如本申请“抗CTLA-4抗体”部分任一实施方式所述。The anti-CTLA-4 antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention is as described in any embodiment of the "anti-CTLA-4 antibody" section of this application.
例如，本发明所述药物组合物中的抗CTLA-4抗体或其抗原结合片段包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的LCDR1、LCDR2和LCDR3，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HCDR1、HCDR2和HCDR3。优选地，上述抗CTLA-4抗体或其抗原结合片段选自鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、人源化抗体或其抗原结合片段，优选为人源化抗体或其抗原结合片段。优选地，上述抗CTLA-4抗体或其抗原结合片段包含如SEQ ID NO:10所示的轻链可变区，和如SEQ ID NO:11所示的重链可变区。更优选地，上述抗CTLA-4抗体包含如SEQ ID NO:16所示的轻链氨基酸序列，和如SEQ ID NO:17所示的重链氨基酸序列。For example, the anti-CTLA-4 antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequence are respectively shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 HCDR1, HCDR2 and HCDR3 are indicated. Preferably, the above-mentioned anti-CTLA-4 antibody or antigen-binding fragment thereof is selected from murine antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, preferably humanized antibodies or its antigen-binding fragment. Preferably, the above-mentioned anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a light chain variable region as shown in SEQ ID NO:10, and a heavy chain variable region as shown in SEQ ID NO:11. More preferably, the above-mentioned anti-CTLA-4 antibody comprises the light chain amino acid sequence shown in SEQ ID NO:16, and the heavy chain amino acid sequence shown in SEQ ID NO:17.
本发明所述药物组合物中的抗CTLA-4抗体或其抗原结合片段的浓度为约1～100mg/mL，优选为约1～50mg/mL，更优选为约5～30mg/mL。The concentration of the anti-CTLA-4 antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention is about 1-100 mg/mL, preferably about 1-50 mg/mL, more preferably about 5-30 mg/mL.
本发明所述药物组合物的pH为约4.5～6.5，优选为约5.0～6.0，更优选为约5.3～5.7。The pH of the pharmaceutical composition of the present invention is about 4.5-6.5, preferably about 5.0-6.0, more preferably about 5.3-5.7.
本发明所述药物组合物中的缓冲液选自醋酸缓冲液、柠檬酸缓冲液、磷酸盐缓冲液和组氨酸缓冲液中的一种或多种；优选地，所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，优选为醋酸-醋酸钠缓冲液。The buffer in the pharmaceutical composition of the present invention is selected from one or more of acetate buffer, citrate buffer, phosphate buffer and histidine buffer; preferably, the buffer is acetate buffer liquid. Preferably, the acetic acid buffer is acetic acid-sodium acetate buffer or acetic acid-potassium acetate buffer, preferably acetic acid-sodium acetate buffer.
优选地，缓冲液的浓度为约5～100mM，优选为约10～50mM，优选为约10～30mM；优选为约15～25mM。优选地，缓冲液的pH为约4.5～6.5，优选为约5.0～6.0，优选为约5.3～5.7。Preferably, the concentration of the buffer is about 5-100 mM, preferably about 10-50 mM, preferably about 10-30 mM; preferably about 15-25 mM. Preferably, the pH of the buffer is about 4.5-6.5, preferably about 5.0-6.0, preferably about 5.3-5.7.
因此，本发明的药物组合物可含有：pH约为5.0～6.0的醋酸缓冲液，其在药物组合物中的浓度约为10～30mM；和约1～50mg/mL的前文任一实施方式所述的抗CTLA-4抗体或其抗原结合片段，尤其是本文所述的人源化抗体huJS007-47或其抗原结合片段。Therefore, the pharmaceutical composition of the present invention may contain: an acetate buffer solution with a pH of about 5.0-6.0, and its concentration in the pharmaceutical composition is about 10-30 mM; Anti-CTLA-4 antibodies or antigen-binding fragments thereof, especially the humanized antibody huJS007-47 or antigen-binding fragments thereof described herein.
在一些方案中，本发明所述药物组合物还包括稳定剂，所述稳定剂选自精氨酸、精氨酸盐、氯化钠、甘露醇、山梨醇、蔗糖和海藻糖中的一种或多种；优选地，上述精氨酸盐为盐酸精氨酸。优选地，上述稳定剂的浓度为约10～400mM，优选为约100～300mM，优选为约130～280mM，优选为约200～260mM。优选地，上述稳定剂为浓度约130～280mM的甘露醇；或上述稳定剂为浓度约130～280mM的蔗糖；或上述稳定剂为浓度约20～80mM的氯化钠与浓度约110-170mM的甘露醇的组合；或上述稳定剂为浓度约20～80mM的氯化钠与浓度约110-170mM的蔗糖的组合；在一些方案中，上述稳定剂为浓度约200～260mM的甘露醇；或上述稳定剂为浓度 约200～260mM的蔗糖；或上述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的甘露醇的组合；或上述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的蔗糖的组合。In some schemes, the pharmaceutical composition of the present invention also includes a stabilizer, and the stabilizer is selected from one of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose and trehalose or more; preferably, the above-mentioned arginine salt is arginine hydrochloride. Preferably, the above stabilizer has a concentration of about 10-400 mM, preferably about 100-300 mM, preferably about 130-280 mM, preferably about 200-260 mM. Preferably, the aforementioned stabilizer is mannitol with a concentration of about 130-280mM; or the aforementioned stabilizer is sucrose with a concentration of about 130-280mM; or the aforementioned stabilizer is sodium chloride with a concentration of about 20-80mM and A combination of mannitol; or the above-mentioned stabilizer is a combination of sodium chloride at a concentration of about 20-80mM and sucrose at a concentration of about 110-170mM; in some embodiments, the above-mentioned stabilizer is mannitol at a concentration of about 200-260mM; or the above-mentioned The stabilizer is the concentration about 200-260mM sucrose; or the above-mentioned stabilizer is a combination of sodium chloride with a concentration of about 30-70mM and mannitol with a concentration of about 120-160mM; or the above-mentioned stabilizer is a combination of sodium chloride with a concentration of about 30-70mM and a concentration of about A combination of 120-160 mM sucrose.
因此，本发明的药物组合物可含有：pH约为5.0～6.0的醋酸缓冲液，其在药物组合物中的浓度约为10～30mM；和约1～50mg/mL的前文任一实施方式所述的抗CTLA-4抗体或其抗原结合片段，尤其是本文所述的人源化抗体huJS007-47或其抗原结合片段；以及约130～280mM的稳定剂，优选地，所述稳定剂选自精氨酸、精氨酸盐、氯化钠、甘露醇、山梨醇、蔗糖和海藻糖中的一种或多种，优选地，上述精氨酸盐为盐酸精氨酸。优选地，上述稳定剂为浓度约200～260mM的蔗糖；或上述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的甘露醇的组合；或上述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的蔗糖的组合。Therefore, the pharmaceutical composition of the present invention may contain: an acetate buffer solution with a pH of about 5.0-6.0, and its concentration in the pharmaceutical composition is about 10-30 mM; anti-CTLA-4 antibody or antigen-binding fragment thereof, especially the humanized antibody huJS007-47 or antigen-binding fragment thereof described herein; and a stabilizer of about 130-280 mM, preferably, the stabilizer is selected from sperm One or more of amino acid, arginine salt, sodium chloride, mannitol, sorbitol, sucrose and trehalose, preferably, the above-mentioned arginine salt is arginine hydrochloride. Preferably, the above-mentioned stabilizer is sucrose with a concentration of about 200-260mM; or the above-mentioned stabilizer is a combination of sodium chloride with a concentration of about 30-70mM and mannitol with a concentration of about 120-160mM; A combination of 70 mM sodium chloride and sucrose at a concentration of about 120-160 mM.
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和泊洛沙姆188中的一种或多种。优选地，以w/v计算，上述表面活性剂浓度为约0.001％～0.1％，优选为约0.01％～0.1％，优选为约0.01％～0.05％。In some embodiments, the above pharmaceutical composition further includes a surfactant, and the surfactant is selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188. Preferably, the above-mentioned surfactant concentration is about 0.001%-0.1%, preferably about 0.01%-0.1%, preferably about 0.01%-0.05%, calculated by w/v.
因此，本发明的药物组合物可含有：pH约为5.0～6.0的醋酸缓冲液，其在药物组合物中的浓度约为10～30mM；和约1～50mg/mL的前文任一实施方式所述的抗CTLA-4抗体或其抗原结合片段，尤其是本文所述的人源化抗体huJS007-47或其抗原结合片段；以及约130～280mM的稳定剂，优选地，所述稳定剂选自精氨酸、精氨酸盐、氯化钠、甘露醇、山梨醇、蔗糖和海藻糖中的一种或多种，优选地，上述精氨酸盐为盐酸精氨酸。优选地，上述稳定剂为浓度约200～260mM的蔗糖；或上述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的甘露醇的组合；或上述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的蔗糖的组合；以及以w/v计，约0.01％～0.1％的聚山梨醇酯80。Therefore, the pharmaceutical composition of the present invention may contain: an acetate buffer solution with a pH of about 5.0-6.0, and its concentration in the pharmaceutical composition is about 10-30 mM; anti-CTLA-4 antibody or antigen-binding fragment thereof, especially the humanized antibody huJS007-47 or antigen-binding fragment thereof described herein; and a stabilizer of about 130-280 mM, preferably, the stabilizer is selected from sperm One or more of amino acid, arginine salt, sodium chloride, mannitol, sorbitol, sucrose and trehalose, preferably, the above-mentioned arginine salt is arginine hydrochloride. Preferably, the above-mentioned stabilizer is sucrose with a concentration of about 200-260mM; or the above-mentioned stabilizer is a combination of sodium chloride with a concentration of about 30-70mM and mannitol with a concentration of about 120-160mM; 70 mM sodium chloride in combination with sucrose at a concentration of about 120-160 mM; and polysorbate 80 at about 0.01%-0.1% w/v.
本发明的药物组合物为液体制剂或冻干制剂。The pharmaceutical composition of the present invention is a liquid formulation or a freeze-dried formulation.
本发明还提供了一种注射剂，其含有本文任一项方案中所述的药物组合物与氯化钠溶液或葡萄糖溶液；优选地，所述氯化钠溶液浓度为约0.85～0.9％(w/v)；优选地，所述葡萄糖溶液浓度为约5～25％(w/v)，更优选为约5～10％(w/v)；优选地，所述注射剂中，所述抗CTLA-4抗体的浓度为约0.05～10.5mg/mL，更优 选为约0.1～5mg/mL或约1～5mg/mL；所述注射剂的pH为约4.5～6.5，优选为约5.0～6.0，更优选为约5.3～5.7。The present invention also provides an injection, which contains the pharmaceutical composition described in any scheme herein and sodium chloride solution or glucose solution; preferably, the concentration of the sodium chloride solution is about 0.85-0.9% (w /v); Preferably, the concentration of the glucose solution is about 5-25% (w/v), more preferably about 5-10% (w/v); Preferably, in the injection, the anti-CTLA -4 antibody concentration is about 0.05-10.5mg/mL, more preferably It is selected as about 0.1-5 mg/mL or about 1-5 mg/mL; the pH of the injection is about 4.5-6.5, preferably about 5.0-6.0, more preferably about 5.3-5.7.
在一些方案中，上述药物组合物或注射剂，其经静脉注射施用。In some aspects, the above-mentioned pharmaceutical composition or injection is administered through intravenous injection.
医药用途和方法Medicinal uses and methods
本发明还提供了本文任一项方案中所述的药物组合物或注射剂在制备用于治疗和/或预防CTLA-4介导的疾病或病症的药物中的用途。The present invention also provides the use of the pharmaceutical composition or injection described in any of the schemes herein in the preparation of medicines for treating and/or preventing CTLA-4-mediated diseases or disorders.
本发明还提供了本文任一项方案中所述的药物组合物或注射剂，其用于治疗和/或预防CTLA-4介导的疾病或病症。The present invention also provides the pharmaceutical composition or injection described in any one of the schemes herein, which is used for treating and/or preventing CTLA-4-mediated diseases or conditions.
本发明还提供了一种治疗和/或预防CTLA-4介导的疾病或病症的方法，其包括向有需要的受试者施用如本文任一项方案中所述的药物组合物或注射剂。The present invention also provides a method for treating and/or preventing CTLA-4-mediated diseases or conditions, which comprises administering the pharmaceutical composition or injection as described in any one of the regimens herein to a subject in need.
本发明中，CTLA-4介导的疾病指在CTLA-4参与了疾病的发生和发展的疾病，包括但不限于癌症。In the present invention, CTLA-4-mediated diseases refer to diseases in which CTLA-4 is involved in the occurrence and development of diseases, including but not limited to cancer.
“癌症”和“癌性”指或描述哺乳动物中特征通常为细胞生长不受调控的生理疾患。此定义中包括良性和恶性癌症以及休眠肿瘤或微转移。癌症的例子包括但不限于癌，淋巴瘤，母细胞瘤，肉瘤，和白血病。此类癌症的更具体例子包括鳞状细胞癌，肺癌(包括小细胞肺癌，非小细胞肺癌，肺的腺癌，和肺的鳞癌)，腹膜癌，肝细胞癌，胃的癌或胃癌(包括胃肠癌)，胰腺癌，成胶质细胞瘤，***，卵巢癌，肝癌，膀胱癌，肝瘤(hepatoma)，乳腺癌，结肠癌，结肠直肠癌，子宫内膜癌或子宫癌，唾液腺癌，肾癌或肾的癌，鼻咽癌，食管鳞状细胞癌，肝癌，***癌，外阴癌，甲状腺癌，肝的癌，及各种类型的头颈癌，以及B细胞淋巴瘤(包括低级/滤泡性非何杰金氏淋巴瘤(NHL)，小淋巴细胞性(SL)NHL，中级/滤泡性NHL，中级弥漫性NHL，高级成免疫细胞性NHL，高级成淋巴细胞性NHL，高级小无核裂细胞性NHL，贮积病(bulky disease)NHL，套细胞淋巴瘤，AIDS相关淋巴瘤，和瓦尔登斯特伦氏(Waldenstrom)巨球蛋白血症)，慢性淋巴细胞性白血病(CLL)，急性成淋巴细胞性白血病(ALL)，毛细胞性白血病，慢性成髓细胞性白血病，和移植后淋巴增殖性病症(PTLD)，以及与瘢痣病(phakomatoses)，水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖。 "Cancer" and "cancerous" refer to or describe the physiological condition in mammals that is often characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal carcinoma, hepatocellular carcinoma, carcinoma of the stomach or gastric cancer ( Including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, Salivary gland cancer, renal cancer or renal carcinoma, nasopharyngeal carcinoma, esophageal squamous cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, and B-cell lymphoma (including Low-grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL , high-grade small anucleocytic NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic Leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplantation lymphoproliferative disorder (PTLD), and in association with phakomatoses, edema (such as associated with brain tumors) and abnormal blood vessel proliferation associated with Meigs' syndrome.
实施例Example
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。本文已经详细描述了本发明，其中也公开了其具体实施方式。对本领域的技术人员而言，在不脱离本发明精神和范围的情况下，针对本发明具体实施方式进行各种变化和改进将是显而易见的的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。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。The present invention will be illustrated below in the form of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The invention has been described in detail herein, and specific embodiments thereof are also disclosed. For those skilled in the art, without departing from the spirit and scope of the present invention, any modification, equivalent replacement, improvement, etc. that will be obvious to the specific embodiments of the present invention for various changes and improvements shall be included in the within the protection scope of the present invention. Methods and materials used in the examples, unless otherwise stated, are conventional methods and materials in the art.
实施例中所用的检测方法The detection method used in the embodiment
(1)外观(1) Appearance
采用目检法来检测外观。确保澄明度检测仪的光照强度保持在1000lx～1500lx之间。将样品保持在眼睛的同一水平面，轻轻摇晃或颠倒以避免产生气泡。分别在黑色背景和白色背景前进行目检。从颜色，乳光和可见异物三个方面记录结果。The appearance is checked by visual inspection. Make sure that the light intensity of the clarity detector is kept between 1000lx and 1500lx. Hold the sample at eye level and shake or invert gently to avoid air bubbles. Visual inspection was performed in front of black and white backgrounds, respectively. The results were recorded in terms of color, opalescence and visible foreign matter.
(2)蛋白含量(2) Protein content
蛋白浓度使用紫外分光光度计(Thermofisher，BIO MATE 3S)来检测。将百分比消光系数(E1％)设定在1.5367(mg/ml)-1cm-1。使用BIO MATE 3S仪器，用超纯水清洗比色皿三次再向皿中加入150μL超纯水，点击测量，以超纯水做空白校正，空白样品吸光度不超出±0.003。使用超纯水稀释至0.3mg/ml～0.7mg/ml，每份终体积不得低于900μL，单次稀释倍数不得超过10倍。每个样品平行测定2份溶液，每份溶液重复测定3次；每份溶液测定之前均需先用150μL溶液润洗比色皿2次，然后取150μL溶液进行测定。通过消光系数及OD值计算出对应检品的浓度。Protein concentration was detected using a UV spectrophotometer (Thermofisher, BIO MATE 3S). The percent extinction coefficient (E1%) was set at 1.5367 (mg/ml)-1 cm-1. Use the BIO MATE 3S instrument, wash the cuvette with ultrapure water three times, then add 150 μL of ultrapure water to the cuvette, click to measure, and use ultrapure water for blank calibration. The absorbance of the blank sample does not exceed ±0.003. Use ultrapure water to dilute to 0.3mg/ml~0.7mg/ml, the final volume of each part should not be less than 900μL, and the single dilution factor should not exceed 10 times. Two solutions were measured in parallel for each sample, and the measurement was repeated three times for each solution; each solution was rinsed with 150 μL solution for 2 times before the measurement, and then 150 μL solution was taken for measurement. The concentration of the corresponding test substance was calculated by the extinction coefficient and OD value.
(3)SEC-HPLC纯度(3) SEC-HPLC purity
SEC-HPLC纯度采用安装了SEC色谱柱(TOSOH G4000sxl SEC 7.8mm ID×30cm,8μm)的HPLC(Waters e2695仪器)进行检测。流动相组成为50mM三羟甲基氨基甲烷(Tris)，150mM NaCl，pH 7.4。采用峰面积归一化对结果进行定量分析。分别计算单体、聚体和片段的峰面积百分比，以单体的峰面积百分比作为样品的纯度，聚体和片段的峰面积百分比作为聚体和片段的含量。色谱参数见下表1。SEC-HPLC purity was detected by HPLC (Waters e2695 instrument) equipped with SEC chromatographic column (TOSOH G4000sxl SEC 7.8mm ID×30cm, 8 μm). The mobile phase consisted of 50 mM Tris (Tris), 150 mM NaCl, pH 7.4. The results were quantified by peak area normalization. The peak area percentages of monomers, aggregates and fragments were calculated respectively, the peak area percentages of monomers were used as the purity of the sample, and the peak area percentages of aggregates and fragments were regarded as the contents of aggregates and fragments. The chromatographic parameters are shown in Table 1 below.
表1：SEC-HPLC色谱参数
Table 1: SEC-HPLC chromatographic parameters
(4)R-CE-SDS纯度(4) R-CE-SDS purity
抗体制剂还原CE-SDS电泳法纯度检测以高压直流电场为驱动力，以毛细管为分离通道。预先填充的凝胶会在毛细管内形成分子筛，十二烷基硫酸钠可消除不同蛋白质分子的电荷效应，还原剂β-巯基乙醇可切除样品中的二硫键，分子大小不同的样品在毛细管内移动速度不同，因而可进行分离。用上样缓冲液(SDS-MWSample Buffer)将样品稀释至1mg/mL；取上样缓冲液(SDS-MW Sample Buffer)95μL，加入β-巯基乙醇5μL，涡旋混匀后作为空白对照。取供试品(1mg/mL)95μL，加入β-巯基乙醇5μL，3000rpm室温离心30sec，70±2℃孵育15±2min，冷却至室温，6000rpm室温离心1min，分离时间40min，使用安装了CE-SDS卡盒的毛细管电泳仪(Maurice仪器)检测。计算重链(HC)，非糖基化重链(NGHC)和轻链(LC)的纯度，三者之和即为样品的纯度。The purity detection of antibody preparation reduction CE-SDS electrophoresis uses a high-voltage direct current electric field as the driving force and a capillary as the separation channel. The pre-filled gel will form a molecular sieve in the capillary, sodium lauryl sulfate can eliminate the charge effect of different protein molecules, the reducing agent β-mercaptoethanol can cut the disulfide bond in the sample, and samples with different molecular sizes are in the capillary. Different speeds of movement allow separation. Dilute the sample to 1 mg/mL with the sample buffer (SDS-MW Sample Buffer); take 95 μL of the sample buffer (SDS-MW Sample Buffer), add 5 μL of β-mercaptoethanol, vortex and mix well as a blank control. Take 95 μL of the test substance (1 mg/mL), add 5 μL of β-mercaptoethanol, centrifuge at room temperature at 3000rpm for 30sec, incubate at 70±2℃ for 15±2min, cool to room temperature, centrifuge at room temperature at 6000rpm for 1min, and separate for 40min. Capillary electrophoresis (Maurice Instruments) detection of SDS cartridges. Calculate the purity of the heavy chain (HC), non-glycosylated heavy chain (NGHC) and light chain (LC), and the sum of the three is the purity of the sample.
(5)NR-CE-SDS纯度(5) Purity of NR-CE-SDS
抗体制剂非还原CE-SDS电泳法纯度检测以高压直流电场为驱动力，以毛细管为分离通道。预先填充的凝胶会在毛细管内形成分子筛，用十二烷基硫酸钠处理样品以消除不同蛋白质分子的电荷效应，使分子大小不同而在毛细管内移动速度不同的样品进行分离。供试品溶液中加入烷基化试剂，能够有效减小组分扩散，使所得峰型尖锐，分离效率高，且可以保证样品保持非还原状态。用上样缓冲液(SDS-MWSample Buffer)将样品稀释至1mg/mL；取上样缓冲液(SDS-MW Sample Buffer)95μL，加入0.8M碘乙酰胺溶液5μL，涡旋混匀后作为空白对照；取供试品(1mg/mL)95μL，加入0.8M碘乙酰胺溶液5μL，3000rpm室温离心30sec，70±2℃孵育5±1min，冷却至室温，6000rpm室温离心1min，使用安装了CE-SDS卡盒的毛细管电泳仪(Maurice仪器)检测。The non-reducing CE-SDS electrophoresis method for purity detection of antibody preparations uses a high-voltage direct current electric field as the driving force and a capillary as the separation channel. The pre-filled gel will form a molecular sieve in the capillary, and the sample is treated with sodium dodecyl sulfate to eliminate the charge effect of different protein molecules, so that samples with different molecular sizes and different moving speeds in the capillary can be separated. Adding an alkylating agent to the test solution can effectively reduce the diffusion of the components, make the obtained peak shape sharp, and the separation efficiency is high, and it can ensure that the sample remains in a non-reducing state. Dilute the sample to 1 mg/mL with SDS-MW Sample Buffer; take 95 μL of SDS-MW Sample Buffer, add 5 μL of 0.8M iodoacetamide solution, vortex and mix well as a blank control ;Take 95μL of the test substance (1mg/mL), add 5μL of 0.8M iodoacetamide solution, centrifuge at room temperature at 3000rpm for 30sec, incubate at 70±2℃ for 5±1min, cool to room temperature, centrifuge at room temperature at 6000rpm for 1min, use CE-SDS installed Cartridge capillary electrophoresis (Maurice Instruments) detection.
(6)CEX-HPLC纯度 (6) CEX-HPLC purity
电荷异质性采用阳离子交换色谱法(CEX-HPLC)进行检测。样品经流动相A混合液稀释处理后，使用HPLC检测。检测方法参数具体见下表。采用峰面积归一化法计算主峰、酸性峰、碱性峰百分比，如果采用自动积分无法获得合理的积分结果，则采用手动积分。详细色谱参数见下表2。Charge heterogeneity was detected by cation exchange chromatography (CEX-HPLC). After the sample was diluted with mobile phase A mixture, it was detected by HPLC. The parameters of the detection method are shown in the table below. Use the peak area normalization method to calculate the percentages of main peaks, acidic peaks, and basic peaks. If automatic integration cannot obtain reasonable integration results, use manual integration. The detailed chromatographic parameters are shown in Table 2 below.
表2：CEX-HPLC色谱参数
Table 2: CEX-HPLC chromatographic parameters
(7)细胞活性(7) Cell activity
实验第一天用huJS007-47分析缓冲液(RPMI 1640 Medium+1％FBS)稀释huJS007-47抗体至80μg/ml(4×分析浓度)，1.8倍梯度稀释，10个浓度。CTLA-4 Fc Protein浓度稀释至8μg/ml(4×分析浓度)备用。将GS-J1/CD28细胞密度调整为2.0×106cells/ml，再加入PHA使其浓度为20μg/ml(4×分析浓度)备用。GS-C1/CD80细胞密度调整为1.0×106cells/ml备用。将huJS007-47抗体、CTLA-4 Fc Protein、GS-J1/CD28细胞和GS-C1/CD80细胞分别按照50μl/well加入到96孔圆底板中，置于37.0℃，5.0％二氧化碳培养箱中孵育24h。第二天每孔吸取16μl培养上清到HTRF 96 well low volume plate中，再加入4μl混合抗体(Cisbio Human IL2 kits)， 室温避光孵育18±2h，最后用多功能酶标仪读板(Tecan，M1000 Pro)和GraphPad Prism软件进行数据分析处理。计算公式为：Ratio 665/620＝OD 665nm/OD 620nm*10000。On the first day of the experiment, the huJS007-47 antibody was diluted to 80 μg/ml (4×analysis concentration) with huJS007-47 assay buffer (RPMI 1640 Medium+1% FBS), 1.8-fold serial dilution, 10 concentrations. The concentration of CTLA-4 Fc Protein was diluted to 8 μg/ml (4×analytical concentration) for use. The GS-J1/CD28 cell density was adjusted to 2.0×10 6 cells/ml, and PHA was added to make the concentration 20 μg/ml (4×analysis concentration) for later use. The density of GS-C1/CD80 cells was adjusted to 1.0×10 6 cells/ml for later use. Add huJS007-47 antibody, CTLA-4 Fc Protein, GS-J1/CD28 cells, and GS-C1/CD80 cells into 96-well round bottom plates at 50 μl/well, and incubate in a 5.0% carbon dioxide incubator at 37.0°C 24h. On the second day, pipette 16 μl of culture supernatant per well into HTRF 96 well low volume plate, then add 4 μl of mixed antibody (Cisbio Human IL2 kits), Incubate at room temperature in the dark for 18±2h, and finally use a multifunctional microplate reader (Tecan, M1000 Pro) and GraphPad Prism software for data analysis and processing. The calculation formula is: Ratio 665/620=OD 665nm/OD 620nm*10000.
(8)结合活性(8) Binding activity
结合ELISA(Binding ELISA)实验：使用酶标仪(Thermo Scientific，Multiskan Go)，用固定浓度的His标签CTLA4抗原(1.0μg/mL)包板，2％BSA封闭后，加入梯度稀释的抗体(1μg/ml起始，2.5倍梯度稀释共12个浓度)，将Anti-Human IgG(Fc Specific)-Peroxidase antibody produced in goat(Sigma，A0170)稀释5000作为检测抗体进行检测，然后用0.1mg/ml TMB显色，最后用2M HCl终止反应，在450nm/620nm下读板。使用四参数对数回归(4PL)模型拟合EC50。Binding ELISA (Binding ELISA) experiment: use a microplate reader (Thermo Scientific, Multiskan Go), coat the plate with a fixed concentration of His-tagged CTLA4 antigen (1.0 μg/mL), block with 2% BSA, and add a serially diluted antibody (1 μg /ml starting, 2.5 times serial dilution, a total of 12 concentrations), Anti-Human IgG (Fc Specific)-Peroxidase antibody produced in goat (Sigma, A0170) was diluted 5000 as the detection antibody for detection, and then 0.1mg/ml TMB The color was developed, and finally the reaction was terminated with 2M HCl, and the plate was read at 450nm/620nm. EC50s were fitted using a four parameter logarithmic regression (4PL) model.
(9)亚可见颗粒检测(9) Sub-visible particle detection
亚可见颗粒检测采用微粒检测仪(MFI5100)进行检测。由于样品为高浓度样品，上样前需将进行一定稀释，稀释后轻轻充分混匀避免气泡后于超净台中用无热原枪头取样1.3mL至上样板中，上样板以洁净的锡箔纸覆盖严密，从超净台移至仪器对应工作板块后，将上样位置输入仪器，运行检测序列，样品流经流通池时，其中亚可见颗粒由***头拍照并进行计数。The detection of sub-visible particles is carried out by a particle detector (MFI5100). Since the sample is a high-concentration sample, it needs to be diluted to a certain extent before loading the sample. After dilution, mix gently and fully to avoid air bubbles, then use a pyrogen-free pipette tip to sample 1.3mL into the sample plate in the ultra-clean bench, and use a clean tin foil paper on the sample plate The coverage is tight. After moving from the ultra-clean bench to the corresponding working plate of the instrument, input the sample loading position into the instrument, run the detection sequence, and when the sample flows through the flow cell, the sub-visible particles in it are photographed and counted by the micro camera.
以下实施例中的缩写说明：“h”表示小时，“W”表示周，“M”表示月，“C”表示冻融循环的次数，“FT”表示冻融循环，“SH”表示振摇，“ST”表示搅拌，“RT”表示室温，“rpm”表示转/分钟，“T0”表示处方样品经放样处理前的起始测试。Explanation of abbreviations in the following examples: "h" for hours, "W" for weeks, "M" for months, "C" for number of freeze-thaw cycles, "FT" for freeze-thaw cycles, "SH" for shaking , "ST" means stirring, "RT" means room temperature, "rpm" means revolutions per minute, and "T0" means the initial test of the prescription sample before it is set out.
实施例1：缓冲液体系和稳定剂初步筛选实验Embodiment 1: buffer system and stabilizer preliminary screening experiment
液体型药物组合物中，缓冲液体系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。本实施例旨在初步筛选出最佳缓冲液体系和稳定剂，使本发明公开的抗CTLA-4抗体具有最佳的稳定性以适宜临床应用。In liquid pharmaceutical compositions, the buffer system closely affects the stability of antibodies, and each antibody with unique physical and chemical properties has the most suitable type of buffer. This example aims to preliminarily screen out the best buffer system and stabilizer, so that the anti-CTLA-4 antibody disclosed in the present invention has the best stability and is suitable for clinical application.
1.1实验步骤1.1 Experimental steps
本实施例以抗体huJS007-47进行。样品使用Millipore Pellicon3 88cm2膜进行UF/DF超滤浓缩至浓度约21mg/mL，再将样品透析至对应的如表3所示的处方中，将最终浓度调至约20mg/mL，再加入对应浓度的聚山梨醇酯80。在超净台无菌灌装到2R西林瓶，2.0mL/瓶，进行稳定性放样和检测。 This example was performed with antibody huJS007-47. The sample was concentrated by UF/DF ultrafiltration using Millipore Pellicon3 88cm2 membrane to a concentration of about 21mg/mL, then the sample was dialyzed into the corresponding formula shown in Table 3, and the final concentration was adjusted to about 20mg/mL, and then the corresponding concentration was added Polysorbate 80. Aseptically fill it into 2R vials, 2.0mL/bottle, in a clean bench, and carry out stability sampling and testing.
表3：第一轮处方筛选-缓冲液体系和稳定剂初步筛选实验中的处方信息
Table 3: The first round of formulation screening - the formulation information in the preliminary screening experiment of buffer system and stabilizer
1.2实验结果1.2 Experimental results
1.2.1外观和浓度结果1.2.1 Appearance and concentration results
根据表4的结果，经加速条件或长期条件，所有样品的蛋白含量均未出现显著变化。According to the results in Table 4, there was no significant change in the protein content of all samples under accelerated or long-term conditions.
根据表5中的结果，所有样品在T0时均未发现明显可见异物，无明显乳光。经加速条件下放置4周，所有样品的外观均无显著变化；经长期条件下放置8周，除FS1-4出现蛋白沉淀外，其它所有样品的外观均未出现显著变化。According to the results in Table 5, no obvious visible foreign matter was found in all samples at T0, and there was no obvious opalescence. After being placed under accelerated conditions for 4 weeks, the appearance of all samples had no significant change; after being placed under long-term conditions for 8 weeks, the appearance of all samples had no significant change except for protein precipitation in FS1-4.
表4：第一轮处方筛选—蛋白含量数据
Table 4: The first round of formulation screening - protein content data
表5：第一轮处方筛选—外观数据

Table 5: First Round of Prescription Screening—Appearance Data

1.2.2 SEC纯度结果1.2.2 SEC purity results
根据表6中的SEC纯度结果，经加速条件下放置4周，7个处方聚体和片段均增加，处方FS1-7纯度下降明显，处方FS1-1、FS1-3和FS1-5表现相对较优；经长期条件放置下放置12周，所有处方均未出现SEC纯度的显著降低。According to the SEC purity results in Table 6, after being placed under accelerated conditions for 4 weeks, the aggregates and fragments of the 7 prescriptions all increased, the purity of the prescription FS1-7 decreased significantly, and the performance of the prescriptions FS1-1, FS1-3 and FS1-5 was relatively high. Excellent; after being placed under long-term conditions for 12 weeks, there was no significant decrease in the purity of SEC in all formulations.
表6：第一轮处方筛选—SEC-HPLC数据
Table 6: The first round of formulation screening—SEC-HPLC data
1.2.3 R-CE-SDS纯度结果1.2.3 R-CE-SDS purity results
根据表7中的R-CE-SDS纯度结果，经加速条件放置4周处方FS1-7纯度下降明显其他组间差异不大，经长期条件放置4周，所有样品均未出现显著变化。According to the R-CE-SDS purity results in Table 7, the purity of prescription FS1-7 decreased significantly after being placed under accelerated conditions for 4 weeks.
表7：第一轮处方筛选—R-CE-SDS数据
Table 7: The first round of prescription screening—R-CE-SDS data
1.2.4 NR-CE-SDS纯度结果1.2.4 NR-CE-SDS purity results
根据表8中的NR-CE-SDS纯度结果，经加速条件放置4周所有样品纯度均出现下降，且处方FS1-7下降明显，其他组间差异不大，经长期条件放置4周，处方FS1-4、FS1-5、FS1-6和FS1-7纯度下降。According to the NR-CE-SDS purity results in Table 8, the purity of all samples decreased after being placed under accelerated conditions for 4 weeks, and the prescription FS1-7 decreased significantly, and there was little difference between other groups. After being placed under long-term conditions for 4 weeks, the prescription FS1 -4, FS1-5, FS1-6 and FS1-7 decreased in purity.
表8：第一轮处方筛选—NR-CE-SDS数据
Table 8: The first round of prescription screening—NR-CE-SDS data
注：HHL是指具有两条重链和一条轻链的抗体碎片。NOTE: HHL refers to antibody fragments with two heavy chains and one light chain.
1.2.5 CEX-HPLC纯度结果 1.2.5 CEX-HPLC purity results
根据表9中的CEX-HPLC纯度结果，经加速条件下放置4周，所有样品均出现酸碱峰增加主峰纯度降低，其中FS1-1、FS1-3、FS1-5和FS1-6四个处方主峰纯度降低较慢，处方FS1-7稳定性表现最差；经长期条件下放置放置8周，所有样品均未出现纯度显著降低。According to the CEX-HPLC purity results in Table 9, after being placed under accelerated conditions for 4 weeks, all samples showed an increase in the acid-base peak and a decrease in the purity of the main peak, of which FS1-1, FS1-3, FS1-5 and FS1-6 were four prescriptions The purity of the main peak decreased slowly, and the stability of the prescription FS1-7 was the worst; after being placed under long-term conditions for 8 weeks, all samples did not show a significant decrease in purity.
表9：第一轮处方筛选—CEX-HPLC数据
Table 9: The first round of formulation screening—CEX-HPLC data
1.2.6结合活性结果1.2.6 Binding activity results
根据表10中的结合活性结果，经加速或长期条件下放置4周，所有处方均未出现活性降低。According to the binding activity results in Table 10, none of the formulations exhibited a decrease in activity after 4 weeks of storage under accelerated or chronic conditions.
表10：第一轮处方筛选—结合活性数据

Table 10: First Round Formulation Screening - Binding Activity Data

1.2.7细胞活性结果1.2.7 Cell Viability Results
根据表11中的细胞活性结果，经加速或长期条件下放置4周，所有处方均未出现活性降低。According to the cell viability results in Table 11, all formulations showed no decrease in activity after being placed under accelerated or long-term conditions for 4 weeks.
表11：第一轮处方筛选—细胞活性数据
Table 11: First round of formulation screening - cell viability data
1.2.8亚可见颗粒结果1.2.8 Sub-visible particle results
根据表12中的亚可见颗粒结果，经加速条件下放置4周，所有处方样品的颗粒数没有显著增加；经长期条件下放置8W，处方FS1-4颗粒明显增多与外观出现蛋白沉淀一致，其他所有处方样品的颗粒数没有显著增加。According to the results of sub-visible particles in Table 12, after being placed under accelerated conditions for 4 weeks, the number of particles in all prescription samples did not increase significantly; after being placed under long-term conditions for 8W, the number of particles in the prescription FS1-4 increased significantly, which was consistent with the appearance of protein precipitation. Others There was no significant increase in particle counts for all formulation samples.
表12：第一轮处方筛选—亚可见颗粒数据

Table 12: First Round Formulation Screening—Subvisible Particle Data

1.3第一轮处方筛选结论1.3 Conclusion of the first round of prescription screening
从蛋白含量、结合活性结果来看，所有处方未出现显著差异。从外观来看，处方FS1-4外观长期8周出现蛋白沉淀；从SEC-HPLC结果来看，处方FS1-7聚体增加明显；从CEX-HPLC结果来看处方FS1-2、FS1-4和FS1-7主峰纯度下降明显；从NR-CE-SDS和R-CE-SDS结果来看，处方FS1-7纯度下降明显。From the results of protein content and binding activity, there was no significant difference among all the formulations. From the appearance, the appearance of prescription FS1-4 showed protein precipitation for 8 weeks; from the results of SEC-HPLC, the 7-mer of prescription FS1-7 increased significantly; from the results of CEX-HPLC, the results of prescription FS1-2, FS1-4 and The purity of the main peak of FS1-7 decreased significantly; from the results of NR-CE-SDS and R-CE-SDS, the purity of prescription FS1-7 decreased significantly.
综上所述，醋酸缓冲液(FS1-3)和枸橼酸缓冲液(FS1-5)表现相对较优。因此选择20Mm醋酸缓冲液(pH5.5)和20Mm枸橼酸缓冲液(pH6.0)作为缓冲体系进入下一轮筛选实验。In summary, acetate buffer (FS1-3) and citrate buffer (FS1-5) performed relatively better. Therefore, 20Mm acetate buffer (pH5.5) and 20Mm citrate buffer (pH6.0) were selected as the buffer system to enter the next round of screening experiments.
实施例2：稳定剂和表面活性剂筛选实验Embodiment 2: stabilizer and surfactant screening experiment
为了进一步探究不同辅料对抗体稳定性影响，我们选取不同的稳定剂和表面活性剂进行了比较测试。考察pH5.5、20mM醋酸缓冲体系和pH6.0、20mM柠檬酸缓冲体系，抗体huJS007-47浓度10mg/mL条件下，上述不同辅料对稳定性的影响。In order to further explore the effect of different excipients on antibody stability, we selected different stabilizers and surfactants for comparative testing. Investigate the effect of the above different excipients on the stability under the conditions of pH5.5, 20mM acetic acid buffer system and pH6.0, 20mM citric acid buffer system, and antibody huJS007-47 concentration of 10mg/mL.
2.1实验步骤2.1 Experimental steps
本实施例以抗体huJS007-47进行。样品使用Millipore Pellicon3 0.11m2膜进行UF/DF超滤浓缩至浓度约10mg/mL，再将样品透析至对应的如表13所示的处方中，将最终浓度调至约10mg/mL，再加入对应浓度的聚山梨醇酯80。在超净台无菌灌装到2R西林瓶，2.0mL/瓶，进行稳定性放样和检测。This example was performed with antibody huJS007-47. The sample was concentrated by UF/DF ultrafiltration using Millipore Pellicon3 0.11m2 membrane to a concentration of about 10mg/mL, and then the sample was dialyzed into the corresponding formula shown in Table 13, and the final concentration was adjusted to about 10mg/mL, and then the corresponding concentration of polysorbate 80. Aseptically fill it into 2R vials, 2.0mL/bottle, in a clean bench, and carry out stability sampling and testing.
表13：第二轮处方筛选-稳定剂和表面活性剂筛选实验中的处方信息

注“/”表示无。Table 13: Second Round of Formulation Screening - Prescription Information in Stabilizer and Surfactant Screening Experiments

Note "/" means none.
2.2实验结果2.2 Experimental results
2.2.1外观和浓度结果2.2.1 Appearance and concentration results
根据表14中的结果，经加速条件和长期条件下放置4周，所有样品的蛋白含量均未出现显著变化。According to the results in Table 14, there was no significant change in the protein content of all samples after being placed under accelerated conditions and long-term conditions for 4 weeks.
根据表15中的结果，所有样品在T0时均未发现明显可见异物，无明显乳光。经加速条件放置4周，FS2-5、FS2-12两组处方外观上出现蛋白沉淀，其余处方外 观正常。经长期条件下放置3个月均未发现明显可见异物。According to the results in Table 15, no visible foreign matter was found in all samples at T0, and no obvious opalescence was found. After being placed under accelerated conditions for 4 weeks, protein precipitation appeared on the appearance of the FS2-5 and FS2-12 prescriptions, and the rest of the prescriptions It looks normal. No obvious foreign matter was found after being placed under long-term conditions for 3 months.
表14：第二轮处方筛选—蛋白含量数据
Table 14: The second round of formulation screening - protein content data
表15：第二轮处方筛选—外观数据

Table 15: Second Round of Prescription Screening—Appearance Data

2.2.2 SEC-HPLC纯度结果2.2.2 SEC-HPLC purity results
根据表16中的SEC纯度结果，所有样品经加速条件放置4W均出现聚集体和片段的增加，处方FS2-3和FS2-4下降速率较慢表现较优，长期条件下放置3M，所有样品单体纯度未出现明显下降。According to the SEC purity results in Table 16, all samples were placed under accelerated conditions for 4W to increase aggregates and fragments, and prescriptions FS2-3 and FS2-4 performed better with slower decline rates, and were placed under long-term conditions for 3M. There was no significant decrease in body purity.
表16：第二轮处方筛选—SEC-HPLC数据


Table 16: The second round of formulation screening—SEC-HPLC data


2.2.3 R-CE-SDS纯度结果2.2.3 R-CE-SDS purity results
根据表17中的R-CE-SDS纯度结果，经加速条件下放置4周，所有样品均出现纯度下降，组间纯度差异不大。According to the R-CE-SDS purity results in Table 17, after being placed under accelerated conditions for 4 weeks, the purity of all samples decreased, and there was little difference in purity between groups.
表17：第二轮处方筛选—R-CE-SDS数据
Table 17: The second round of prescription screening—R-CE-SDS data
2.2.4 NR-CE-SDS纯度结果2.2.4 NR-CE-SDS purity results
根据表18中的NR-CE-SDS纯度结果，经加速条件下放置4周，所有样品均出现纯度下降，组间纯度差异不大。According to the NR-CE-SDS purity results in Table 18, after being placed under accelerated conditions for 4 weeks, the purity of all samples decreased, and there was little difference in purity between groups.
表18：第二轮处方筛选—NR-CE-SDS数据
Table 18: Second Round of Prescription Screening—NR-CE-SDS Data
2.2.5 CEX-HPLC纯度结果2.2.5 CEX-HPLC purity results
根据表19中的CEX-HPLC纯度结果，经长期条件下放置4周，所有样品均未出现纯度的显著变化。经加速条件下放置4周，所有样品均出现主峰纯度下降，处方FS2-2、FS2-5、FS2-6和FS2-10酸碱峰增加明显，但未表现出显著的组间差异。According to the CEX-HPLC purity results in Table 19, there was no significant change in the purity of all samples after being placed under long-term conditions for 4 weeks. After being placed under accelerated conditions for 4 weeks, all samples showed a decrease in the purity of the main peak, and the acid-base peaks of prescriptions FS2-2, FS2-5, FS2-6 and FS2-10 increased significantly, but there was no significant difference between the groups.
表19：第二轮处方筛选—CEX-HPLC数据


Table 19: The second round of formulation screening—CEX-HPLC data


2.2.6细胞活性结果2.2.6 Cell Viability Results
根据表20中的细胞活性结果，经加速条件或长期条件下放置4周，所有样品细胞活性结果均未出现显著变化。According to the cell viability results in Table 20, the cell viability results of all samples did not change significantly after being placed under accelerated conditions or long-term conditions for 4 weeks.
表20：第二轮处方筛选—细胞活性数据

Table 20: Second Round of Formulation Screening—Cell Viability Data

2.2.7结合活性结果2.2.7 Binding activity results
根据表21中的结合，经加速条件或长期条件下放置4周，所有样品结合活性结果均未出现显著变化。According to the binding in Table 21, there was no significant change in the binding activity results of all samples after being placed under accelerated conditions or long-term conditions for 4 weeks.
表21：第二轮处方筛选—结合活性数据

Table 21: Second Round Formulation Screening - Binding Activity Data

2.2.8亚可见颗粒结果2.2.8 Sub-visible particle results
根据表22中的亚可见颗粒结果，经加速条件条件下放置4周，所有样品均出现大小颗粒增多，FS2-4表现最佳；经长期条件下放置4周无明显颗粒变化。According to the results of sub-visible particles in Table 22, after being placed under accelerated conditions for 4 weeks, all samples showed an increase in the size of particles, and FS2-4 performed the best; after being placed under long-term conditions for 4 weeks, there was no obvious particle change.
表22：第二轮处方筛选—亚可见颗粒数据

Table 22: Second Round Formulation Screening—Subvisible Particle Data

2.3第二轮处方筛选结论2.3 Conclusion of the second round of prescription screening
从蛋白含量、NR-CE-SDS、R-CE-SDS纯度、细胞活性和结合活性结果来看，所有处方未出现显著差异。从外观结果，可以得出处方中含辅料二水海藻糖的样品稳定性较差，吐温20和吐温80含量增高能提高溶解性。从SEC-HPLC和亚可见颗粒结果来看，可以得出醋酸缓冲体系中，含辅料蔗糖有较好稳定性。综上所述，20mM醋酸缓冲液(FS2-4)pH5.5含220mM蔗糖表现最优。From the results of protein content, NR-CE-SDS, R-CE-SDS purity, cell viability and binding activity, there were no significant differences among all formulations. From the appearance results, it can be concluded that the stability of the samples containing trehalose dihydrate as an excipient in the prescription is poor, and the solubility can be improved by increasing the content of Tween 20 and Tween 80. From the results of SEC-HPLC and sub-visible particles, it can be concluded that in the acetic acid buffer system, the excipient sucrose has better stability. In summary, 20mM acetate buffer (FS2-4) pH5.5 containing 220mM sucrose performed optimally.
实施例3：影响因素实验Embodiment 3: Influencing factor experiment
3.1实验步骤3.1 Experimental steps
根据第二轮加速实验结果处方显示处方FS2-3、FS2-4、FS2-7和FS2-8四个处方稳定性较好，选择候选四个处方进行影响因素实验，本实施例以抗体huJS007-47进行。具体方案见表23。According to the results of the second round of accelerated experiments, the prescriptions showed that the four prescriptions FS2-3, FS2-4, FS2-7 and FS2-8 were more stable, and the four candidate prescriptions were selected for the experiment of influencing factors. In this example, the antibody huJS007- 47 carried out. See Table 23 for specific plans.
表23：影响因素实验方案
Table 23: Experimental scheme of influencing factors
3.2实验结果3.2 Experimental results
表24：影响因素检测结果汇总
Table 24: Summary of Influencing Factor Test Results
影响因素实验结论：在四个处方中，经搅拌、冻融和震荡等影响因素考察后，SEC-HPLC、外观和亚可见颗粒结果均无明显差异，均表现出较好稳定性。Experimental conclusion of influencing factors: Among the four formulations, after the investigation of influencing factors such as stirring, freezing and thawing, and shaking, there was no significant difference in the results of SEC-HPLC, appearance and sub-visible particles, and all showed good stability.
实施例4：配伍稳定性实验Embodiment 4: Compatibility stability experiment
实验步骤Experimental procedure
选取处方FS2-4考察0.9％氯化钠注射液和5％葡萄糖注射液两种介质稀释后的稳定性，本实施例以抗体huJS007-47进行。按如下方案稀释至相应介质不同浓度(表25)，放置于25℃恒温箱24h后检测。Prescription FS2-4 was selected to investigate the stability of 0.9% Sodium Chloride Injection and 5% Glucose Injection after dilution in two media. This embodiment was carried out with antibody huJS007-47. Dilute to different concentrations of the corresponding medium according to the following scheme (Table 25), and place in a 25°C incubator for 24 hours before testing.
表25：相容性方案

Table 25: Compatibility Scheme

表26：相容性检测结果汇总
Table 26: Summary of Compatibility Test Results
注：样品编号“FS2-4-Glu”表示用5％葡萄糖注射液稀释，样品编号“FS2-4-NaCl”表示用0.9％氯化钠注射液稀释。Note: The sample number "FS2-4-Glu" means diluted with 5% glucose injection, and the sample number "FS2-4-NaCl" means diluted with 0.9% sodium chloride injection.
相容性实验结论：以0.9％氯化钠注射液作为稀释溶媒时，在0.1mg/ml～5mg/ml的浓度范围都有良好的稳定性。以5％葡萄糖注射液作为溶媒时，稀释样品在低浓度0.1mg/ml时，SEC-HPLC结果出现片段增加，在1.0mg/ml～5.0mg/ml浓度范围表现良好稳定性。Compatibility test conclusion: when 0.9% sodium chloride injection is used as the dilution medium, it has good stability in the concentration range of 0.1mg/ml～5mg/ml. When 5% glucose injection is used as the solvent, when the diluted sample is at a low concentration of 0.1mg/ml, the SEC-HPLC results show increased fragments, and show good stability in the concentration range of 1.0mg/ml～5.0mg/ml.
综上所述，我们通过对不同缓冲体系，不同pH条件、不同抗体浓度和不同辅料组成进行考察，目标pH范围控制在5.0～6.0，渗透压范围在250～350mOsm/kg，选择了最优制剂配方：约20mM醋酸钠缓冲液(pH约为5.5)，约220mM蔗糖和约0.02％聚山梨醇酯80；为使渗透压处于300mOsm/kg左右，蔗糖的浓度可以为约228mM。To sum up, we investigated different buffer systems, different pH conditions, different antibody concentrations, and different excipient compositions. The target pH range was controlled at 5.0-6.0, and the osmotic pressure range was 250-350mOsm/kg, and the optimal preparation was selected. Recipe: about 20mM sodium acetate buffer (pH about 5.5), about 220mM sucrose and about 0.02% polysorbate 80; to make the osmotic pressure about 300mOsm/kg, the concentration of sucrose can be about 228mM.
实施例5：抗CTLA-4药物组合物的抗原亲和力Example 5: Antigen Affinity of Anti-CTLA-4 Pharmaceutical Compositions
使用GE医疗生命科学公司的T200型分子相互作用分析仪Biacore，利用 表面等离子共振技术，将40μg/mL羊抗人Fc片断抗体(Jackson ImmunoResearch)偶联在CM5芯片(GE医疗生命科学，货号：BR100530)表面，然后捕获抗CTLA-4药物组合物(按处方FS2-4的配方配制，抗体huJS007-47浓度0.5μg/mL)，再进样梯度稀释的His标签的重组人CTLA-4抗原蛋白(君盟自制)，检测结合信号。CTLA-4抗原蛋白梯度稀释浓度为24nM、12nM、6nM、3nM、1.5nM、0.75nM，其中24nM为重复。使用Biacore分析软件Biacore T200 Evaluation Software 3.0的动力学模型分析，拟合得到亲和力KD值。Using the T200 molecular interaction analyzer Biacore of GE Healthcare Life Sciences, using Using surface plasmon resonance technology, 40 μg/mL goat anti-human Fc fragment antibody (Jackson ImmunoResearch) was coupled to the surface of a CM5 chip (GE Healthcare Life Sciences, article number: BR100530), and then the anti-CTLA-4 pharmaceutical composition (by prescription FS2- 4, the concentration of the antibody huJS007-47 was 0.5 μg/mL), and the His-tagged recombinant human CTLA-4 antigen protein (manufactured by Junmeng) was injected in a gradient dilution to detect the binding signal. The gradient dilution concentration of CTLA-4 antigen protein is 24nM, 12nM, 6nM, 3nM, 1.5nM, 0.75nM, wherein 24nM is repeated. Using the kinetic model analysis of Biacore analysis software Biacore T200 Evaluation Software 3.0, the affinity KD value was obtained by fitting.
实验结果如表27和图1所示，抗CTLA-4药物组合物与人CTLA-4蛋白有较高的亲和力，KD数值为2.06E-10M。The experimental results are shown in Table 27 and Figure 1. The anti-CTLA-4 pharmaceutical composition has a high affinity with human CTLA-4 protein, and the K D value is 2.06E-10M.
表27：抗CTLA-4药物组合物与重组人CTLA-4蛋白亲和力结果表
Table 27: Affinity result table of anti-CTLA-4 pharmaceutical composition and recombinant human CTLA-4 protein
实施例6：抗CTLA-4药物组合物对hCTLA4人源化小鼠移植EMT6肿瘤生长的抑制作用Example 6: Inhibitory Effect of Anti-CTLA-4 Pharmaceutical Composition on EMT6 Tumor Growth Transplanted in hCTLA4 Humanized Mice
将小鼠乳腺癌EMT6细胞(0.5×106)(ATCC CRL-2755)接种到hCTLA-4人源化小鼠(上海南方模式生物科技有限公司)右后背皮下。当肿瘤大小约为99mm3时，将小鼠随机分为5组，每组6只小鼠。小鼠腹腔注射10mg/kg的anti-KLH hIgG1、1、3或10mg/kg抗CTLA-4药物组合物(按处方FS2-4的配方配制，抗体为抗体huJS007-47)，每周给药两次，共5次。实验结果如图2所示，接种肿瘤后第24天，与anti-KLH hIgG1相比，10mg/kg的抗CTLA-4药物组合物显著性抑制肿瘤生长，TGI值为96.4％(p<0.05)。此外，动物对抗CTLA-4药物组合物的耐受性良好，研究期间小鼠体重逐渐增加。其中，肿瘤抑制率TGI％(TGI％＝[1-(Ti-T0)/(Vi-V0)]×100％)，(Ti：治疗组在给药第i天的肿瘤体积均值，T0：治疗组在给药第0天的肿瘤体积均值；Vi：阴性对照组在给药第i天的肿瘤体积均值，V0：阴性对照组在给药第0天的肿瘤体积均值)。Mouse breast cancer EMT6 cells (0.5×10 6 ) (ATCC CRL-2755) were inoculated subcutaneously on the right back of hCTLA-4 humanized mice (Shanghai Southern Model Biotechnology Co., Ltd.). When the tumor size was about 99 mm, the mice were randomly divided into 5 groups with 6 mice in each group. Anti-KLH hIgG1, 1, 3 or 10 mg/kg anti-CTLA-4 pharmaceutical composition (prepared according to prescription FS2-4, antibody is antibody huJS007-47) of intraperitoneal injection of 10 mg/kg, administration twice a week times, a total of 5 times. The experimental results are shown in Figure 2. On the 24th day after tumor inoculation, compared with anti-KLH hIgG1, the 10 mg/kg anti-CTLA-4 pharmaceutical composition significantly inhibited tumor growth, and the TGI value was 96.4% (p<0.05) . In addition, the anti-CTLA-4 pharmaceutical composition was well tolerated by the animals, and the mice gradually gained weight during the study period. Among them, the tumor inhibition rate TGI% (TGI%=[1-(Ti-T0)/(Vi-V0)]×100%), (Ti: the mean value of the tumor volume of the treatment group on the i day of administration, T0: the treatment The average tumor volume of the group on the 0th day of administration; Vi: the average tumor volume of the negative control group on the i-th day of administration, V0: the average tumor volume of the negative control group on the 0th day of administration).
实施例7：抗CTLA-4抗体的药物组合物的生物学活性 Example 7: Biological activity of pharmaceutical compositions of anti-CTLA-4 antibodies
检测人源化抗CTLA-4抗体与huCTLA-4的结合、阻断huCTLA-4与CD80/CD86结合的能力以及拮抗huCTLA-4的生物学活性。本实施例人源化抗CTLA-4抗体按处方FS2-4的配方配制，实验方法和结果如下所示。The binding of humanized anti-CTLA-4 antibody to huCTLA-4, the ability to block the binding of huCTLA-4 to CD80/CD86, and the biological activity of antagonizing huCTLA-4 were detected. In this example, the humanized anti-CTLA-4 antibody was prepared according to the formula of prescription FS2-4, and the experimental methods and results are as follows.
7.1 ELISA法检测人源化抗CTLA-4抗体与huCTLA-4的结合7.1 ELISA method to detect the binding of humanized anti-CTLA-4 antibody to huCTLA-4
使用PBS(Hyclone)稀释HX1 hCTLA4 his至1.0μg/mL，以100μl/孔加入酶标板，37℃恒温培养箱中静置包被90min；洗板；加入200μl/孔2％BSA至板内，置37℃恒温培养箱内孵育90min；洗板。将人源化抗CTLA-4抗体及对照抗体Ipilimumab(君实自制，参见专利WO2001014424A2和CN1371416A)用稀释液(2％BSA)稀释至1000ng/ml，每次稀释倍数不高于10倍。然后，在样品稀释板上以2.5倍梯度依次稀释人源化抗体及对照抗体。所有人源化抗体及对照抗体溶液以100μl/孔加入酶标板中，置37℃恒温培养箱内孵育60min；洗板；将HRP偶联的羊抗人抗体IgG(Fc特异性)(Sigma，货号：A0170)用2％BSA稀释5000倍，以100μl/孔加入酶标板，置37℃恒温培养箱内孵育60min；洗板；加入显色液TMB，100μl/孔，避免气泡，37℃避光显色15min；最后加2M的盐酸溶液终止反应，100μl/孔，避免气泡，10min内完成酶标仪读数(波长：450/620nm)。图3为人源化抗CTLA-4抗体对比对照抗体的相对结合活性。Use PBS (Hyclone) to dilute HX1 hCTLA4 his to 1.0 μg/mL, add 100 μl/well to the ELISA plate, and keep it in a constant temperature incubator at 37°C for 90 minutes; wash the plate; add 200 μl/well 2% BSA to the plate, Place in a constant temperature incubator at 37°C and incubate for 90 minutes; wash the plate. The humanized anti-CTLA-4 antibody and the control antibody Ipilimumab (manufactured by Junshi, refer to patents WO2001014424A2 and CN1371416A) were diluted to 1000 ng/ml with diluent (2% BSA), and each dilution factor was not higher than 10 times. Then, the humanized antibody and the control antibody were sequentially diluted in a 2.5-fold gradient on the sample dilution plate. All humanized antibody and control antibody solutions were added to the microtiter plate at 100 μl/well, incubated in a constant temperature incubator at 37°C for 60 min; the plate was washed; HRP-coupled goat anti-human antibody IgG (Fc specific) (Sigma, Product number: A0170) was diluted 5000 times with 2% BSA, added 100 μl/well to the microplate, incubated in a 37°C constant temperature incubator for 60 minutes; washed the plate; Light color development for 15 minutes; finally add 2M hydrochloric acid solution to terminate the reaction, 100 μl/well, avoid air bubbles, and complete the microplate reader reading within 10 minutes (wavelength: 450/620nm). Figure 3 shows the relative binding activities of humanized anti-CTLA-4 antibodies compared to control antibodies.
如图3所示，人源化抗CTLA-4抗体具有与huCTLA-4良好的结合，与对照抗体相当或优于对照抗体Ipilimumab。As shown in Figure 3, the humanized anti-CTLA-4 antibody has good binding to huCTLA-4, which is comparable to or better than the control antibody Ipilimumab.
7.2 ELISA法检测人源化抗CTLA-4抗体阻断huCTLA-4与CD80结合的能力7.2 ELISA method to detect the ability of humanized anti-CTLA-4 antibody to block the binding of huCTLA-4 and CD80
使用PBS(Hyclone)稀释HX1 hCTLA4-his至1.0μg/mL，以100μl/孔加入酶标板，37℃恒温培养箱中静置孵育90min；洗板；加入200μl/孔2％BSA至板内，置37℃恒温培养箱内孵育90min；洗板；取MX2 hCD80 Fc(君盟自制)用2％BSA稀释至5.0μg/mL，并以此浓度的MX2 hCD80 Fc来稀释抗体。将人源化抗CTLA-4抗体及对照抗体Ipilimumab(君实自制，参见专利WO2001014424A2和CN1371416A)稀释至100μg/ml，每次稀释倍数不高于10倍。然后，在样品稀释板上以2.5倍梯度依次稀释人源化抗体及对照抗体。所有人源化抗体及对照抗体溶液以100μl/孔加入酶标板中，置37℃恒温培养 箱内孵育90min；洗板；将HRP偶联的羊抗鼠抗体IgG(Fc特异性)用2％BSA稀释5000倍，以100μl/孔加入酶标板，置37℃恒温培养箱内孵育60min；洗板；加入显色液TMB，100μl/孔，避免气泡，37℃避光显色15min；最后加2M的盐酸溶液终止反应，100μl/孔，避免气泡，10min内完成酶标仪读数(波长：450/620nm)。图4为人源化抗CTLA-4抗体对比对照抗体的相对抑制活性。Use PBS (Hyclone) to dilute HX1 hCTLA4-his to 1.0 μg/mL, add 100 μl/well to the ELISA plate, and incubate at 37°C for 90 minutes; wash the plate; add 200 μl/well 2% BSA to the plate, Incubate in a constant temperature incubator at 37°C for 90 min; wash the plate; dilute MX2 hCD80 Fc (manufactured by Junmeng) with 2% BSA to 5.0 μg/mL, and dilute the antibody with this concentration of MX2 hCD80 Fc. Dilute the humanized anti-CTLA-4 antibody and control antibody Ipilimumab (manufactured by Junshi, refer to patents WO2001014424A2 and CN1371416A) to 100 μg/ml, and each dilution factor is not more than 10 times. Then, the humanized antibody and the control antibody were sequentially diluted in a 2.5-fold gradient on the sample dilution plate. All humanized antibody and control antibody solutions were added to the microtiter plate at 100 μl/well, and incubated at a constant temperature of 37°C Incubate in the box for 90 minutes; wash the plate; dilute the HRP-coupled goat anti-mouse antibody IgG (Fc specific) 5000 times with 2% BSA, add 100 μl/well to the microtiter plate, and incubate in a 37°C constant temperature incubator for 60 minutes; Wash the plate; add chromogenic solution TMB, 100 μl/well, avoid air bubbles, develop color at 37°C in the dark for 15 minutes; finally add 2M hydrochloric acid solution to terminate the reaction, 100 μl/well, avoid air bubbles, and complete the microplate reader reading within 10 minutes (wavelength: 450/620nm). Figure 4 shows the relative inhibitory activity of humanized anti-CTLA-4 antibodies compared to control antibodies.
如图4所示，人源化抗CTLA-4抗体具有良好的阻断huCTLA-4与CD80结合的能力，与对照抗体相当或优于对照抗体Ipilimumab。As shown in Figure 4, the humanized anti-CTLA-4 antibody has a good ability to block the binding of huCTLA-4 to CD80, which is comparable to or better than the control antibody Ipilimumab.
7.3荧光素酶法检测人源化抗CTLA-4抗体拮抗huCTLA-4的生物学活性7.3 Luciferase method to detect the biological activity of humanized anti-CTLA-4 antibody antagonizing huCTLA-4
根据上述结合活性、阻断结合活性和人源化程度选择人源化抗CTLA-4抗体huJS007-46、47、48、49、55、56、73、79、82、88、100和106进行生物学活性分析。Humanized anti-CTLA-4 antibodies huJS007-46, 47, 48, 49, 55, 56, 73, 79, 82, 88, 100 and 106 were selected for biological Activity analysis.
将表达huCTLA-4的Jurkat细胞按每孔6×104个细胞铺板，每孔加入3×104个Raji APC细胞以及不同浓度的人源化抗CTLA-4抗体或对照抗体(Ipilimumab，君实自制，参见专利WO2001014424A2和CN1371416A)，孵育6小时后，采用荧光素酶法测定T细胞激活活性。The Jurkat cells expressing huCTLA-4 were plated at 6× 104 cells per well, and 3× 104 Raji APC cells and different concentrations of humanized anti-CTLA-4 antibodies or control antibodies (Ipilimumab, Junshi Self-made, see patents WO2001014424A2 and CN1371416A), after incubation for 6 hours, T cell activation activity was measured by luciferase method.
如图5所示，12种人源化抗CTLA-4抗体均具有很高的生物学活性，其EC50均明显优于对照抗体Ipilimumab。As shown in Figure 5, all 12 humanized anti-CTLA-4 antibodies have high biological activity, and their EC50 are significantly better than the control antibody Ipilimumab.
实施例8：人源化抗CTLA-4抗体的ADCC活性Example 8: ADCC activity of humanized anti-CTLA-4 antibodies
293T-CTLA4细胞用CFSE标记，按1:25的靶细胞:效应细胞(Target:effector)比例添加外周血单核细胞(PBMC2144896)和不同浓度的人源化抗CTLA-4抗体或对照抗体(Ipilimumab，君实自制，参见专利WO2001014424A2和CN1371416A)，孵育过夜。细胞用碘化丙啶(PI)染色，用流式细胞仪进行分析。ADCC杀伤(ADCC Killing,％)表示为死亡靶细胞(PI和CFSE阳性)占总靶细胞(CFSE阳性)的百分比。本实施例人源化抗CTLA-4抗体按处方FS2-4的配方配制。293T-CTLA4 cells were labeled with CFSE, and peripheral blood mononuclear cells (PBMC2144896) and different concentrations of humanized anti-CTLA-4 antibodies or control antibodies (Ipilimumab , made by Junshi, see patents WO2001014424A2 and CN1371416A), incubated overnight. Cells were stained with propidium iodide (PI) and analyzed by flow cytometry. ADCC killing (ADCC Killing,%) was expressed as the percentage of dead target cells (PI and CFSE positive) in total target cells (CFSE positive). In this example, the humanized anti-CTLA-4 antibody was prepared according to the formula of prescription FS2-4.
如图6(分成两幅小图仅仅是为了方便显示)所示，12种人源化抗CTLA-4抗体均具有ADCC活性，其中人源化抗CTLA-4抗体46、47、48、73、79及 106的ADCC活性与对照抗体相当或优于对照抗体，特别是抗体46和48。As shown in Figure 6 (divided into two panels for convenience), all 12 humanized anti-CTLA-4 antibodies have ADCC activity, among which humanized anti-CTLA-4 antibodies 46, 47, 48, 73, 79 and The ADCC activity of 106 was comparable to or better than control antibodies, especially antibodies 46 and 48.
实施例9：人源化抗CTLA-4抗体的CDC活性Example 9: CDC Activity of Humanized Anti-CTLA-4 Antibodies
293T-CTLA4细胞用不同浓度(0.8-100μg/mL)的12种人源化抗CTLA-4抗体或对照抗体(Ipilimumab，君实自制，参见专利WO2001014424A2和CN1371416A)在37℃活化15分钟，其中本实施例人源化抗CTLA-4抗体按处方FS2-4的配方配制，然后加入不同稀释梯度(1:5、1:10、1:20)的人血清补体并培养1小时。培养结束后，用碘化丙啶(PI)染色细胞，用BD FACSCalibur流式细胞仪进行分析。CDC杀伤(CDC Killing,％)表示为PI阳性靶细胞占总靶细胞的百分比。结果如图7所述，表明12个人源化抗CTLA-4抗体没有CDC活性或者CDC活性可以忽略不计。293T-CTLA4 cells were activated at 37°C for 15 minutes with 12 humanized anti-CTLA-4 antibodies or control antibodies (Ipilimumab, made by Junshi, see patents WO2001014424A2 and CN1371416A) at different concentrations (0.8-100 μg/mL), in which this Example Humanized anti-CTLA-4 antibody was prepared according to the formula of prescription FS2-4, and then human serum complement with different dilution gradients (1:5, 1:10, 1:20) was added and incubated for 1 hour. After culturing, cells were stained with propidium iodide (PI) and analyzed with a BD FACSCalibur flow cytometer. CDC Killing (CDC Killing,%) was expressed as the percentage of PI-positive target cells in the total target cells. The results are shown in Figure 7, showing that 12 humanized anti-CTLA-4 antibodies had no or negligible CDC activity.
实施例10：抗CTLA-4抗体药物组合物对小鼠肿瘤生长的抑制Example 10: Inhibition of tumor growth in mice by anti-CTLA-4 antibody pharmaceutical composition
取50只6-8周龄雌性B-hCTLA4人源化小鼠(百奥赛图)，将小鼠结肠癌细胞MC38 WT细胞(上海舜冉生物科技有限公司)以1×106个/0.1mL浓度接种小鼠的右侧皮下，待肿瘤生长到约138mm3时将小鼠按肿瘤体积随机分组，每组6只，共6组，分别为：Take 50 6-8-week-old female B-hCTLA4 humanized mice (Biocytogen), and use 1× 106 /0.1mL mouse colon cancer cell MC38 WT cells (Shanghai Sunran Biotechnology Co., Ltd.) The concentration was inoculated subcutaneously on the right side of the mouse, and when the tumor grew to about 138mm3 , the mice were randomly divided into groups according to the tumor volume, with 6 mice in each group, a total of 6 groups, respectively:
G1 KLH IgG1(0.3mg/kg)阴性对照组、G1 KLH IgG1 (0.3mg/kg) negative control group,
G2 Ipilimumab(0.3mg/kg)阳性对照组、G2 Ipilimumab (0.3mg/kg) positive control group,
G3 huJS007-47(0.3mg/kg)治疗组、G3 huJS007-47 (0.3mg/kg) treatment group,
G4 huJS007-48(0.3mg/kg)治疗组、G4 huJS007-48 (0.3mg/kg) treatment group,
G5 huJS007-79(0.3mg/kg)治疗组，和G5 huJS007-79 (0.3mg/kg) treatment group, and
G6 huJS007-106(0.3mg/kg)治疗组。G6 huJS007-106 (0.3mg/kg) treatment group.
本实施例人源化抗CTLA-4抗体按处方FS2-4的配方配制。In this example, the humanized anti-CTLA-4 antibody was prepared according to the formula of prescription FS2-4.
所有组的给药途径均为腹腔注射，给药剂量为0.3mg/kg，给药浓度为0.03mg/ml。每周给药2次，连续给药5次，末次给药3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。实验结束时将小鼠安乐死，计算相对肿瘤抑制率TGI％＝(1-(Ti-T0)/(Vi-V0))×100％。Ti：治疗组和阳性对照组在给药第i天的肿瘤体积均值；T0：治疗组和阳性对照组在给药第0天的肿 瘤体积均值；Vi：阴性对照组在给药第i天的肿瘤体积均值；V0：阴性对照组在给药第0天的肿瘤体积均值。The administration route of all groups was intraperitoneal injection, the administration dose was 0.3mg/kg, and the administration concentration was 0.03mg/ml. The administration was given twice a week, 5 consecutive administrations, and the experiment was ended 3 days after the last administration. The tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the mice were euthanized, and the relative tumor inhibition rate TGI%=(1-(Ti-T0)/(Vi-V0))×100% was calculated. Ti: the average tumor volume of the treatment group and the positive control group on the i-th day of administration; T0: the tumor volume of the treatment group and the positive control group on the 0th day of administration The average tumor volume; Vi: the average tumor volume of the negative control group on day i of administration; V0: the average tumor volume of the negative control group on day 0 of administration.
如图8所示，在小鼠接种肿瘤细胞后第25天，KLH IgG1阴性对照组平均肿瘤体积为975±115mm3，Ipilimumab(君实自制，参见专利WO2001014424A2和CN1371416A)阳性对照组平均肿瘤体积为824±267mm3，与KLH IgG1相比，相对肿瘤抑制率为18.1％；huJS007-47治疗组、huJS007-48治疗组、huJS007-79治疗组和huJS007-106治疗组平均肿瘤体积分别为229±85mm3、313±197mm3、550±229mm3和472±125mm3，与KLH IgG1相比，相对肿瘤抑制率分别为89.2％、79.1％、50.9％和60.1％，表明上述人源化抗CTLA-4抗体能够体内抑制B-hCTLA4人源化小鼠MC38-WT细胞皮下移植瘤的生长，且明显优于对照抗体Ipilimumab。As shown in Figure 8, on the 25th day after mice were inoculated with tumor cells, the average tumor volume of the KLH IgG1 negative control group was 975±115mm 3 , and the average tumor volume of the Ipilimumab (made by Junshi, see patents WO2001014424A2 and CN1371416A) positive control group was 975±115mm 3 . 824±267mm 3 , compared with KLH IgG1, the relative tumor inhibition rate was 18.1%; the average tumor volume of huJS007-47 treatment group, huJS007-48 treatment group, huJS007-79 treatment group and huJS007-106 treatment group were 229±85mm 3 , 313±197mm 3 , 550±229mm 3 and 472±125mm 3 , compared with KLH IgG1, the relative tumor inhibition rates were 89.2%, 79.1%, 50.9% and 60.1%, respectively, indicating that the above-mentioned humanized anti-CTLA-4 The antibody can inhibit the growth of B-hCTLA4 humanized mouse MC38-WT cell subcutaneously transplanted tumor in vivo, and it is significantly better than the control antibody Ipilimumab.
实施例11：Fortebio结合实验鉴定抗原表位Example 11: Identification of epitopes by Fortebio binding assay
使用Protein A探针(Fortebio)先分别捕获全长抗体，即2.7μg/mL的人源化抗CTLA-4抗体huJS007-47与2μg/mL的对照抗体(Ipilimumab，君实自制，参见专利WO2001014424A2和CN1371416A)。再将探针浸入55nM的人源CTLA(huCTLA)抗原溶液中，使全长抗体与抗原结合。最后将探针浸入600nM Fab溶液中，包括人源化Fab huJS00-47与对照Fab(Ipilimumab)，检测抗原与Fab是否发生结合。Use the Protein A probe (Fortebio) to first capture the full-length antibody, namely 2.7 μg/mL humanized anti-CTLA-4 antibody huJS007-47 and 2 μg/mL control antibody (Ipilimumab, made by Junshi, see patents WO2001014424A2 and CN1371416A). Then the probe was immersed in 55nM human CTLA (huCTLA) antigen solution to make the full-length antibody bind to the antigen. Finally, the probes were immersed in 600nM Fab solution, including humanized Fab huJS00-47 and control Fab (Ipilimumab), to detect whether the antigen combined with the Fab.
如图9所示，全长抗体huJS007-47与抗原huCTLA-4结合后，huCTLA-4可以继续与对照Fab(Ipilimumab)结合，但按照Fortebio结合实验检测结果，huJS007-47与Ipilimumab结合于huCTLA-4的不同表位。As shown in Figure 9, after the full-length antibody huJS007-47 binds to the antigen huCTLA-4, huCTLA-4 can continue to bind to the control Fab (Ipilimumab). 4 different epitopes.
实施例12：huJS007-47药物组合物对hCTLA4人源化小鼠移植MC38肿Example 12: Effect of huJS007-47 pharmaceutical composition on hCTLA4 humanized mice transplanted with MC38
瘤生长的抑制作用inhibition of tumor growth
取6-8周龄雌性hCTLA4人源化小鼠(百奥赛图江苏基因生物技术有限公司)，于右侧背部皮下接种1×106小鼠结肠癌细胞MC38细胞(上海舜冉生物科技有限公司)(0.1ml/只(含细胞的培养基RMPI1640(Gibco)))。待平均肿瘤体积约为119mm3时，挑选40只动物，根据肿瘤体积随机分为5组， 每组8只动物。分别为6-8 weeks old female hCTLA4 humanized mice (Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were subcutaneously inoculated with 1×10 6 mouse colon cancer MC38 cells (Shanghai Sunran Biotechnology Co., Ltd. ) (0.1ml/only (cell-containing medium RMPI1640 (Gibco))). When the average tumor volume was about 119 mm, 40 animals were selected and randomly divided into 5 groups according to the tumor volume. 8 animals per group. respectively
Anti-KLH hIgG1阴性对照组，1mg/kg；Anti-KLH hIgG1 negative control group, 1mg/kg;
Ipilimumab阳性对照组，1mg/kg；Ipilimumab positive control group, 1mg/kg;
huJS007-47治疗组，0.1mg/kg；huJS007-47 treatment group, 0.1mg/kg;
huJS007-47治疗组，0.3mg/kg；huJS007-47 treatment group, 0.3mg/kg;
huJS007-47治疗组，1mg/kg；huJS007-47 treatment group, 1mg/kg;
本实施例人源化抗CTLA-4抗体按处方FS2-4的配方配制。In this example, the humanized anti-CTLA-4 antibody was prepared according to the formula of prescription FS2-4.
分组当天给药，所有组给药途径均为腹腔注射，每周给药2次，连续给药6次，末次给药3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，将小鼠安乐死，计算肿瘤抑制率TGI％(TGI％＝[1-(Ti-T0)/(Vi-V0)]×100％)。(Ti：治疗组在给药第i天的肿瘤体积均值，T0：治疗组在给药第0天的肿瘤体积均值；Vi：阴性对照组在给药第i天的肿瘤体积均值，V0：阴性对照组在给药第0天的肿瘤体积均值)。The administration was administered on the day of grouping, and the administration route of all groups was intraperitoneal injection, administered twice a week, for 6 consecutive administrations, and the experiment ended 3 days after the last administration. The tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the mice were euthanized, and the tumor inhibition rate TGI% was calculated (TGI%=[1-(Ti-T0)/(Vi-V0)]×100%). (Ti: the average tumor volume of the treatment group on day i of administration, T0: the average tumor volume of the treatment group on day 0 of administration; Vi: the average tumor volume of the negative control group on day i of administration, V0: negative The average tumor volume of the control group on the 0th day of administration).
如图10所示，在开始给药后第21天，anti-KLH hIgG1阴性对照组在1mg/kg的剂量下的平均肿瘤体积为1116±106mm3。Ipilimumab(君实自制，参见专利WO2001014424A2和CN1371416A)阳性对照组在1mg/kg的剂量下的平均肿瘤体积为255±88mm3，TGI％为86.36％。huJS007-47在0.1，0.3和1mg/kg的剂量下，平均肿瘤体积分别为736±203mm3，47±12mm3，33±15mm3，TGI％分别为38.11％、107.22％和108.63％。表明huJS007-47在0.1，0.3和1mg/kg的剂量下，显著抑制hCTLA4人源化小鼠移植MC38肿瘤体积增长，并呈现良好的剂量效应，且在等剂量条件下(1mg/kg)，huJS007-47抑瘤作用显著优于Ipilimumab。As shown in FIG. 10 , the average tumor volume of the anti-KLH hIgG1 negative control group at a dose of 1 mg/kg was 1116±106 mm 3 on day 21 after the start of administration. Ipilimumab (made by Junshi, refer to patents WO2001014424A2 and CN1371416A) in the positive control group had an average tumor volume of 255±88 mm 3 and a TGI% of 86.36% at a dose of 1 mg/kg. For huJS007-47 at doses of 0.1, 0.3 and 1 mg/kg, the average tumor volumes were 736±203mm 3 , 47±12mm 3 , 33±15mm 3 , and TGI% were 38.11%, 107.22% and 108.63%, respectively. It was shown that huJS007-47 significantly inhibited the growth of MC38 tumor volume in hCTLA4 humanized mice at doses of 0.1, 0.3 and 1 mg/kg, and showed a good dose effect, and at the same dose (1 mg/kg), huJS007 The anti-tumor effect of -47 was significantly better than that of Ipilimumab.
实施例13：huJS007-47药物组合物对hCTLA4人源化小鼠移植H22肿瘤生长的抑制作用Example 13: Inhibitory Effect of huJS007-47 Pharmaceutical Composition on H22 Tumor Growth Transplanted in hCTLA4 Humanized Mice
取6-8周龄雌性hCTLA4人源化小鼠(上海南方模式生物科技股份有限公司)，于右侧背部皮下接种1×106小鼠肝癌细胞H22细胞(上海诺百生物科技有限公司，货号C01-FV)(0.1ml/只(含细胞的培养基RMPI1640(Gibco)))。待平均肿瘤体积约为119mm3时，挑选35只动物，根据肿瘤体积随机分为5 组，每组7只动物。分别为Take 6-8 week-old female hCTLA4 humanized mice (Shanghai Nanfang Model Biotechnology Co., Ltd.), and subcutaneously inoculate 1× 106 mouse liver cancer cell H22 cells (Shanghai Nuobai Biotechnology Co., Ltd., Cat. No. C01-FV) (0.1 ml/one (cell-containing medium RMPI1640 (Gibco))). When the average tumor volume was about 119 mm, 35 animals were selected and randomly divided into 5 animals according to the tumor volume. groups, with 7 animals in each group. respectively
Anti-KLH hIgG1阴性对照组，0.3mg/kg；Anti-KLH hIgG1 negative control group, 0.3mg/kg;
Ipilimumab阳性对照组，0.1mg/kg；Ipilimumab positive control group, 0.1mg/kg;
huJS007-47治疗组，0.03mg/kg；huJS007-47 treatment group, 0.03mg/kg;
huJS007-47治疗组，0.1mg/kg；huJS007-47 treatment group, 0.1mg/kg;
huJS007-47治疗组，0.3mg/kg；huJS007-47 treatment group, 0.3mg/kg;
本实施例人源化抗CTLA-4抗体按处方FS2-4的配方配制。In this example, the humanized anti-CTLA-4 antibody was prepared according to the formula of prescription FS2-4.
分组当天给药，所有组给药途径均为腹腔注射，每周给药2次，连续给药6次，末次给药3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，将小鼠安乐死，计算肿瘤抑制率TGI％，计算方式与实施例15相同。The administration was administered on the day of grouping, and the administration route of all groups was intraperitoneal injection, administered twice a week, for 6 consecutive administrations, and the experiment ended 3 days after the last administration. The tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the mice were euthanized, and the tumor inhibition rate TGI% was calculated in the same way as in Example 15.
如图11所示，在开始给药后第21天，anti-KLH hIgG1阴性对照组在0.3mg/kg的剂量下的平均肿瘤体积为2304±402mm3。Ipilimumab(君实自制，参见专利WO2001014424A2和CN1371416A)阳性对照组在0.1mg/kg的剂量下的平均肿瘤体积为837±397mm3，TGI％为67.14％。huJS007-47在0.03，0.1和0.3mg/kg的剂量下，平均肿瘤体积分别为1674±508mm3，466±171mm3，271±155mm3，TGI％分别为28.83％、84.12％和93.04％。表明huJS007-47在0.1和0.3mg/kg的剂量下，显著抑制hCTLA4人源化小鼠移植H22肿瘤体积增长，并呈现良好的剂量效应，且在等剂量条件下(0.1mg/kg)，huJS007-47抑瘤作用显著优于Ipilimumab。



As shown in FIG. 11 , on day 21 after the start of administration, the average tumor volume of the anti-KLH hIgG1 negative control group at a dose of 0.3 mg/kg was 2304±402 mm 3 . Ipilimumab (made by Junshi, see patents WO2001014424A2 and CN1371416A) in the positive control group at a dose of 0.1 mg/kg had an average tumor volume of 837±397mm 3 and a TGI% of 67.14%. At the doses of 0.03, 0.1 and 0.3 mg/kg of huJS007-47, the average tumor volumes were 1674±508mm 3 , 466±171mm 3 , 271±155mm 3 , and the TGI% were 28.83%, 84.12% and 93.04%, respectively. It showed that huJS007-47 significantly inhibited the growth of hCTLA4 humanized mouse transplanted H22 tumor volume at the dose of 0.1 and 0.3 mg/kg, and showed a good dose effect, and at the same dose (0.1 mg/kg), huJS007 The anti-tumor effect of -47 was significantly better than that of Ipilimumab.



Claims (10)

  1. 一种药物组合物，包含：A pharmaceutical composition comprising:
    (1)缓冲液；和(1) Buffer; and
    (2)抗CTLA-4抗体或其抗原结合片段；(2) Anti-CTLA-4 antibody or antigen-binding fragment thereof;
    其中，所述抗CTLA-4抗体或其抗原结合片段包含的LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3分别为：Wherein, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 contained in the anti-CTLA-4 antibody or its antigen-binding fragment are respectively:
    LCDR1：RASQNVGTYVA(SEQ ID NO:1)；LCDR1: RASQNVGTYVA (SEQ ID NO: 1);
    LCDR2：STSYRYS(SEQ ID NO:2)；LCDR2: STSYRYS (SEQ ID NO: 2);
    LCDR3：HQYDTYPLT(SEQ ID NO:3)；LCDR3: HQYDTYPLT (SEQ ID NO: 3);
    HCDR1：SGYYWN(SEQ ID NO:4)；HCDR1: SGYYWN (SEQ ID NO: 4);
    HCDR2：YIGYDGSNX1YNPSLKX2，其中，X1为N或Y，X2为S或N；HCDR2: YIGYDGSNX1YNPSLKX2, where X1 is N or Y, X2 is S or N;
    HCDR3：X3YYSGYFDS，X3为D或N；HCDR3: X3YYSGYFDS, X3 is D or N;
    优选地，所述抗CTLA-4抗体或其抗原结合片段的浓度为约1～100mg/mL，优选为约1～50mg/mL，更优选为约5～30mg/mL；Preferably, the concentration of the anti-CTLA-4 antibody or antigen-binding fragment thereof is about 1-100 mg/mL, preferably about 1-50 mg/mL, more preferably about 5-30 mg/mL;
    优选地，所述药物组合物的pH为约4.5～6.5，优选为约5.0～6.0；Preferably, the pH of the pharmaceutical composition is about 4.5-6.5, preferably about 5.0-6.0;
    优选地，所述药物组合物的渗透压为约250～350mOsm/kg；Preferably, the osmotic pressure of the pharmaceutical composition is about 250-350 mOsm/kg;
    优选地，所述药物组合物经静脉注射施用。Preferably, the pharmaceutical composition is administered intravenously.
  2. 如权利要求1所述的药物组合物，其中所述抗CTLA-4抗体或其抗原结合片段包含：The pharmaceutical composition of claim 1, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises:
    氨基酸序列如SEQ ID NO:1所示的LCDR1；The amino acid sequence is LCDR1 shown in SEQ ID NO:1;
    氨基酸序列如SEQ ID NO:2所示的LCDR2；The amino acid sequence is LCDR2 shown in SEQ ID NO:2;
    氨基酸序列如SEQ ID NO:3所示的LCDR3；The amino acid sequence is LCDR3 shown in SEQ ID NO:3;
    氨基酸序列如SEQ ID NO:4所示的HCDR1；The amino acid sequence is HCDR1 as shown in SEQ ID NO:4;
    氨基酸序列如SEQ ID NO:5或7或9所示的HCDR2；The amino acid sequence is HCDR2 as shown in SEQ ID NO:5 or 7 or 9;
    氨基酸序列如SEQ ID NO:6或8所示的HCDR3；The amino acid sequence is HCDR3 as shown in SEQ ID NO:6 or 8;
    优选地，所述抗CTLA-4抗体或其抗原结合片段包含：Preferably, the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises:
    (1)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的LCDR1、LCDR2和LCDR3，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HCDR1、HCDR2和HCDR3；或 (1) Amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, and amino acid sequences such as SEQ ID NO:4, SEQ ID NO:5 and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 6; or
    (2)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:7和SEQ ID NO:8所示的HCDR1、HCDR2和HCDR3；或(2) Amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, and amino acid sequences such as SEQ ID NO:4, SEQ ID NO:7 and HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO:8; or
    (3)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:9和SEQ ID NO:6所示的HCDR1、HCDR2和HCDR3；(3) Amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and amino acid sequences such as SEQ ID NO:4, SEQ ID NO:9 and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 6;
    优选地，所述抗CTLA-4抗体或其抗原结合片段选自鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、人源化抗体或其抗原结合片段，优选为人源化抗体或其抗原结合片段。Preferably, the anti-CTLA-4 antibody or antigen-binding fragment thereof is selected from murine antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, preferably humanized antibodies or an antigen-binding fragment thereof.
  3. 如权利要求1或2所述的药物组合物，其中所述抗CTLA-4抗体或其抗原结合片段包含：The pharmaceutical composition of claim 1 or 2, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises:
    (1)氨基酸序列如SEQ ID NO:10所示的轻链可变区，和氨基酸序列如SEQ ID NO:11所示的重链可变区；或(1) a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 10, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 11; or
    (2)氨基酸序列如SEQ ID NO:12所示的轻链可变区，和氨基酸序列如SEQ ID NO:11所示的重链可变区；或(2) a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 12, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 11; or
    (3)氨基酸序列如SEQ ID NO:13所示的轻链可变区，和氨基酸序列如SEQ ID NO:14所示的重链可变区；或(3) a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 13, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 14; or
    (4)氨基酸序列如SEQ ID NO:13所示的轻链可变区，和氨基酸序列如SEQ ID NO:15所示的重链可变区；(4) a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 13, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 15;
    优选地，所述抗CTLA-4抗体包含：Preferably, the anti-CTLA-4 antibody comprises:
    (1)氨基酸序列如SEQ ID NO:16所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO:17所示的重链氨基酸序列；(1) the amino acid sequence of the light chain amino acid sequence shown in SEQ ID NO: 16, and the heavy chain amino acid sequence of the amino acid sequence shown in SEQ ID NO: 17;
    (2)氨基酸序列如SEQ ID NO:18所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO:19所示的重链氨基酸序列；(2) the amino acid sequence of the light chain amino acid sequence shown in SEQ ID NO: 18, and the heavy chain amino acid sequence of the amino acid sequence shown in SEQ ID NO: 19;
    (3)氨基酸序列如SEQ ID NO:20所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO:21所示的重链氨基酸序列；或(3) the amino acid sequence of the light chain amino acid sequence shown in SEQ ID NO: 20, and the heavy chain amino acid sequence of the amino acid sequence shown in SEQ ID NO: 21; or
    (4)氨基酸序列如SEQ ID NO:22所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO:23所示的重链氨基酸序列。(4) Amino acid sequence such as the light chain amino acid sequence shown in SEQ ID NO:22, and the amino acid sequence such as the heavy chain amino acid sequence shown in SEQ ID NO:23.
  4. 如权利要求1-3中任一项所述的药物组合物，其中所述缓冲液选自醋酸缓 冲液、柠檬酸缓冲液、磷酸盐缓冲液和组氨酸缓冲液中的一种或多种；优选地，所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，优选为醋酸-醋酸钠缓冲液；优选地，所述缓冲液的浓度为约5～100mM，优选为约10～50mM，优选为约10～30mM；优选地，所述缓冲液的pH为约4.5～6.5，优选为约5.0～6.0。The pharmaceutical composition according to any one of claims 1-3, wherein said buffer is selected from acetate buffer One or more of washing solution, citrate buffer, phosphate buffer and histidine buffer; preferably, the buffer is acetate buffer; preferably, the acetate buffer is acetic acid-acetic acid Sodium buffer solution or acetic acid-potassium acetate buffer solution, preferably acetic acid-sodium acetate buffer solution; preferably, the concentration of the buffer solution is about 5-100mM, preferably about 10-50mM, preferably about 10-30mM; preferably Preferably, the pH of the buffer is about 4.5-6.5, preferably about 5.0-6.0.
  5. 如权利要求1-4中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，所述稳定剂选自精氨酸、精氨酸盐、氯化钠、甘露醇、山梨醇、蔗糖和海藻糖中的一种或多种；优选地，所述精氨酸盐为盐酸精氨酸；优选地，所述稳定剂的浓度为约100～300mM，优选为约130～280mM，优选为约200～260mM。The pharmaceutical composition according to any one of claims 1-4, wherein the pharmaceutical composition also includes a stabilizer selected from the group consisting of arginine, arginine salt, sodium chloride, mannitol, One or more of sorbitol, sucrose and trehalose; preferably, the arginine salt is arginine hydrochloride; preferably, the concentration of the stabilizer is about 100 to 300 mM, preferably about 130 to 280mM, preferably about 200-260mM.
  6. 如权利要求5所述的药物组合物，其中，所述稳定剂为浓度约130～280mM的甘露醇；或所述稳定剂为浓度约130～280mM的蔗糖；或所述稳定剂为浓度约20～80mM的氯化钠与浓度约110-170mM的甘露醇的组合；或所述稳定剂为浓度约20～80mM的氯化钠与浓度约110-170mM的蔗糖的组合；优选地，所述稳定剂为浓度约200～260mM的甘露醇；或所述稳定剂为浓度约200～260mM的蔗糖；或所述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的甘露醇的组合；或所述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的蔗糖的组合。The pharmaceutical composition according to claim 5, wherein the stabilizer is mannitol with a concentration of about 130 to 280 mM; or the stabilizer is sucrose with a concentration of about 130 to 280 mM; or the stabilizer is a concentration of about 20 mM A combination of ~80mM sodium chloride and mannitol at a concentration of about 110-170mM; or the stabilizer is a combination of sodium chloride at a concentration of about 20~80mM and sucrose at a concentration of about 110-170mM; preferably, the stabilizer The agent is mannitol with a concentration of about 200-260mM; or the stabilizer is sucrose with a concentration of about 200-260mM; or the stabilizer is a mixture of sodium chloride with a concentration of about 30-70mM and mannitol with a concentration of about 120-160mM combination; or the stabilizer is a combination of sodium chloride at a concentration of about 30-70 mM and sucrose at a concentration of about 120-160 mM.
  7. 如权利要求1-6中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和泊洛沙姆188中的一种或多种；优选地，以w/v计算，所述表面活性剂浓度为约0.01％～0.1％，更优选地，所述表面活性剂浓度为约0.01％～0.05％。The pharmaceutical composition according to any one of claims 1-6, wherein said pharmaceutical composition also comprises a surfactant selected from the group consisting of polysorbate 80, polysorbate 20 and poloxa One or more of M188; preferably, the surfactant concentration is about 0.01% to 0.1%, more preferably, the surfactant concentration is about 0.01% to 0.05% in w/v calculation .
  8. 如权利要求1-7中任一项所述的药物组合物，其分别包含如下(1)～(13)中任一项所示的组分：The pharmaceutical composition according to any one of claims 1-7, which respectively comprises the components shown in any one of the following (1) to (13):
    (1)(a)约1～50mg/mL的所述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)130～280mM的甘露醇；以及(d)约0.01％～0.1％(w/v)的聚山梨醇酯80；或(1) (a) about 1-50 mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) 130-280 mM mannitol; and (d) about 0.01% to 0.1% (w/v) polysorbate 80; or
    (2)(a)约1～50mg/mL的所述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)130～280mM的蔗糖；以及(d)约0.01％～0.1％(w/v)的聚山梨醇酯80；或 (2) (a) about 1 to 50 mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer with a pH of about 5.0 to 6.0; (c) 130 to 280 mM sucrose; and (d) about 0.01% to 0.1% (w/v) polysorbate 80; or
    (3)(a)约1～50mg/mL的所述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)稳定剂，所述稳定剂为浓度约20～80mM的氯化钠与浓度约110-170mM的甘露醇的组合；以及(d)约0.01％～0.1％(w/v)的聚山梨醇酯80；或(3) (a) about 1 to 50 mg/mL of said anti-CTLA-4 antibody or an antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer with a pH of about 5.0 to 6.0; (c) a stabilizer, The stabilizer is a combination of sodium chloride at a concentration of about 20-80 mM and mannitol at a concentration of about 110-170 mM; and (d) polysorbate 80 at about 0.01%-0.1% (w/v); or
    (4)(a)约1～50mg/mL的所述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)稳定剂，所述稳定剂为浓度约20～80mM的氯化钠与浓度约110-170mM的蔗糖的组合；以及(d)约0.01％～0.1％(w/v)的聚山梨醇酯80；或(4) (a) about 1 to 50 mg/mL of said anti-CTLA-4 antibody or an antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer with a pH of about 5.0 to 6.0; (c) a stabilizer, The stabilizer is a combination of sodium chloride at a concentration of about 20-80 mM and sucrose at a concentration of about 110-170 mM; and (d) polysorbate 80 at about 0.01%-0.1% (w/v); or
    (5)(a)约5～30mg/mL的所述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)200～260mM的甘露醇；以及(d)约0.01％～0.05％(w/v)的聚山梨醇酯80；或(5) (a) about 5-30 mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) 200-260 mM mannitol; and (d) about 0.01% to 0.05% (w/v) polysorbate 80; or
    (6)(a)约5～30mg/mL的所述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)200～260mM的蔗糖；以及(d)约0.01％～0.05％(w/v)的聚山梨醇酯80；或(6) (a) about 5-30 mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) 200-260 mM sucrose; and (d) about 0.01% to 0.05% (w/v) polysorbate 80; or
    (7)(a)约5～30mg/mL的所述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)稳定剂，所述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的甘露醇的组合；以及(d)约0.01％～0.05％(w/v)的聚山梨醇酯80；或(7) (a) about 5-30 mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) a stabilizer, The stabilizer is a combination of sodium chloride at a concentration of about 30-70 mM and mannitol at a concentration of about 120-160 mM; and (d) polysorbate 80 at about 0.01%-0.05% (w/v); or
    (8)(a)约5～30mg/mL的所述抗CTLA-4抗体或其抗原结合片段；(b)约10～30mM醋酸缓冲液，pH为约5.0～6.0；(c)稳定剂，所述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的蔗糖的组合；以及(d)约0.01％～0.05％(w/v)的聚山梨醇酯80；或(8) (a) about 5-30 mg/mL of said anti-CTLA-4 antibody or an antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) a stabilizer, The stabilizer is a combination of sodium chloride at a concentration of about 30-70 mM and sucrose at a concentration of about 120-160 mM; and (d) polysorbate 80 at about 0.01%-0.05% (w/v); or
    (9)(a)约10mg/mL的抗CTLA-4抗体，所述抗CTLA-4抗体包含如SEQ ID NO:16所示的轻链氨基酸序列和如SEQ ID NO:17所示的重链氨基酸序列；(b)约20mM醋酸缓冲液，pH为约5.5；(c)约240mM的甘露醇；以及(d)约0.02％(w/v)的聚山梨醇酯80；或(9) (a) an anti-CTLA-4 antibody of about 10 mg/mL, said anti-CTLA-4 antibody comprising a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain as shown in SEQ ID NO: 17 the amino acid sequence; (b) about 20 mM acetate buffer, pH about 5.5; (c) about 240 mM mannitol; and (d) about 0.02% (w/v) polysorbate 80; or
    (10)(a)约10mg/mL的抗CTLA-4抗体，所述抗CTLA-4抗体包含如SEQ ID NO:16所示的轻链氨基酸序列和如SEQ ID NO:17所示的重链氨基酸序列；(b) 约20mM醋酸缓冲液，pH为约5.5；(c)约220mM的蔗糖；以及(d)约0.02％(w/v)的聚山梨醇酯80；或(10) (a) an anti-CTLA-4 antibody of about 10 mg/mL, said anti-CTLA-4 antibody comprising the light chain amino acid sequence shown in SEQ ID NO: 16 and the heavy chain shown in SEQ ID NO: 17 amino acid sequence; (b) about 20 mM acetate buffer, pH about 5.5; (c) about 220 mM sucrose; and (d) about 0.02% (w/v) polysorbate 80; or
    (11)(a)约10mg/mL的抗CTLA-4抗体，所述抗CTLA-4抗体包含如SEQ ID NO:16所示的轻链氨基酸序列和如SEQ ID NO:17所示的重链氨基酸序列；(b)约20mM醋酸缓冲液，pH为约5.5；(c)约228mM的蔗糖；以及(d)约0.02％(w/v)的聚山梨醇酯80；或(11) (a) an anti-CTLA-4 antibody of about 10 mg/mL, said anti-CTLA-4 antibody comprising a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain as shown in SEQ ID NO: 17 Amino acid sequence; (b) about 20 mM acetate buffer, pH about 5.5; (c) about 228 mM sucrose; and (d) about 0.02% (w/v) polysorbate 80; or
    (12)(a)约10mg/mL的抗CTLA-4抗体，所述抗CTLA-4抗体包含如SEQ ID NO:16所示的轻链氨基酸序列和如SEQ ID NO:17所示的重链氨基酸序列；(b)约20mM醋酸缓冲液，pH为约5.5；(c)稳定剂，所述稳定剂为浓度约50mM的氯化钠与浓度约140mM的甘露醇的组合；以及(d)约0.02％(w/v)的聚山梨醇酯80；或(12) (a) an anti-CTLA-4 antibody of about 10 mg/mL, said anti-CTLA-4 antibody comprising a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain as shown in SEQ ID NO: 17 Amino acid sequence; (b) about 20 mM acetate buffer, pH is about 5.5; (c) stabilizer, the combination of the sodium chloride of concentration about 50 mM and the mannitol of concentration about 140 mM; And (d) about 0.02% (w/v) polysorbate 80; or
    (13)(a)约10mg/mL的抗CTLA-4抗体，所述抗CTLA-4抗体包含如SEQ ID NO:16所示的轻链氨基酸序列和如SEQ ID NO:17所示的重链氨基酸序列；(b)约20mM醋酸缓冲液，pH为约5.5；(c)稳定剂，所述稳定剂为浓度约50mM的氯化钠与浓度约140mM的蔗糖的组合；以及(d)约0.02％(w/v)的聚山梨醇酯80。(13) (a) an anti-CTLA-4 antibody of about 10 mg/mL, said anti-CTLA-4 antibody comprising a light chain amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain as shown in SEQ ID NO: 17 Amino acid sequence; (b) about 20 mM acetate buffer at a pH of about 5.5; (c) a stabilizer comprising a combination of sodium chloride at a concentration of about 50 mM and sucrose at a concentration of about 140 mM; and (d) about 0.02 % (w/v) polysorbate 80.
  9. 一种注射剂，其含有如权利要求1-8中任一项所述的药物组合物与氯化钠溶液或葡萄糖溶液；优选地，所述氯化钠溶液浓度为约0.85～0.9％(w/v)；优选地，所述葡萄糖溶液浓度为约5～25％(w/v)，优选为约5～10％(w/v)；优选地，所述注射剂中，所述抗CTLA-4抗体的浓度为约0.05～10.5mg/mL，更优选为约0.1～5mg/mL或约1～5mg/mL；优选地，所述注射剂的pH为约4.5～6.5，更优选为约5.0～6.0。A kind of injection, it contains the pharmaceutical composition as described in any one in the claim 1-8 and sodium chloride solution or glucose solution; Preferably, described sodium chloride solution concentration is about 0.85～0.9% (w/ v); Preferably, the concentration of the glucose solution is about 5-25% (w/v), preferably about 5-10% (w/v); Preferably, in the injection, the anti-CTLA-4 The concentration of the antibody is about 0.05-10.5 mg/mL, more preferably about 0.1-5 mg/mL or about 1-5 mg/mL; preferably, the pH of the injection is about 4.5-6.5, more preferably about 5.0-6.0 .
  10. 如权利要求1-8中任一项所述的药物组合物或如权利要求9所述的注射剂在制备用于治疗和/或预防CTLA-4介导的疾病或病症的药物中的用途；优选地，所述疾病或病症为癌症。 Use of the pharmaceutical composition according to any one of claims 1-8 or the injection according to claim 9 in the preparation of medicines for treating and/or preventing CTLA-4-mediated diseases or disorders; preferably Preferably, the disease or condition is cancer.
PCT/CN2023/072509 2022-01-17 2023-01-17 Pharmaceutical composition of anti-ctla-4 antibody and use thereof WO2023134771A1 (en)

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