WO2023134771A1 - Composition pharmaceutique d'anticorps anti-ctla-4 et son utilisation - Google Patents

Composition pharmaceutique d'anticorps anti-ctla-4 et son utilisation 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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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/fr

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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

La présente invention concerne une composition pharmaceutique stable d'un anticorps anti-CTLA-4 et son utilisation. La composition pharmaceutique contient une solution tampon et un anticorps anti-CTLA-4 ou un fragment de liaison à l'antigène de celui-ci, la concentration de l'anticorps anti-CTLA-4 ou du fragment de liaison à l'antigène de celui-ci étant d'environ 1 à 100 mg/mL. Selon la présente invention, au moyen de la sélection d'un système tampon et d'un pH appropriés et de l'optimisation d'un stabilisant et d'un tensioactif, la préparation d'anticorps développée peut être utilisée pour une injection intraveineuse.
PCT/CN2023/072509 2022-01-17 2023-01-17 Composition pharmaceutique d'anticorps anti-ctla-4 et son utilisation WO2023134771A1 (fr)

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Citations (5)

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DE102005019845A1 (de) * 2005-04-28 2006-11-02 Li-Te Chin Neue aus humanen Keimbahnsequenzen gewonnene Antikörperstrukturen
CN110545844A (zh) * 2017-02-21 2019-12-06 瑞美德生物医药科技有限公司 使用结合细胞毒性t淋巴细胞抗原-4(ctla-4)的抗体的癌症治疗
CN110840830A (zh) * 2019-10-25 2020-02-28 北京东方百泰生物科技有限公司 一种抗ctla-4单克隆抗体的注射制剂
US20200262921A1 (en) * 2015-11-19 2020-08-20 Zeling Cai Ctla-4 antibodies and uses thereof
WO2022017428A1 (fr) * 2020-07-21 2022-01-27 上海君实生物医药科技股份有限公司 Anticorps anti-ctla-4 et son utilisation

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DE102005019845A1 (de) * 2005-04-28 2006-11-02 Li-Te Chin Neue aus humanen Keimbahnsequenzen gewonnene Antikörperstrukturen
US20200262921A1 (en) * 2015-11-19 2020-08-20 Zeling Cai Ctla-4 antibodies and uses thereof
CN110545844A (zh) * 2017-02-21 2019-12-06 瑞美德生物医药科技有限公司 使用结合细胞毒性t淋巴细胞抗原-4(ctla-4)的抗体的癌症治疗
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