WO2021083271A1 - Préparation stable contenant un anticorps anti-pd-l1 - Google Patents

Préparation stable contenant un anticorps anti-pd-l1 Download PDF

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WO2021083271A1
WO2021083271A1 PCT/CN2020/124804 CN2020124804W WO2021083271A1 WO 2021083271 A1 WO2021083271 A1 WO 2021083271A1 CN 2020124804 W CN2020124804 W CN 2020124804W WO 2021083271 A1 WO2021083271 A1 WO 2021083271A1
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antibody
buffer
stabilizer
trehalose
antigen
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PCT/CN2020/124804
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English (en)
Chinese (zh)
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刘洪川
刘沛想
张静
孟琴
姚盛
冯辉
武海
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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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Priority to CN202080076651.2A priority Critical patent/CN114616249B/zh
Publication of WO2021083271A1 publication Critical patent/WO2021083271A1/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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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

Definitions

  • the invention relates to the field of therapeutic pharmaceutical preparations.
  • the present invention relates to the field of pharmaceutical preparations, which contain a humanized antibody that specifically binds to programmed cell death protein ligand 1 (PD-L1).
  • PD-L1 programmed cell death protein ligand 1
  • Programmed cell death protein ligand 1 (Programmed death-ligand 1, PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7 homolog1, B7-H1), It belongs to the tumor necrosis factor superfamily. It is a type I transmembrane glycoprotein composed of 290 amino acid residues, including an IgV-like region, an IgC-like region, a transmembrane hydrophobic region, and an intracellular tail of 30 amino acids. The molecular weight is 40kDa.
  • PD-L1 mRNA is expressed in almost all tissues, but PD-L1 protein is only continuously expressed in a small number of tissues, including liver, lungs, tonsils, and immune amnesty tissues such as eyes and placenta. PD-L1 is also expressed on activated T cells, B cells, monocytes, dendritic cells, macrophages, etc.
  • the receptor of PD-L1 is PD-1, which is mainly expressed on the surface of immune cells such as CD4+ T cells, CD8+ T cells, NKT cells, B cells and activated monocytes.
  • the combination of PD-L1 and PD-1 can initiate the phosphorylation of ITIM (immunoreceptor tyrosine inhibitory action module) tyrosine residues in the cytoplasm of PD-1, which promotes the binding of tyrosine phospholipase to SHP2 and activates SHP2.
  • ITIM immunomunoreceptor tyrosine inhibitory action module
  • the downstream Syk and PI3K are dephosphorylated to transmit the termination signal and limit the interaction between antigen-presenting cells or dendritic cells and T cells.
  • This combination can further inhibit the metabolism of T cells, inhibit the secretion of anti-apoptotic protein Bcl-X2, reduce the secretion of effector cytokines IL-2 and IFN-r, induce T cell exhaustion and apoptosis, thereby reducing immune T cells Participating in the immune response exercises a negative regulatory function.
  • T cells After T cells recognize the antigen and activate, they secrete IFN-r.
  • IFN-r derived from T cells can expand and maintain T cell functions, such as up-regulating MHC molecules, enhancing the antigen processing and presentation of target cells, and promoting T cell differentiation.
  • IFN-r also induces the expression of PD-L1 in the tissues of immune inflammation, preventing excessive immunity from causing damage to the tissues.
  • IFN-r can induce the expression of PD-L1 on the surface of conventional epithelial cells, vascular endothelial cells, myeloid cells, and naive T cells.
  • the interferon regulatory factor 1 (IRF-1) induced by IFN-r can also bind to the interferon regulatory factor binding sites 200bp and 320bp before the transcription start site of PD-L1 to regulate PD-L1 at the transcription level.
  • PD-L1 can combine with PD-1 on the surface of T cells to exercise a negative regulatory function, thereby protecting the inflammatory site.
  • PD-L1 The negative regulatory function of PD-L1 plays an important role in tumor immunity.
  • Konishi et al. were the first to find the expression of PD-L1 in tissue samples from patients with non-small cell lung cancer. Subsequently, PD-L1 was found to be expressed in the tissues of various tumor patients, including gastric cancer, lung cancer, liver cancer, and intrahepatic cholangiocarcinoma.
  • new protein molecules will be produced due to gene mutation, foreign gene (virus) expression or quiescent gene activation. After these proteins are degraded in the cell, certain degraded peptides can be expressed on the cell surface and become Tumor antigen.
  • the immune system can recognize tumor antigens and eliminate tumor cells through immune surveillance, and tumor cells use PD-L1 to escape immune attack.
  • TIL Tumor infiltrating lymphocytes
  • the PD-L1 of tumor cells can bind to PD-1 on TIL, inhibit the activation of TIL cells, and further lead to their apoptosis.
  • Tumor-related PD-L1 can increase tumor-specific T cell apoptosis, and PD-L1 monoclonal antibody can attenuate this effect.
  • Tumor-related PD-L1 can promote the expression of IL-10 by T cells and further suppress the immune response.
  • PD-L1 is not only a ligand for PD-1, it can also act as a receptor to transmit reverse signals to protect tumor cells from apoptosis induced by FAS-FASL and other anti-tumor pathways.
  • the present invention provides pharmaceutical preparations containing human antibodies that specifically bind to programmed cell death protein ligand 1 (PD-L1).
  • PD-L1 programmed cell death protein ligand 1
  • the present invention provides an anti-PD-L1 antibody pharmaceutical preparation, comprising: (1) a buffer; (2) a stabilizer; (3) an anti-PD-L1 antibody or an antigen-binding fragment thereof.
  • the pharmaceutical preparation may also contain a non-ionic surfactant.
  • the buffer is one of acetate buffer, citrate buffer, histidine buffer, or a combination thereof.
  • the buffer is a histidine buffer; preferably, the histidine buffer is selected from histidine-hydrochloride buffer or histidine-acetate buffer , Preferably histidine-hydrochloride buffer.
  • the histidine buffer is made of L-histidine and L-histidine monohydrochloride.
  • the concentration of the buffer is about 1 mM to 50 mM, preferably 5 mM to 40 mM, more preferably 10 mM to 30 mM.
  • the buffer is a histidine buffer, wherein the concentration of the histidine buffer is about 10 mM to 30 mM, preferably 20 mM to 30 mM. In some embodiments, the concentration of the histidine buffer is about 10 mM. In some embodiments, the concentration of the histidine buffer is about 20 mM. In some embodiments, the concentration of the histidine buffer is about 30 mM.
  • the above-mentioned histidine buffer is a histidine-acetate buffer, preferably, the molar ratio of the two is 1:1 to 1.5:1, and preferably, the pH of such a buffer is 5.5 ⁇ 0.3, preferably about 5.5.
  • this type of buffer contains 15-20 mM histidine and 12-15 mM acetic acid.
  • the above-mentioned buffer is an acetate buffer.
  • the acetate buffer is an acetic acid-sodium acetate buffer or an acetic acid-potassium acetate buffer, preferably an acetic acid-sodium acetate buffer.
  • the above-mentioned buffer is a citric acid buffer, and preferably, the citric acid buffer is a citric acid-sodium citrate buffer.
  • the above-mentioned buffer is a succinic acid buffer, and preferably, the succinic acid buffer is a succinic acid-sodium succinate buffer.
  • the pH of the aforementioned buffer is about 5.0-6.5, preferably about 5.0-6.0, preferably about 5.5-6.5, preferably about 5.0-5.5, preferably about 5.5-6.0, preferably about 6.0-6.5 ,
  • a non-limiting example of the pH of the above buffer is about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, preferably about 5.0, 5.5 or 6.0.
  • the stabilizer includes one or a combination selected from sodium chloride, arginine hydrochloride, mannitol, sorbitol, sucrose, and trehalose. In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is a combination of trehalose and sodium chloride.
  • the concentration of the stabilizer is about 50 mM to 300 mM, preferably 100 mM to 300 mM, more preferably 200 mM to 250 mM.
  • the stabilizer is sodium chloride at a concentration of about 30-200 mM; or the stabilizer is mannitol at a concentration of about 100-300 mM; or the stabilizer is sorbitol at a concentration of about 100-300 mM Or said stabilizer is sucrose with a concentration of about 100-300mM; or said stabilizer is trehalose with a concentration of about 100-300mM; or said stabilizer is arginine hydrochloride with a concentration of about 30-200mM; or said The stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM mannitol; or the stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose; or the stabilizer The agent is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose; or the stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM trehalose; or the
  • the stabilizer is about 100-300 mM trehalose.
  • the aforementioned stabilizer is sodium chloride.
  • the aforementioned stabilizer is sodium chloride at a concentration of about 30-200 mM, and the concentration of the aforementioned sodium chloride is preferably about 50-190 mM, preferably about 100-180 mM, preferably about 120-170 mM, preferably about 130. -150mM, non-limiting examples of the above sodium chloride concentration are about 100mM, 110mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 170mM, 180mM, 190mM, 200mM, preferably 135mM or 140mM .
  • the aforementioned stabilizer is mannitol.
  • the aforementioned stabilizer is mannitol at a concentration of about 100-300 mM, the concentration of the aforementioned mannitol is preferably about 150-300 mM, preferably about 200-280 mM, and a non-limiting example of the aforementioned mannitol concentration is about 200 mM. , 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240mM.
  • the aforementioned stabilizer is sorbitol.
  • the aforementioned stabilizer is sorbitol at a concentration of about 100-300 mM, the concentration of the aforementioned sorbitol is preferably about 150-300 mM, preferably about 200-280 mM, and a non-limiting example of the aforementioned sorbitol concentration is about 200 mM. , 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240mM.
  • the aforementioned stabilizer is sucrose. In some embodiments, the aforementioned stabilizer is sucrose at a concentration of about 100-300 mM. The concentration of the aforementioned sucrose is preferably about 150-300 mM, preferably about 200-280 mM. A non-limiting example of the aforementioned sucrose concentration is about 200 mM, 210 mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240mM.
  • the aforementioned stabilizer is trehalose.
  • the aforementioned stabilizer is trehalose at a concentration of about 100-300 mM, the concentration of the aforementioned trehalose is preferably about 150-300 mM, preferably about 200-250 mM, and a non-limiting example of the aforementioned trehalose concentration is about 180 mM. , 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 200mM, 220mM or 240mM.
  • the aforementioned stabilizer is arginine hydrochloride.
  • the aforementioned stabilizer is arginine hydrochloride at a concentration of about 30-200 mM, and the concentration of the aforementioned arginine hydrochloride is preferably about 50-190 mM, preferably about 100-180 mM, preferably about 120-170 mM, preferably about It is 130-150mM, non-limiting examples of the above-mentioned arginine hydrochloride concentration are about 100mM, 110mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 170mM, 180mM, 190mM, 200mM, preferably 135mM or 140mM.
  • the aforementioned stabilizer is a combination of sodium chloride and mannitol. In some aspects, the aforementioned stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM mannitol, preferably a combination of about 40-150 mM sodium chloride and about 40-180 mM mannitol, preferably about A combination of 40-120 mM sodium chloride and about 40-150 mM mannitol, a non-limiting example of the above stabilizer is a combination of about 59 mM sodium chloride and about 135 mM mannitol, and about 50 mM sodium chloride and A combination of about 135 mM mannitol, a combination of about 101 mM sodium chloride and about 60 mM mannitol, a combination of about 28 mM sodium chloride and about 190 mM mannitol.
  • the aforementioned stabilizer is a combination of sodium chloride and sucrose. In some aspects, the aforementioned stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM sucrose, preferably a combination of about 30-150 mM sodium chloride and about 50-190 mM sucrose, preferably about 40-200 mM sodium chloride and about 50-190 mM sucrose. A combination of 100 mM sodium chloride and about 60-150 mM sucrose. A non-limiting example of the above stabilizer is a combination of about 59 mM sodium chloride and about 135 mM sucrose, and about 28 mM sodium chloride and about 190 mM sucrose. The combination of about 101mM sodium chloride and about 60mM sucrose.
  • the aforementioned stabilizer is a combination of sodium chloride and trehalose. In some aspects, the aforementioned stabilizer is a combination of about 30-200 mM sodium chloride and about 30-200 mM trehalose, preferably about 30-150 mM sodium chloride and about 50-190 mM trehalose, preferably about A combination of 40-100 mM sodium chloride and about 60-150 mM trehalose, a non-limiting example of the above stabilizer is about a combination of 59 mM sodium chloride and about 135 mM sucrose.
  • the aforementioned stabilizer is a combination of arginine hydrochloride and sucrose.
  • the aforementioned stabilizer is a combination of about 30-200 mM arginine hydrochloride and about 30-200 mM sucrose, preferably a combination of about 40-150 mM arginine hydrochloride and about 40-150 mM sucrose, preferably about 40-150 mM sucrose.
  • a non-limiting example of the above stabilizer is a combination of about 59 mM arginine hydrochloride and about 135 mM sucrose.
  • the pH of the pharmaceutical formulation is about 5.0 to 6.5. In some embodiments, the pH of the pharmaceutical formulation is about 5.5 to 6.5. In some embodiments, the pH of the pharmaceutical formulation is about 5.0. In some embodiments, the pH of the pharmaceutical formulation is about 5.5. In some embodiments, the pH of the pharmaceutical formulation is about 6.0. In some embodiments, the pH of the pharmaceutical formulation is about 6.5.
  • the concentration of the antibody or antigen-binding fragment thereof is about 10-200 mg/mL, preferably about 20-100 mg/mL, more preferably about 30-80 mg/mL; more preferably, the above-mentioned anti-PD-
  • the concentration of the L1 antibody or its antigen-binding fragment is about 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL or 80 mg/mL, preferably about 40 mg/mL, 50 mg/mL or 60 mg/mL.
  • the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: the amino acid sequence of HCDR1 is as shown in SEQ ID NO:1; the amino acid sequence of HCDR2 is As shown in SEQ ID NO: 2; HCDR3 has an amino acid sequence as SEQ ID NO: 3; LCDR1 has an amino acid sequence as SEQ ID NO: 4; LCDR2 has an amino acid sequence as SEQ ID NO: 5 ; And the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequence of the VH is as shown in SEQ ID NO: 7. Show; and the amino acid sequence of VL is shown in SEQ ID NO: 8.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is shown in SEQ ID NO: 9; and The amino acid sequence is shown in SEQ ID NO: 10.
  • the concentration of the antibody or antigen-binding fragment thereof is about 30 mg/mL to about 80 mg/mL.
  • the concentration of the antibody or antigen-binding fragment thereof is about 40 mg/mL to about 60 mg/mL. In a specific embodiment, the concentration of the antibody or antigen-binding fragment thereof is about 40 mg/mL. In a specific embodiment, the concentration of the antibody or antigen-binding fragment thereof is about 50 mg/mL. In a specific embodiment, the concentration of the antibody or antigen-binding fragment thereof is about 60 mg/mL.
  • the pharmaceutical preparation provided by the present invention further comprises a surfactant, which is selected from one of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. Or a combination.
  • the pharmaceutical formulation provided by the present invention contains polysorbate 20.
  • the concentration of the above-mentioned surfactant 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 surfactant concentration is about 0.001%-0.1%, preferably about 0.01%-0.1%, preferably about 0.01%-0.05%; The concentration of the surfactant is about 0.02%, 0.04% or 0.08%, preferably 0.02%.
  • the pharmaceutical preparation provided by the present invention contains polysorbate 20 at a concentration of about 0.01% to about 0.05%. In a specific embodiment, the pharmaceutical preparation provided by the present invention contains polysorbate 20 at a concentration of about 0.02%.
  • the pharmaceutical preparations provided by the present invention comprise: (1) about 10-30 mM histidine buffer; (2) about 200 mM to about 250 mM trehalose stabilizer; (3) about 30 mg/mL to about About 80 mg/mL of an anti-PD-L1 antibody or antigen-binding fragment thereof; and (4) about 0.01% to about 0.05% polysorbate 20; wherein the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, where: the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1; the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2; the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 4; the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5; and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6.
  • the pharmaceutical preparation contains the following components shown in any one of (1) to (5):
  • the pharmaceutical preparation comprises:
  • amino acid sequence of the heavy chain of the anti-PD-L1 antibody is shown in SEQ ID NO: 9 and the amino acid sequence of the light chain is shown in SEQ ID NO: 10.
  • the pharmaceutical formulation is a liquid formulation or a lyophilized formulation.
  • the pharmaceutical formulation is a liquid formulation.
  • the above-mentioned liquid formulation or lyophilized formulation is stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.
  • the above-mentioned liquid formulation or lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.
  • the present invention provides the application of any of the above-mentioned pharmaceutical preparations in the preparation of drugs for the treatment of PD-L1 related diseases; preferably, the diseases include breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, and melanoma Tumors are preferably non-small cell lung cancer, melanoma and kidney cancer.
  • Figure 1 Binding of humanized antibody to human PD-L1.
  • Figure 2 Binding of humanized antibody to PD-L1 on 293F cells.
  • Figure 3 Humanized antibody inhibits the binding of human PD-L1 to PD-1 on 293F cells.
  • FIG. 4 Jurkat fluorescein analysis of humanized antibodies.
  • Figure 5 In vivo experiments to detect the inhibitory effect of humanized antibodies on tumor growth.
  • the present invention is characterized by a stable aqueous liquid pharmaceutical formulation containing an anti-PD-L1 antibody or an antigen-binding portion thereof, which has improved properties compared with formulations recognized in the art.
  • the preparation provided by the invention has high concentration and high stability.
  • the "about” used in this application when referring to a measurable value is intended to cover a variation of ⁇ 20% or ⁇ 10% relative to a specific value, including ⁇ 5%, ⁇ 1%, and ⁇ 0.1 %, because these changes are suitable for carrying out the disclosed method.
  • “Therapeutically active antibody” or “therapeutic antibody” refers to an antibody that can be used for therapeutic purposes, that is, to treat a disorder in a subject. It should be noted that although therapeutic proteins can be used for therapeutic purposes, the present invention is not limited to such uses, as the proteins can also be used in in vitro studies.
  • pharmaceutical preparation or “preparation” is a product that adopts a form that makes the biological activity of the active ingredient effective and does not contain other ingredients that have unacceptable toxicity to the subject to which the formulation is administered.
  • the preparation is sterile.
  • liquid formulation refers to a formulation in a liquid state, and is not intended to refer to a lyophilized formulation that is resuspended by weight.
  • the liquid formulation of the present invention is stable during storage, and its stability does not depend on lyophilization (or other state change methods, such as spray drying).
  • aqueous liquid preparation refers to a liquid preparation using water as a solvent.
  • the aqueous liquid formulation is a formulation that does not require lyophilization, spray drying, and/or freezing to maintain stability (e.g., chemical and/or physical stability and/or biological activity).
  • excipient refers to an agent that can be added to a formulation to provide desired characteristics (e.g., consistency, increased stability) and/or to adjust osmotic pressure.
  • desired characteristics e.g., consistency, increased stability
  • excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.
  • the term "buffer with a pH of about 5.0 to about 6.5” refers to a reagent whose acid/base conjugated component acts to make a solution containing the reagent resistant to pH changes.
  • the buffer used in the formulation of the present invention may have a pH in the range of about 5.0 to about 6.5, or a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.5 to about 6.0. In some embodiments, the pH is about 6.0.
  • examples of the "buffer” for controlling the pH within this range include acetate (e.g. sodium acetate), succinate (e.g. sodium succinate), gluconic acid, histidine, methionine, lemon Acid salts, phosphates, citrate/phosphate, imidazole, acetic acid, acetate, citrate, combinations thereof and other organic acid buffers.
  • the buffer is not a protein.
  • the buffer is histidine.
  • the concentration of the buffer is about 5-100 mM, such as 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM or any two values within these ranges are used as the range formed by the endpoints.
  • the buffer concentration is about 20 mM. In some embodiments, the buffer concentration is about 30 mM.
  • histidine buffer is a buffer containing histidine.
  • histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like.
  • the present invention preferably uses a histidine buffer with a pH of about 5.5-6.0.
  • the histidine buffer is a histidine buffer made of 1-20 mM L-histidine and 1-20 mM L-histidine monohydrochloride.
  • the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1:4.
  • the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:1. In some embodiments, the histidine buffer is composed of histidine and histidine hydrochloride in a molar ratio of 1:3. In some embodiments, the histidine preparation is: a histidine buffer with a pH of 5.5 made of 4.5 mM L-histidine and 15.5 mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is: a histidine buffer with a pH of 6.0 made of 15 mM histidine and 15 mM histidine hydrochloride. In some embodiments, the histidine preparation is: a histidine buffer with a pH of 6.0 made of 10 mM histidine and 10 mM histidine hydrochloride.
  • surfactant generally includes protection of proteins such as antibodies from stresses induced by the air/solution interface, solution/surface-induced stresses to reduce antibody aggregation or minimize the formation of particulates in the formulation Chemical reagents.
  • exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, poly Ethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ether, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene- Polyoxypropylene copolymer (Pluronic), sodium dodecyl sulfate (SDS).
  • nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copoly
  • the nonionic surfactant is polysorbate 20.
  • the concentration of polysorbate 20 is about 0 to 0.1% (w/v). In some embodiments, the concentration of polysorbate 20 is 0.01% to about 0.05% (w/v). In some embodiments, the concentration of polysorbate 20 is about 0.02% (w/v).
  • stabilizer can reduce aggregation of antibodies and other proteins.
  • exemplary stabilizers include, but are not limited to: human serum albumin (hsa), bovine serum albumin (bsa), ⁇ -casein, globulin, ⁇ -lactalbumin, LDH, lysozyme, myoglobin, egg white Protein and RNAaseA.
  • Stabilizers also include amino acids, sugars, polyhydric alcohols and their metabolites, such as: sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, arginine, glycine, propyl Acid ( ⁇ -alanine, ⁇ -alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine , ⁇ -aminobutyric acid (GABA), opines (alanine, octopine, glycinine (strombine)) and trimethylamine N-oxide (TMAO).
  • amino acids such as: sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, arginine, glycine, propyl Acid ( ⁇ -alanine,
  • the concentration of the stabilizer may be in the range of 20-300 mM, such as 50-300 mM or 50-250 mM.
  • the stabilizer is a sugar.
  • the stabilizer is trehalose.
  • the trehalose concentration is about 20 to 300 mM.
  • the trehalose concentration is about 100 to 250 mM.
  • the trehalose concentration is about 200 to 250 mM.
  • the concentration of trehalose is about 200 mM, 220 mM, 240 mM, or 250 mM.
  • the formulation of the present invention contains such stabilizers, preferably a combination of such stabilizers and trehalose, such as a combination of sodium chloride and trehalose.
  • the stabilizer of the present invention is sodium chloride and trehalose, wherein the concentration of sodium chloride in the formulation is 30-80 mM, preferably 40-70 mM, and the concentration of trehalose is 100-200 mM, preferably 100- 150mM.
  • the concentration of trehalose may be in the range of 100-200mM, preferably 100-150mM, and the concentration of the stabilizer having the function of controlling osmotic pressure may be In the range of 30-80 mM, preferably 40-70 mM.
  • the formulations of the present invention do not contain such stabilizers that can control osmotic pressure.
  • Isotonic means that the preparation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. The isotonicity can be measured using a vapor pressure or freezing point drop osmometer.
  • a “stable” formulation is one in which the antibody substantially maintains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or during storage. Even if the contained antibody fails to maintain 100% of its chemical structure or biological function after a certain period of storage, the pharmaceutical preparation can be stable. In some cases, after a certain period of storage, it can maintain about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the structure or function of the antibody, which can also be considered as " stable”.
  • Various analytical techniques for measuring protein stability are available in this technical field, and are reviewed in "Peptide and Protein Drug Delivery” 247-301, Vincent Lee, Editor-in-Chief, Marcel Dekker, Inc. ., New York, NY, Pubs. (1991)), and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90 (both are incorporated by reference).
  • the stability of the protein is determined by the percentage of monomeric protein in a solution that has a low percentage of degradation (eg, fragmentation) and/or aggregated protein.
  • the formulation can be stored stably at room temperature, about 25-30°C or 40°C for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months , At least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months , Or longer, no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregate form at most.
  • an acceptable degree of stability means that when the formulation is stored at a certain temperature for a certain period of time, the acidic form of the antibody that can be detected therein does not exceed about 49%, 45%, 40%, 35.
  • the certain period of storage before measuring stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer.
  • a certain temperature that allows the storage of the pharmaceutical preparation can be any temperature in the range of about -80°C to about 45°C, for example, storage at about -80°C, about -30°C, about -20°C, or about 0°C , About 2-8°C, about 5°C, about 25°C, or about 40°C.
  • the antibody does not substantially show signs of aggregation, precipitation, and/or denaturation during visual inspection of color and/or clarity or by UV light scattering or measurement by pore exclusion chromatography, the antibody is in the pharmaceutical preparation "Maintain its physical stability". Aggregation is the process by which single molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the point where visible precipitates are formed.
  • the stability of the formulation can be evaluated by methods known in the art, including measuring the apparent extinction (absorbance or optical density) of the sample. Such extinction measurement is related to the turbidity of the formulation.
  • the turbidity of a formulation is partly an inherent property of the protein dissolved in the solution, and is usually measured by turbidimetry and measured in turbidity units (NTU).
  • the level of turbidity that varies with, for example, the concentration of one or more components in the solution (eg, protein and/or salt concentration) is also referred to as the "opacity" or "opaque appearance" of the formulation.
  • the turbidity level can be calculated with reference to a standard curve generated using a suspension of known turbidity.
  • the reference standard used to determine the turbidity level of the pharmaceutical composition can be based on the "European Pharmacopoeia” standard ("European Pharmacopoeia”, fourth edition, "Directorate for the Quality of Medicine” of the Council of Europe (EDQM), France).
  • a clear solution is defined as a solution with a turbidity lower than or equal to the turbidity of a reference suspension of about 3 according to the "European Pharmacopoeia” standard.
  • Turbidity measurement by turbidimetry can detect Rayleigh scattering in the absence of association or non-ideal effects, which usually varies linearly with concentration. Other methods for assessing physical stability are well known in the art.
  • the antibody "retains its chemical stability" in the pharmaceutical formulation.
  • the chemical stability can be assessed by, for example, detecting or quantifying the form of chemical changes in the antibody.
  • Chemical changes can include size changes (e.g., cropping), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS).
  • Other types of chemical changes include charge changes (for example, occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.
  • the antibody in the pharmaceutical formulation is biologically active for its intended purpose, the antibody "retains its biological activity" in the pharmaceutical formulation.
  • the preparation is stored at a temperature such as 5°C, 25°C, 45°C for a certain period of time (for example, 1 to 12 months)
  • the binding affinity of the anti-PD-L1 antibody contained in the preparation to PD-L1 is the storage
  • the binding affinity of the previous antibody is at least 90%, 95% or more, it can be considered that the formulation of the present invention is stable.
  • the binding affinity can also be determined by techniques such as ELISA or plasmon resonance.
  • the "therapeutically effective amount” or “effective amount” of the antibody refers to an amount effective in the prevention or treatment or alleviation of symptoms of disorders that the antibody can effectively treat.
  • 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.
  • antibody as used herein should be understood to include intact antibody molecules and antigen-binding fragments thereof.
  • antigen-binding portion or “antigen-binding fragment” (or simply “antibody portion” or “antibody fragment”) of an antibody as used herein refers to the antibody that retains the ability to specifically bind to human PD-L1 or its epitope One or more fragments of.
  • full-length antibody refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70kDa in full length) and two light (L) chains (about 50-70kDa in full length). 25kDa) are connected to each other through disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region (abbreviated as CH herein).
  • the heavy chain constant region is composed of three structural domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • VH and VL regions can be further subdivided into complementarity determining regions (CDR) with high variability and more conservatively spaced regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors (including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system).
  • CDR refers to the complementarity determining region within the variable sequence of an antibody.
  • HCDR1, HCDR2, and HCDR3 or LCDR1, LCDR2, and LCDR3 are 3 CDRs in each variable region of the heavy chain and light chain.
  • the exact boundaries of these CDRs have different definitions according to different systems.
  • variable region CDRs of the antibodies of the present invention can be determined using any of many well-known schemes, including Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention includes any one of the anti-PD-L1 antibodies described in the international publication number WO2018153320A1, and the entire content disclosed herein is incorporated herein by way of introduction.
  • the antibodies used in the methods and compositions of the invention include CDR sequences from JS003.
  • antigen-binding fragment includes fragments or derivatives of antibodies, usually including at least one fragment of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody, which retains at least some of the binding of the parent antibody Specificity.
  • antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments And multispecific antibodies.
  • the binding fragment or derivative When the antigen-binding activity is expressed on a molar concentration basis, the binding fragment or derivative usually retains at least 10% of its antigen-binding activity.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also expected that the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as “conservative variants” or “functionally conservative variants” of the antibody).
  • the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: HCDR1 has an amino acid sequence as shown in SEQ ID NO:1, The amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, and the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH has SEQ ID NO: 7
  • VH heavy chain variable region
  • VL light chain variable region
  • the amino acid sequence of VL has the amino acid sequence shown in SEQ ID NO: 8.
  • JS003 is a humanized fully humanized antibody that specifically binds to human PD-L1, which comprises a heavy chain and a light chain, where the heavy chain
  • the amino acid sequence is SEQ ID NO: 9
  • the light chain amino acid sequence is SEQ ID NO: 10.
  • the CDR sequences of the heavy and light chains of clone 30 (JS003) and clone 38 are shown in the following table; further preferably, the light chain variable region and heavy chain variable region sequences of clone or antibody 30 are respectively As shown in SEQ ID NOs: 42 and 44 in WO2018153320A1; the light chain variable region and heavy chain variable region sequences of clone or antibody 38 are shown in SEQ ID NO: 46 and 48 in WO2018153320A1, respectively.
  • the amino acid sequence boundaries of the CDRs in the table below adopt the IMGT scheme.
  • the preparation of the present invention is a liquid preparation containing a high concentration of antibodies and having high stability.
  • the present invention found that the addition of a single trehalose can significantly improve the stability of the formulation.
  • the formulation of the present invention comprises: (1) an anti-PD-L1 antibody or antigen-binding fragment thereof; (2) a buffer with a pH of about 5.0-6.5; and (3) a stabilizer.
  • the pH of the formulation of the present invention is about 5.0 to 6.5.
  • the anti-PD-L1 antibody contained in the preparation of the present invention is the anti-PD-L1 antibody according to any embodiment of the present invention, and preferably contains SEQ ID NO: 1-3 as LCDR1, LCDR2, LCDR3 and SEQ ID NO: 4, respectively -6 as a humanized antibody of HCDR1, HCDR2, and HCDR3; more preferably, a humanized antibody with the amino acid sequence of VH as SEQ ID NO: 7, and the amino acid sequence of VL as SEQ ID NO:; further preferably a heavy chain antibody
  • the amino acid sequence is shown in SEQ ID NO: 9 and the amino acid sequence of the light chain is shown in SEQ ID NO: 10.
  • the concentration of the antibody or antigen-binding fragment thereof in the formulation may be in the range of 30 mg/mL to 80 mg/mL, preferably 40-60 mg/mL.
  • the buffer contained in the preparation of the present invention may be selected from one of acetate buffer, citrate buffer, and histidine buffer, or a combination thereof.
  • the pH of the buffer may be in the range of 5.0-6.5, such as in the range of 5.5-6.5, or in the range of 5.5-6.0.
  • a preferred buffer contains histidine and acetic acid or histidine salt.
  • the buffer in the formulation of the present invention contains histidine and acetic acid; optionally or preferably, the molar ratio of the two is 1:1 to 1.5:1; optionally or preferably, such
  • the pH of the buffer is 5.5 ⁇ 0.3, preferably about 5.5; optionally or preferably, this type of buffer contains 15-20 mM histidine and 12-15 mM acetic acid.
  • the buffer in the formulation of the present invention contains histidine and histidine salt (this type of buffer is also referred to as histidine buffer in the present invention), and the preferred histidine salt is histidine salt.
  • Acid salts such as histidine monohydrochloride.
  • the molar ratio of histidine to histidine salt is 1:1 to 1:4.
  • such a buffer contains 1-20 mM histidine and 1-20 mM histidine monohydrochloride.
  • the concentration of the buffer in the formulation of the present invention is about 10-30 mM.
  • the stabilizer in the formulation of the present invention can be as described above, and can be one of sodium chloride, arginine hydrochloride, mannitol, sorbitol, sucrose, trehalose, or a combination thereof.
  • the concentration of the stabilizer in the formulation may be in the range of 50-300 mM, such as 50-250 mM.
  • the stabilizer in the formulation of the present invention is trehalose and/or sodium chloride.
  • the concentration of sodium chloride in the formulation does not exceed 140 mM, preferably in the range of 40-70 mM.
  • the concentration of sodium chloride in the formulation is in the range of 40-70 mM, and the concentration of trehalose is in the range of 100-200 mM.
  • the concentration of trehalose in the formulation is 200-250 mM.
  • the formulations of the present invention may also contain nonionic surfactants.
  • nonionic surfactants include, but are not limited to, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or a combination thereof, preferably polysorbate 20.
  • concentration of the surfactant in the formulation is about 0.01% to about 0.05%.
  • the formulation of the present invention comprises: (1) about 30 mg/mL to about 80 mg/mL of the anti-PD-L1 antibody described herein or an antigen-binding fragment thereof; (2) about 10-25 mM herein
  • the histidine buffer has a pH of about 5.0-6.5; (3) about 100 mM to about 250 mM trehalose; and (4) about 0% to about 0.1% of the nonionic surfactant described herein.
  • the formulation of the present invention comprises: (1) about 30 mg/mL to about 80 mg/mL of the anti-PD-L1 antibody or antigen-binding fragment thereof described herein; (2) about 20 mM as described herein The histidine buffer, pH is about 5.5-6.5; (3) about 100mM to about 250mM trehalose; (4) about 20mM to about 200mM sodium chloride; and (5) about 0% to about 0.1% Of the nonionic surfactants described herein.
  • the formulation of the present invention comprises: (1) about 50 mg/mL of the anti-PD-L1 antibody described herein or an antigen-binding fragment thereof; (2) about 20 mM histidine buffer described herein Liquid, pH is about 5.5-6.5; (3) about 220 mM trehalose; and (4) about 0.02% polysorbate 20.
  • the formulation of the present invention contains: (1) about 60 mg/mL of the anti-PD-L1 antibody or antigen-binding fragment thereof described herein; (2) about 20 mM histidine buffer, pH about It is 5.5-6.5; (3) about 240 mM trehalose; and (4) about 0.02% polysorbate 20.
  • the formulation of the present invention contains: (1) about 60 mg/mL of the anti-PD-L1 antibody or antigen-binding fragment thereof described herein; (2) about 20 mM histidine buffer, pH about (3) about 135 mM trehalose; (4) about 59 mM sodium chloride; and (5) about 0.02% polysorbate 80.
  • the formulation of the present invention comprises: (1) about 50 mg/mL of an anti-PD-L1 antibody or antigen-binding fragment thereof as described herein, wherein the antibody comprises HCDR1, HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3, where: HCDR1 has an amino acid sequence such as SEQ ID NO: 1, HCDR2 has an amino acid sequence such as SEQ ID NO: 2, HCDR3 has an amino acid sequence such as SEQ ID NO: 3, LCDR1 has an amino acid sequence such as SEQ ID NO: 4, LCDR2 has an amino acid sequence such as SEQ ID NO: 5, and LCDR3 has an amino acid sequence such as SEQ ID NO: 6; (2) About 20mM histidine buffer, pH is about 5.5-6.5; (3) ) About 220 mM trehalose; and (4) about 0.02% polysorbate 20.
  • the antibody comprises HCDR1, HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3, where: HCDR1 has an amino acid sequence such
  • the formulation of the present invention has a pH of 5.5-6.5 and contains: (1) about 50 mg/mL of an anti-PD-L1 antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain Variable region (VH) and light chain variable region (VL), where VH has an amino acid sequence such as SEQ ID NO: 7; and VL amino acid sequence SEQ ID NO: 8; (2) about 20 mM histidine buffer, The pH is about 5.5-6.5; (3) about 220 mM trehalose; and (4) about 0.02% polysorbate 20.
  • VH heavy chain Variable region
  • VL light chain variable region
  • the formulation of the present invention comprises: (1) about 50 mg/mL of an anti-PD-L1 antibody or antigen-binding fragment thereof, wherein the antibody is a full-length antibody, wherein the amino acid sequence of the heavy chain It is SEQ ID NO: 9 and the amino acid sequence of the light chain SEQ ID NO: 10; (2) about 20 mM histidine buffer, pH about 5.5-6.5; (3) about 220 mM arginine; and (4) About 0.02% polysorbate 20.
  • the pharmaceutical preparations of the present invention can be used to prevent or treat PD-L1 mediated diseases or conditions, and the disease is preferably cancer; more preferably, PD-L1 expressing cancer.
  • PD-L1 mediated cancers include breast cancer, lung cancer, gastric cancer, bowel cancer, kidney cancer, and melanoma, preferably non-small cell lung cancer, melanoma, and kidney cancer.
  • the pharmaceutical preparations of the present invention can be used to prepare medicines for preventing or treating PD-1 mediated diseases or disorders.
  • the disease is preferably cancer; more preferably, it is a cancer that expresses PD-L1.
  • Exemplary PD-L1 mediated cancers include breast cancer, lung cancer, gastric cancer, bowel cancer, kidney cancer, and melanoma, preferably non-small cell lung cancer, melanoma, and kidney cancer.
  • the buffer system and pH closely affect the stability of the antibody.
  • Each antibody with unique physical and chemical properties has the most suitable buffer type and pH. This example aims to screen an optimal buffer system and pH, so that the anti-PD-L1 antibody disclosed in the present invention has the best stability for clinical application.
  • This example was performed with JS003 at a concentration of about 40 mg/mL and about 60 mg/mL.
  • the samples were placed in an environment of 40°C, and were taken out in the 0th week, the 2nd week and the 4th week for analysis and testing.
  • the main pathways of protein degradation are the formation of aggregates, lysate products and charged variants.
  • SEC-HPLC Size exclusion chromatography
  • CEX-HPLC cation exchange chromatography
  • the antibody in the CEX-HPLC test, the antibody remained relatively stable within the pH range of 5.5 to 6.5. After being placed at a high temperature of 40°C for 4 weeks, the monomer content of the sample decreased at a rate of 3%/ Less than a week: In the SEC-HPLC test, the main charge of the sample decreases at a rate of less than 6%/week.
  • the buffer system is histidine buffer and the pH is 6.0 (prescription numbers 7 and 8)
  • the average drop rate of monomer purity of the sample after being placed at high temperature of 40°C for 4 weeks is only 0.29%/week, which is approximately sodium acetate buffer (Prescription 2) 10%. Based on these results, a histidine buffer with a pH of 5.5-6.0 was selected for further research.
  • the JS003 monomer content change was detected by size exclusion high performance liquid chromatography (SEC-HPLC), and the main charge peak content of JS003 was detected by weak cation high performance liquid chromatography (CEX-HPLC). The results are shown in Table 5.
  • the prescription 18 containing trehalose is the most stable.
  • the formulation group containing only trehalose after being placed at a high temperature of 40°C for 2 weeks (prescription number 18): (1)
  • the antibody monomer The rate of decrease in purity is obviously the lowest, as low as 1.2%/week, which is about 49% of the sucrose group (prescription 17), and the purity of antibody monomer is 97.60%;
  • the main charge of antibody The rate of decline is relatively low, 10.75%/week, and the main charge is 78.5%.
  • the formulation group containing trehalose and sodium chloride also had excellent stability, and the rate of decrease in antibody monomer purity was about 1.5%/week, approximately It was 51% of the highest sucrose + arginine hydrochloride group (prescription 23), and the decline rate of the main charge of the antibody was only 10.05%/week.
  • Surfactants added to liquid formulations are often used to protect proteins such as antibodies from the stress induced by the air/solution interface and the stress induced by the solution/surface during storage to reduce the aggregation of antibodies or minimize the formation of particulate matter in the formulation.
  • Reagent which is beneficial to the stability of the physical and chemical properties of the antibody.
  • 20mM histidine buffer the molar ratio of histidine to histidine salt is 1:1, pH is 6.0
  • 50mg/ml of JS003 different concentrations of stabilizers, (0-0.5 %) Polysorbate 20 or Polysorbate 80, placed at 40°C for 4 weeks and then analyzed and tested. The results are shown in Table 6.
  • the stability evaluation of the JS003 antibody prescription was carried out for 24 months.
  • the anti-PD-L1 antibody prescription contains 20mM histidine buffer (the molar ratio of histidine to histidine salt is 1:1, and the pH is 6.0), 50mg/ml JS003 antibody, 220mM trehalose and 0.02% poly Sorbate 20.
  • Evaluation indicators include: 1. Appearance (visual inspection); 2. Visible foreign matter (visual inspection); 3. Protein content (ultraviolet spectrophotometry); 4. Size exclusion chromatography (SEC-HPLC) to measure antibody monomer and polymer Content of body and fragment; 5. Cation exchange chromatography (CEX-HPLC) to measure the content of antibody main peak, acidic peak and basic peak; 6. Non-reducing/reducing capillary gel electrophoresis (NR/R-CE-SDS); 7 . Insoluble particles (photoresistance method); 8. ELISA method to detect the relative binding activity and relative blocking activity of the antibody.
  • Example 5 ELISA to detect the binding of humanized antibody to human PD-L1
  • the EC 50 values of humanized antibody binding to PD-L1 were 558 pg/mL and 837 pg/mL, respectively.
  • the CHO cells expressing PD-L1 were digested and resuspended in FACS buffer by centrifugation , and added to a 1.5ml EP tube with a cell mass of ⁇ 2.5 ⁇ 10 4 and a volume of 50ul.
  • Add 50ul of antibodies of different concentrations (prescription number: 30) Mix the diluent and incubate at room temperature for 30 minutes; wash the cells twice with FACS buffer, add 100ul goat anti-human IgG-PE antibody, and incubate for 30 minutes in the dark; wash twice with FACS buffer and perform FACS detection.
  • humanized antibodies 30 and 38 can specifically bind to PD-L1 on 293F cells.
  • Example 7 Humanized antibody inhibits the binding of human PD-L1 to PD-1 on 293F cells
  • humanized antibody (10ug/ml, prepared with prescription number 30) and mix with biotin-labeled human PD-L1 (1ug/ml), and incubate at room temperature for 30 minutes. Then the mixture was incubated with the stable 293F PD-1 cell line (1.5 ⁇ 10 5 cells) at 37 degrees Celsius for 15 minutes, eluted with PBS three times, and 5 ⁇ g/ml SA-APC was added and incubated at 4 degrees Celsius for 15 minutes. After eluting with PBS three times, it was tested by flow cytometry to verify whether the humanized antibody can inhibit the binding of human PD-L1 to PD-1 on the surface of 293F cells.
  • the humanized antibody can specifically inhibit the binding of human PD-L1 to PD-1 on the surface of 293F cells.
  • CHO cells expressing PD-L1 were plated on a 96-well plate, with a cell volume of 5 ⁇ 10 4 per well, cultured overnight at 37°C and 7% CO 2 , the cell supernatant was removed, and 40 ul of antibody 30 and 38 ( Prepared with prescription number 30) (initial concentration 60ug/ml, 3 times concentration gradient dilution), add 40ul Jurkat reporter cells that can continuously express PD-1 and NFAT-luciferase reporter gene, the total cell number is Incubate 1 ⁇ 10 5 cells at 37° C., 7% CO 2 for 6 hours, add luciferase reagent, and detect the luminescence value with a microplate reader.
  • humanized antibodies 30 and 38 can specifically inhibit the binding of human PD-L1 to PD-1 and promote the expression of reporter genes.
  • humanized antibodies can significantly inhibit the growth of tumors induced by MC38-B7H1.

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Abstract

La présente invention concerne une préparation pharmaceutique stable contenant un anticorps anti-PD-L1 (ligand de mort programmée 1) et une application médicale correspondante. La préparation contient un anticorps anti-PD-L1 et une solution tampon, peut en outre contenir au moins un stabilisant, et peut éventuellement aussi contenir un tensioactif. La préparation pharmaceutique fournie par la présente invention peut inhiber efficacement l'agrégation d'anticorps et empêcher la dégradation de produits d'anticorps. Elle présente en outre une stabilité élevée.
PCT/CN2020/124804 2019-10-31 2020-10-29 Préparation stable contenant un anticorps anti-pd-l1 WO2021083271A1 (fr)

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WO2023006055A1 (fr) * 2021-07-29 2023-02-02 Shanghai Junshi Biosciences Co., Ltd. Composition pharmaceutique d'anticorps anti-pd-1 et son utilisation
WO2023040999A1 (fr) * 2021-09-18 2023-03-23 江苏康宁杰瑞生物制药有限公司 Composition comprenant un fragment de liaison à l'antigène pd-l1 et son utilisation
WO2023198115A1 (fr) * 2022-04-14 2023-10-19 Beigene Switzerland Gmbh Formulations stables de chlorure de sodium à haute concentration contenant un anticorps pd-1 et leurs procédés d'utilisation
WO2023217294A1 (fr) * 2022-05-09 2023-11-16 浙江特瑞思药业股份有限公司 Formulation de nano-anticorps anti-pd-1 et son utilisation

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WO2024017241A1 (fr) * 2022-07-18 2024-01-25 Suzhou Transcenta Therapeutics Co., Ltd. Formulation pharmaceutique stable comprenant un anticorps anti-gremlin1
CN116271015B (zh) * 2022-12-16 2023-10-24 北京东方略生物医药科技股份有限公司 一种IgM抗体制剂及其用途

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WO2023040999A1 (fr) * 2021-09-18 2023-03-23 江苏康宁杰瑞生物制药有限公司 Composition comprenant un fragment de liaison à l'antigène pd-l1 et son utilisation
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