WO2021143767A1 - Préparation d'anticorps bispécifique combinant pd-1 et pd-l1 et son utilisation - Google Patents

Préparation d'anticorps bispécifique combinant pd-1 et pd-l1 et son utilisation Download PDF

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WO2021143767A1
WO2021143767A1 PCT/CN2021/071757 CN2021071757W WO2021143767A1 WO 2021143767 A1 WO2021143767 A1 WO 2021143767A1 CN 2021071757 W CN2021071757 W CN 2021071757W WO 2021143767 A1 WO2021143767 A1 WO 2021143767A1
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seq
antibody
amino acid
cancer
histidine
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PCT/CN2021/071757
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Chinese (zh)
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谢瑞霞
马丽强
汪音爵
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信达生物制药(苏州)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
    • A61J1/00—Containers specially adapted for medical or pharmaceutical purposes
    • 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
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/26—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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/178—Syringes
    • A61M5/24—Ampoule syringes, i.e. syringes with needle for use in combination with replaceable ampoules or carpules, e.g. automatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of antibody preparations. More specifically, the present invention relates to the stabilization of bispecific antibodies (also known as anti-PD-1/PD-L1 antibodies) that bind human programmed cell death 1 (PD-1) and human PD-1 ligand 1 Preparations, and the therapeutic and/or preventive uses of the preparations.
  • bispecific antibodies also known as anti-PD-1/PD-L1 antibodies
  • PD-1 programmed cell death 1
  • Preparations and the therapeutic and/or preventive uses of the preparations.
  • Immune checkpoint pathways are used to maintain self-tolerance and control T cell activation, but cancer cells can use these pathways to inhibit anti-tumor responses and prevent their destruction.
  • the PD-1/PD-L1 pathway is one such immune checkpoint.
  • Human PD-1 is found on T cells, and human PD-L1 is abnormally expressed by a variety of tumor types; the combination of PD-L1 and PD-1 inhibits T cell proliferation and cytokine production.
  • the PD-1/PD-L1 inhibitory axis has been conquered by tumors as part of the natural selection process that shapes tumor evolution in the context of anti-tumor immune responses.
  • PCT application number PCT/US2018/041205 discloses an exemplary therapeutic antibody specific for human PD-1/PD-L1, the disclosure of which is hereby incorporated by reference in its entirety.
  • anti-PD-1/PD-L1 antibody preparations that can be used to treat, prevent or delay various cancers and immune-related diseases.
  • Antibodies used in human subjects must be stored and transported to the site of administration before use. Reproducibly obtaining the desired antibody drug level in the subject requires that the drug be stored in a formulation that maintains the biological activity of the drug.
  • subcutaneous administration of high-concentration preparations is more convenient and safer. It can realize self-administration by patients at home, which is convenient for long-term treatment of chronic diseases.
  • high-concentration preparations can meet high-dose administration requirements and reduce Cost of production.
  • the development of high-concentration preparations has become the development trend of monoclonal antibody preparations.
  • lyophilized formulations can bring superior formulation stability, and higher concentration liquid formulations can be prepared by adding less liquid during reconstitution.
  • the present invention meets the above-mentioned needs by providing stable formulations containing antibodies that specifically bind to PD-1/PD-L1.
  • the present invention relates to a lyophilized preparation of an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, which comprises: (i) an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof; ii) a buffer system; (iii) a stabilizer including polyols and/or amino acids; and (iv) a surfactant, wherein the formulation has a pH between 5.5-6.5 when reconstituted, for example, pH Approximately 5.5, 6.0 or 6.5.
  • the lyophilized formulation is capable of reconstituted the antibody or antigen-binding fragment thereof at a concentration between about 1 mg/mL to about 200 mg/mL.
  • the surfactant is selected from polysorbate 80, polysorbate 20, poloxamer, polyethylene glycol polysorbate 80, or a combination thereof, and is at a rate of about 0.01% (w/v ) Exist in a weight ratio of -1% (w/v).
  • the buffer system is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, citric acid and sodium citrate buffer system, sodium acetate buffer system, phosphate buffer system
  • the system is present in a weight ratio of about 0.01% (w/v) to 1% (w/v).
  • the polyol is selected from sorbitol, mannitol, or a combination thereof
  • the amino acid is selected from arginine, arginine hydrochloride, methionine, glycine, proline, or a combination thereof;
  • the stabilizer includes sorbitol and arginine; more preferably, the sorbitol is at about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, The weight ratio of 8, 9, 10% (w/v) is present, and the arginine is present at about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10% (w/v) weight ratio exists;
  • the stabilizer includes sorbitol and arginine hydrochloride; more preferably, the sorbitol is at a concentration of about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10% (w/v) weight ratio exists, about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 The weight ratio of %(w/v) exists.
  • the stabilizer further includes methionine; more preferably, the methionine is present in a weight ratio of about 0.1-10% (w/v).
  • the present invention relates to a lyophilized preparation of an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, which is prepared by lyophilizing an aqueous solution containing: (i) anti-PD-1/PD -L1 antibody or its antigen-binding fragment; (ii) buffer system; (iii) stabilizer, said stabilizer including polyol and/or amino acid; and (iv) surfactant, said liquid formulation has a concentration of about 5.5-6.5
  • the pH for example, the pH is about 5.5, 6.0 or 6.5; preferably, the liquid formulation has a pH of about 6.0.
  • the present invention relates to a liquid pharmaceutical preparation of an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, which comprises an aqueous solution comprising: (i) an anti-PD-1/PD-L1 antibody or The antigen-binding fragment thereof; (ii) a buffer system; (iii) a stabilizer, the stabilizer includes a polyol and/or amino acid; and (iv) a surfactant, the liquid formulation has a pH of about 5.5-6.5, for example, The pH is about 5.5, 6.0 or 6.5; preferably, the liquid formulation has a pH of about 6.0.
  • the anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof is present in an aqueous solution at a concentration of about 1 mg/mL to about 200 mg/mL, For example, about 1, 5, 10, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg
  • concentration of /ml exists in the aqueous solution.
  • the surfactant is selected from polysorbate 80, polysorbate 20, poloxamer, polyethylene glycol polysorbate 80, or a combination thereof , And exist at a concentration of about 0.1-10 mg/ml, for example, about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml.
  • the buffer system is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, citric acid and sodium citrate buffer system , Sodium acetate acetate buffer system, phosphate buffer system, and exist in a concentration of about 1-100 mM, for example, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM.
  • the buffer system is a histidine buffer system; more preferably, the histidine is at a concentration of about 0.1-10 mg/mL, such as about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10mg/ml concentration exists;
  • the buffer system is a histidine and histidine hydrochloride buffer system; more preferably, the histidine and the histidine hydrochloride are respectively about 0.1-10 mg/mL, such as about 0.1, 0.5, Concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml exist.
  • the polyol is selected from sorbitol, mannitol or a combination thereof, and the amino acid is selected from arginine, arginine hydrochloride, and methionine , Glycine, proline or a combination thereof; preferably, the stabilizer includes sorbitol and arginine; more preferably, the sorbitol is about 10-100 mg/mL, such as about 10, 20, 30, 40, The arginine is present at a concentration of 50, 60, 70, 80, 90, 100 mg/mL, and the arginine is present at, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL;
  • the stabilizer includes sorbitol and arginine hydrochloride; more preferably, the sorbitol and the arginine hydrochloride are respectively about 10-100 mg/mL, such as about 10, 20, 30, 40, 50, Concentrations of 60, 70, 80, 90, 100 mg/mL exist.
  • the stabilizer further includes methionine; preferably, the methionine is at a concentration of about 1-100 mg/mL, such as about 1, 5 , 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL.
  • the anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof binds to human PD-L1 (SEQ ID NO: 1) and human PD-1 (SEQ ID NO: 2).
  • the anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof comprises: a first heavy chain (HCl), which comprises a first heavy chain variable region (HCVR1) and a constant region; a first light chain ( LC1), which includes a first light chain variable region (LCVR1) and a constant region; a second heavy chain (HC2), which includes a second heavy chain variable region (HCVR2) and a constant region; and a second light chain (LC2 ), which contains the second light chain variable region (LCVR2) and the constant region, where:
  • the HCVR1 includes the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 3, and the LCVR1 includes the light chain variable region shown in SEQ ID NO: 4 LCDR1, LCDR2 and LCDR3 included; and
  • the HCVR2 includes the three complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 5, and the LCVR2 includes the light chain shown in SEQ ID NO: 8
  • the variable region contains three complementary determining regions LCDR1, LCDR2 and LCDR3.
  • the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention may include: a first heavy chain (HCl), which includes a first heavy chain variable region (HCVR1) and a constant region;
  • the light chain (LC1) which includes the first light chain variable region (LCVR1) and the constant region;
  • the second heavy chain (HC2) which includes the second heavy chain variable region (HCVR2) and the constant region;
  • the second light chain Chain (LC2) which includes the second light chain variable region (LCVR2) and constant region, where:
  • the HCVR1 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 16, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and the amino acid sequence shown in SEQ ID NO: 18 Sequence complementarity determining region CDR3;
  • the LCVR1 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 19, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 20, and the amino acid sequence shown in SEQ ID NO: 21 Sequence complementarity determining region CDR3;
  • the HCVR2 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 22, and the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24 and having SEQ ID NO : The complementarity determining region CDR3 of the amino acid sequence shown in 25; and
  • the LCVR2 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 26, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 27, and the amino acid sequence shown in SEQ ID NO: 28 The complementarity of the sequence determines the region CDR3.
  • the antibody or antigen-binding fragment thereof comprises HCVR1 that is at least 90%, 95%, 98%, or 99% or more identical to SEQ ID NO: 3; and/or is identical to SEQ ID NO: : 4 LCVR1 with at least 90%, 95%, 98% or 99% or higher identity; and/or at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 5 HCVR2; and/or LCVR2 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 8;
  • the HCVR1 includes the amino acid sequence shown in SEQ ID NO: 3; b) the LCVR2 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 4; c) the HCVR2 includes the amino acid sequence shown in SEQ ID NO: 5; and d) the LCVR2 includes the amino acid sequence shown in SEQ ID NO: 8.
  • the antibody or antigen-binding fragment thereof comprises HC1 that is at least 90%, 95%, 98%, or 99% or more identical to SEQ ID NO: 49 or SEQ ID NO: 29; and / Or LC1 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 30; and/or with SEQ ID NO: 33 or SEQ ID NO: 31 or SEQ ID NO: 32 HC2 with at least 90%, 95%, 98% or 99% or higher identity; and/or LC2 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 34 ;
  • the HC1, the LC1, the HC2 and the LC2 respectively comprise SEQ ID NO: 49, SEQ ID NO: 30, The amino acid sequence shown in SEQ ID NO: 31 and SEQ ID NO: 34; or the amino acid sequence shown in SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 34 respectively; or They respectively include the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 34.
  • the anti-PD-1/PD-L1 antibody is the anti-PD-1/PD-L1 antibody disclosed in PCT application number PCT/US2018/041205.
  • the entire content of the PCT application is hereby incorporated herein by reference.
  • the antibody is selected from: PD-1/PD-L1 bispecific Ab v13844, Ab v3.0 or Ab v3.2.
  • the antibody in the formulation of the present invention exhibits stability for at least about 12 months, preferably, the antibody exhibits stability for at least about 24 months, more preferably The antibody exhibits stability for at least about 36 months.
  • the formulation is stable after storage, for example, after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 6 months, or after storage at 40°C ⁇ 2°C for 1 month.
  • the formulation is stable after storage, for example, after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 6 months, or after storage at 40°C ⁇ 2°C for 1 month.
  • Preferably has one or more of the following features:
  • the preparation has a purity greater than 90%, preferably greater than 92%, 94%, 96%, 98%, 99% purity;
  • the preparation has a purity greater than 90%, preferably greater than 92%, 94%, 96%, 98% purity;
  • the main component is ⁇ 35%, preferably greater than or equal to 35%, 40%, 45% or 50%.
  • the present invention provides a method for preparing the formulation of the present invention, including the following steps:
  • step (2) Using the solution prepared in step (1) to exchange the PD-1/PD-L1 bispecific antibody or its antigen-binding fragment by ultrafiltration, and then concentrate it to the target concentration;
  • the invention provides a delivery device comprising an effective amount of the liquid antibody preparation or lyophilized preparation of the invention.
  • the delivery device of the present invention is provided in the form of a pre-filled syringe containing an effective amount of the liquid antibody formulation or lyophilized formulation of the present invention, for example for intravenous, subcutaneous, intradermal or intramuscular injection, or Intravenous infusion.
  • the present invention provides a method for delivering anti-PD-1/PD-L1 antibody protein to a subject, such as a mammal, comprising administering to the subject an effective amount of the liquid antibody preparation or lyophilized preparation of the present invention
  • the delivery is carried out, for example, by a delivery device using a pre-filled syringe.
  • the present invention provides the use of the liquid antibody preparation or lyophilized preparation of the present invention to prepare a delivery device (eg, a pre-filled syringe) or a drug for treating or preventing tumors in a subject.
  • a delivery device eg, a pre-filled syringe
  • a drug for treating or preventing tumors in a subject e.g, cancer, including but not limited to solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions.
  • the cancers described herein include, but are not limited to, Hodgkin's or non-Hodgkin's lymphoma, melanoma, renal cell carcinoma, kidney cancer, lung cancer, bladder cancer, gastric and esophageal cancer, colorectal cancer Cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, breast cancer, triple negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung (NSCLC), mesothelioma , Squamous cell carcinoma of the head and neck (SCCHN), soft tissue sarcoma or glioblastoma multiforme.
  • the lung cancer is NSCLC (squamous and non-squamous), small cell lung cancer or mesothelioma.
  • the present invention also provides a liquid antibody preparation or lyophilized preparation of the present invention or a delivery device (such as a pre-filled syringe) or a drug containing the liquid antibody preparation or lyophilized preparation of the present invention by administering the liquid antibody preparation or lyophilized preparation of the present invention to a subject.
  • a delivery device such as a pre-filled syringe
  • a drug containing the liquid antibody preparation or lyophilized preparation of the present invention by administering the liquid antibody preparation or lyophilized preparation of the present invention to a subject.
  • the present invention also provides a liquid antibody preparation or lyophilized preparation of the present invention or a delivery device (e.g., pre-filled syringe) or medicine containing the liquid antibody preparation or lyophilized preparation of the present invention to prevent and/ Or a method for treating a subject's disease, such as the above-mentioned tumor.
  • a delivery device e.g., pre-filled syringe
  • medicine containing the liquid antibody preparation or lyophilized preparation of the present invention to prevent and/ Or a method for treating a subject's disease, such as the above-mentioned tumor.
  • freeze-dried and liquid antibody preparations provided by the present invention are stable in nature, and can be stable for at least 24 months at 5°C ⁇ 3°C.
  • the prescription provided by the present invention can not only ensure the stability of low-concentration liquid preparations, but also can ensure the stability of high-concentration liquid preparations without adding auxiliary materials, providing the possibility of subcutaneous administration of high-concentration liquid preparations.
  • the high-concentration liquid preparation of the present invention can be directly prepared into a freeze-dried preparation without optimizing the prescription, and can maintain its structural and functional integrity after freeze-drying.
  • the freeze-dried preparation provided by the present invention is easily reconstituted, is within the acceptable injection viscosity range of the liquid preparation ( ⁇ 10cP), and all the detection indicators have not changed after being placed at room temperature for one day.
  • a higher concentration (for example, 170 mg/ml) liquid preparation can be prepared by reducing the volume of the reconstituted solution. The same amount of administration reduces the volume of administration and improves the compliance of clinical administration.
  • Figure 1 shows a graph of the protein purity in each sample measured by the SEC-HPLC method over time in the pre-prescription test. Met in the figure represents methionine.
  • Figure 2 shows the changes in the main components and acidic components of the charge variants in each sample measured by the iCIEF method over time in the pre-prescription test.
  • Figure 3 shows the variation of protein purity in each sample measured by SEC-HPLC method over time under the condition of 40°C ⁇ 2°C in the prescription screening test
  • Figure 4 shows a graph of the protein purity in each sample measured by SEC-HPLC at 25°C ⁇ 2°C over time in the prescription screening test.
  • Figure 5 shows a graph of the protein purity in each sample measured by the non-reduced CE-SDS method over time under the condition of 40°C ⁇ 2°C in the prescription screening test.
  • Figure 6 shows a graph of the main components and acidic components of the charge variants in each sample measured by the CEX-HPLC method at 40°C ⁇ 2°C in the prescription screening test over time.
  • Figure 7 shows a graph of the main components and acidic components of the charge variants in each sample measured by the CEX-HPLC method under the conditions of 25°C ⁇ 2°C in the prescription screening test over time.
  • the term “comprising” or “including” means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprises” or “includes” when used, unless otherwise specified, it also encompasses the situation consisting of the stated elements, integers or steps.
  • an antibody variable region that "comprises” a specific sequence when referring to an antibody variable region that "comprises” a specific sequence, it is also intended to encompass the antibody variable region composed of the specific sequence.
  • immune checkpoint molecule refers to a type of inhibitory signal molecule present in the immune system, which avoids tissue damage by regulating the persistence and intensity of immune response in peripheral tissues, and participates in maintaining tolerance to self-antigens (Pardoll DM. , The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer,2012,12(4):252-264).
  • Immune checkpoint molecules include but are not limited to programmed death 1 (PD-1), PD-L1, PD-L2, cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3 and TIM-3.
  • the terms "programmed cell death ligand 1", “PD-L1”, “programmed cell death ligand 1", “cluster of differentiation 274", “CD274" or “B7 homolog 1” refer to any Any natural PD-L1 of vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats).
  • the term encompasses "full length", unprocessed PD-L1 and any form of PD-L1 produced by processing in the cell.
  • PD-L1 can exist as a transmembrane protein or as a soluble protein.
  • the term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants.
  • the basic structure of PD-L1 includes 4 domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain. Additional information about the human PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 29126. Additional information about mouse PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 60533.
  • an exemplary full-length human PD-L1 protein can be found, for example, under NCBI accession number NP_001254653 or UniProt accession number Q9NZQ7, and an exemplary full-length mouse PD-L1 protein can be found, for example, under NCBI accession number NP_068693 or Uniprot accession number Q9EP73.
  • L1 protein sequence As used herein, the term "antibody” is used in the broadest sense and refers to a protein containing an antigen-binding site, encompassing natural antibodies and artificial antibodies of various structures, including but not limited to intact antibodies and antigen-binding fragments of antibodies.
  • the terms “whole antibody”, “full-length antibody”, “full antibody” and “whole antibody” are used interchangeably herein to refer to at least two heavy chains (H) and two Light chain (L) glycoprotein.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region.
  • 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.
  • the VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDR)), with more conservative regions (framework regions (FR)) interposed between them.
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four
  • the FR composition is arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant region does not directly participate in the binding of the antibody to the antigen, but displays a variety of effector functions.
  • multispecific antibody refers to an antibody having at least two antigen binding sites, each of the at least two antigen binding sites is different from a different epitope of the same antigen or is different from a different epitope of the same antigen. Different epitopes of the antigen bind.
  • the antibodies provided herein are generally multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are antibodies that have binding specificities for at least two different epitopes.
  • provided herein are bispecific antibodies that have binding specificities for a first antigen and a second antigen.
  • the first antigen is PD-L1
  • the second antigen is PD-1.
  • humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human HVR and amino acid residues from human FR.
  • the humanized antibody comprises all or substantially all of the HVR (eg, CDR) corresponding to those of the non-human antibody and all or substantially all of the FR regions corresponding to those of the human antibody.
  • the humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carbonyl end of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, which is also called the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • variable region refers to the domain of the heavy or light chain of an antibody that participates in the binding of an antibody to an antigen.
  • the variable domains of the heavy and light chains of natural antibodies usually have similar structures, where each domain contains four conserved framework regions (FR) and three complementarity determining regions (see, for example, Kindt et al. Kuby Immunology, 6th ed., WHFreeman and Co. 91 page (2007)).
  • a single VH or VL domain may be sufficient to give antigen binding specificity.
  • VH or VL domains from antibodies that bind to a specific antigen can be used to isolate antibodies that bind to the antigen to screen libraries of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • Variable regions generally exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, which are also referred to as complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair are usually aligned by the framework regions, which allow the binding of specific epitopes.
  • the variable regions of the two light and heavy chains usually comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from N-terminus to C-terminus.
  • CDR region or “CDR” or “hypervariable region” (herein can be used interchangeably with hypervariable region “HVR”) is an antibody variable domain that is hypervariable in sequence and A structurally defined loop ("hypervariable loop") and/or a region containing antigen contact residues ("antigen contact point”) is formed.
  • CDR is mainly responsible for binding to antigen epitopes.
  • the CDRs of the heavy and light chains are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • the CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3.
  • the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, which include For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al.
  • the CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (for example, any of the exemplary CDRs of the present invention).
  • variable region residues in an antibody refers to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues).
  • the CDR of the antibody of the present invention is bounded by North definition rules, for example, as shown in Table 1b and Table 1c below.
  • the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when it comes to defining antibodies with specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences include the specific CDR sequences, but due to the application of different schemes (for example, Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • Antibodies with different specificities have different CDRs.
  • CDRs are different from antibody to antibody, there are only a limited number of amino acid positions within the CDR that directly participate in antigen binding.
  • the minimum overlap area can be determined, thereby providing the "minimum binding unit" for antigen binding.
  • the minimum binding unit can be a sub-portion of the CDR.
  • the structure of the antibody and protein folding can determine the residues of the rest of the CDR sequence. Therefore, the present invention also considers any CDR variants given herein. For example, in a CDR variant, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined by Kabat or Chothia can be replaced by conserved amino acid residues.
  • antibody preparation refers to a preparation in a form that allows the biological activity of the antibody as an active ingredient to be effectively exerted, and does not contain unacceptable toxicity to the subject to which the preparation is to be administered. Other components. Such antibody preparations are usually sterile. Generally, pharmaceutically acceptable excipients are included in antibody preparations.
  • a "pharmaceutically acceptable" excipient is an agent that can be reasonably administered to a tested mammal so that an effective dose of the active ingredient used in the formulation can be delivered to the subject. The concentration of the excipient is adapted to the mode of administration, for example, it may be acceptable for injection.
  • anti-PD-1/PD-L1 antibody preparation is also referred to as "antibody preparation of the present invention” herein, meaning that it contains anti-PD-1/PD-L1 antibody protein as an active ingredient and contains pharmaceutically acceptable excipients. Preparation of the agent. After the anti-PD-1/PD-L1 antibody protein is combined with a pharmaceutically acceptable excipient, the anti-PD-1/PD-L1 antibody protein as an active ingredient is suitable for therapeutic or preventive administration to human or non-human animals.
  • the antibody preparation of the present invention can be prepared, for example, as an aqueous liquid preparation, for example, a ready-to-use pre-filled syringe, or prepared as a lyophilized preparation, which is carried out by dissolving and/or suspending in a physiologically acceptable solution immediately before use. Reconstitution (ie, reconstitution).
  • the anti-PD-1/PD-L1 antibody protein preparation is in the form of a liquid preparation.
  • a “stable” antibody preparation is when the antibody in the preparation retains an acceptable degree of physical and/or chemical stability after storage under specific conditions. Although the antibody contained in the antibody preparation may not maintain 100% of its chemical structure after storage for a specific time, it usually maintains about 90%, about 95%, about 96%, about 97%, about 98% after storage for a specific time. Or about 99% of the structure or function of the antibody, the antibody preparation is considered “stable.”
  • the anti-PD-1/PD-L1 antibody protein preparations of the present invention exhibit undetectable antibody aggregation or degradation or chemical modification during manufacturing, preparation, transportation and long-term storage, thereby There is little or no loss of the biological activity of the anti-PD-1/PD-L1 antibody protein, showing a high degree of stability.
  • the anti-PD-1/PD-L1 antibody protein formulation of the present invention substantially retains its physical and chemical stability after storage.
  • the liquid formulation of the present invention can be stable at room temperature for at least 6 months or stored at 40°C ⁇ 2°C for 1 month, and/or stable at 25°C ⁇ 2°C for at least 3 months, and/or at 2-8°C Stable for at least 24 months.
  • the stability can be measured at a selected temperature and selected storage time. For example, the storage time can be selected based on the expected shelf life of the formulation. Alternatively, an accelerated stability test can be used. In some embodiments, the stability test is performed by performing various stress tests on the antibody preparation.
  • the formulated anti-PD-1/PD-L1 antibody protein preparation can be filled into a glass vial to test the antibody stability under high temperature stress.
  • the preparation After a period of storage time, the preparation does not show aggregation, precipitation, turbidity and/or denaturation; or shows very little aggregation, precipitation, turbidity and/or denaturation, it can be considered that the antibody "maintains its physical stability" in the preparation. Since the accumulation of antibodies in the preparation can potentially lead to an increased immune response in patients, it leads to safety issues. Therefore, there is a need to minimize or prevent aggregation of the antibody in the formulation.
  • the light scattering method can be used to determine the visible aggregates in the preparation. SEC can be used to determine soluble aggregates in the formulation.
  • the stability of the preparation can be indicated by visually inspecting the appearance, color, and/or clarity of the preparation, or detecting the turbidity of the preparation by the OD 350nm method, or measuring the purity of the preparation by the non-reducing CE-SDS method.
  • the stability of the formulation is measured by determining the percentage of antibody monomers in the formulation after storage at a specific temperature for a specific time, where the higher the percentage of antibody monomers in the formulation, the higher the stability of the formulation .
  • an "acceptable degree" of physical stability can mean that at least about 90% of the anti-PD-1/PD-L1 antibody protein monomer is detected in the preparation after storage at a specific temperature for a specific time.
  • an acceptable degree of physical stability indicates At least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-PD-1/PD-L1 antibody protein monomer.
  • the specific temperature at which the pharmaceutical preparation is stored may be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C, about -20°C, about 0°C, About 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C.
  • the pharmaceutical preparation is considered stable; if stored at about 25°C for 2 months, at least about 90%, 91%, 92%, 93%, 94% are detected %, 95%, 96%, 97%, 98%, 99% of the anti-PD-1/PD-L1 antibody protein monomer, the pharmaceutical preparation is considered stable; if stored at about 5°C for 9 months, At least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-PD-1/PD-L1 antibody protein monomer is detected, then the pharmaceutical preparation Considered to be stable.
  • the antibody in the preparation After a period of storage, if the antibody in the preparation does not show a significant chemical change, it can be considered that the antibody "maintains its chemical stability" in the preparation.
  • Most chemical instabilities result from the formation of covalently modified forms of antibodies (for example, charge variants of antibodies).
  • charge variants of antibodies for example, by aspartic acid isomerization, N- and C-terminal modification, basic variants can be formed; by deamidation, sialylation and saccharification, acidic variants can be generated.
  • Chemical stability can be assessed by detecting and/or quantifying the chemically altered form of the antibody.
  • the charge variant of the antibody in the preparation can be detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing electrophoresis (iCIEF).
  • CEX cation exchange chromatography
  • iCIEF imaging capillary isoelectric focusing electrophoresis
  • the stability of the formulation is measured by determining the percentage change in the charge
  • the "acceptable degree" of chemical stability can mean that the percentage change value of the charge variant (such as the main component or acidic component or alkaline component) in the preparation after storage at a specific temperature for a specific time does not exceed 50%, for example, does not exceed 30%, not more than 20%.
  • an acceptable degree of chemical stability can be The percentage change value of the main component charge variant does not exceed about 50%, 40%, 30%, 20%, 15%.
  • the storage temperature of the pharmaceutical preparation can be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, or about 45°C.
  • the pharmaceutical preparation can be considered stable; if the percentage change value of the principal component charge variant is less than about 20%, 19%, 18%, 17%, 16% after storage at 25°C for 2 months %, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%, the pharmaceutical preparation can be considered stable; if the percentage change value of the principal component charge variant is less than about 20%, 19%, 18%, 17%, 16% after storage at 25°C for 2 months %, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%, The pharmaceutical preparation can also be regarded as stable; if the percentage change value of the principal component charge variant is less than about 50%, 40%, 30%, 20%, 10%, after storage at 40°C for 1 month, 5% or 4%, the pharmaceutical preparation can also
  • lyophilized preparation refers to a composition obtained or obtainable by freeze-drying a liquid preparation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.
  • reconstituted preparation refers to a liquid preparation obtained by dissolving and/or suspending a solid preparation (for example, a lyophilized preparation) in a physiologically acceptable solution.
  • room temperature refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, more preferably 25°C.
  • Stress conditions refer to environments that are chemically and/or physically unfavorable to the antibody protein, which can lead to unacceptable instability of the antibody protein, for example, high temperature, shaking, freezing and thawing, and light.
  • High temperature stress refers to storing the antibody preparation at room temperature or even at a higher temperature (for example, 40°C ⁇ 2°C) for a period of time. Through the accelerated test of high temperature stress, the stability of the antibody preparation can be checked.
  • parenteral administration means administration methods other than enteral and local administration, usually by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, and intrathecal , Intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspine, epidural and intrasternal injections and infusions .
  • the stabilized anti-PD-1/PD-L1 antibody protein formulation of the present invention is administered to a subject parenterally.
  • the anti-PD-1/PD-L1 antibody protein formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular or intravenous injection.
  • One aspect of the present invention provides a stable freeze-dried formulation, which comprises (i) anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof, (ii) buffer system, (iii) stabilizer, said stabilizer includes multiple An alcohol and/or amino acid, and (iv) a surfactant, wherein the formulation has a pH between 5.5 and 6.5 when reconstituted, for example, the pH is about 5.5, 6.0, or 6.5.
  • a stable lyophilized preparation which is prepared by lyophilizing an aqueous solution, the aqueous solution comprising: (i) an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, (ii) a buffer system, (iii) a stabilizer, the stabilizer includes a polyol and/or amino acid, and (iv) a surfactant, the liquid formulation has a pH of about 5.5-6.5, for example, the pH is about 5.5, 6.0 or 6.5; preferably The liquid formulation has a pH of about 6.0.
  • a stable liquid antibody preparation which comprises an aqueous solution comprising (i) an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, (ii) a buffer system, and (iii) a stabilizer
  • the stabilizer includes a polyol and/or amino acid, and (iv) a surfactant
  • the liquid formulation has a pH of about 5.5-6.5, for example, the pH is about 5.5, 6.0 or 6.5; preferably, the liquid formulation Has a pH of about 6.0.
  • liquid antibody preparation of the present invention is in the form of an injection preparation.
  • Anti-PD-1/PD-L1 antibody or "bispecific antibody that binds PD-1 and PD-L1” or “antibody that binds PD-1 and PD-L1” refers to an antibody that can It binds to PD-1 and PD-L1 molecules with sufficient affinity so that the antibody can be used as a therapeutic and/or preventive agent that simultaneously targets PD-1 and PD-L1 molecules.
  • binding means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) or biofilm optical interference technology (ForteBio) or other conventional binding assays known in the art.
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • FormeBio biofilm optical interference technology
  • an antibody or antigen-binding fragment preferably recognizes its target antigen in a complex mixture of proteins and/or macromolecules, the antibody or antigen-binding fragment is referred to as a specific binding antigen.
  • binding refers to the affinity of an antibody for human PD-L1 (SEQ ID NO: 1), human PD-1 (SEQ ID NO: 2), or both, unless Otherwise, it means that K D is less than about 1 ⁇ 10 -6 M, preferably less than about 1 ⁇ 10 -9 M, as measured at 37° C. by the above-mentioned common method known in the industry.
  • the antibody in the present invention is a heterodimeric bispecific antibody that simultaneously targets PD-L1 and PD-1, and is formed by pairing two different heavy chains and two different light chains to form an IgG-like antibody.
  • the antibody of the present invention provides anti-human PD-L1 and anti-human PD-1 heterodimer bispecific antibodies having one or more of the following characteristics: blocking all three interactions of the PD axis (PD -L1 binds to PD-1, PD-L2 binds to PD-1 and PD-L1 binds to CD80), bridges PD-L1 and PD-1 overexpressing cells, increases T cells due to the proximity of bound T cells and tumor cells Activate and kill tumor cells, exhibit significant anti-tumor activity at low doses in tumor cells with moderate to high PD ligand expression levels, and exhibit physical and chemical stability, including but not limited to in vivo stability and thermal stability , Solubility, low self-association and pharmacokinetic characteristics.
  • the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention comprises: a) the first heavy chain variable region (HCVR1) comprising the amino acid sequence of SEQ ID NO: 3 Chain (HC1); b) the first light chain (LC1) comprising the light chain variable region (LCVR1) containing the amino acid sequence of SEQ ID NO: 4; c) the heavy chain comprising the amino acid sequence of SEQ ID NO: 5 The second heavy chain (HC2) of the variable region (HCVR2); and d) the second light chain (LC2) including the light chain variable region (LCVR2) containing the amino acid sequence of SEQ ID NO: 8.
  • the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention includes HC1, LC1, HC2, and LC2, wherein: a) the HC1 includes an amino acid sequence of SEQ ID NO: 16 in the HCVR CDR1, CDR1 having the amino acid sequence of SEQ ID NO: 17 and CDR3 having the amino acid sequence of SEQ ID NO: 18; b) the LC1 includes CDR1 having the amino acid sequence of SEQ ID NO: 19 in the LCVR, and having SEQ ID NO CDR2 with the amino acid sequence of: 20 and CDR3 with the amino acid sequence of SEQ ID NO: 21; c) The HC2 includes CDR1 with the amino acid sequence of SEQ ID NO: 22 in the HCVR, and has SEQ ID NO: 23 or SEQ ID NO CDR2 with the amino acid sequence of SEQ ID NO: 25 and CDR3 with the amino acid sequence of SEQ ID NO: 25; d) The LC2 includes CDR1 with the amino acid sequence of SEQ ID NO:
  • the CH3 domain of HC2 contains amino acid substitutions of T350V, T366L, K392L and T394W; or the CH3 domain of HC1 contains amino acid substitutions of T350V, T366L, K392L and T394W, and the CH3 domain of HC2 contains amino acids of T350V, L351Y, F405A and Y407V Substitute; and j) where HC1 and HC2 are human IgG1Fc variants with ineffective immune effector functions, where the numbering is based on the EU index.
  • HC1 and HC2 comprise amino acid substitutions of L234A, L235A and D265S.
  • the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention includes: a) HC1, which sequentially includes HCVR and CH1 containing the amino acid sequence of SEQ ID NO: 3 from N-terminus to C-terminus The amino acid sequence of SEQ ID NO: 9 in the domain, the amino acid sequence of SEQ ID NO: 10 in the CH2 domain, and the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12 in the CH3 domain; b) LC1, which extends from the N-terminus to The C-terminus includes the LCVR containing the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 14 in the constant region in sequence; c) HC2, which sequentially includes the amino acid sequence of SEQ ID NO: 5 from the N-terminus to the C-terminus The amino acid sequence of HCVR, the amino acid sequence of SEQ ID NO: 13 in the CH1 domain, the amino acid sequence of SEQ ID NO: 10 in the CH2 domain
  • the amino acid sequence of SEQ ID NO: 12 is stored in the CH3 domain of HC2; or when the amino acid sequence of SEQ ID NO: 12 is stored in the CH3 domain of HC1, the amino acid sequence of SEQ ID NO: 11 The amino acid sequence is stored in the CH3 domain of HC2.
  • the HC1 sequentially includes the HCVR1 and CH1 domains containing the amino acid sequence of SEQ ID NO: 3 from the N-terminus to the C-terminus
  • the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention : a) HC1 comprising the amino acid sequence of SEQ ID NO: 49; b) LC1 comprising the amino acid sequence of SEQ ID NO: 30 ; C) HC2 comprising the amino acid sequence of SEQ ID NO: 31; and d) LC2 comprising the amino acid sequence of SEQ ID NO: 34.
  • the present invention provides antibodies that bind to human PD-L1 (SEQ ID NO: 1) and human PD-1 (SEQ ID NO: 2), which comprise: a) HC1 comprising the amino acid sequence of SEQ ID NO: 49; b) comprising LC1 of the amino acid sequence of SEQ ID NO: 30; c) HC2 of the amino acid sequence of SEQ ID NO: 32; and d) LC2 of the amino acid sequence of SEQ ID NO: 34.
  • the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention : a) HC1 comprising the amino acid sequence of SEQ ID NO: 29; b) LC1 comprising the amino acid sequence of SEQ ID NO: 30 ; C) HC2 comprising the amino acid sequence of SEQ ID NO: 33; and d) LC2 comprising the amino acid sequence of SEQ ID NO: 34.
  • the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention may comprise HCVR1 having at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 3; And/or LCVR1 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 4; and/or with SEQ ID NO: 5 (ie SEQ ID NO: 6 or SEQ ID NO :7) HCVR2 with at least 90%, 95%, 98% or 99% or higher identity; and/or at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 8 Sexual LCVR2.
  • sequence identity refers to the degree of sequence identity on a nucleotide-by-nucleotide or amino acid-by-amino-acid basis in the comparison window.
  • the “percent sequence identity” can be calculated in the following way: the two best aligned sequences are compared in the comparison window, and the same nucleic acid bases (for example, A, T, C, G, I, etc.) are present in the two sequences.
  • sequence identity percentage e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met
  • the optimal alignment to determine the percent sequence identity can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithms required to achieve the maximum alignment within the full-length sequence being compared or within the target sequence region.
  • the HCVR1 of the anti-PD-1/PD-L1 antibody in the formulation of the present invention has no more than 10, preferably no more than 5, 4, or 3 different residues compared with SEQ ID NO: 3.
  • the different residues are conservative amino acid substitutions.
  • the LCVR1 of the anti-PD-1/PD-L1 antibody in the formulation of the present invention has no more than 10, preferably no more than 5, 4, or 3 different residues compared with SEQ ID NO: 4.
  • the different residues are conservative amino acid substitutions.
  • the anti-PD-1/PD-L1 antibody in the formulation of the present invention has no more than 10 HCVR2 compared with SEQ ID NO: 5 (ie SEQ ID NO: 6 or SEQ ID NO: 7), Preferably no more than 5, 4 or 3 different residues, preferably said different residues are conservative amino acid substitutions.
  • the LCVR2 of the anti-PD-1/PD-L1 antibody in the formulation of the present invention has no more than 10, preferably no more than 5, 4, or 3 different residues compared with SEQ ID NO: 8.
  • the different residues are conservative amino acid substitutions.
  • Consservative substitution refers to an amino acid change that results in the substitution of a certain amino acid with a chemically similar amino acid.
  • the conservatively substituted residues are derived from the conservative substitution table A below, preferably the preferred substituted residues shown in Table A.
  • the antibodies of the present invention are engineered non-naturally occurring polypeptide complexes.
  • the DNA molecule of the present invention is a non-naturally occurring DNA molecule that contains a polynucleotide sequence encoding a polypeptide having the amino acid sequence of a polypeptide in the antibody of the present invention.
  • the antibody of the present invention is an IgG type antibody, which has a heavy chain and a light chain, which are cross-linked by intra-chain and inter-chain disulfide bonds.
  • Each heavy chain consists of an N-terminal HCVR and a heavy chain constant region ("HCCR").
  • Each light chain consists of an N-terminal LCVR and a light chain constant region (“LCCR”).
  • the light chains each form a disulfide bond with the heavy chain, and the two heavy chains form two disulfide bonds between each other.
  • the constant region of the heavy chain contains CH1, CH2 and CH3 domains.
  • CH1 is derived from HCVR; CH1 and HCVR form the heavy chain part of the Fab.
  • CH2 is located after the hinge area and before CH3.
  • CH3 is located after CH2, at the carboxyl end of the heavy chain.
  • the constant region of the light chain contains a domain CL.
  • CL is derived from LCVR; CL and LCVR form the light chain portion of the Fab.
  • the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention is the anti-PD-L1 antibody disclosed in PCT Application No. PCT/US2018/041205 (International Filing Date: July 9, 2018).
  • 1/PD-L1 antibody the entire content is incorporated herein by reference.
  • the sequence of the anti-PD-1/PD-L1 antibody continued to use the sequence number in the patent application without renumbering.
  • the antibody is selected from: PD-1/PD-L1 bispecific Ab v13844, Ab v3.0 or Ab v3.2. More preferably, the antibody is PD-1/PD-L1 bispecific Ab v3.2.
  • the anti-PD-1/PD-L1 antibody is a purified antibody produced by recombinant expression such as 293 cells or CHO cells, and contains a variant of the Fc portion derived from human IgG1.
  • the antibody in the liquid preparation of the present invention exhibits significant anti-tumor activity.
  • the antibody of the present invention such as PD-1/PD-L1 bispecific Ab (v13844, v3.0 or v3. 2)
  • CR complete remission
  • parental PD-L1 antibody + parental PD-1 antibody (CR is 2/ 8) More effective.
  • the antibody of the present invention is a heterodimer because each arm of the antibody exhibits selective monovalent binding to its homologous antigen due to two different heavy chains and two different light chains that form the antibody.
  • one arm of the antibody binds human PD-L1 (SEQ ID NO: 1), and the other arm binds human PD-1 (SEQ ID NO: 2).
  • the CH2 and/or CH3 domains of these polypeptide chains do not need to be identical in sequence, and are advantageously modified to promote the complexation between the two polypeptide chains.
  • amino acid substitutions preferably with amino acid substitutions containing bulky side groups forming a "knob", such as tryptophan
  • the domain will force the mutation domain to pair with the domain for which complementary or regulatory mutations have been designed, that is, "holes" (for example, replacement with glycine).
  • holes for example, replacement with glycine
  • Protein engineering methods that favor heterodimerization rather than homodimerization are well known in the art, especially regarding the engineering of immunoglobulin-like molecules (see, for example, WO96/27011, WO98/050431, EP1870459, WO2007 /110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO2012/058768, WO2013/157954, WO2013/096291, EP1870459A1, and Ridgway etc. (1996)” 'Knobs-Into-Holes' engineered antibody CH3 heavy chain domain heterodimerization, "Protein Engineering 9: 617-621, Atwell et al.
  • the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are both engineered in a complementary manner, so that the heavy chain containing an engineered CH3 domain is no longer It can be homodimerized with another heavy chain of the same structure (for example, the first heavy chain of CH3-engineering can no longer be homodimerized with the first heavy chain of another CH3 engineering; and CH3 -The second engineered heavy chain can no longer be homodimerized with another CH3-engineered second heavy chain.
  • the other heavy chain is heterodimerized and the CH3 domain is engineered in a complementary manner.
  • the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are complementary by amino acid substitutions
  • the method is engineered so that the first heavy chain and the second heavy chain are forced to heterodimerize.
  • the first heavy chain and the second heavy chain can no longer homodimerize (for example, for spatial reasons).
  • mutations are incorporated into the heavy chain sequence in the CH1 and CH3 regions and the light chain sequence in the light chain constant region.
  • CH1 and LC mutations facilitate the natural pairing of the necessary light and heavy chain pairs and are not conducive to light chain mismatches.
  • Mutation of CH3 is conducive to the pairing of heterodimers of two different heavy chains and is not conducive to the formation of homodimers.
  • the antibodies of the invention contain variants of the Fc portion derived from human IgG1. It is well known that IgG1 binds to the Fc- ⁇ receptor family (Fc ⁇ R) and C1q. Interactions with these receptors can induce antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Therefore, for the antibodies of the present invention, certain amino acid substitutions are introduced into the human IgG1 Fc region to eliminate immune effector functions. Mutations in the CH2 region of the antibody heavy chain can include positions 234, 235, and 265 in the EU numbering to reduce or eliminate immune effector functions.
  • the isolated DNA encoding the HCVR region can be converted into a full-length heavy chain gene.
  • the sequences of human and other mammalian heavy chain constant region genes are known in the art. DNA fragments containing these regions can be obtained, for example, by standard PCR amplification.
  • the heavy chain constant region of the heavy chain is a variant of human IgG1.
  • the isolated DNA encoding the LCVR region can be converted into a full-length light chain gene.
  • the sequences of human and other mammalian light chain constant region genes are known in the art. DNA fragments containing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region.
  • the light chain constant region of the anti-PD-L1 portion of the antibody is a variant of the lambda light chain
  • the light chain constant region of the anti-PD-1 portion of the antibody is a variant of the kappa light chain.
  • the polynucleotide of the present invention can be expressed in the host cell.
  • An expression vector can usually be used as an episome or as an attachment to replicate a component of the host chromosomal DNA in the host organism.
  • expression vectors contain selectable markers such as tetracycline, neomycin, glutamine synthetase and dihydrofolate reductase to allow detection of those cells transformed with the desired DNA sequence.
  • the amount of the antibody or antigen-binding fragment thereof contained in the antibody preparation of the present invention may vary according to the specific purpose characteristics of the preparation, the specific environment, and the specific purpose for which the preparation is used.
  • the antibody preparation is a liquid preparation, which may contain about 1-200 mg/ml, such as about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/ml, preferably about 10-100 mg/mL, for example about 10, 15, 20, 25, 30, 35, 40, 50, 60 , 70, 80, 90 or 100mg/ml of anti-PD-1/PD-L1 antibody.
  • the antibody formulation is a lyophilized formulation that can be at a concentration between about 1 mg/mL to about 200 mg/mL, such as about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/ml, preferably about 10-100 mg/mL, for example about 10, 15, 20 , 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 mg/ml concentration to reconstitute the antibody or antigen-binding fragment thereof.
  • the buffer system is a system that can maintain the pH of the solution in an acceptable range.
  • the buffer system used in the formulation of the present invention can control the pH of the formulation of the present invention in a pH range of about 5.5-6.5, such as a pH of about 5.5, 6.0, or 6.5.
  • the antibody preparations of the invention have a pH of about 6.
  • the buffer system is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, citric acid and sodium citrate buffer system , Sodium acetate acetate buffer system, phosphate buffer system, and the weight ratio of about 0.01% (w/v)-1% (w/v), such as about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1% (w/v) by weight ratio; preferably, the buffer system is present in a weight ratio of about 0.01% (w/v)-0.2% (w/v),
  • the buffer system is a histidine buffer system.
  • the histidine content is about 0.01-1% (w/v), such as 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1% (w/v)
  • the weight ratio exists.
  • the histidine is present in a weight ratio of about 0.155% (w/v);
  • the buffer system is a histidine and histidine hydrochloride buffer system.
  • the histidine and the histidine hydrochloride are respectively about 0.01-1% (w/v), such as about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, It exists in a weight ratio of 0.9 and 1% (w/v).
  • the histidine and histidine hydrochloride are present in a weight ratio of about 0.085% (w/v) and 0.1% (w/v), respectively.
  • the buffer system in the liquid antibody preparation of the present invention is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, citric acid and sodium citrate buffer system, sodium acetate acetate Buffer system, phosphate buffer system, and exist in a concentration of about 1-100 mM, for example, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM.
  • the buffer system is present at a concentration of about 1-20 mM, for example, 1, 5, 10, 15, 20 mM; more preferably, it is present at a concentration of about 10 mM.
  • the buffer system in the liquid antibody preparation of the present invention is a histidine buffer system.
  • the histidine is present at a concentration of about 0.1-10 mg/mL, for example about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml. In a more preferred embodiment, the histidine is present at a concentration of about 1.55 mg/mL;
  • the buffer system in the liquid antibody preparation of the present invention is a histidine and histidine hydrochloride buffer system.
  • the histidine and the histidine hydrochloride are respectively about 0.1-10 mg/mL, for example about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/mL.
  • the concentration of ml exists.
  • the buffering agent used in the formulation of the present invention is histidine and histidine hydrochloride at a total concentration of about 10 mM, for example, the histidine and the histidine hydrochloride are respectively about 0.85 There are concentrations of mg/mL and about 1.00 mg/mL.
  • suitable stabilizers used in the present invention may be selected from polyols, amino acids, and any combination thereof.
  • the polyol as a stabilizer can be selected from but not limited to: mannitol, sorbitol or a combination thereof. In one embodiment, the polyol used as a stabilizer is sorbitol.
  • the amino acid as a stabilizer can be selected from but not limited to: arginine, arginine hydrochloride, methionine, glycine, proline, or a combination thereof.
  • the amino acid used as a stabilizer is arginine and/or arginine hydrochloride.
  • the stabilizer in the lyophilized formulation of the present invention, includes sorbitol and arginine.
  • the sorbitol is present in a weight ratio of about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v)
  • the arginine is present in a weight ratio of about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v).
  • the sorbitol and the arginine are present in a weight ratio of about 3% (w/v) and about 1.742% (w/v), respectively.
  • the stabilizer in the lyophilized formulation of the present invention, includes sorbitol and arginine hydrochloride.
  • the sorbitol is present in a weight ratio of about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v) , Present in a weight ratio of about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v).
  • the sorbitol and the arginine hydrochloride are present in a weight ratio of about 3% (w/v) and about 2.107% (w/v), respectively.
  • the stabilizer in the lyophilized formulation of the present invention, further includes methionine.
  • the methionine may be present in a weight ratio of about 0.1-10% (w/v), for example, in a weight ratio of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10% (w/v) weight ratio exists. In one embodiment, the methionine is present in a weight ratio of about 0.749% (w/v).
  • the liquid formulation of the present invention contains sorbitol and arginine as stabilizers.
  • concentration of the sorbitol and the arginine in the liquid preparation of the present invention may be about 10-100 mg/mL, for example about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL, respectively. mL.
  • the sorbitol and the arginine in the liquid formulation of the present invention may be present at a concentration of about 10-50 mg/mL, for example, about 10, 20, 30, 40, 50 mg/mL, respectively.
  • the sorbitol and the arginine are present at a concentration of about 30 mg/mL and about 17.42 mg/mL, respectively;
  • the liquid formulation of the present invention contains sorbitol and arginine hydrochloride as stabilizers.
  • the sorbitol and the arginine hydrochloride in the liquid preparation of the present invention may be about 10-100 mg/mL, for example about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL, respectively.
  • the concentration exists.
  • the sorbitol and the arginine hydrochloride in the liquid formulation of the present invention may be present at a concentration of about 10-50 mg/mL, for example, about 10, 20, 30, 40, 50 mg/mL, respectively.
  • sorbitol and arginine hydrochloride are present at a concentration of about 30 mg/mL and about 21.07 mg/mL, respectively.
  • the stabilizer in the liquid formulation of the present invention further includes methionine.
  • the methionine may be about 1-100 mg/mL, for example, about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL The concentration exists. More preferably, the methionine is at about 1-50 mg/mL. In one embodiment, the methionine is present at a concentration of about 7.49 mg/mL.
  • surfactant refers to an organic substance with an amphiphilic structure; that is, they are composed of groups with opposite solubility tendencies, usually oil-soluble hydrocarbon chains and water-soluble ionic groups. group.
  • suitable stabilizers for use in the present invention may be non-ionic surfactants, for example, alkyl poly(ethylene oxide).
  • suitable nonionic surfactants include, for example, polysorbate 80, polysorbate 20, poloxamer, polyethylene glycol polysorbate 80, and the like.
  • the formulation of the present invention contains polysorbate-80 as a surfactant.
  • the amount of the surfactant contained in the antibody preparation of the present invention may vary depending on the specific purpose characteristics of the preparation, the specific environment, and the specific purpose for which the preparation is used.
  • the surfactant in the lyophilized formulation of the present invention, is at a concentration of about 0.01% (w/v) to 1% (w/v), such as 0.01, 0.05, 0.1, 0.2, There are weight ratios of 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1% (w/v). Preferably, it is present in a weight ratio of about 0.01% (w/v)-0.5% (w/v), for example 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5% (w/v).
  • the surfactant is polysorbate 80, more preferably, the polysorbate 80 is about 0.02% (w/v) by weight Than exist.
  • the surfactant in the liquid formulation of the present invention, is about 0.1-10 mg/ml, for example about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, Exist at concentrations of 8, 9, 10 mg/ml. Preferably, it is present at a concentration of about 0.1-5 mg/ml, for example about 0.1, 0.5, 1, 2, 3, 4, 5 mg/ml.
  • the formulation may contain about 0.1-10 mg/ml, for example about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml of polysorbates Surfactant (for example, polysorbate-80). More preferably, the polysorbate 80 is present at a concentration of about 0.2 mg/ml.
  • excipients are optionally included in the antibody formulations of the present invention.
  • the other excipients include, for example, antimicrobial agents, antistatic agents, antioxidants, chelating agents, gelatin, and the like.
  • antimicrobial agents include, for example, antimicrobial agents, antistatic agents, antioxidants, chelating agents, gelatin, and the like.
  • antistatic agents include, for example, antistatic agents, antioxidants, chelating agents, gelatin, and the like.
  • chelating agents include, for example, antimicrobial agents, antistatic agents, antioxidants, chelating agents, gelatin, and the like.
  • These and other known pharmaceutical excipients and/or additives suitable for the formulation of the present invention are well known in the art, for example, listed in "The Handbook of Pharmaceutical Excipients, 4th Edition, edited by Rowe et al., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippincott Williams & Wilkins (2005)
  • the antibody lyophilized preparation of the present invention contains: about 2-17% (w/v) recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibody, about 0.294% (w/v), about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 5.5.
  • PD-1 fully human anti-programmed death receptor 1
  • PD-L1 anti-programmed death ligand 1
  • the antibody lyophilized preparation of the present invention contains: about 2-17% (w/v) recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibody, about 0.155% (w/v) histidine, about 3% (w/v) sorbitol, about 0.749% (w/v) methionine, about 1.742% (w/v) Arginine, about 0.20 mg/ml polysorbate 80, the formulation has a pH of 6.0 when reconstituted.
  • PD-1 fully human anti-programmed death receptor 1
  • P-L1 anti-programmed death ligand 1
  • the antibody lyophilized preparation of the present invention contains: about 2-17% (w/v) recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibody, about 0.085% (w/v) histidine, about 0.1% (w/v) histidine hydrochloride, about 3.00% (w/v) sorbitol, about 0.749% (w/v) methionine, about 2.107% (w/v) arginine hydrochloride, about 0.02% (w/v) polysorbate 80; the formulation has a pH of 6.0 when reconstituted.
  • PD-1 fully human anti-programmed death receptor 1
  • P-L1 anti-programmed death ligand 1
  • the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody, about 2.94 mg/ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 5.5.
  • PD-1 fully human anti-programmed death receptor 1
  • PD -L1 Bispecific antibody about 2.94 mg/ml sodium citrate
  • about 50 mg/ml sorbitol about 0.20 mg/ml polysorbate 80
  • the aqueous solution has a pH of about 5.5.
  • the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody, about 2.94 mg/ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0.
  • PD-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody about 2.94 mg/ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80
  • the aqueous solution has a pH of about 6.0.
  • the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody, about 2.94mg/ml sodium citrate, about 50mg/ml sorbitol, about 2.94mg/ml methionine, about 0.20mg/ml polysorbate 80, the aqueous solution has about The pH of 6.0.
  • PD-1 fully human anti-programmed death receptor 1
  • PD -L1 Bispecific antibody about 2.94mg/ml sodium citrate
  • 50mg/ml sorbitol about 2.94mg/ml methionine
  • 0.20mg/ml polysorbate 80 the aqueous solution has about The pH of 6.0.
  • the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody, about 2.94 mg/ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.5.
  • PD-1 fully human anti-programmed death receptor 1
  • PD -L1 Bispecific antibody about 2.94 mg/ml sodium citrate
  • about 50 mg/ml sorbitol about 0.20 mg/ml polysorbate 80
  • the aqueous solution has a pH of about 6.5.
  • the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody, about 2.94 mg/ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0.
  • PD-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody about 2.94 mg/ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80
  • the aqueous solution has a pH of about 6.0.
  • the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody, about 1.55 mg/ml histidine, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0.
  • PD-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody about 1.55 mg/ml histidine, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80
  • the aqueous solution has a pH of about 6.0.
  • the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody, about 1.55 mg/ml histidine, about 50 mg/ml sorbitol, about 7.49 mg/ml methionine, about 0.20 mg/ml polysorbate 80, the aqueous solution has about 6.0 ⁇ pH.
  • PD-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody about 1.55 mg/ml histidine, about 50 mg/ml sorbitol, about 7.49 mg/ml methionine, about 0.20 mg/ml polysorbate 80, the aqueous solution has about 6.0 ⁇ pH.
  • the aqueous solution in the antibody liquid formulation of the present invention contains: 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibody, about 1.55mg/ml histidine, about 30mg/ml sorbitol, about 7.49mg/ml methionine, about 17.42mg/ml arginine, about 0.20mg/ml poly Sorbate 80, the aqueous solution has a pH of about 6.0.
  • PD-1 fully human anti-programmed death receptor 1
  • PD-L1 anti-programmed death ligand 1
  • the aqueous solution contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibody, about 0.85mg/ml histidine, about 1.00mg/ml histidine hydrochloride, about 30.00mg/ml sorbitol, about 7.49mg/ml methionine, about 21.07 mg/ml arginine hydrochloride, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0.
  • PD-1 fully human anti-programmed death receptor 1
  • PD-L1 anti-programmed death ligand 1
  • the present invention provides a stable formulation containing anti-PD-1/PD-L1 antibody protein.
  • the anti-PD-1/PD-L1 antibody protein used in the formulation of the present invention can be prepared using techniques known in the art for antibody production. For example, antibodies can be produced recombinantly.
  • the antibodies of the present invention can be produced in CHO, NSO, HEK293 or COS cells.
  • the antibody of the present invention can be prepared in CHO or HEK293 cells.
  • the vector containing the polynucleotide sequence of interest (for example, the polynucleotide encoding the polypeptide of the antibody and the expression control sequence) can be transferred to the host cell by a well-known method, and the method varies according to the type of the host cell.
  • recombinantly produced monoclonal antibodies can be purified by conventional purification methods to provide pharmaceutical substances with sufficient reproducibility and moderate purity for the preparation of antibody preparations.
  • a commercially available protein concentration filter such as Amicon's ultrafiltration device can be used to concentrate the supernatant from the expression system.
  • the antibody can be purified using methods such as chromatography, dialysis, and affinity purification.
  • Protein A is suitable as an affinity ligand for the purification of IgG1, IgG2 and IgG4 type antibodies.
  • Other antibody purification methods such as ion exchange chromatography, can also be used.
  • a preparation containing the antibody can be prepared according to methods known in the art.
  • the following steps can be used for preparation: (1) After the fermentation, the fermentation broth is centrifuged to clarify impurities such as cells to obtain the supernatant; (2) affinity chromatography (for example, specific for IgG1, IgG2 and IgG4 antibodies Affinity protein A column) capture antibody; (3) virus inactivation; (4) purification and purification (usually CEX cation exchange chromatography can be used) to remove impurities in the protein; (4) virus filtration (to make the virus titer Reduce, for example, 4 log10 or more); (5) Ultrafiltration/diafiltration (which can be used to replace the protein in a formulation buffer that is conducive to its stability and concentrate it to a suitable concentration for injection). See, for example, B. Minow, P. Rogge, K. Thompson, BioProcess International, Vol. 10, No. 6, 2012, pp. 48-57.
  • affinity chromatography for example, specific for IgG1, IgG2 and IgG4 antibodies Affinity protein A column
  • virus inactivation
  • antibodies may undergo aggregation, degradation or chemical modification, resulting in antibody heterogeneity (including size heterogeneity and charge heterogeneity), aggregates and fragments, etc., thereby affecting the quality of antibody preparations. Therefore, it is necessary to monitor the stability of antibody preparations.
  • Various methods are known in the art that can be used to test the stability of antibody preparations.
  • methods such as reduced CE-SDS, non-reduced CE-SDS, and SEC-HPLC can be used to analyze the purity of antibody preparations and evaluate the aggregation level of antibodies; capillary isoelectric focusing (cIEF), imaging capillary, etc.
  • Focused electrophoresis (iCIEF) and ion exchange chromatography (IEX) are used to analyze charge variants in antibody preparations.
  • the stability of the preparation can be quickly judged by visually inspecting the appearance of the preparation.
  • the OD 350nm method can also be used to detect changes in the turbidity of the preparation, which can give information about the amount of soluble and insoluble aggregates.
  • ultraviolet spectrophotometry UV method
  • UV method ultraviolet spectrophotometry
  • the non-reduced CE-SDS method is a method of antibody purity determination using capillary as a separation channel.
  • CE-SDS protein migration is driven by the surface charge caused by SDS binding, and the surface charge is proportional to the molecular weight of the protein. Since all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on molecular size or hydrodynamic radius can be achieved in the molecular sieve gel matrix of the capillary. This method has been widely used to monitor the purity of denatured intact antibodies.
  • the test sample is mixed with SDS sample buffer and iodoacetamide.
  • the mixture can be incubated at 68-72°C for about 10-15 minutes, and the supernatant centrifuged after cooling to room temperature is used for analysis.
  • a UV detector is used to detect the migration of the protein and obtain an electrophoresis spectrum.
  • the purity of the antibody preparation can be calculated as the percentage of the peak area of the main IgG peak to the sum of all peak areas.
  • Size exclusion high performance liquid chromatography is another important method for antibody standards and quality control. This method is mainly based on the size of the molecule or the difference in hydrodynamic radius to separate the molecules.
  • SEC-HPLC antibodies can be separated into three main forms: high molecular weight form (HMMS), main peak (mainly antibody monomer), and low molecular weight form (LMMS).
  • HMMS high molecular weight form
  • LMMS low molecular weight form
  • Antibody purity can be calculated as the percentage of the main peak area on the chromatogram to the sum of all peak areas.
  • the SEC-HPLC method the percentage of antibody monomers in the preparation product can be measured, and information about the content of soluble aggregates and shears can be given.
  • Imaging capillary isoelectric focusing electrophoresis can be used to analyze the charge heterogeneity of antibodies. This method can provide a quantitative distribution of charge variants.
  • iCIEF achieves molecular separation based on the charge difference (apparent pI value) of molecules in the pH gradient.
  • the separation column is usually a short capillary (for example, a silica capillary with a length of 5 cm and an inner diameter of 100 ⁇ m).
  • the protein is focused in the capillary column under high voltage, and the focusing is performed by a whole column imaging detection system operating at 280 nM. Real-time online monitoring.
  • One advantage of this technology is that the whole column detection system can simultaneously record various charge variants of antibody samples.
  • icIEF the sample is mixed with urea and icIEF buffer, where the buffer contains methyl cellulose, pi molecular weight standards and amphoteric electrolytes.
  • an iCIEF column such as an iCIEF column assembled by ProtionSimple on an iCIEF analyzer such as iCE280 analyzer (Protein Simple, Santa Clara, CA).
  • iCE280 analyzer Protein Simple, Santa Clara, CA
  • the protein-related peaks eluted before the main peak are classified as acidic components; in contrast, the protein-related peaks eluted after the main peak are classified as basic components.
  • the relative amounts of the main components, acidic components, and basic components can be expressed as a percentage of the total peak area.
  • the charge variant of the antibody in the antibody preparation can also be determined by cation exchange high performance liquid chromatography (CEX-HPLC).
  • CEX-HPLC cation exchange high performance liquid chromatography
  • Accelerated stability studies can be used to check the stability properties of products, which is conducive to the screening of stable pharmaceutical formulations.
  • a sample of the formulation can be placed at an elevated temperature, such as about 40°C ⁇ 2°C, 25°C ⁇ 2°C, for accelerated stability studies.
  • Detection indicators can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (iCIEF method, CEX-HPLC method).
  • the anti-PD-1/PD-L1 antibody protein preparation of the present invention is stable.
  • the anti-PD-1/PD-L1 antibody protein formulation of the present invention is stored, for example, after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 6 months, or after 40 It is stable after storage at °C ⁇ 2°C for 1 month.
  • the anti-PD-1/PD-L1 antibody protein purity in the antibody preparation of the present invention is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, as determined by size exclusion chromatography or by non-reduced CE-SDS.
  • the anti-PD-1/PD-L1 antibody protein in the antibody preparation of the present invention is in the non-basic and non-acidic form (that is, the main peak or the main charge form), such as by CEX-HPLC Measured by law.
  • the antibody preparation of the present invention containing the anti-PD-1/PD-L1 antibody protein of the present invention can be used to treat or prevent tumors and the like.
  • the present invention further provides the use of the antibody preparation of the present invention for the manufacture of a drug for the treatment or prevention of cancer, wherein the drug can be simultaneously, separately or sequentially with one or more anti-tumor agents selected from the group consisting of Administration: Cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and more Rubicin, navelbine, eribulin, paclitaxel, paclitaxel protein-binding particles for injectable suspension, ixabepilone, capecitabine (capecitabine), FOLFOX (leucovorin, fluorouracil and oxaliplatin), FOLFIRI (calcium folinate, fluorouracil and irinotecan), gemcitabine, Topo Topotecan, liposomal irinotecan, pemetrexed, cetuximab, nivolumab, ipilimum
  • the present invention provides the use of the antibody preparation of the present invention for the manufacture of drugs for the treatment of cancer, wherein the drugs can be administered simultaneously, separately or sequentially with ionizing radiation.
  • cancers to be prevented or treated include, but are not limited to, Hodgkin's or non-Hodgkin's lymphoma, melanoma, renal cell carcinoma, kidney cancer, lung cancer, bladder cancer, stomach and esophageal cancer , Colorectal cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, breast cancer, triple negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), Mesothelioma, squamous cell carcinoma of the head and neck (SCCHN), soft tissue sarcoma, or glioblastoma multiforme.
  • the cancer is a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, and the like.
  • the present invention also provides the use of the preparation of the present invention in the preparation of medicines, wherein the medicines are used to deliver anti-PD-1/PD-L1 antibody proteins to mammals.
  • the present invention also provides a method for the preparation of the present invention to treat or prevent one or more of the above-mentioned diseases and disorders.
  • the mammal is a human.
  • the antibody formulation of the present invention can be administered to a subject or patient in a variety of ways.
  • administration can be by infusion or by syringe.
  • the present invention provides a delivery device (e.g., a syringe) comprising the antibody preparation of the present invention (e.g., a pre-filled syringe).
  • the patient will receive an effective amount of anti-PD-1/PD-L1 antibody protein as the main active ingredient, that is, an amount sufficient to treat, ameliorate or prevent the target disease or condition.
  • the present invention further provides a method for preventing and/or treating tumors, which comprises administering to a subject an effective amount of the formulation of the present invention.
  • the tumor is cancer.
  • the cancer is Hodgkin or non-Hodgkin lymphoma, melanoma, renal cell carcinoma, kidney cancer, lung cancer, bladder cancer, gastric and esophageal cancer, colorectal cancer, Liver cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, breast cancer, triple negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), mesothelioma, head and neck Squamous cell carcinoma (SCCHN), soft tissue sarcoma or glioblastoma multiforme; more preferably, the lung cancer is NSCLC, small cell lung cancer or mesothelioma.
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • the method of the present invention further includes simultaneous, separate or sequential administration of one or more anti-tumor agents selected from the group consisting of cisplatin (cisplatin), carboplatin (carboplatin), and Carbachol (dacarbazine), liposomal doxorubicin (liposomal doxorubicin), docetaxel (docetaxel), cyclophosphamide (cyclophosphamide) and doxorubicin, navelbine (navelbine), eribulin ( eribulin, paclitaxel, paclitaxel protein-binding particles for injectable suspension, ixabepilone, capecitabine, FOLFOX (leucovorin), fluorouracil ) And oxaliplatin), FOLFIRI (leucovorin, fluorouracil and irinotecan), gemcitabine, topotecan, liposomal irinotecan, pemetre
  • the therapeutic effect may include reducing physical symptoms.
  • the optimal effective amount and concentration of antibodies for any particular subject will depend on many factors, including the age, weight, health and/or gender of the patient, the nature and extent of the disease, the activity of the particular antibody, and the Its clearance rate, and also includes any possible other treatments administered in combination with the antibody formulation.
  • the effective amount delivered can be determined within the judgment of the clinician.
  • the application of known antibody-based drugs can provide certain guidance.
  • the dosage can be a single-dose schedule or a multiple-dose schedule.
  • treatment refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • the desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis.
  • the antibody molecules of the present invention are used to delay the progression of a disease or to slow the progression of a disease.
  • prevention includes the inhibition of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition.
  • subjects with a family history of cancer are candidates for prophylactic regimens.
  • prevention refers to the administration of drugs before the onset of signs or symptoms of cancer, especially in subjects at risk of cancer.
  • Therapeutically effective amount refers to the amount that is effective to achieve the desired therapeutic result at the required dose and for the required period of time.
  • the therapeutically effective amount of the antibody or antibody fragment or its conjugate or composition can vary according to various factors such as disease state, the age, sex and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual.
  • a therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or its conjugate or composition are not as good as the therapeutically beneficial effects.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (such as tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%. % And still more preferably at least about 80%.
  • a compound to inhibit a measurable parameter e.g., cancer
  • this property of the composition can be evaluated by testing the compound's ability to inhibit, said inhibition in vitro by an assay known to the skilled artisan.
  • “Prophylactically effective amount” refers to an amount that effectively achieves the desired preventive result at the required dose and for the required period of time.
  • Table 1a The amino acid sequence numbers of the light chain, heavy chain, light chain variable region and heavy chain variable region of antibodies v3.2, v3.0 and v13844
  • Table 1b The amino acid sequence numbers of the CDRs of the PD-L1 binding variable regions of antibodies v3.2, v3.0 and v13844 (determined according to the North rule)
  • Table 1c The amino acid sequence numbers of the CDRs of the PD-1 binding variable regions of antibodies v3.2, v3.0 and v13844 (determined according to the North rule)
  • CE-SDS Sodium Lauryl Sulfate Capillary Gel Electrophoresis
  • iCIEF Imaging Capillary Isoelectric Focusing Electrophoresis
  • the mobile phase is phosphate buffer (weigh 3.12g sodium dihydrogen phosphate dihydrate, 8.77g sodium chloride and 34.84g arginine, after dissolving in ultrapure water, adjust the pH to 6.8 with hydrochloric acid And dilute to 1000ml), the column protection solution is 0.05% (w/v) NaN 3 , the injection volume is 50 ⁇ l, the flow rate is 0.5ml/min, the collection time is 30 minutes, the column temperature is 25°C, and the detection wavelength is 280nm. Take the sample to be tested and dilute it to 2mg/ml with ultrapure water as the test solution. Take the preparation buffer solution and dilute it with the same treatment method as above and use it as a blank solution. Take 50 ⁇ l each of the blank solution and the test solution into the liquid chromatograph to start the test.
  • phosphate buffer weigh 3.12g sodium dihydrogen phosphate dihydrate, 8.77g sodium chloride and 34.84g arginine, after dissolving in ultrapure water
  • the capillary is an uncoated capillary with an inner diameter of 50 ⁇ m, a total length of 30.2cm, and an effective length of 20.2cm. Wash the capillary column with 0.1mol/L sodium hydroxide, 0.1mol/L hydrochloric acid, ultrapure water, and 70psi electrophoresis gel before electrophoresis.
  • Sample injection conditions -5kV for 20 seconds; separation voltage: -15kV for 35 minutes.
  • the capillary column temperature is controlled at 25°C, and the detection wavelength is 220nm.
  • the capillary is an uncoated capillary with an inner diameter of 50 ⁇ m, a total length of 30.2cm, and an effective length of 20.2cm. Wash the capillary column with 0.1mol/L sodium hydroxide, 0.1mol/L hydrochloric acid, ultrapure water, and 70psi electrophoresis gel before electrophoresis.
  • Sample injection conditions -5kV for 20 seconds; separation voltage: -15kV for 35 minutes.
  • the capillary column temperature is controlled at 25°C, and the detection wavelength is 220nm.
  • the capillary has an inner diameter of 100 ⁇ m and a total length of 5cm.
  • 0.5% methyl cellulose solution hereinafter also abbreviated as MC solution
  • ultrapure water should be used to rinse the capillary column respectively.
  • the vacuum sampling method is adopted.
  • the pre-focusing voltage and time are 1.5kV for 1 minute, the focusing voltage and time are 3kV for 8 minutes, the sampling time is 55 seconds, the sample tray temperature is 10°C, and the detection wavelength is 280nm.
  • Cathodic Stabilizer is 500mmol/L arginine solution, 0.5% MC solution reduces the adhesion between protein and capillary.
  • the test product Dilute the test product to 1.0 mg/ml with water, take 20 ⁇ l of the diluted test product solution, and add 78 ⁇ l of the pre-mixed solution to it (the pre-mixed solution is as follows: 70 ⁇ l pI 0.5% MC solution, 4 ⁇ l amphoteric electrolyte (pH 3-10) ), 2 ⁇ l cathode stabilizer, 1 ⁇ l pI 5.85 marker, 1 ⁇ l pI 9.99 marker), mix well to prepare the sample solution to be tested.
  • Sample injection analysis according to the area normalization method, calculate the main component, acidic component and alkaline component content.
  • ion exchange chromatography (CEX-HPLC method).
  • the mobile phase is 10mmol/L phosphate buffer (weigh 1.15g sodium dihydrogen phosphate dihydrate, 0.95g disodium hydrogen phosphate dodecahydrate, dissolve in 800ml ultrapure water and dilute to 1000ml )
  • 10mmol/L phosphate+200mmol/L sodium chloride buffer weigh 1.15g sodium dihydrogen phosphate dihydrate, 0.95g disodium hydrogen phosphate dodecahydrate, 11.69g sodium chloride dissolved in 800ml ultrapure water Constant volume to 1000ml
  • flow rate 0.5ml/min
  • detection wavelength 280nm column temperature 25°C.
  • the light chain of new antibodies v3.2, v3.0 and v13844, and antibodies v3.2, v3.0 and v13844 that specifically bind to PD-1 and PD-L1 were obtained
  • the SEQ ID NOs of the amino acid sequences of the heavy chain, light chain variable region, and heavy chain variable region are shown in Table 1(a) above.
  • the SEQ ID NOs of the amino acid sequences of the CDRs of the PD-L1 and PD-1 binding variable regions of antibodies v3.2, v3.0, and v13844 are shown in Table 1(b) and Table 1(c), respectively.
  • the antibodies of the present invention include but are not limited to antibodies v3.2, v3.0 and v13844, and are basically prepared and purified as follows.
  • a quadruple vector ie, a single vector encoding the expression of two light chains and two heavy chains
  • a dual vector ie, two vectors encoding two different light chains and two different heavy chains together
  • a quadruple vector can be used.
  • a single vector two of which code for different light chains and two of which code for different heavy chains
  • the medium in which the purified antibody is secreted can be used by any of many commonly used techniques.
  • the culture medium can be applied to a MabSelect column (GE Healthcare) or a KappaSelect column (GE Healthcare) for Fab fragments, and the column has been equilibrated with a compatible buffer (for example, phosphate buffered saline (pH 7.4)).
  • a compatible buffer for example, phosphate buffered saline (pH 7.4)
  • the column can be washed to remove non-specific binding components.
  • the bound can be eluted by a pH gradient (for example, 20mM Tris buffer (pH 7) to 10mM citrate buffer (pH 3.0), or physiological saline (pH 7.4) to 100mM glycine buffer (pH 3.0)).
  • a pH gradient for example, 20mM Tris buffer (pH 7) to 10mM citrate buffer (pH 3.0), or physiological saline (pH 7.4) to 100mM glycine buffer (pH 3.0)).
  • Antibody for example, 20m
  • the antibody antibody fragments can be detected, for example, by SDS-PAGE, and can then be pooled. Soluble aggregates and multimers can be effectively removed by common techniques, including size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, multimodal chromatography, or hydroxyapatite chromatography. Common techniques can be used to concentrate and/or sterile filter the antibody.
  • the product can be frozen immediately at -70°C or can be lyophilized.
  • the heavy chain of PD-1 and the heavy chain of PD-L1 are respectively connected to the eukaryotic plasmid expression vector PEE6.4, and the light chain of PD-1 and the light chain of PD-L1 are respectively connected to the eukaryotic plasmid expression vector PEE12.4.
  • the intermediate expression vector containing the heavy chain of PD-1 and the intermediate expression vector containing the light chain of PD-1 are combined to obtain a recombinant expression vector containing the nucleotide sequences of the heavy chain and light chain of PD-1.
  • the intermediate expression vector containing the heavy chain of PD-L1 and the intermediate expression vector containing the light chain of PD-L1 are combined to obtain a recombinant expression vector containing the nucleotide sequences of the heavy chain and light chain of PD-L1.
  • two recombinant expression vectors were finally obtained for cell transfection to 269M cells (Lonza Biologics), and the selection pressure was LM7300 medium without glutamine (from the main medium).
  • the cell harvest liquid is filtered with an adsorption depth filter membrane bag to filter the harvest liquid.
  • the filtration process keep the aeration and stirring parameter settings of the bioreactor unchanged, and then rinse the membrane package with the affinity balance solution, combine the rinse solution and the filtrate, and name the clarified collection solution.
  • the pH of the affinity collection solution was adjusted to about 6.0, and it was named the virus inactivation collection solution.
  • the virus inactivation collection solution uses a depth filtration membrane package to perform adsorption and deep filtration of the virus inactivation collection solution, and collects the filtrate and the top washing solution, which is named as the absorption depth filtration collection solution.
  • Pack the Poros XQ column balance the column with an anion balance solution, then start to load the sample, collect the flow-through solution, and name it as the anion collection solution.
  • Pack the Poros HS column equilibrate with the cation balance solution, load the sample, rinse with the balance solution after the sample is loaded, eluate with the eluent, collect the eluent, and name it as the cation collector.
  • the sample has an anti-PD-1/PD-L1 antibody v3.2 protein content of about 5.0-25.0 mg/ml.
  • This example examines the stability of the formulation containing the anti-PD-1/PD-L1 antibody at pH 5.5 to 6.5.
  • a total of 3 pH values were designed, namely 5.5, 6.0 and 6.5.
  • Pre-prescription test results show that the protein is stable at pH 5.5-6.5, especially at pH 6.0. Based on the above results, the pH was selected as 6.0 for the next round of prescription screening test.
  • This example examines the effects of different buffer systems and stabilizers on the stability of anti-PD-1/PD-L1 antibody formulations.
  • Prescription 1 uses citric acid to adjust pH
  • prescriptions 2 to 4 use hydrochloric acid to adjust pH.
  • prescription 4 was selected as the IBI318 formulation prescription.
  • the buffer system was adjusted to 10mmol/L histidine and histidine hydrochloride, that is, the prescription of IBI318: 20mg/ml recombinant fully human anti-programmed death receptor 1 (PD- 1) And anti-programmed death ligand 1 (PD-L1) bispecific antibody, 0.85mg/ml histidine, 1.00mg/ml histidine hydrochloride, 30.00mg/ml sorbitol, 7.49mg/ml methyl sulfide Acid, 21.07mg/ml arginine hydrochloride, 0.20mg/ml polysorbate 80, pH 6.0.
  • PD-1 fully human anti-programmed death receptor 1
  • PD-L1 anti-programmed death ligand 1
  • Example 4 Stability test of IBI318 high-concentration liquid preparation and its freeze-dried preparation
  • This experiment is used to investigate the stability of IBI318 high-concentration liquid preparation and its freeze-dried preparation under this prescription.
  • the inventors unexpectedly discovered that through this prescription, not only a stable low concentration, for example, 20 mg/ml liquid antibody preparation, but also a stable high concentration, such as 100 mg/ml, liquid preparation and its lyophilized preparation can be prepared.
  • Example 3 Change the antibody by ultrafiltration to the buffer in Example 3 (20mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific Antibody, 0.85mg/ml histidine, 1.00mg/ml histidine hydrochloride, 30.00mg/ml sorbitol, 7.49mg/ml methionine, 21.07mg/ml arginine hydrochloride, pH 6.0), After being concentrated to about 100 mg/ml, polysorbate 80 was added to make the final concentration 0.2 mg/ml. Filter and dispense into vials, 0.5ml each, 22 bottles in total.
  • PD-1 fully human anti-programmed death receptor 1
  • P-L1 anti-programmed death ligand 1
  • the detection indicators are appearance, protein content, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (CEX-HPLC method).
  • ND means that the check item is not set, - means that the test is in progress, the same below.
  • K20180504 is a 20mg/ml liquid preparation
  • PD20191109-1 is a 100mg/ml high-concentration liquid preparation
  • PD20191109-2 is a 100mg/ml high-concentration lyophilized preparation.
  • Non-reduced CE-SDS method The purity of high-concentration liquid preparations decreases at 40°C, and the rate of decrease is equivalent to that of 20mg/ml liquid preparations. At 40°C, the purity is still greater than 90% for 1 month, and the stability is good. accept. Compared with high-concentration liquid preparations, freeze-dried preparations show better stability, and the purity (non-reduced CE-SDS method) does not change significantly after being placed at 40°C for 1 month. See Table 20 for details.
  • the high-concentration preparation prescription 100mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies , 0.85mg/ml histidine, 1.00mg/ml histidine hydrochloride, 30.00mg/ml sorbitol, 7.49mg/ml methionine, 21.07mg/ml arginine hydrochloride, 0.20mg/ml polysorbate 80, pH 6.0)
  • PD-1 fully human anti-programmed death receptor 1
  • P-L1 anti-programmed death ligand 1 bispecific antibodies , 0.85mg/ml histidine, 1.00mg/ml histidine hydrochloride, 30.00mg/ml sorbitol, 7.49mg/ml methionine, 21.07mg/ml arginine hydrochloride, 0.20mg/ml polysorbate 80, pH
  • the lyophilized preparation can be reconstituted to prepare a higher concentration liquid preparation by adding less water.
  • the IBI318 freeze-dried preparation PD20191109-2 can be reconstituted to form a liquid preparation with a protein concentration as high as 170mg/ml, with a viscosity lower than 10cP, and within the acceptable injection viscosity range of the liquid preparation. And when placed at room temperature (25°C ⁇ 2°C, 60%RH ⁇ 5%RH, 500Lux ⁇ 50Lux) for one day, there is no change in all the detection indicators. The above results indicate that it is feasible to reconstitute a lyophilized preparation to prepare a high-concentration liquid preparation for subcutaneous injection.

Abstract

Une préparation pharmaceutique d'un anticorps anti-PD-1/PD-L1, un tampon, un stabilisateur et un tensioactif sont divulgués. De plus, l'invention concerne également l'utilisation de la préparation pour le traitement ou la prévention de maladies.
PCT/CN2021/071757 2020-01-15 2021-01-14 Préparation d'anticorps bispécifique combinant pd-1 et pd-l1 et son utilisation WO2021143767A1 (fr)

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