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WO2021143767A1 - 结合pd-1和pd-l1的双特异性抗体的制剂及其用途 - Google Patents

结合pd-1和pd-l1的双特异性抗体的制剂及其用途 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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English (en)
French (fr)
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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.

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Abstract

提供抗PD-1/PD-L1抗体、缓冲剂、稳定剂和表面活性剂的药物制剂。此外，还提供制剂的疾病治疗或预防用途。

Description

结合PD-1和PD-L1的双特异性抗体的制剂及其用途 技术领域
本发明涉及抗体制剂领域。更具体而言，本发明涉及包含结合人程序性细胞死亡1(PD-1)和人PD-1配体1的双特异性抗体(也称为抗PD-1/PD-L1抗体)的稳定的制剂，以及所述制剂的治疗和/或预防用途。
背景技术
免疫检查点路径用于维持自身耐受性及控制T细胞活化，但癌细胞可使用该等路径来抑制抗肿瘤反应并防止其破坏。PD-1/PD-L1路径是一种该免疫检查点。在T细胞上发现人类PD-1，且人类PD-L1由多种肿瘤类型异常表达；PD-L1与PD-1的结合抑制T细胞增殖及细胞介素产生。PD-1/PD-L1抑制性轴已经被肿瘤征服，作为在抗肿瘤免疫反应的背景下使肿瘤进化成形的自然选择过程的一部分。
尽管靶向PD-1/PD-L1路径的治疗剂在临床上得到了验证且已经在治疗癌症方面取得了显著临床进展，但仅一部分患者受益于该治疗(参见，例如，Sharma,P.及Allison,J.P.,Immune checkpoint targeting in cancer therapy:toward combination strategies with curative potential.Cell.2015；161:2015-14)。举例而言，仅约20％患有非小细胞肺癌(NSCLC)的患者对PD-1抗体治疗有反应。
尽管目前正在进行涉及共同给药PD-L1抗体及PD-1抗体的临床试验(参见，例如，EUROPEAN SOCIETY FOR MEDICAL ONCOLOGY(ESMO)，摘要编号2130；2016年10月)，但该治疗方案涉及输注两种单独的抗体产品，每种抗体的剂量相对较高。此外，尚未知与单一疗法相比，该组合疗法是否会提供效能的改良而不会加剧不良事件特性。
因此，需要以高亲和性结合PD-L1及PD-1、有效地中和由所有PDx家族配体的PD-L1及PD-1活化及/或相对于已知靶向PD-1/PD-L1路径的治疗剂提供优异活性或甚至其组合的双特异性抗体作为某些癌症的更有效的药理学介入。例如，PCT申请号PCT/US2018/041205中公开了一种对人PD-1/PD-L1特异性的示例性的治疗性抗体，其公开内容特此通过引用整体并入。同时，本领域中需要能够用来治疗、预防或延缓各种癌症和免疫相关疾病的抗PD-1/PD-L1抗体制剂。
用于人类受试者的抗体在使用之前必须储存和运输至施用地点。在受试者中可再现地获得期望的抗体药物水平要求所述药物储存在维持药物的生物活性的制剂中。同时，相较于静脉输注，高浓度制剂皮下给药更方便安全，能实现病人在家自主给药，便于慢性病的长期给药治疗；同时高浓度制剂可以满足高剂量的给药要求，能够降低生产成本。开发高浓度制剂已然成为单抗类制剂的发展趋势。同时，冻干制剂能够带来更优越的制剂稳定性，且通过复溶时加入更少的液体能够制备更高浓度的液体制剂。
因此，在本领域中对于含有足够稳定且适于以各种形式施用给受试者的抗PD-1/PD-L1抗体的新颖药物制剂仍存在需要。特别需要这样的制剂，其能够表现出长的贮存期限，在储存 和运输时是稳定的，并且适合以高浓度(例如用于皮下施用)和低浓度(例如用于静脉内施用)施用。
发明概述
本发明通过提供含有特异结合至PD-1/PD-L1的抗体的稳定制剂来满足上述需求。
在第一方面中，本发明涉及抗PD-1/PD-L1抗体或其抗原结合片段的冻干制剂，其包含：(i)抗PD-1/PD-L1抗体或其抗原结合片段；(ii)缓冲体系；(iii)稳定剂，所述稳定剂包括多元醇和/或氨基酸；和(iv)表面活性剂，其中所述制剂当重构时具有5.5-6.5之间的pH，例如，pH约为5.5、6.0或6.5。
在某些实施方案中，所述冻干制剂能够在约1mg/mL至约200mg/mL之间的浓度重构所述抗体或其抗原结合片段。
在某些实施方案中，所述表面活性剂选自聚山梨酯80、聚山梨酯20、泊洛沙姆、聚乙二醇聚山梨酯80或其组合，且以约0.01％(w/v)-1％(w/v)的重量比存在。
在某些实施方案中，所述缓冲体系选自组氨酸缓冲体系、组氨酸和盐酸组氨酸缓冲体系、枸橼酸和枸橼酸钠缓冲体系、醋酸醋酸钠缓冲体系、磷酸盐缓冲体系，且以约0.01％(w/v)-1％(w/v)的重量比存在。
在某些实施方案中，所述多元醇选自山梨醇、甘露醇或其组合，所述氨基酸选自精氨酸、盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸或其组合；
优选地，所述稳定剂包括山梨醇和精氨酸；更优选地，所述山梨醇以约1-10％(w/v)，例如约1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在,所述精氨酸以约1-10％(w/v)，例如约1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在；
优选地，所述稳定剂包括山梨醇和盐酸精氨酸；更优选地，所述山梨醇以约1-10％(w/v)，例如约1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在,以约1-10％(w/v)，例如约1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在。
在某些实施方案中，所述稳定剂还包括甲硫氨酸；更优选地，所述甲硫氨酸以约0.1-10％(w/v)的重量比存在。
在第二方面中，本发明涉及抗PD-1/PD-L1抗体或其抗原结合片段的冻干制剂，其通过冻干水溶液制成，所述水溶液包含：(i)抗PD-1/PD-L1抗体或其抗原结合片段；(ii)缓冲体系；(iii)稳定剂，所述稳定剂包括多元醇和/或氨基酸；和(iv)表面活性剂，所述液体制剂具有约5.5-6.5的pH，例如，pH约为5.5、6.0或6.5；优选地，所述液体制剂具有约6.0的pH。
在第三方面中，本发明涉及抗PD-1/PD-L1抗体或其抗原结合片段的液体药物制剂，其包含水溶液，所述水溶液包含：(i)抗PD-1/PD-L1抗体或其抗原结合片段；(ii)缓冲体系；(iii)稳定剂，所述稳定剂包括多元醇和/或氨基酸；和(iv)表面活性剂，所述液体制剂具有约5.5-6.5的pH，例如，pH约为5.5、6.0或6.5；优选地，所述液体制剂具有约6.0的pH。
在某些实施方案中，上述的冻干制剂或液体制剂中，所述抗PD-1/PD-L1抗体或其抗原结 合片段以约1mg/mL至约200mg/mL的浓度存在于水溶液中，例如以约1、5、10、20、25、30、35、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或200mg/ml的浓度存在于水溶液中。
在某些实施方案中，上述的冻干制剂或液体制剂中，所述表面活性剂选自聚山梨酯80、聚山梨酯20、泊洛沙姆、聚乙二醇聚山梨酯80或其组合，且以约0.1-10mg/ml，例如约0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的浓度存在。
在某些实施方案中，上述的冻干制剂或液体制剂中，所述缓冲体系选自组氨酸缓冲体系、组氨酸和盐酸组氨酸缓冲体系、枸橼酸和枸橼酸钠缓冲体系、醋酸醋酸钠缓冲体系、磷酸盐缓冲体系，且以约1-100mM，例如，1、5、10、15、20、30、40、50、60、70、80、90、100mM的浓度存在。
优选地，所述缓冲体系为组氨酸缓冲体系；更优选地，所述组氨酸以约0.1-10mg/mL，例如约0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的浓度存在；
优选地，所述缓冲体系为组氨酸和盐酸组氨酸缓冲体系；更优选地，所述组氨酸和所述盐酸组氨酸分别以约0.1-10mg/mL，例如约0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的浓度存在。
在某些实施方案中，上述的冻干制剂或液体制剂中，所述多元醇选自山梨醇、甘露醇或其组合，所述氨基酸选自精氨酸、盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸或其组合；优选地，所述稳定剂包括山梨醇和精氨酸；更优选地，所述山梨醇以约10-100mg/mL，例如约10、20、30、40、50、60、70、80、90、100mg/mL的浓度存在，所述精氨酸以例如约10、20、30、40、50、60、70、80、90、100mg/mL存在；
优选地，所述稳定剂包括山梨醇和盐酸精氨酸；更优选地，所述山梨醇和所述盐酸精氨酸分别以约10-100mg/mL，例如约10、20、30、40、50、60、70、80、90、100mg/mL的浓度存在。
在某些实施方案中，上述的冻干制剂或液体制剂中，所述稳定剂还包括甲硫氨酸；优选地，所述甲硫氨酸以约1-100mg/mL，例如约1、5、10、20、30、40、50、60、70、80、90、100mg/mL的浓度存在。
在本文中描述的任意制剂中，所述抗PD-1/PD-L1抗体或其抗原结合片段结合人类PD-L1(SEQ ID NO:1)及人类PD-1(SEQ ID NO:2)。优选地，所述抗PD-1/PD-L1抗体或其抗原结合片段包含：第一重链(HCl)，其包含第一重链可变区(HCVR1)和恒定区；第一轻链(LC1)，其包含第一轻链可变区(LCVR1)和恒定区；第二重链(HC2)，其包含第二重链可变区(HCVR2)和恒定区；以及第二轻链(LC2)，其包含第二轻链可变区(LCVR2)和恒定区，其中：
所述HCVR1包含SEQ ID NO:3所示的重链可变区所含的三个互补决定区域HCDR1、HCDR2和HCDR3，并且所述LCVR1包含SEQ ID NO:4所示的轻链可变区所含的LCDR1、LCDR2和LCDR3；以及
所述HCVR2包含SEQ ID NO:5所示的重链可变区中所含的三个互补决定区域(CDR)HCDR1、HCDR2和HCDR3，并且所述LCVR2包含SEQ ID NO:8所示的轻链可变区所含的 三个互补决定区域LCDR1、LCDR2和LCDR3。
在一些实施方案中，本发明抗体制剂中的抗PD-1/PD-L1抗体可以包含：第一重链(HCl)，其包含第一重链可变区(HCVR1)和恒定区；第一轻链(LC1)，其包含第一轻链可变区(LCVR1)和恒定区；第二重链(HC2)，其包含第二重链可变区(HCVR2)和恒定区；以及第二轻链(LC2)，其包含第二轻链可变区(LCVR2)和恒定区，其中：
a)所述HCVR1包含具有SEQ ID NO:16所示的氨基酸序列的互补决定区域CDR1、具有SEQ ID NO:17所示的氨基酸序列的互补决定区域CDR2及具有SEQ ID NO:18所示的氨基酸序列的互补决定区域CDR3；
b)所述LCVR1包含具有SEQ ID NO:19所示的氨基酸序列的互补决定区域CDR1、具有SEQ ID NO:20所示的氨基酸序列的互补决定区域CDR2及具有SEQ ID NO:21所示的氨基酸序列的互补决定区域CDR3；
c)所述HCVR2包含具有SEQ ID NO:22所示的氨基酸序列的互补决定区域CDR1、具有SEQ ID NO:23或SEQ ID NO:24所示的氨基酸序列的互补决定区域CDR2及具有SEQ ID NO:25所示的氨基酸序列的互补决定区域CDR3；以及
d)所述LCVR2包含具有SEQ ID NO:26所示的氨基酸序列的互补决定区域CDR1、具有SEQ ID NO:27所示的氨基酸序列的互补决定区域CDR2及具有SEQ ID NO:28所示的氨基酸序列的互补决定区域CDR3。
在某些实施方案中，所述抗体或其抗原结合片段包含与SEQ ID NO:3具有至少90％,95％,98％或99％或更高同一性的HCVR1；和/或与SEQ ID NO:4具有至少90％,95％,98％或99％或更高同一性的LCVR1；和/或与SEQ ID NO:5具有至少90％,95％,98％或99％或更高同一性的HCVR2；和/或与SEQ ID NO:8具有至少90％,95％,98％或99％或更高同一性的LCVR2；
优选地，在上述抗PD-1/PD-L1抗体或其抗原结合片段中，a)所述HCVR1包含具有SEQ ID NO:3所示的氨基酸序列；b)所述LCVR2包含具有SEQ ID NO:4所示的氨基酸序列；c)所述HCVR2包含具有SEQ ID NO:5所示的氨基酸序列；及d)所述LCVR2包含具有SEQ ID NO:8所示的氨基酸序列。
在某些实施方案中，所述抗体或其抗原结合片段包含与SEQ ID NO:49或SEQ ID NO:29具有至少90％,95％,98％或99％或更高同一性的HC1；和/或与SEQ ID NO:30具有至少90％,95％,98％或99％或更高同一性的LC1；和/或与SEQ ID NO:33或SEQ ID NO:31或SEQ ID NO:32具有至少90％,95％,98％或99％或更高同一性的HC2；和/或与SEQ ID NO:34具有至少90％,95％,98％或99％或更高同一性的LC2；
优选地，在上述抗PD-1/PD-L1抗体或其抗原结合片段中，所述HC1、所述LC1、所述HC2及所述LC2分别包含SEQ ID NO:49、SEQ ID NO:30、SEQ ID NO:31及SEQ ID NO:34所示的氨基酸序列；或者分别包含SEQ ID NO:49、SEQ ID NO:30、SEQ ID NO:32及SEQ ID NO:34所示的氨基酸序列；或者分别包含SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:33及SEQ ID NO:34所示的氨基酸序列。
在某些实施方案中，所述抗PD-1/PD-L1抗体为PCT申请号PCT/US2018/041205中公开 的抗PD-1/PD-L1抗体。为了本申请的目的，该PCT申请的全部内容特此并入本文作为参考。优选地，所述抗体选自：PD-1/PD-L1双特异性Ab v13844、Ab v3.0或Ab v3.2。
在某些实施方案中，本发明所述的制剂中所述抗体在至少约12个月内表现出稳定性，优选地，所述抗体在至少约24个月内表现出稳定性，更优选地，所述抗体在至少约36个月内表现出稳定性。
在某些实施方案中，制剂在储存后，例如在2-8℃储存至少24个月后，或在室温储存至少6个月后，或在40℃±2℃储存1个月后，是稳定的，优选地具有如下特征之一或多项：
(i)通过SEC-HPLC法测量，制剂具有大于90％的纯度，优选大于92％、94％、96％、98％、99％的纯度；
(ii)通过还原型或非还原型CE-SDS法测量，制剂具有大于90％的纯度，优选大于92％、94％、96％、98％的纯度；
(iii)通过iCIEF或CEX-HPLC法测量，主成分≥35％，优选大于或等于35％、40％、45％或50％。
在一个方面，本发明提供了一种制备本发明的制剂的方法，包括以下步骤：
(1)将除表面活性剂之外的成分配制成溶液；
(2)通过超滤，采用步骤(1)制备的溶液对所述PD-1/PD-L1双特异性抗体或其抗原结合片段进行换液，然后浓缩至目标浓度；
(3)添加所述表面活性剂到步骤(2)制备的液体中。
在一个方面，本发明提供了一种递送装置，其包含有效量的本发明的液体抗体制剂或冻干制剂。在一个实施方案中，本发明的递送装置以包含有效量的本发明的液体抗体制剂或冻干制剂的预填装注射器形式提供，例如用于静脉内、皮下、皮内或者肌内注射，或静脉内输注。
在又一方面，本发明提供向受试者，例如哺乳动物递送抗PD-1/PD-L1抗体蛋白的方法，包括给予所述受试者有效量的本发明的液体抗体制剂或冻干制剂的步骤，所述递送是例如通过使用预填装注射器的递送装置实施的。
在又一方面，本发明提供本发明的液体抗体制剂或冻干制剂的用途，用于制备在受试者中治疗或预防肿瘤的递送装置(如，预填装注射器)或药物，所述肿瘤为例如癌症，包括但不限于实体瘤、血液学癌、软组织肿瘤和转移性病灶。在一些实施方案中，本文所述的癌症包括但不限于霍奇金氏或非霍奇金氏淋巴瘤、黑色素瘤、肾细胞癌、肾癌、肺癌、膀胱癌、胃及食道癌、结肠直肠癌、肝癌、肝细胞癌、胆管癌、胰脏癌、乳癌、三阴性乳癌、卵巢癌、子宫内膜癌、前列腺癌、小细胞肺癌(SCLC)、非小细胞肺(NSCLC)、间皮瘤、头颈鳞状细胞癌(SCCHN)、软组织肉瘤或多形性神经胶母细胞瘤。优选地，所述肺癌为NSCLC(鳞状及非鳞状)、小细胞肺癌或间皮瘤。
本发明还提供了一种通过向受试者施用本发明的液体抗体制剂或冻干制剂或包含该液体抗体制剂或冻干制剂的递送装置(如，预填装注射器)或药物，在受试者中增强免疫效应细胞反应和/或减少免疫抑制的方法。
本发明还提供了一种通过向受试者施用本发明的液体抗体制剂或冻干制剂或包含该液体抗体制剂或冻干制剂的递送装置(如，预填装注射器)或药物,预防和/或治疗受试者的疾病，例如上述肿瘤的方法。
本发明制剂的有益效果：
1)本发明提供的冻干及液体抗体制剂性质稳定，在5℃±3℃条件下可稳定至少24个月。
2)本发明提供的处方不仅可以保证低浓度液体制剂的稳定，还可以保证高浓度液体制剂的稳定而不额外添加辅料，为高浓度液体制剂皮下给药提供可能。在实施案例中,本发明高浓度液体制剂能够直接制备成冻干制剂无需优化处方，冻干后能保持其结构与功能的完整性。
3)本发明提供的冻干制剂易复溶，在液体制剂可接受的注射粘度范围内(≤10cP)，且在常温下放置一天后各项检测指标均未发生变化。通过减少复溶液体积可以制备更高浓度(例如170mg/ml)液体制剂。相同的给药量，减少给药体积，提高临床给药的顺应性。
本发明的其它实施方案将通过参阅此后的详细说明而清楚明了。
附图简述
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1显示了处方前试验中，通过SEC-HPLC法测定的各样品中蛋白纯度随时间变化图。图中Met表示甲硫氨酸。
图2显示了处方前试验中，通过iCIEF法测定的各样品中电荷变异体主成分和酸性组分随时间变化图。
图3显示了处方筛选试验中，在40℃±2℃条件下，通过SEC-HPLC法测定的各样品中蛋白纯度随时间变化图
图4显示了处方筛选试验中，在25℃±2℃条件下，通过SEC-HPLC法测定的各样品中蛋白纯度随时间变化图。
图5显示了处方筛选试验中，在40℃±2℃条件下，通过非还原型CE-SDS法测定的各样品中蛋白纯度随时间变化图。
图6显示了处方筛选试验中，在40℃±2℃条件下，通过CEX-HPLC法测定的各样品中电荷变异体主成分和酸性组分随时间变化图。
图7显示了处方筛选试验中，在25℃±2℃条件下，通过CEX-HPLC法测定的各样品中电荷变异体主成分和酸性组分随时间变化图。
发明详述
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说明特定实施方案之用， 而不意欲为限制性的。
定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
术语“免疫检查点分子”意指免疫系统中存在的一类抑制性信号分子，通过调节外周组织中免疫反应的持续性和强度避免组织损伤，并参与维持对于自身抗原的耐受(Pardoll DM.,The blockade of immune checkpoints in cancer immunotherapy.Nat Rev Cancer,2012,12(4):252-264)。研究发现，肿瘤细胞能够逃避体内免疫系统而失控增殖的原因之一是利用了免疫检查点分子的抑制性信号通路，由此抑制了T淋巴细胞活性，使得T淋巴细胞不能有效发挥对肿瘤的杀伤效应(Yao S,Zhu Y和Chen L.，Advances in targeting cell surface signaling molecules for immune modulation.Nat Rev Drug Discov,2013,12(2):130-146)。免疫检查点分子包括但不限于程序性死亡1(PD-1)、PD-L1、PD-L2、细胞毒T淋巴细胞抗原4(CTLA-4)、LAG-3和TIM-3。
如本文所用的术语“程序性细胞死亡配体1”、“PD-L1”、“程序性死亡配体1”、“分化簇274”、“CD274”或“B7同系物1”是指来自任何脊椎动物来源的任何天然PD-L1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人)和啮齿类(例如，小鼠和大鼠)。所述术语涵盖“全长”、未加工的PD-L1以及由细胞中的加工所产生的任何形式的PD-L1。PD-L1可作为跨膜蛋白或作为可溶性蛋白存在。所述术语还涵盖天然存在的PD-L1的变体，例如剪接变体或等位基因变体。PD-L1的基本结构包括4个结构域：胞外Ig样V型结构域和Ig样C2型结构域、跨膜结构域以及细胞质结构域。可在NCBI Gene ID No.29126下找到关于人PD-L1基因(包括基因组DNA序列)的另外信息。可在NCBI Gene ID No.60533下找到关于小鼠PD-L1基因(包括基因组DNA序列)的另外信息。示例性全长人PD-L1蛋白的氨基酸序列可例如在NCBI登录号NP_001254653或UniProt登录号Q9NZQ7下找到，而可例如在NCBI登录号NP_068693或Uniprot登录号Q9EP73下找到示例性全长小鼠PD-L1蛋白序列。在本文中，术语“抗体”以最广意义使用，指包含抗原结合位点的蛋白质，涵盖各种结构的天然抗体和人工抗体，包括但不限于完整抗体和抗体的抗原结合片段。
术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换地用来指包含由二硫键相互连接的至少两条重链(H)和两条轻链(L)的糖蛋白。每条重链由重链可变区(本文中缩 写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH区和VL区可以进一步再划分为超变区(为互补决定区(CDR)，其间插有较保守的区域(为构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。
如本文所用，术语“多特异性”抗体指具有至少两个抗原结合位点的抗体，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。本文提供的抗体通常是多特异性抗体，例如双特异性抗体。多特异性抗体是对至少两个不同抗原表位具有结合特异性的抗体。在一个实施方案中，本文提供了这样的双特异性抗体，其具有针对第一抗原和第二抗原的结合特异性。例如第一抗原为PD-L1，第二抗原为PD-1。
术语“人源化”抗体指包含来自非人类HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在一些实施方案中，人源化抗体包含全部或基本上全部的HVR(例如，CDR)与非人抗体的那些HVR对应并且全部或基本上全部的FR区与人抗体的那些FR对应。人源化抗体任选地可以包含从人抗体衍生的抗体恒定区的至少一部分。抗体(例如非人抗体)的“人源化形式”指已经历过人源化的抗体。
术语“Fc区”在本文中用于定义免疫球蛋白重链的C端区域，所述区域包含至少一部分的恒定区。该术语包括天然序列Fc区和变体Fc区。在某些实施方案中，人IgG重链Fc区从Cys226或Pro230延伸至重链的羰基端。然而，Fc区的C端赖氨酸(Lys447)可以存在或者可以不存在。除非另外说明，Fc区或恒定区中的氨基酸残基的编号是根据EU编号系统，其也被称为EU索引，如在Kabat等，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，MD，1991中所述。
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的框架区(FR)和三个互补决定区(参见，例如，Kindt等Kuby Immunology，6th ed.，W.H.Freeman and Co.91页(2007))。单个VH或VL结构域可以足以给予抗原结合特异性。此外，可以使用来自与特定抗原结合的抗体的VH或VL结构域来分离结合所述抗原的抗体，以分别筛选互补VL或VH结构域的文库。参见，例如，Portolano等，J.Immunol.150：880-887(1993)；Clarkson等，Nature 352：624-628(1991)。
可变区通常表现出由三个高变区连接的相对保守的构架区(FR)的相同的一般结构，所述高变区也被称为互补决定区或CDR。通常通过构架区定位(align)来自每对的两条链的CDR，所述CDR使得可结合特异性表位。两条轻链和重链可变区从N-末端到C-末端通常包含结构域FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。
“互补决定区”或“CDR区”或“CDR”或“高变区”(在本文中与超变区“HVR”可以互换使用)，是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR 通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义(North等，“A New Clustering of Antibody CDR Loop Conformations”，Journal of Molecular Biology，406，228-256(2011))。
例如，使用Kabat和Chothia编号的CDR区域的不同定义范围。
Figure PCTCN2021071757-appb-000001
CDR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))的编号位置。
在一个实施方案中，本发明抗体的CDR通过North定义规则确定边界，例如下文表1b和表1c所示。
然而，应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia,AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。
术语“抗体制剂”指一种制备物，所述制备物处于允许作为活性成分的抗体的生物活性可以有效发挥的形式，并且不含有对于待施用该制剂的受试者而言具有不可接受毒性的其它组分。这类抗体制剂通常是无菌的。通常，抗体制剂中包含可药用赋形剂。“可药用”赋形剂是可以合理地施用至受试哺乳动物以便制剂中所用活性成分的有效剂量可以递送至受试者的试剂。赋形剂的浓度与施用模式相适应，例如可以是注射可接受的。
术语“抗PD-1/PD-L1抗体制剂”在本文中也简称为“本发明的抗体制剂”，意指包含抗PD-1/PD-L1抗体蛋白作为活性成分并包含可药用赋形剂的制备物。将抗PD-1/PD-L1抗体蛋白与可药用赋形剂组合后，作为活性成分的抗PD-1/PD-L1抗体蛋白适于治疗性或预防性施与人类或非人类动物。本发明的抗体制剂可以例如制备成水性形式的液体制剂，例如，即用式预填装注射器，或者制备成冻干制剂，在即将使用前通过溶解和/或悬浮于生理可接受的溶液中进行重构(即，复溶)。在一些实施方案中，抗PD-1/PD-L1抗体蛋白制剂是液体制剂形式。
“稳定的”抗体制剂是制剂中的抗体在储存于特定条件下之后保有可接受程度的物理稳定性和/或化学稳定性。尽管抗体制剂中所含的抗体在储存特定时间之后可能不会100％维持其化学结构，但通常在储存特定时间之后维持约90％、约95％、约96％、约97％、约98％或约99％的抗体结构或功能，则认为抗体制剂是“稳定的”。在一些具体的实施方案中，本发明的抗PD-1/PD-L1抗体蛋白制剂在制造、制备、运输和长期储存过程中表现出低至检测不到的抗体聚集或降解或化学修饰，从而极少或甚至是没有抗PD-1/PD-L1抗体蛋白的生物活性损 失，表现出高度稳定性。在一些实施方案中，本发明的抗PD-1/PD-L1抗体蛋白制剂在储存后，基本上保留其物理和化学稳定性。优选地，本发明液体制剂可以在室温稳定至少6个月或在40℃±2℃储存1个月，和/或在25℃±2℃稳定至少3个月，和/或在2-8℃稳定至少24个月。
本领域已知多种分析技术可以用于测定蛋白质的稳定性，参见例如Peptide and Protein Drug Delivery,247-301,Vincent Lee Ed.,Marcel Dekker,Inc.,New York,N.Y.,Pubs(1991)and Jones,A.Adv.Drug Delivery Rev.10:29-90(1993)。可以在选定的温度和选定的储存时间测量稳定性。例如，可以基于预期的制剂货架期来选择储存时间。备选地，可以使用加速稳定性试验。在一些实施方案中，通过对抗体制剂进行各种胁迫测试来进行稳定性测试。这些测试可以代表调配的抗体制剂在制造、储存或运输期间可能遭遇到的极端条件，也可以代表在非制造、储存或运输期间可能使抗体制剂中的抗体的不稳定性加速的条件。例如，可以将经调配的抗PD-1/PD-L1抗体蛋白制剂充填至玻璃小瓶中以检验在高温胁迫下的抗体稳定性。
经一段储存时间后，制剂不显示聚集、沉淀、混浊和/或变性；或显示非常少的聚集、沉淀、混浊和/或变性，则可以认为抗体在制剂中“保持其物理稳定性”。由于制剂中抗体的聚集可以潜在地导致患者增加的免疫反应，从而导致安全性问题。因此，需要使在制剂中的抗体聚集最小化或防止聚集。光散射法可以用于测定制剂中的可见聚集物。SEC可以用于测定制剂中的可溶性聚集物。此外，可以通过目视检查制剂的外观、颜色和/或澄清度、或者通过OD 350nm法检测制剂的浊度、或者通过非还原型CE-SDS法测定制剂的纯度，来指示制剂的稳定性。在一个实施方案中，通过测定在特定温度下储存特定时间之后制剂中的抗体单体的百分比来测量制剂的稳定性，其中制剂中的抗体单体的百分比越高，则制剂的稳定性越高。
“可接受程度的”物理稳定性可以表示于特定温度下储存特定时间之后，在制剂中检测到至少约90％的抗PD-1/PD-L1抗体蛋白单体。在一些实施方案中，在特定温度储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月或更久后，可接受程度的物理稳定性表示至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗PD-1/PD-L1抗体蛋白单体。当评估物理稳定性时，药物制剂储存的特定温度可为约-80℃至约45℃的任一温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约4℃-8℃、约5℃、约25℃、约35℃、约37℃、约40℃、约42℃或约45℃。例如，若储存于约40℃±2℃1个月之后，检测到至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗PD-1/PD-L1抗体蛋白单体，则药物制剂视为是稳定的；若储存于约25℃2个月之后，检测到至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗PD-1/PD-L1抗体蛋白单体，则药物制剂视为是稳定的；若储存于约5℃9个月之后，检测到至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗PD-1/PD-L1抗体蛋白单体，则药物制剂视为是稳定的。
经一段储存时间后，如果制剂中的抗体不显示显著的化学改变，则可以认为抗体在制剂中“保持其化学稳定性”。大多数化学不稳定性源自于形成了抗体的共价修饰形式(例如，抗体的电荷变异体)。例如由天冬氨酸异构化、N和C末端修饰，可以形成碱性变异体；由脱酰胺 化、唾液酸化和糖化，可以产生酸性变异体。化学稳定性可以通过检测和/或定量抗体的化学改变形式来评估。例如，可以通过阳离子交换色谱(CEX)或成像毛细管等电聚焦电泳(iCIEF)检测制剂中抗体的电荷变异体。在一个实施方案中，通过测定在特定温度下储存特定时间之后制剂中抗体的电荷变异体百分比变化值来测量制剂的稳定性，其中该变化值越小，则制剂的稳定性越高。
“可接受程度”的化学稳定性可以表示于特定温度下储存特定时间之后制剂中电荷变异体(例如主成分或酸性组分或碱性组分)的百分比变化值不超过50％，例如不超过30％、不超过20％。在一些实施方案中，在特定温度储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月或更久后，可接受程度的化学稳定性可以表现为主成分电荷变异体的百分比变化值不超过约50％、40％、30％、20％、15％。当评估化学稳定性时，储存药物制剂的温度可为约-80℃至约45℃的任一温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约4℃-8℃、约5℃、约25℃或约45℃。例如，若在储存于5℃24个月之后，主成分电荷变异体的百分比变化值少于约25％、24％、23％、22％、21％、20％、19％、18％、17％、16％、15％、14％、13％、12％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、0.5％或0.1％，则药物制剂可被视为是稳定的；若在储存于25℃2个月后，主成分电荷变异体的百分比变化值少于约20％、19％、18％、17％、16％、15％、14％、13％、12％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、0.5％或0.1％，则药物制剂亦可被视为是稳定的；若在储存于40℃1个月之后，主成分电荷变异体的百分比变化值少于约50％、40％、30％、20％、10％、5％或4％，则药物制剂亦可被视为是稳定的。
术语“冻干制剂”是指通过液体制剂的冷冻干燥处理得到或能够得到的组合物。优选地，其为具有少于5％、优选少于3％水含量的固体组合物。
术语“重构制剂”是指将固体制剂(例如冻干制剂)溶解和/或悬浮于生理可接受的溶液中得到的液体制剂。
文中使用的术语“室温”是指15℃至30℃、优选20℃至27℃、更优选25℃的温度。
“胁迫条件”是指在化学和/或物理上不利于抗体蛋白的环境，所述环境可以导致不可接受的抗体蛋白失稳定，例如，高温、振荡、冻融、光照。“高温胁迫”是指，将抗体制剂置于室温或甚至于更高温度(例如40℃±2℃)储存一段时间。通过高温胁迫加速试验，可以检查抗体制剂的稳定性。
如本文所使用，术语“肠胃外施用”意指肠内和局部给药以外的给药方式，通常通过注射或输注方式，并且包括但不限于，静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下(subcuticular)、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射以及输注。在一些实施方案中，本发明的稳定抗PD-1/PD-L1抗体蛋白制剂肠胃外施用于受试者。在一个实施方案中，本发明的抗PD-1/PD-L1抗体蛋白制剂以皮下、皮内、肌内或静脉内注射方式施用于受试者。
I.抗体制剂
本发明的一方面提供稳定的冻干制剂，其包含(i)抗PD-1/PD-L1抗体或其抗原结合片段，(ii)缓冲体系，(iii)稳定剂，所述稳定剂包括多元醇和/或氨基酸，和(iv)表面活性剂，其中所述制剂当重构时具有5.5-6.5之间的pH，例如，pH约为5.5、6.0或6.5。
本发明的另一方面提供稳定的冻干制剂，其通过冻干水溶液制成，所述水溶液包含：(i)抗PD-1/PD-L1抗体或其抗原结合片段，(ii)缓冲体系，(iii)稳定剂，所述稳定剂包括多元醇和/或氨基酸，和(iv)表面活性剂，所述液体制剂具有约5.5-6.5的pH，例如，pH约为5.5、6.0或6.5；优选地，所述液体制剂具有约6.0的pH。
本发明的再一方面提供稳定的液体抗体制剂，其包含水溶液，所述水溶液包含(i)抗PD-1/PD-L1抗体或其抗原结合片段，(ii)缓冲体系，(iii)稳定剂，所述稳定剂包括多元醇和/或氨基酸，和(iv)表面活性剂，所述液体制剂具有约5.5-6.5的pH，例如，pH约为5.5、6.0或6.5；优选地，所述液体制剂具有约6.0的pH。
在一个优选方案中，本发明的液体抗体制剂是注射制剂形式。
(i)抗PD-1/PD-L1抗体
“抗PD-1/PD-L1抗体”或“结合PD-1和PD-L1的双特异性抗体”或“结合PD-1和PD-L1的抗体”是指这样的抗体，所述抗体能够以足够的亲和力结合PD-1以及PD-L1分子，以致所述抗体可以用作同时靶向PD-1和PD-L1分子的治疗剂和/或预防剂。
如本文所用，术语“结合”或“特异性结合”意指结合作用对抗原是选择性的，并且可以与不想要的或非特异的相互作用区别开。抗体与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)、表面等离子共振法(SPR)或生物膜层光学干涉技术(ForteBio)或本领域已知的其它常规结合测定法测定。作为本发明的实施例，指抗体或抗原结合片段在体外测定法中，优选地在采用纯化的野生型抗原的生物膜层光学干涉测量中与抗原表位结合。在某些实施方案中，在抗体或抗原结合片段优选地识别蛋白质和/或大分子的复杂混合物中的其靶抗原时，将抗体或抗原结合片段称作特异性结合抗原。
如本文所用“结合”或“特异性结合”在提及抗体对人类PD-L1(SEQ ID NO:1)、人类PD-1(SEQ ID NO:2)或二者的亲和性时，除非另有指示，否则指K D小于约1×10 -6M、较佳小于约1×10 -9M，如借由上述业内已知的常见方法于37℃下所测定。
本发明中的抗体为同时靶向PD-L1及PD-1的异二聚体双特异性抗体，经由使两条不同重链及两条不同轻链配对成一个IgG样抗体而成。此外，本发明中的抗体提供具有以下特征中之一或多个的抗人类PD-L1及抗人类PD-1异二聚体双特异性抗体：阻断PD轴的所有三种相互作用(PD-L1结合至PD-1，PD-L2结合至PD-1及PD-L1结合至CD80)，桥接PD-L1及PD-1过表达细胞，由于靠近结合的T细胞及肿瘤细胞而增加T细胞活化及肿瘤细胞杀死，在具有中等至高PD配体表达程度的肿瘤细胞中于低剂量下展现显著抗肿瘤活性，并展现物理及化学稳定性，包括但不限于活体内稳定性、热稳定性、溶解性、低自缔合及药物动力学特征。
因此，在一些实施方案中，本发明抗体制剂中的抗PD-1/PD-L1抗体包含：a)包含含有SEQ ID NO:3的氨基酸序列的重链可变区(HCVR1)的第一重链(HC1)；b)包含含有SEQ ID NO:4的氨基酸序列的轻链可变区(LCVR1)的第一轻链(LC1)；c)包含含有SEQ ID NO:5的 氨基酸序列的重链可变区(HCVR2)的第二重链(HC2)；及d)包含含有SEQ ID NO:8的氨基酸序列的轻链可变区(LCVR2)的第二轻链(LC2)。
在一些实施方案中，本发明抗体制剂中的抗PD-1/PD-L1抗体包含HC1、LC1、HC2及LC2，其中：a)该HC1在HCVR中包含具有SEQ ID NO:16的氨基酸序列的CDR1、具有SEQ ID NO:17的氨基酸序列的CDR2及具有SEQ ID NO:18的氨基酸序列的CDR3；b)该LC1在LCVR中包含具有SEQ ID NO:19的氨基酸序列的CDR1、具有SEQ ID NO:20的氨基酸序列的CDR2及具有SEQ ID NO:21的氨基酸序列的CDR3；c)该HC2包含在HCVR中具有SEQ ID NO:22的氨基酸序列的CDR1、具有SEQ ID NO:23或SEQ ID NO:24的氨基酸序列的CDR2及具有SEQ ID NO:25的氨基酸序列的CDR3；d)该LC2在LCVR中包含具有SEQ ID NO:26的氨基酸序列的CDR1、具有SEQ ID NO:27的氨基酸序列的CDR2及具有SEQ ID NO:28的氨基酸序列的CDR3；e)HC1的CH1域包含S183K的氨基酸取代；f)LC1的恒定区包含S176E及Y178E的氨基酸取代的人类λ变体；g)HC2的CH1域包含L128E、K147T及Q175E的氨基酸取代；h)LC2的恒定区包含S131R、V133G及S176R的氨基酸取代的人类κ变体；且i)HC1的CH3域包含T350V、L351Y、F405A及Y407V的氨基酸取代，且HC2的CH3域包含T350V、T366L、K392L及T394W的氨基酸取代；或HC1的CH3域包含T350V、T366L、K392L及T394W的氨基酸取代，且HC2的CH3域包含T350V、L351Y、F405A及Y407V的氨基酸取代；且j)其中HC1及HC2是免疫效应功能无效的人类IgG1Fc变体，其中编号是根据EU索引。优选地，HC1及HC2包含L234A、L235A及D265S的氨基酸取代。
在一些实施方案中，本发明抗体制剂中的抗PD-1/PD-L1抗体包含：a)HC1，其自N端至C端依序包含含有SEQ ID NO:3的氨基酸序列的HCVR、CH1域中SEQ ID NO:9的氨基酸序列、CH2域中SEQ ID NO:10的氨基酸序列及CH3域中SEQ ID NO:11或SEQ ID NO:12的氨基酸序列；b)LC1，其自N端至C端依序包含含有SEQ ID NO:4的氨基酸序列的LCVR及恒定区中SEQ ID NO:14的氨基酸序列；c)HC2，其自N端至C端依序包含含有SEQ ID NO:5的氨基酸序列的HCVR、CH1域中SEQ ID NO:13的氨基酸序列、CH2域中SEQ ID NO:10的氨基酸序列及CH3域中SEQ ID NO:12的氨基酸序列或SEQ ID NO:11的氨基酸序列；及d)LC2，其自N端依序包含含有SEQ ID NO:8的氨基酸序列的LCVR及恒定区中SEQ ID NO:15的氨基酸序列，条件是当SEQ ID NO:11的氨基酸序列存于该HC1的CH3域中时，SEQ ID NO:12的氨基酸序列存于该HC2的CH3域中；或当SEQ ID NO:12的氨基酸序列存于该HC1的CH3域中时，SEQ ID NO:11的氨基酸序列存于该HC2的CH3域中。
在一些实施方案中，本发明抗体制剂中的抗PD-1/PD-L1抗体中：a)该HC1自N端至C端依序包含含有SEQ ID NO:3的氨基酸序列的HCVR1、CH1域中SEQ ID NO:9的氨基酸序列、CH2域中SEQ ID NO:10的氨基酸序列及CH3域中SEQ ID NO:11的氨基酸序列；b)该LC1，其自N端至C端依序包含含有SEQ ID NO:4的氨基酸序列的LCVR1及恒定区中SEQ ID NO:14的氨基酸序列；及c)该HC2，其自N端至C端依序包含含有SEQ ID NO:6的氨基酸序列的HCVR2、CH1域中SEQ ID NO:13的氨基酸序列、CH2域的区中SEQ ID NO:10 的氨基酸序列及CH3域中SEQ ID NO:12的氨基酸序列；及d)该LC2，其自N端依序包含含有SEQ ID NO:8的氨基酸序列的LCVR2及恒定区中SEQ ID NO:15的氨基酸序列。
在一些实施方案中，本发明抗体制剂中的抗PD-1/PD-L1抗体中：a)包含SEQ ID NO:49的氨基酸序列的HC1；b)包含SEQ ID NO:30的氨基酸序列的LC1；c)包含SEQ ID NO 31的氨基酸序列的HC2；及d)包含SEQ ID NO:34的氨基酸序列的LC2。
本发明提供结合人类PD-L1(SEQ ID NO:1)及人类PD-1(SEQ ID NO:2)的抗体，其包含：a)包含SEQ ID NO:49的氨基酸序列的HC1；b)包含SEQ ID NO:30的氨基酸序列的LC1；c)包含SEQ ID NO 32的氨基酸序列的HC2；及d)包含SEQ ID NO:34的氨基酸序列的LC2。
在一些实施方案中，本发明抗体制剂中的抗PD-1/PD-L1抗体中：a)包含SEQ ID NO:29的氨基酸序列的HC1；b)包含SEQ ID NO:30的氨基酸序列的LC1；c)包含SEQ ID NO:33的氨基酸序列的HC2；及d)包含SEQ ID NO:34的氨基酸序列的LC2。
在一些实施方案中，本发明抗体制剂中的抗PD-1/PD-L1抗体可以包含与SEQ ID NO:3具有至少90％,95％,98％或99％或更高同一性的HCVR1；和/或与SEQ ID NO:4具有至少90％,95％,98％或99％或更高同一性的LCVR1；和/或与SEQ ID NO:5(即SEQ ID NO:6或SEQ ID NO:7)具有至少90％,95％,98％或99％或更高同一性的HCVR2；和/或与SEQ ID NO:8具有至少90％,95％,98％或99％或更高同一性的LCVR2。在本文中，“序列同一性”是指在比较窗中以逐个核苷酸或逐个氨基酸为基础的序列相同的程度。可以通过以下方式计算“序列同一性百分比”：将两条最佳比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同一性百分比。为了确定序列同一性百分数而进行的最佳比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。
在一些实施方案中，本发明制剂中的抗PD-1/PD-L1抗体的HCVR1与SEQ ID NO：3相比具有不超过10个，优选地不超过5个、4个或3个不同残基，优选地所述不同残基为保守氨基酸替代。在一些实施方案中，本发明制剂中的抗PD-1/PD-L1抗体的LCVR1与SEQ ID NO：4相比具有不超过10个，优选地不超过5个、4个或3个不同残基，优选地所述不同残基为保守氨基酸替代。在一些实施方案中，本发明制剂中的抗PD-1/PD-L1抗体的HCVR2与SEQ ID NO:5(即SEQ ID NO:6或SEQ ID NO:7)相比具有不超过10个，优选地不超过5个、4个或3个不同残基，优选地所述不同残基为保守氨基酸替代。在一些实施方案中，本发明制剂中的抗PD-1/PD-L1抗体的LCVR2与SEQ ID NO：8相比具有不超过10个，优选地不超过5个、4个或3个不同残基，优选地所述不同残基为保守氨基酸替代。“保守性取代”是指导致某个氨基酸置换为化学上相似的氨基酸的氨基酸改变。提供功能上相似氨基酸的保守性 置换表是本领域熟知的。在本发明任一实施方案中，在一个优选的方面，保守取代残基来自以下的保守替代表A，优选地为表A中所示优选置换残基。
表A
原始残基 示例性取代 优选的保守氨基酸取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp；Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；正亮氨酸 Leu
Leu(L) 正亮氨酸；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸 Leu
本发明的抗体是工程化的非天然存在的多肽复合物。本发明的DNA分子是非天然存在的DNA分子，其包含编码具有本发明抗体中一种多肽的氨基酸序列的多肽的多核苷酸序列。
本发明的抗体是IgG型抗体，具有重链和轻链，它们通过链内和链间二硫键交联。每条重链由N-末端HCVR和重链恒定区(“HCCR”)组成。每条轻链由N末端LCVR和轻链恒定区(“LCCR”)组成。轻链各自与重链形成二硫键，并且两条重链在彼此之间形成两个二硫键。
重链的恒定区含有CH1，CH2和CH3结构域。CH1来自HCVR；CH1和HCVR形成Fab的重链部分。CH2位于铰链区之后和CH3之前。CH3位于CH2之后，位于重链的羧基末端。轻链的恒定区含有一个结构域CL。CL来自LCVR；CL和LCVR形成Fab的轻链部分。
在一个优选的实施方案中，本发明抗体制剂中的抗PD-1/PD-L1抗体是PCT申请号PCT/US2018/041205(国际申请日：2018年7月9日)中公开的抗PD-1/PD-L1抗体，通过引用将全部内容并入本文。所述抗PD-1/PD-L1抗体的序列延用该专利申请中的序列号，没有重新进行编号。优选地，所述抗体选自：PD-1/PD-L1双特异性Ab v13844、Ab v3.0或Ab v3.2。更优选地，所述抗体为PD-1/PD-L1双特异性Ab v3.2。在一个实施方案中，该抗PD-1/PD-L1 抗体是由例如293细胞或CHO细胞等重组表达产生并经纯化的抗体，其含有衍生自人IgG1的Fc部分变体。在本发明液体制剂中的所述抗体表现出显著的抗肿瘤活性。例如，在植入NCI-H292肿瘤细胞与PBMC的混合物的来自Jackson Laboratories的NSG小鼠中，施用本发明的抗体例如PD-1/PD-L1双特异性Ab(v13844、v3.0或v3.2)以10mg/kg qw进行治疗，完全缓解(CR)分别为7/8、5/8及5/8，比组合疗法(亲代PD-L1抗体+亲代PD-1抗体)(CR为2/8)更有效。
本发明的抗体是异二聚体，因为抗体的每个臂由于形成抗体的两条不同的重链和两条不同的轻链而表现出与其同源抗原的选择性单价结合。在本发明中，抗体的一个臂结合人PD-L1(SEQ ID NO：1)，另一个臂结合人PD-1(SEQ ID NO：2)。这些多肽链的CH2和/或CH3结构域在序列上不需要相同，并且有利地被修饰以促进两条多肽链之间的复合。例如，可以将氨基酸取代(优选用包含形成“旋钮”的庞大侧基的氨基酸取代，例如色氨酸)引入CH2或CH3结构域，使得空间干扰将阻止与类似突变的相互作用。结构域将强制突变结构域与已经设计了互补或调节突变的结构域配对，即“空穴”(例如，用甘氨酸取代)。可以将这些突变组工程化为包含形成Fc区的CH2-CH3结构域的任何多肽对。有利于异二聚化而不是同型二聚化的蛋白质工程方法是本领域熟知的，特别是关于免疫球蛋白样分子的工程化(参见例如WO96/27011，WO 98/050431，EP 1870459，WO 2007/110205，WO 2007/147901，WO 2009/089004，WO 2010/129304，WO 2011/90754，WO 2011/143545，WO2012/058768，WO2013/157954，WO2013/096291，EP1870459A1，以及Ridgway等。(1996)“'Knobs-Into-Holes'工程抗体CH3重链结构域异二聚化，“蛋白质工程9：617-621，Atwell等人(1997)”使用噬菌体展示文库改造Homodimer的结构域的稳定的异二聚体，“J.Mol.Biol.270：26-35，和Xie等人(2005)“双特异性抗体的新形式：高效异源二聚化，表达和肿瘤细胞裂解”，J.Immunol.Methods 296：95-101)。通常，在本领域已知的方法中，CH3第一重链的结构域和第二重链的CH3结构域均以互补的方式工程化，使得包含一个工程化的CH3结构域的重链不再能够与另一个相同结构的重链同源二聚体化(例如CH3-工程化的第一条重链不能再与另一条CH3工程化的第一条重链同源二聚体化；而CH3-工程化的第二条重链不能再与另一条CH3-工程化的第二条重链同源二聚体化。由此迫使包含一个工程化CH3结构域的重链与包含CH3结构域的另一条重链异二聚化，所述CH3结构域以互补方式工程化。对于本发明的该实施方案，第一重链的CH3结构域和第二重链的CH3结构域通过氨基酸取代以互补方式工程化，使得第一重链和第二重链被迫异二聚化。而第一条重链和第二条重链不能再同源二聚化(例如出于空间原因)。
支持本领域已知的用于支持重链异二聚化的不同方法，作为在根据本发明的双特异性抗体中使用的不同替代方案。在本发明的一些实施方案中，突变掺入CH1和CH3区内的重链序列和轻链恒定区内的轻链序列中。CH1和LC突变有利于必需的轻链和重链对的天然配对并且不利于轻链错配。使CH3突变有利于两个不同重链的异二聚体配对并且不利于同型二聚体的形成。
在一些实施方案中，本发明的抗体含有衍生自人IgG1的Fc部分变体。众所周知，IgG1 与Fc-γ受体家族(FcγR)以及C1q结合。与这些受体的相互作用可以诱导抗体依赖性细胞毒性(ADCC)和补体依赖性细胞毒性(CDC)。因此，对于本发明的抗体，某些氨基酸取代被引入人IgG1Fc区以消除免疫效应子功能。抗体重链的CH2区域中的突变可以包括EU编号中的位置234,235和265，以减少或消除免疫效应子功能。
通过将编码HCVR的DNA可操作地连接到编码重链恒定区或其变体的另一DNA分子，可以将编码HCVR区的分离的DNA转化为全长重链基因。人类以及其他哺乳动物重链恒定区基因的序列是本领域已知的。包含这些区域的DNA片段可以例如通过标准PCR扩增获得。优选地，对于本发明的抗体，重链的重链恒定区是人IgG1的变体。
通过将编码LCVR的DNA可操作地连接到编码轻链恒定区或其变体的另一DNA分子，可以将编码LCVR区的分离的DNA转化为全长轻链基因。人类以及其他哺乳动物轻链恒定区基因的序列是本领域已知的。包含这些区域的DNA片段可通过标准PCR扩增获得。轻链恒定区可以是κ或λ恒定区。优选地，对于本发明的抗体，抗体的抗PD-L1部分的轻链恒定区是λ轻链的变体，并且抗体的抗PD-1部分的轻链恒定区是κ轻链的变体。
在序列与表达控制序列可操作地连接后，本发明的多核苷酸可以在宿主细胞中表达。表达载体通常可以作为附加体或作为附着体在宿主生物中复制宿主染色体DNA的组成部分。通常，表达载体含有选择标记，例如四环素，新霉素，谷氨酰胺合成酶和二氢叶酸还原酶，以允许检测用所需DNA序列转化的那些细胞。
本发明的抗体制剂中所包含的抗体或其抗原结合片段的量可随着制剂的特定目的特性、特定环境、和使用制剂的特定目的而改变。在一些实施方案中，抗体制剂为液体制剂，其可含有约1-200mg/ml，例如约10、15、20、25、30、35、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或200mg/ml，优选地约10-100mg/mL，例如约10、15、20、25、30、35、40、50、60、70、80、90或100mg/ml的抗PD-1/PD-L1抗体。在一些实施方案中，抗体制剂为冻干制剂，所述冻干制剂能够在约1mg/mL至约200mg/mL之间的浓度，例如约10、15、20、25、30、35、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或200mg/ml，优选地约10-100mg/mL，例如约10、15、20、25、30、35、40、50、60、70、80、90或100mg/ml的浓度来重构所述抗体或其抗原结合片段。
(ii)缓冲体系
缓冲体系是可以将溶液的pH维持在可接受范围的体系。在一些实施方案中，用于本发明制剂中的缓冲体系可以将本发明制剂的pH控制在大约5.5-6.5的pH范围，例如约5.5、6.0或6.5的pH。在一些具体的实施方案中，本发明的抗体制剂具有约6的pH。
在一些实施方案中，在本发明所述的冻干制剂中，所述缓冲体系选自组氨酸缓冲体系、组氨酸和盐酸组氨酸缓冲体系、枸橼酸和枸橼酸钠缓冲体系、醋酸醋酸钠缓冲体系、磷酸盐缓冲体系，且以约0.01％(w/v)-1％(w/v)的重量比，例如约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)的重量比存在；优选地，所述缓冲体系以约0.01％(w/v)-0.2％(w/v)的重量比存在,
在一些实施方案中，所述缓冲体系为组氨酸缓冲体系。优选地，所述组氨酸以约0.01-1％(w/v)，例如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)的重量比存在。在一些实施方案中，所述组氨酸以约0.155％(w/v)的重量比存在；
在一些实施方案中，所述缓冲体系为组氨酸和盐酸组氨酸缓冲体系。优选地，所述组氨酸和所述盐酸组氨酸分别以约0.01-1％(w/v)，例如约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)的重量比存在。在一些实施方案中，所述组氨酸和盐酸组氨酸分别以约0.085％(w/v)和0.1％(w/v)的重量比存在。
在一些实施方案中，本发明的液体抗体制剂中所述缓冲体系选自组氨酸缓冲体系、组氨酸和盐酸组氨酸缓冲体系、枸橼酸和枸橼酸钠缓冲体系、醋酸醋酸钠缓冲体系、磷酸盐缓冲体系，且以约1-100mM，例如，1、5、10、15、20、30、40、50、60、70、80、90、100mM的浓度存在。优选地，所述缓冲体系以约1-20mM，例如，1、5、10、15、20mM的浓度存在；更优选地，以约10mM的浓度存在。
在一个实施方案中，本发明的液体抗体制剂中的所述缓冲体系为组氨酸缓冲体系。选地，所述组氨酸以约0.1-10mg/mL，例如约0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的浓度存在。在更优选的实施方案中，所述组氨酸以约1.55mg/mL的浓度存在；
本发明的液体抗体制剂中的所述缓冲体系为组氨酸和盐酸组氨酸缓冲体系。优选地，所述组氨酸和所述盐酸组氨酸分别以约0.1-10mg/mL，例如约0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的浓度存在。在更优选的实施方案中，用于本发明制剂中的缓冲剂是总浓度约10mM的组氨酸和盐酸组氨酸，例如，所述组氨酸和所述盐酸组氨酸分别以约0.85mg/mL和约1.00mg/mL的浓度存在。
(iii)稳定剂
无论是用于冻干制剂或液体制剂，用于本发明的合适的稳定剂可以选自多元醇、氨基酸和它们的任意组合。
作为稳定剂的多元醇可以选自但不限于：甘露醇、山梨醇或其组合。在一个实施方案中，作为稳定剂的多元醇是山梨醇。
作为稳定剂的氨基酸可以选自但不限于：精氨酸、盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸或其组合。在一些实施方案中，作为稳定剂的氨基酸是精氨酸和/或盐酸精氨酸。
在一些实施方案中，在本发明所述的冻干制剂中，所述稳定剂包括山梨醇和精氨酸。优选地，所述山梨醇以约1-10％(w/v)，例如约1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在,所述精氨酸以约1-10％(w/v)，例如约1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在。在一个更优选的实施方案中，所述山梨醇和所述精氨酸分别以约3％(w/v)和约1.742％(w/v)的重量比存在。
在一些实施方案中，在本发明所述的冻干制剂中，所述稳定剂包括山梨醇和盐酸精氨酸。优选地，所述山梨醇以约1-10％(w/v)，例如约1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在,以约1-10％(w/v)，例如约1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在。在一个更优选的实施方案中，所述山梨醇和所述盐酸精氨酸分别以约3％(w/v)和约2.107％ (w/v)的重量比存在。
在一些实施方案中，在本发明所述的冻干制剂中，所述稳定剂还包括甲硫氨酸。在本发明液体制剂中，所述甲硫氨酸可以以约0.1-10％(w/v)的重量比存在,例如以约0.1、0.5、1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在。在一个实施方案中，所述甲硫氨酸以约0.749％(w/v)的重量比存在。
在一些实施方案中，本发明液体制剂包含山梨醇和精氨酸作为稳定剂。在本发明液体制剂中的所述山梨醇和所述精氨酸的浓度分别可以是约10-100mg/mL，例如约10、20、30、40、50、60、70、80、90、100mg/mL。优选地，在本发明液体制剂中的所述山梨醇和所述精氨酸分别可以以约10-50mg/mL，例如约10、20、30、40、50mg/mL的浓度存在。在一个实施方案中，所述山梨醇和所述精氨酸分别以约30mg/mL和约17.42mg/mL的浓度存在；
在一些实施方案中，本发明液体制剂包含山梨醇和盐酸精氨酸作为稳定剂。在本发明液体制剂中的所述山梨醇和所述盐酸精氨酸分别可以以约10-100mg/mL，例如约10、20、30、40、50、60、70、80、90、100mg/mL的浓度存在。优选地，在本发明液体制剂中的所述山梨醇和所述盐酸精氨酸分别可以以约10-50mg/mL，例如约10、20、30、40、50mg/mL的浓度存在。在一个实施方案中，山梨醇和盐酸精氨酸分别以约30mg/mL和约21.07mg/mL的浓度存在。
在一些实施方案中，本发明液体制剂中所述稳定剂还包括甲硫氨酸。在本发明液体制剂中，所述甲硫氨酸可以以约1-100mg/mL，例如约1、5、10、20、30、40、50、60、70、80、90、100mg/mL的浓度存在。更优选地，所述甲硫氨酸以约1-50mg/mL。在一个实施方案中，所述甲硫氨酸以约7.49mg/mL的浓度存在。
(iv)表面活性剂
如本文所使用的，术语“表面活性剂”是指具有两亲结构的有机物质；即，它们由相反的溶解性倾向的基团所组成，通常是油溶性的烃链和水溶性的离子基团。
无论是用于冻干制剂或液体制剂，用于本发明的合适的稳定剂可以是非离子型表面活性剂，例如，烷基聚(环氧乙烯)。可包括在本发明制剂中的特定非离子型表面活性剂包括，例如聚山梨酯80、聚山梨酯20、泊洛沙姆、聚乙二醇聚山梨酯80等。在一个优选实施方案中，本发明的制剂中包含聚山梨酯-80作为表面活性剂。
本发明抗体制剂中所含的表面活性剂的量可随制剂的特定目的特性、特定环境、和使用制剂的特定目的而改变。
在一些实施方案中，在本发明的所述的冻干制剂中，所述表面活性剂以约0.01％(w/v)-1％(w/v)，例如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)的重量比存在。优选地，以约0.01％(w/v)-0.5％(w/v)，例如0.01、0.05、0.1、0.2、0.3、0.4、0.5％(w/v)的重量比存在。
在一些实施方案中，在本发明的所述的冻干制剂中，所述表面活性剂为聚山梨酯80，更优选地，所述聚山梨酯80以约0.02％(w/v)的重量比存在。
在一些实施方案中，在本发明的所述的液体制剂中，所述表面活性剂以约0.1-10mg/ml， 例如约0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的浓度存在。优选地，约0.1-5mg/ml，例如约0.1、0.5、1、2、3、4、5mg/ml的浓度存在。
在优选的一些实施方案中，制剂可含有约0.1-10mg/ml，例如约0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的聚山梨酯类表面活性剂(例如，聚山梨酯-80)。更优选地，所述聚山梨酯80以约0.2mg/ml的浓度存在。
(v)其它赋形剂
在一些实施方案中，本发明的抗体制剂中任选地包含其它赋形剂。所述其它赋形剂包括，例如，抗微生物剂、抗静电剂、抗氧化剂、螯合剂、明胶等等。这些和另外已知的药物赋形剂和/或适用于本发明制剂的添加剂是本领域公知的，例如，列出于“The Handbook of Pharmaceutical Excipients，第4版，Rowe等人编，American Pharmaceuticals Association(2003)；和Remington:the Science and Practice of Pharmacy，第21版，Gennaro编，Lippincott Williams&Wilkins(2005)”。
在一些实施方案中，本发明的抗体冻干制剂包含：约2-17％(w/v)重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约0.294％(w/v)、约50mg/ml山梨醇、约0.20mg/ml聚山梨酯80，所述水溶液具有约5.5的pH。
在一些优选的实施方案中，本发明的抗体冻干制剂包含：约2-17％(w/v)重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约0.155％(w/v)组氨酸、约3％(w/v)山梨醇、约0.749％(w/v)甲硫氨酸、约1.742％(w/v)精氨酸、约0.20mg/ml聚山梨酯80，所述制剂当重构时具有6.0的pH。
在一些优选的实施方案中，本发明的抗体冻干制剂包含：约2-17％(w/v)重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约0.085％(w/v)组氨酸、约0.1％(w/v)盐酸组氨酸、约3.00％(w/v)山梨醇、约0.749％(w/v)甲硫氨酸、约2.107％(w/v)盐酸精氨酸、约0.02％(w/v)聚山梨酯80；所述制剂当重构时具有6.0的pH。
在一些实施方案中，本发明的抗体液体制剂中所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约2.94mg/ml枸橼酸钠、约50mg/ml山梨醇、约0.20mg/ml聚山梨酯80，所述水溶液具有约5.5的pH。
在一些实施方案中，本发明的抗体液体制剂中所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约2.94mg/ml枸橼酸钠、约50mg/ml山梨醇、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH。
在一些实施方案中，本发明的抗体液体制剂中所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约2.94mg/ml枸橼酸钠、约50mg/ml山梨醇、约2.94mg/ml甲硫氨酸、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH。
在一些实施方案中，本发明的抗体液体制剂中所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约2.94mg/ml枸橼酸钠、约50mg/ml山梨醇、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.5的pH。
在一些实施方案中，本发明的抗体液体制剂中所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约2.94mg/ml枸橼酸钠、约50mg/ml山梨醇、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH。
在一些实施方案中，本发明的抗体液体制剂中所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约1.55mg/ml组氨酸、约50mg/ml山梨醇、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH。
在一些实施方案中，本发明的抗体液体制剂中所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约1.55mg/ml组氨酸、约50mg/ml山梨醇、约7.49mg/ml甲硫氨酸、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH。
在一些优选的实施方案中，本发明的抗体液体制剂中所述水溶液包含：20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约1.55mg/ml组氨酸、约30mg/ml山梨醇、约7.49mg/ml甲硫氨酸、约17.42mg/ml精氨酸、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH。
在一些优选的实施方案中，本发明的抗体液体制剂中，所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约0.85mg/ml组氨酸、约1.00mg/ml盐酸组氨酸、约30.00mg/ml山梨醇、约7.49mg/ml甲硫氨酸、约21.07mg/ml盐酸精氨酸、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH。
II.制剂的制备
本发明提供了包含抗PD-1/PD-L1抗体蛋白的稳定制剂。在本发明制剂中使用的抗PD-1/PD-L1抗体蛋白可以使用本领域已知的用于生产抗体的技术进行制备。例如，可以重组制备抗体。在一些实施方案中，本发明的抗体可以在CHO,NSO,HEK293或COS细胞中制备。优选地，本发明的抗体可以在CHO或HEK293细胞中制备。可借由熟知方法将含有所关注多核苷酸序列(例如编码抗体的多肽的多核苷酸及表达控制序列)的载体转移至宿主细胞中，该方法根据宿主细胞类型而有所变化。
抗体作为药物的活性成分的应用现在已经很广泛。用于将治疗性抗体纯化至药用级的技术是本领域公知的。例如，Tugcu等(Maximizing productivity of chromatography steps for purification of monoclonal antibodies,Biotechnology and Bioengineering 99(2008)599–613.)描述在蛋白A捕获步骤后使用离子交换色谱(阴离子IEX和/或阳离子CEX色谱)的单克隆抗体三柱纯化方法。Kelley等(Weak partitioning chromatography for anion exchange purification of monoclonal antibodies,Biotechnology and Bioengineering 101(2008)553–566)描述了两柱纯化法，其中在蛋白A亲和色谱后使用弱分配阴离子交换树脂。
一般，重组产生的单克隆抗体可以利用常规的纯化方法纯化，以提供具有足够的可重复性和适度纯度的药物物质用于抗体制剂的配制。例如，在抗体从重组表达细胞分泌至培养基中后，可以使用商业可得的蛋白浓缩过滤器例如Amicon的超滤装置，浓缩来自该表达系统 的上清液。之后，可以使用例如色谱、透析和亲和纯化等方式进行抗体的纯化。蛋白A适应于作为亲和配体用于纯化IgG1、IgG2和IgG4型抗体。也可以使用其它抗体纯化方法，例如离子交换色谱。在获得足够纯度的抗体后，可以按照本领域已知的方法，制备包含抗体的制剂。
例如，可以采用如下步骤进行制备：(1)在发酵结束后将发酵液离心澄清去除细胞等杂质以获得上清；(2)使用亲和层析(例如对IgG1、IgG2和IgG4型抗体具有特异亲和力的蛋白A柱)捕获抗体；(3)进行病毒灭活；(4)精制纯化(一般可以采用CEX阳离子交换层析)，以去除蛋白中的杂质；(4)病毒过滤(使病毒滴度降低例如4log10以上)；(5)超滤/渗滤(可以用于将蛋白置换于利于其稳定的制剂缓冲液中并浓缩至合适的浓度供注射用)。参见例如，B.Minow,P.Rogge,K.Thompson,BioProcess International,Vol.10,No.6,2012,pp.48–57。
III.制剂的分析方法
在抗体制剂的储存过程中，抗体可能会发生聚集、降解或化学修饰，导致抗体异质性(包括大小异质性和电荷异质性)以及聚集物和片段等，从而影响抗体制剂的质量。因此，有必要进行抗体制剂稳定性的监测。
在本领域中已知多种方法可以用于检测抗体制剂的稳定性。例如，可以通过还原型CE-SDS、非还原型CE-SDS和SEC-HPLC等方法，分析抗体制剂的纯度和评估抗体的聚集水平；可以通过毛细管等电聚焦电泳(cIEF)、成像毛细管等电聚焦电泳(iCIEF)和离子交换色谱(IEX)等，分析抗体制剂中的电荷变异体。此外，可以通过目视检测制剂外观，快速地判断制剂的稳定性。也可以使用OD 350nm法检测制剂的浊度改变，该方法可以给出有关可溶性和不溶性聚集物量的信息。此外，可以使用紫外分光光度法(UV法)检测制剂中的蛋白质含量变化。
非还原型CE-SDS法是一种以毛细管为分离通道进行的抗体纯度测定方法。在CE-SDS中，蛋白迁移由SDS结合引起的表面电荷来驱动，而该表面电荷与蛋白质的分子量成正比。由于所有的SDS-蛋白质复合物都具有相似的质量-电荷比，故可以在毛细管的分子筛凝胶基质中，实现基于分子的大小或流体动力学半径的电泳分离。该方法已经被广泛地用于监测变性的完整抗体的纯度。一般，在非还原型CE-SDS法中，供试样品与SDS样品缓冲液和碘乙酰胺混合。之后，混合物可以于68-72℃孵育约10-15分钟，冷却至室温后离心的上清液用于分析。采用紫外检测器检测蛋白的迁移，获得电泳谱图。抗体制剂纯度可以计算为IgG主峰的峰面积占所有峰面积之和的百分比。关于CE-SDS法的进一步描述，可以参见例如Richard R.等,Application of CE SDS gel in development of biopharmaceutical antibody-based products,Electrophoresis,2008,29,3612-3620。
尺寸排阻高效液相色谱法，即SEC-HPLC法，是用于抗体标准和质控的另一重要方法。该方法主要依据分子的尺寸大小或流体动力学半径差异来进行分子的分离。通过SEC-HPLC，抗体可以分离出三种主要形式：高分子量形式(HMMS)、主峰(主要是抗体单体)、和低分子量形式(LMMS)。抗体纯度可以计算为色谱图上主峰面积占所有峰面积之和的百分比。通过SEC-HPLC法，可以测量制剂产品中抗体单体的百分数，给出可溶性聚集物和剪切物的含量 信息。关于SEC-HPLC法的进一步描述，可以参见例如，J.Pharm.Scien.,83:1645-1650,(1994)；Pharm.Res.,11:485(1994)；J.Pharm.Bio.Anal.,15:1928(1997)；J.Pharm.Bio.Anal.,14:1133-1140(1986)。此外，也可以参见例如，R.Yang等，High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography(SE-UHPLC),Journal of Pharmaceutical and Biomedical Analysis(2015),http://dx.doi.org/10.1016/j.jpba.2015.02.032；和Alexandre Goyon等,Protocols for the analytical characterization of therapeutic monoclonal antibodies.I–Non-denaturing chromatographic techniques,Journal of Chromatography，http://dx.doi.org/10.1016/j.jchromb.2017.05.010。
成像毛细管等电聚焦电泳(iCIEF)可以用于分析抗体的电荷异质性。该方法可以提供电荷变异体的定量分布情况。iCIEF基于分子在pH梯度中的电荷差异(表观pI值)来实现分子分离的目的。在iCIEF中，分离柱通常是短毛细管(例如,5cm长,100μm内径的二氧化硅毛细管)，蛋白质在高电压下在毛细管柱中聚焦，并通过在280nM操作的全柱成像检测系统对聚焦进行实时在线监测。该技术的一个优点是，可以通过该全柱检测系统同时记录抗体样品的各种电荷变异体。一般而言，在icIEF中，将样品与尿素和icIEF缓冲液混合，其中所述缓冲液含有甲基纤维素、pI分子量标准和两性电解质。然后，可以在iCIEF分析仪例如iCE280分析仪(Protein Simple,Santa Clara,CA)上，使用iCIEF柱例如ProtionSimple组装的iCIEF柱，在样品聚焦一定时间后，测定280nm的吸光度，获得聚焦抗体电荷变异体的谱图。在iCEIF谱图中，在主峰(即主成分)之前洗脱的蛋白相关峰被分类为酸性组分；相对地，在主峰之后洗脱的蛋白相关峰被分类为碱性组分。主成分、酸性组分和碱性组分的相对量可以表示为占总峰面积的百分数。关于iCIEF的进一步描述，可以参见例如，Salas-Solano O等,Robustness of iCIEF methodology for the analysis of monoclonal antibodies:an interlaboratory study,J Sep Sci.2012 Nov；35(22):3124-9.doi:10.1002/jssc.201200633.Epub 2012 Oct 15；和Dada OO等,Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation,Electrophoresis.2015 Nov；36(21-22):2695-2702.doi:10.1002/elps.201500219.Epub 2015 Sep 18.
也可以通过阳离子交换高效液相色谱法(CEX-HPLC)测定抗体制剂中抗体的电荷变异体。在该测定法中，以比主峰的保留时间更早从CEX-HPLC柱洗脱出的峰被标记为“酸性峰”，而那些以比主峰的保留时间更晚从CEX-HPLC柱洗脱出的峰被标记为“碱性峰”。
加速稳定性研究可以用于检查产品的稳定性性质，有利于筛选稳定药物制剂形式。例如，可以将制剂样品放置于升高的温度，例如约40℃±2℃、25℃±2℃条件下进行加速稳定性研究。检测指标可以包括外观、可见异物、蛋白含量、浊度、纯度(SEC-HPLC法、非还原型CE-SDS法)和电荷变异体(iCIEF法、CEX-HPLC法)。
本发明的抗PD-1/PD-L1抗体蛋白制剂是稳定的。在一些实施方案中，本发明的抗PD-1/PD-L1抗体蛋白制剂在储存后，例如在2-8℃储存至少24个月后，或在室温储存至少6个月后，或在40℃±2℃储存1个月后，是稳定的。在一些实施方案中，于约5℃、25℃、37℃、40℃、或45℃储存至少1个月、2个月或3个月后，例如，在5℃±3℃储存3个月后， 本发明的抗体制剂中的抗PD-1/PD-L1抗体蛋白纯度是至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％以上，如通过尺寸排阻色谱法或通过非还原型CE-SDS所测定。在一个实施方案中，于约5℃、25℃、37℃、40℃、或45℃储存至少1个月、2个月或3个月后，例如，在5℃±3℃储存3个月后，本发明的抗体制剂中抗PD-1/PD-L1抗体蛋白的至少55％，优选至少60％是非碱性及非酸性形式(亦即，主峰或主要电荷形式)，如通过CEX-HPLC法所测定。
IV.制剂的用途
本发明的包含抗PD-1/PD-L1抗体蛋白的本发明抗体制剂能够用于治疗或预防肿瘤等。
本发明进一步提供本发明抗体制剂的用途，其用于制造用于治疗或预防癌症的药物，其中该药物可与一或多种选自由以下组成的群组的抗肿瘤剂同时、分开或依序给药：顺铂(cisplatin)、卡铂(carboplatin)、达卡巴嗪(dacarbazine)、脂质体多柔比星(liposomal doxorubicin)、多西他赛(docetaxel)、环磷酰胺(cyclophosphamide)及多柔比星、诺维本(navelbine)、艾瑞布林(eribulin)、太平洋紫杉醇(paclitaxel)、用于可注射悬浮液的太平洋紫杉醇蛋白质结合颗粒、伊沙匹隆(ixabepilone)、卡培他滨(capecitabine)、FOLFOX(亚叶酸钙(leucovorin)、氟尿嘧啶(fluorouracil)及奥沙利铂(oxaliplatin))、FOLFIRI(亚叶酸钙、氟尿嘧啶及伊立替康(irinotecan))、吉西他滨(gemcitabine)、托泊替康(topotecan)、脂质体伊立替康、培美曲塞(pemetrexed)、西妥昔单抗(cetuximab)、尼沃鲁单抗(nivolumab)、伊匹单抗(ipilimumab)、匹利珠单抗(pidilizumab)、派姆单抗(pembrolizumab)、曲美木单抗(tremelimumab)、乌瑞鲁单抗(urelumab)、利丽单抗(lirilumab)、阿替珠单抗(atezolizumab)、爱帕司他(epacadostat)及德瓦鲁单抗(durvalumab)。
本发明提供本发明抗体制剂的用途，其用于制造用于治疗癌症的药物，其中该药物可与电离辐射同时、分开或依次施用。
在一些实施方案中，预防或治疗的癌症包括但不限于霍奇金氏(Hodgkin’s)或非霍奇金氏淋巴瘤、黑色素瘤、肾细胞癌、肾癌、肺癌、膀胱癌、胃及食道癌、结肠直肠癌、肝癌、肝细胞癌、胆管癌、胰脏癌、乳癌、三阴性乳癌、卵巢癌、子宫内膜癌、前列腺癌、小细胞肺癌(SCLC)、非小细胞肺癌(NSCLC)、间皮瘤、头颈鳞状细胞癌(SCCHN)、软组织肉瘤或多形性神经胶母细胞瘤。在一些实施方案中，癌症为胃肠道癌症，例如胃癌、直肠癌、结肠癌、结肠直肠癌等。
本发明也提供了本发明的制剂在制备药物中的用途，其中所述药物用于向哺乳动物递送抗PD-1/PD-L1抗体蛋白。本发明还提供了本发明的制剂用于治疗或预防一种或多种上述疾病和病症的方法。优选地，哺乳动物是人。
可以以多种途径将本发明的抗体制剂施用于受试者或患者。例如，施用可以通过输注或通过注射器进行。因此，在一个方面，本发明提供了一种递送装置(例如注射器)，其包含本发明的抗体制剂(例如，预填装注射器)。患者将接受有效量的抗PD-1/PD-L1抗体蛋白作为主要活性成分，即足以治疗、改善或预防目的疾病或病症的量。
本发明进一步提供一种预防和/或治疗肿瘤的方法，包括向受试者施用有效量的本发明所述的制剂。例如，所述肿瘤是癌症，优选地，所述癌症为霍奇金或非霍奇金淋巴瘤、黑色素 瘤、肾细胞癌、肾癌、肺癌、膀胱癌、胃及食道癌、结肠直肠癌、肝癌、肝细胞癌、胆管癌、胰脏癌、乳癌、三阴性乳癌、卵巢癌、子宫内膜癌、前列腺癌、小细胞肺癌(SCLC)、非小细胞肺癌(NSCLC)、间皮瘤、头颈的鳞状细胞癌(SCCHN)、软组织肉瘤或多形性神经胶母细胞瘤；更优选地，该肺癌是NSCLC、小细胞肺癌或间皮瘤。
在一些实施方案中，本发明所述的方法，还包括同时，分开或依次给予一种或多种抗肿瘤剂，所述抗肿瘤剂选自顺铂(cisplatin)、卡铂(carboplatin)、达卡巴嗪(dacarbazine)、脂质体多柔比星(liposomal doxorubicin)、多西他赛(docetaxel)、环磷酰胺(cyclophosphamide)及多柔比星、诺维本(navelbine)、艾瑞布林(eribulin)、太平洋紫杉醇(paclitaxel)、用于可注射悬浮液的太平洋紫杉醇蛋白质结合颗粒、伊沙匹隆(ixabepilone)、卡培他滨(capecitabine)、FOLFOX(亚叶酸钙(leucovorin)、氟尿嘧啶(fluorouracil)及奥沙利铂(oxaliplatin))、FOLFIRI(亚叶酸钙、氟尿嘧啶及伊立替康(irinotecan))、吉西他滨(gemcitabine)、托泊替康(topotecan)、脂质体伊立替康、培美曲塞(pemetrexed)、西妥昔单抗(cetuximab)、尼沃鲁单抗(nivolumab)、伊匹单抗(ipilimumab)、匹利珠单抗(pidilizumab)、派姆单抗(pembrolizumab)、曲美木单抗(tremelimumab)、乌瑞鲁单抗(urelumab)、利丽单抗(lirilumab)、阿替珠单抗(atezolizumab)、爱帕司他(epacadostat)及德瓦鲁单抗(durvalumab)。
治疗效果可包括减少生理症状。用于任何特定受试者的抗体的最佳有效量和浓度将取决于多种因素，包括患者的年龄、体重、健康状况和/或性别、疾病的性质和程度、特定抗体的活性，身体对其清除率，并且也包括与所述抗体制剂组合施用的任何可能的其它治疗。对于具体的情况，所递送的有效量可以在临床医师的判断范围内来确定。在这方面，已知的基于抗体的药物的应用可以提供一定的指导。剂量可以是单剂量方案或多剂量方案。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，本发明的抗体分子用来延缓疾病发展或用来减慢疾病的进展。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。抗体或抗体片段或其缀合物或组合物的治疗有效量可以根据多种因素如疾病状态、个体的年龄、性别和重量和抗体或抗体部分在个体中激发所需反应的能力而变动。治疗有效量也是这样的一个量，其中抗体或抗体片段或其缀合物或组合物的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率)至少约20％、更优选地至少约40％、甚至更优选地至少约50％、60％或70％和仍更优选地至少约80％。可以在预示人肿瘤中的功效的动物模型系统中评价化合物抑制可度量参数(例如，癌症)的能力。 可选地，可以通过检验化合物抑制的能力评价组合物的这种特性，所述抑制在体外通过熟练技术人员已知的测定法。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。
V.本发明的示例性双特异性抗体
表1a抗体v3.2、v3.0及v13844的轻链、重链、轻链可变区及重链可变区的氨基酸序列编号
  抗体v3.2 抗体v3.0 抗体v13844
第一重链可变区HCVR1-抗PD-L1 3 3 3
第二重链可变区HCVR2-抗PD-1 6 7 7
第一轻链可变区LCVR1-抗PD-L1 4 4 4
第二轻链可变区LCVR2-抗PD-1 8 8 8
第一重链HC1-抗PD-L1 49 49 29
第二重链HC2-抗PD-1 31 32 33
第一轻链LC1-抗PD-L1 30 30 30
第二轻链L2-抗PD-1 34 34 34
表1b抗体v3.2、v3.0及v13844的PD-L1结合可变区的CDR的氨基酸序列编号(根据North规则确定)
Figure PCTCN2021071757-appb-000002
表1c抗体v3.2、v3.0及v13844的PD-1结合可变区的CDR的氨基酸序列编号(根据North规则确定)
Figure PCTCN2021071757-appb-000003
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
缩略词描述
CEX-HPLC：阳离子交换高效液相色谱
CE-SDS：十二烷基硫酸钠毛细管凝胶电泳
iCIEF：成像毛细管等电聚焦电泳
SEC-HPLC：尺寸排阻高效液相色谱法
实施例
为了开发出抗PD-1/PD-L1抗体注射液以及冻干制剂长期稳定储存的制剂处方，确保产品在有效期内(至少24个月)的质量可控，设计了处方筛选试验，考察了不同辅料对于抗PD-1/PD-L1抗体制剂稳定性的影响。试验所用材料和方法如下：
材料和方法
1.1.本发明的制剂研究中使用的材料
Figure PCTCN2021071757-appb-000004
1.2.本发明的制剂研究中使用的仪器设备
Figure PCTCN2021071757-appb-000005
1.3.制剂稳定性的检测项目和检测方法
对抗体制剂检测了以下项目：(1)检测外观以及是否存在可见异物；(2)通过紫外法(UV法)测定制剂中的蛋白质含量；(3)通过检测在350nm处的吸光度，测定浊度；(4)通过尺 寸排阻色谱法，例如，尺寸排阻高效液相色谱法(size-exclusion chromatography-HPLC；SEC-HPLC)测定抗体制剂的纯度，表示为单体的面积占所有峰面积之和的百分数；(5)通过还原型十二烷基硫酸钠毛细管电泳(还原型CE-SDS)和/或非还原型十二烷基硫酸钠毛细管电泳(非还原型CE-SDS)测定抗体制剂的纯度，表示为单体的面积占所有峰面积之和的百分数；(6)通过成像毛细管等电聚焦电泳法(iCIEF法)和阳离子交换高效液相色谱法(CEX-HPLC)测定抗体制剂中电荷变异体，表示为主成分、酸性组分和碱性组分的百分数。
可见异物检测
按照国家药典委员会，中华人民共和国药典(2015年版，四部通则0904“可见异物检查法”，北京：中国医药科技出版社.2015)中所记载的方法，采用澄明度检测仪(天津天大天发生产，型号YB-2)，检查样品中的可见异物。
蛋白含量测定
使用紫外分光光度计(日本岛津生产，型号UV-1800)测定样品中的蛋白质含量。
浊度测定
使用紫外分光光度计(日本岛津生产，型号UV-1800)，测定样品在350nm的吸光度，确定样品浊度。
纯度(SEC-HPLC法)
使用体积排阻色谱柱分离，流动相为磷酸盐缓冲液(称取3.12g二水合磷酸二氢钠，8.77g氯化钠和34.84g精氨酸，超纯水溶解后用盐酸调节pH至6.8并定容至1000ml)，色谱柱保护液为0.05％(w/v)NaN 3，进样量50μl，流速0.5ml/分钟，采集时间30分钟，柱温25℃，检测波长280nm。取待测样品用超纯水稀释至2mg/ml，作为供试品溶液。取制剂缓冲液用上述相同处理方式稀释后做为空白溶液。取空白溶液、供试品溶液各50μl注入液相色谱仪，开始检测。
纯度(还原型CE-SDS法)
采用毛细管凝胶电泳法检测。毛细管为无涂层毛细管，内径50μm，总长30.2cm，有效长度20.2cm。电泳前分别使用0.1mol/L氢氧化钠、0.1mol/L盐酸、超纯水、电泳胶70psi冲洗毛细管柱。将待测样品用适量超纯水稀释至2.0mg/ml，取以上稀释后的样品50μl于1.5ml离心管中，分别向其中加入45μl pH 6.5的样品缓冲液(称取一水柠檬酸0.32g，十二水合磷酸氢二钠2.45g，溶于45ml超纯水中，定容至50ml，制得柠檬酸-磷酸盐缓冲液，精密量取该缓冲液200μl，加10％(w/v)十二烷基硫酸钠溶液80μl，加水至1ml，混匀，即得)、1μl内标(10kDa蛋白质，5mg/mL)(Beckman Coulter，货号：390953)和5μlβ-巯基乙醇，充分混匀后70±2℃加热10±2分钟，冷却至室温后转移至样品瓶作为供试品溶液。取与供试品相同体积的制剂缓冲液，按上述方法同样操作，制得空白溶液。样品进样条件：-5kV 20秒；分离电压：-15kV 35分钟。毛细管柱温控制在25℃，检测波长为220nm。
纯度(非还原型CE-SDS法)
采用毛细管凝胶电泳法检测。毛细管为无涂层毛细管，内径50μm，总长30.2cm，有效长度20.2cm。电泳前分别使用0.1mol/L氢氧化钠、0.1mol/L盐酸、超纯水、电泳胶70psi 冲洗毛细管柱。将待测样品用适量超纯水稀释至2.0mg/ml，取以上稀释后的样品50μl于1.5ml离心管中，分别向其中加入45μl pH 6.5的样品缓冲液(称取一水柠檬酸0.32g，十二水合磷酸氢二钠2.45g，溶于45ml超纯水中，定容至50ml，制得柠檬酸-磷酸盐缓冲液，精密量取该缓冲液200μl，加10％(w/v)十二烷基硫酸钠溶液80μl，加水至1ml，混匀，即得)、1μl内标(10kDa蛋白质，5mg/mL)(Beckman Coulter，货号：390953)和5μl 250mmol/L NEM溶液(称取N-乙基顺丁稀二酰亚胺62mg，溶于2ml超纯水中)，充分混匀后70±2℃加热10±2分钟，冷却至室温后转移至样品瓶作为供试品溶液。取与供试品相同体积的制剂缓冲液，按上述方法同样操作，制得空白溶液。样品进样条件：-5kV 20秒；分离电压：-15kV 35分钟。毛细管柱温控制在25℃，检测波长为220nm。
电荷变异体(iCIEF)
采用成像毛细管等电聚焦电泳(iCIEF法)检测。毛细管内径100μm，总长5cm。样品电泳前需分别使用0.5％甲基纤维素溶液(下文中也缩写为MC溶液)、超纯水冲洗毛细管柱。采用真空进样方式，预聚焦电压及时间为1.5kV 1分钟，聚焦电压及时间为3kV 8分钟，进样时间55秒，样品盘温度为10℃，检测波长为280nm。阴极稳定剂(Cathodic Stabilizer)为500mmol/L精氨酸溶液，0.5％MC溶液降低蛋白与毛细管之间的粘附。将供试品用水稀释至1.0mg/ml，取稀释后的供试品溶液20μl，向其中加入78μl预混液(预混液配比如下：70μl pI 0.5％MC溶液，4μl两性电解质(pH 3-10)，2μl阴极稳定剂，1μl pI 5.85标志物，1μl pI 9.99标志物)，充分混匀制得待测样品溶液。进样分析，根据面积归一化法，计算主成分、酸性组分及碱性组分含量。
电荷变异体(CEX-HPLC法)
采用离子交换色谱法(CEX-HPLC法)检测。使用离子交换色谱柱分离，流动相为10mmol/L磷酸盐缓冲液(称取1.15g二水合磷酸二氢钠，0.95g十二水合磷酸氢二钠，用800ml超纯水溶解后定容至1000ml)及10mmol/L磷酸盐+200mmol/L氯化钠缓冲液(称取1.15g二水合磷酸二氢钠，0.95g十二水合磷酸氢二钠，11.69g氯化钠用800ml超纯水溶解后定容至1000ml)，流速0.5ml/分钟，检测波长280nm，柱温25℃。用流动相将样品稀释至2mg/ml，作为供试品溶液；将工作参比品用流动相稀释至2mg/ml，作为系统适用性溶液。取制剂缓冲液用流动相稀释相同倍数做为空白溶液。取空白溶液、系统适用性溶液、供试品溶液各50μl注入液相色谱仪，流动相流速1.0ml/min，采集时间35分钟，柱温35℃，检测波长280nm。
1.4判断标准
根据对产品的认识以及仪器和方法的精密度，设定了样品检测指标数值与初始值相比质量未发生变化的判断标准，用以判断样品是否发生了变化，具体见下表。
质量未发生变化的判断标准
检测项目 未发生变化的判定标准
外观(液体制剂及冻干制剂复溶后) 澄明至微乳光，无色至微黄色液体，无异物
外观(冻干制剂) 白色至淡黄色块状疏松体
可见异物(液体制剂及冻干制剂复溶 符合《中华人民共和国药典》(2015年版，四部)
检测项目 未发生变化的判定标准
后) 通则0904
蛋白含量(UV法) 变化率≤10％
纯度(SEC-HPLC法) 主峰纯度≥95％
纯度(还原型CE-SDS法) 主峰纯度≥90％
纯度(非还原型CE-SDS法) 主峰纯度≥90％
电荷变异体(iCIEF法/CEX-HPLC法) 主成分≥35％
实施例1.制备和纯化抗PD-1/PD-L1抗体
根据PCT申请号PCT/US2018/041205所述，获得特异性结合PD-1和PD-L1的新型抗体抗体v3.2、v3.0及v13844,抗体v3.2、v3.0及v13844的轻链、重链、轻链可变区及重链可变区的氨基酸序列的SEQ ID NO示于上表1(a)中。此外，抗体v3.2、v3.0及v13844的PD-L1及PD-1结合可变区的CDR的氨基酸序列的SEQ ID NO分别示于表1(b)及表1(c)中。
本发明抗体包括但不限于抗体v3.2、v3.0及v13844，且基本上如下制备及纯化。可使用四倍载体(即，编码两条轻链及两条重链的表达的单一载体)、双重载体(即，一起编码两条不同轻链及两条不同重链的两个载体)或四个单一载体(其中的两个编码不同轻链且其中的两个编码不同重链)用分泌抗体的表达系统瞬时或稳定转染适当宿主细胞(例如CHO)。可使用许多常用技术中的任一纯化抗体分泌于其中的培养基。举例而言，可将培养基施加至MabSelect柱(GE Healthcare)或KappaSelect柱(GE Healthcare)用于Fab片段，该柱已经用相容缓冲液(例如磷酸盐缓冲盐水(pH 7.4))平衡。可洗涤柱以移除非特异性结合组分。可例如借由pH梯度(例如20mM Tris缓冲液(pH 7)至10mM柠檬酸缓冲液(pH 3.0)，或生理盐水(pH 7.4)至100mM甘胺酸缓冲液(pH 3.0))洗脱结合的抗体。可例如借由SDS-PAGE检测抗体抗体片段，且随后可汇集。可溶性聚集体及多聚体可借由常见技术有效移除，该技术包括尺寸排除色谱、疏水相互作用色谱、离子交换色谱、多峰(multimodal)色谱或羟磷灰石色谱等。可使用常见技术浓缩及/或无菌过滤抗体。可将产物立即于-70℃下冷冻或可经冻干。
以下描述了v3.2的一种示例性制备方法。
具体而言，合成编码人源化抗体PD-1的重链的核苷酸序列和轻链核苷酸序列；合成编码人源化抗体PD-L1的重链的核苷酸序列和轻链核苷酸序列。PD-1的重链、PD-L1的重链分别连接入真核质粒表达载体PEE6.4，PD-1的轻链、PD-L1的轻链分别连接入真核质粒表达载体PEE12.4。含有PD-1重链的中间表达载体和含有PD-1轻链的中间表达载体合并，得到含有PD-1重链和轻链的核苷酸序列的重组表达载体。含有PD-L1重链的中间表达载体和含有PD-L1轻链的中间表达载体合并，得到含有PD-L1重链和轻链的核苷酸序列的重组表达载体。培养，最后得到两个重组表达载体用于细胞转染至269M细胞(Lonza Biologics公司)，筛选压力为无谷氨酰胺的LM7300培养基(礼来自主培养基)。采用生物反应器培养，接种密度不小于1.0×10 6个/ml；培养至第12～16天或细胞活率＜60％时，进行收获，细胞收获液用于下 游纯化。
细胞收获液用吸附深层过滤膜包过滤，过滤收获液。过滤过程中，保持生物反应器的通气及搅拌参数设置不变，再用亲和平衡液冲洗膜包，将冲洗液与滤过液合并，命名为澄清收集液。填装Mabselect Sure LX层析柱，用亲和平衡液平衡，平衡后将澄清收集液加入层析柱，再用平衡液冲洗，随后再用亲和冲洗液冲洗，再亲和平衡液冲洗。然后开始洗脱，收集洗脱液，命名为亲和收集液。亲和收集液调节pH至约6.0，命名为病毒灭活收集液。病毒灭活收集液使用深层过滤膜包对病毒灭活收集液进行吸附深层过滤，收集滤出液及顶洗液，命名为吸附深层过滤收集液。填装Poros XQ层析柱，层析柱用阴离子平衡液平衡，然后开始上样，收集流穿液，命名为阴离子收集液。填装Poros HS柱，用阳离子平衡液平衡，上样，完成上样后用平衡液冲洗，用洗脱液洗脱，收集洗脱液，命名为阳离子收集液。样品具有约5.0－25.0mg/ml的抗PD-1/PD-L1抗体v3.2蛋白含量。
以下实施例以抗体v3.2为例，其产品代号为IBI318。
实施例2.pH对制剂的稳定性影响试验
本实施例考察了包含抗PD-1/PD-L1抗体的制剂在pH 5.5至6.5的稳定性。共设计了3个pH值，分别为5.5、6.0和6.5。
2.1 实验步骤
按照表5配制各个处方的缓冲液，用稀盐酸将pH分别调节为5.5、6.0和6.5，将实施例1的经纯化的抗PD-1/PD-L1抗体超滤置换到所述不同pH值的溶液中。置换完成后，调节样品中的抗PD-1/PD-L1抗体蛋白含量至约20mg/ml；然后加入聚山梨酯80，使聚山梨酯80的终浓度为0.20mg/ml；过滤分装至西林瓶中，加塞压盖后根据实验方案见表6进行研究考察。检测指标为外观、蛋白含量、纯度(SEC-HPLC法、非还原型CE-SDS法)和电荷变异体(iCIEF法)。
表5.处方前试验处方表
Figure PCTCN2021071757-appb-000006
表6.处方前试验方案
Figure PCTCN2021071757-appb-000007
2.2 实验结果
(1)外观
在40℃±2℃的条件下放置2周，各组样品外观均合格。
(2)蛋白含量
在40℃±2℃条件下，各组样品的蛋白含量均未发生变化(见表7)。
表7.处方前试验蛋白含量结果(UV法，mg/ml)
Figure PCTCN2021071757-appb-000008
(3)纯度
纯度(SEC-HPLC法)：40℃±2℃条件下加速2周，各组样品的纯度均发生明显变化，随着pH升高，样品纯度下降速率越快，表现为聚体增多；加入甲硫氨酸能有减缓纯度下降的速率。(见表8、图1)。
纯度(非还原型CE-SDS法)：40℃±2℃条件下加速2周，各pH条件下样品的纯度均未发生明显变化。(见表9)。
表8.处方前试验纯度结果(SEC-HPLC法，％)
Figure PCTCN2021071757-appb-000009
表9.处方前试验纯度结果(非还原型CE-SDS法，％)
Figure PCTCN2021071757-appb-000010
(4)电荷变异体
40℃±2℃条件下，各处方电荷变异体-酸性组分和主成分均发生明显变化，各处方间趋势 一致，无明显差异。(见表10和图2)
表10.处方前试验电荷变异体结果(iCIEF法)
Figure PCTCN2021071757-appb-000011
处方前试验结果表明蛋白在pH5.5-6.5间稳定，尤其是pH 6.0时较稳定。综合以上结果，选定pH为6.0，进行下一轮处方筛选试验。
实施例3.处方确定实验(一)
3.1 实验步骤
本实施例考察了不同缓冲体系和稳定剂对包含抗PD-1/PD-L1抗体制剂稳定性的影响。
共设计了4个处方，详细处方信息见表11。
按照表11配制各个处方的缓冲液，将IBI318蛋白超滤置换到各自的处方溶液中。置换完成后，将各处方蛋白浓度稀释至约20mg/ml，并加入聚山梨酯80，使终浓度为0.2mg/ml。过滤分装至西林瓶，加塞轧盖后在40℃±2℃、25℃±2℃条件下进行加速稳定性考察，并考察其振荡和冻融稳定性。检测指标为外观、可见异物、蛋白含量、纯度(SEC-HPLC法、非还原型CE-SDS法)和电荷变异体(CEX-HPLC法)。
表11.处方筛选试验备选处方信息表
Figure PCTCN2021071757-appb-000012
注：处方1用枸橼酸调节pH，处方2～4用盐酸调节pH。
详细试验条件及取样计划见表12。
表12.试验条件及取样表
Figure PCTCN2021071757-appb-000013
3.2 试验结果
(1)外观、可见异物
在40℃±2℃的条件下观察至1个月，25℃±2℃条件下观察至3个月，振荡5天，冻融6次，四组处方外观、可见异物均合格。
(2)蛋白含量
在40℃±2℃和25℃±2℃以及振荡和冻融条件下，4组处方的蛋白含量均未发生变化(见表13)。
表13.处方筛选试验蛋白含量结果(UV法，mg/ml)
Figure PCTCN2021071757-appb-000014
(3)纯度
纯度(SEC-HPL C法)：40℃±2℃条件下1个月、25℃±2℃条件下3个月，处方1(枸橼酸缓冲体系)纯度发生下降，主要表现为聚体增多，其余处方纯度均未发生明显变化。振荡和冻融条件下各处方纯度均未发生变化。详见表14、图3和图4。
纯度(非还原型CE-SDS法)：40℃±2℃条件下，各处方纯度均有下降趋势，处方间无差异；25℃±2℃条件下，各处方纯度没有发生明显变化。详见表15、图5。
表14.处方筛选试验纯度结果(SEC-HPLC法，％)
Figure PCTCN2021071757-appb-000015
Figure PCTCN2021071757-appb-000016
表15.处方筛选试验纯度结果(非还原型CE-SDS法，％)
Figure PCTCN2021071757-appb-000017
(4)电荷变异体
40℃±2℃和25℃±2℃条件下，各处方电荷变异体-酸性组分和主成分均发生明显变化，变化速度处方1＞处方2＞处方3＞处方4，表明组氨酸体系优于枸橼酸和枸橼酸钠体系，同时IBI318蛋白在山梨醇、甲硫氨酸、精氨酸的共同作用下稳定性最优(见表16、图6～7)。振荡、冻融条件下，各处方间电荷变异体无明显差异。
表16.处方筛选试验电荷变异体结果(CEX-HPLC法，％)
Figure PCTCN2021071757-appb-000018
综合上述各个试验结果，选定处方4为IBI318制剂处方。为生产时避免使用浓盐酸调节pH，将缓冲体系调整为10mmol/L的组氨酸和盐酸组氨酸，即IBI318处方为：20mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、0.85mg/ml组氨酸、1.00mg/ml盐酸组氨酸、30.00mg/ml山梨醇、7.49mg/ml甲硫氨酸、21.07mg/ml盐酸精氨酸、0.20mg/ml聚山梨酯80，pH6.0。
实施例4：IBI318高浓度液体制剂及其冻干制剂稳定性实验
本实验用于考察该处方下IBI318高浓度液体制剂及其冻干制剂的稳定性。发明人意外地发现，通过该处方，不仅可以制备稳定的低浓度例如20mg/ml的液体抗体制剂，还可以用于制备稳定的高浓度例如100mg/ml的液体制剂及其冻干制剂。
4.1 试验步骤
将抗体超滤换液至实施例3中的缓冲液(20mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、0.85mg/ml组氨酸、1.00mg/ml盐酸组氨酸、30.00mg/ml山梨醇、7.49mg/ml甲硫氨酸、21.07mg/ml盐酸精氨酸、pH6.0)中，浓缩至约100mg/ml后，加入聚山梨酯80，使其终浓度为0.2mg/ml。过滤分装至西林瓶，每瓶0.5ml，共分装22瓶。取11瓶，加塞压盖标记为PD20191109-1。另取11瓶，进行冻干后，加塞压盖标记为PD20191109-2。根据实验方案见表17进行稳定性考察。检测指标为外观、蛋白含量、纯度(SEC-HPLC法、非还原型CE-SDS法)和电荷变异体(CEX-HPLC法)。
表17.稳定性研究方案
Figure PCTCN2021071757-appb-000019
4.2 试验结果
(1)外观、可见异物
在40℃±2℃的条件下考察1个月，25℃±2℃条件下考察至1个月，高浓度制剂处方外观、可见异物均合格；冻干制剂外观合格，复溶后外观和可见异物均合格。
(2)蛋白含量
在40℃±2℃和25℃±2℃条件下1个月，高浓度液体制剂蛋白含量均未发生变化；冻干制剂复溶后进行蛋白含量检测，均未发生变化。
表18.蛋白含量结果(UV法，mg/ml)
Figure PCTCN2021071757-appb-000020
注：ND表示未设置该检项，--表示试验进行中，下同。
K20180504为20mg/ml液体制剂；PD20191109-1为100mg/ml高浓度液体制剂；PD20191109-2为100mg/ml高浓度冻干制剂。
(3)纯度
纯度(SEC-HPL C法)：40℃和25℃条件下高浓度液体制剂纯度均发生下降，主要表现为聚体增多，其下降速度略快于20mg/ml液体制剂，但40℃条件下1个月纯度仍大于95％，稳定性可以接受。相较于高浓度液体制剂，冻干制剂表现出更佳的稳定性。详见表19。
纯度(非还原型CE-SDS法)：40℃条件下高浓度液体制剂纯度发生下降，其下降速度与20mg/ml液体制剂相当，40℃条件下1个月纯度仍大于90％，稳定性可以接受。相较于高浓度液体制剂，冻干制剂表现出更佳的稳定性，在40℃条件下放置1个月，纯度(非还原型CE-SDS法)无明显变化。详见表20。
表19.稳定性研究纯度结果(SEC-HPLC法，％)
Figure PCTCN2021071757-appb-000021
表20.稳定性研究纯度结果(非还原型CE-SDS法，％)
Figure PCTCN2021071757-appb-000022
(4)电荷变异体
40℃条件下高浓度液体制剂酸性组分增加，主成分下降，其变化速度与20mg/ml液体制剂相当，冻干制剂电荷变异体未发生明显变化。25℃条件下高浓度液体制剂和冻干制剂电荷变异体均未发生明显变化。详见表21。
表21.稳定性研究电荷变异体结果(CEX-HPLC法，％)
Figure PCTCN2021071757-appb-000023
试验结论
综合上述各个试验现有结果可知，高浓度制剂处方(100mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、0.85mg/ml组氨酸、1.00mg/ml盐酸组氨酸、30.00mg/ml山梨醇、7.49mg/ml甲硫氨酸、21.07mg/ml盐酸精氨酸、0.20mg/ml聚山梨酯80，pH6.0)制备的液体制剂和冻干制剂稳定性均符合要求。
实施例5：IBI318冻干制剂复溶实验以及粘度测试
冻干制剂通过加入更少的水可以复溶制备更高浓度的液体制剂。IBI318冻干制剂PD20191109-2可以复溶形成蛋白浓度高至170mg/ml的液体制剂，粘度低于10cP，在液体制剂可接受的注射粘度范围内。且在常温下(25℃±2℃，60％RH±5％RH,500Lux±50Lux)放置一天，各项检测指标均未发生变化。以上结果表明，以冻干制剂复溶制备高浓度液体制剂进行皮下注射是可行的。
表22 IBI318冻干制剂复溶实验稳定性研究结果
Figure PCTCN2021071757-appb-000024
Figure PCTCN2021071757-appb-000025
以上描述了本发明的示例性实施方案，本领域技术人员应当理解的是，这些公开内容仅是示例性的，在本发明的范围内可以进行各种其它替换、适应和修改。因此，本发明不限于文中列举的具体实施方案。

Claims (27)

  1. 抗PD-1/PD-L1抗体或其抗原结合片段的冻干制剂，其包含：
    (i)抗PD-1/PD-L1抗体或其抗原结合片段；
    (ii)缓冲体系；
    (iii)稳定剂，所述稳定剂包括多元醇和/或氨基酸；和
    (iv)表面活性剂，
    其中所述制剂当重构时具有5.5-6.5之间的pH，例如，pH约为5.5、6.0或6.5。
  2. 根据权利要求1所述的冻干制剂，其中所述制剂当重构时具有约6.0的pH。
  3. 根据权利要求1-2任一项所述的冻干制剂，所述制剂能够在约1mg/mL至约150mg/mL之间的浓度重构所述抗体或其抗原结合片段，例如所述制剂能够在约1mg/mL至约200mg/mL的浓度，例如约1、10、15、20、25、30、35、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或200mg/ml的浓度重构所述抗体或其抗原结合片段；优选地，所述制剂能够在约20mg/mL至约170mg/mL之间的浓度重构所述抗体或其抗原结合片段。
  4. 根据权利要求1-3任一项所述的冻干制剂，其中所述表面活性剂选自聚山梨酯80、聚山梨酯20、泊洛沙姆、聚乙二醇聚山梨酯80或其组合，且以约0.01％(w/v)-1％(w/v)，例如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)的重量比存在；优选地，所述表面活性剂为聚山梨酯80，更优选地，所述聚山梨酯80以约0.02％(w/v)的重量比存在。
  5. 根据权利要求1-4任一项所述的冻干制剂，其中所述缓冲体系选自组氨酸缓冲体系、组氨酸和盐酸组氨酸缓冲体系、枸橼酸和枸橼酸钠缓冲体系、醋酸醋酸钠缓冲体系、磷酸盐缓冲体系，且以约0.01％(w/v)-1％(w/v)的重量比存在，例如约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)的重量比存在；优选地，所述缓冲体系以约0.01％(w/v)-0.2％(w/v)的重量比存在,
    优选地，所述缓冲体系为组氨酸缓冲体系；更优选地，所述组氨酸以约0.01-1％(w/v)，例如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)的重量比存在；更优选地，所述组氨酸以约0.155％(w/v)的重量比存在；
    优选地，所述缓冲体系为组氨酸和盐酸组氨酸缓冲体系；更优选地，所述组氨酸和所述盐酸组氨酸分别以约0.01-1％(w/v)，例如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)的重量比存在；更优选地，所述组氨酸和盐酸组氨酸分别以约0.085％(w/v)和约0.1％(w/v)的重量比存在。
  6. 根据权利要求1-5任一项所述的冻干制剂，其中所述多元醇选自山梨醇、甘露醇或其组合，所述氨基酸包括精氨酸、盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸或其组合；
    优选地，所述稳定剂包括山梨醇和精氨酸；更优选地，所述山梨醇以约1-10％(w/v)，例如约1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在,所述精氨酸以约1-10％(w/v)，例如约1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在；更优选地，所述山梨醇和所述精氨酸分别以约3％(w/v)和约1.742％(w/v)的重量比存在；
    优选地，所述稳定剂包括山梨醇和盐酸精氨酸；更优选地，所述山梨醇以约1-10％(w/v)，例如约1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在,以约1-10％(w/v)，例如约1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在；更优选地，所述山梨醇和所述盐酸精氨酸分别以约3％(w/v)和约2.107％(w/v)的重量比存在。
  7. 根据权利要求1-6任一项所述的冻干制剂，其中所述稳定剂还包括甲硫氨酸；优选地，所述甲硫氨酸以约0.1-10％(w/v)的重量比存在,例如以约0.1、0.5、1、2、3、4、5、6、7、8、9、10％(w/v)的重量比存在；更优选地，所述甲硫氨酸以约0.749％(w/v)的重量比存在。
  8. 抗PD-1/PD-L1抗体或其抗原结合片段的冻干制剂，其通过冻干水溶液制成，所述水溶液包含：
    (i)抗PD-1/PD-L1抗体或其抗原结合片段；
    (ii)缓冲体系；
    (iii)稳定剂，所述稳定剂包括多元醇和/或氨基酸；和
    (iv)表面活性剂，
    所述液体制剂具有约5.5-6.5的pH，例如，pH约为5.5、6.0或6.5；优选地，所述液体制剂具有约6.0的pH。
  9. 抗PD-1/PD-L1抗体或其抗原结合片段的液体制剂，其包含水溶液，所述水溶液包含：
    (i)抗PD-1/PD-L1抗体或其抗原结合片段；
    (ii)缓冲体系；
    (iii)稳定剂，所述稳定剂包括多元醇和/或氨基酸；和
    (iv)表面活性剂，
    所述液体制剂具有约5.5-6.5的pH，例如，pH约为5.5、6.0或6.5；优选地，所述液体制剂具有约6.0的pH。
  10. 根据权利要求8所述的冻干制剂或根据权利要求9所述的液体制剂，其中所述抗PD-1/PD-L1抗体或其抗原结合片段以约1mg/mL至约200mg/mL的浓度存在于水溶液中，例如以约1、5、10、20、25、30、35、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或200mg/ml的浓度存在于水溶液中；优选地，所述抗PD-1/PD-L1 抗体或其抗原结合片段以约20mg/mL至约170mg/mL之间的浓度存在于水溶液中。
  11. 根据权利要求8所述的冻干制剂或根据权利要求9-10任一项所述的液体制剂，其中所述表面活性剂选自聚山梨酯80、聚山梨酯20、泊洛沙姆、聚乙二醇聚山梨酯80或其组合，且以约0.1-10mg/ml，例如约0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的浓度存在；优选地，所述表面活性剂为聚山梨酯80，更优选地，所述聚山梨酯80以约0.2mg/ml的浓度存在。
  12. 根据权利要求8所述的冻干制剂或根据权利要求9-11任一项所述的液体制剂，其中所述缓冲体系选自组氨酸缓冲体系、组氨酸和盐酸组氨酸缓冲体系、枸橼酸和枸橼酸钠缓冲体系、醋酸醋酸钠缓冲体系、磷酸盐缓冲体系，且以约1-100mM，例如，约1、5、10、15、20、30、40、50、60、70、80、90、100mM的浓度存在；优选地，所述缓冲体系以约1-20mM，例如，1、5、10、15、20mM的浓度存在；
    优选地，所述缓冲体系为组氨酸缓冲体系；更优选地，所述组氨酸以约0.1-10mg/mL，例如约0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的浓度存在；更优选地，所述组氨酸以约1.55mg/mL的浓度存在；
    优选地，所述缓冲体系为组氨酸和盐酸组氨酸缓冲体系；更优选地，所述组氨酸和所述盐酸组氨酸分别以约0.1-10mg/mL，例如约0.1、0.5、1、2、3、4、5、6、7、8、9、10mg/ml的浓度存在；更优选地，所述组氨酸和所述盐酸组氨酸分别以约0.85mg/mL和约1.00mg/mL的浓度存在。
  13. 根据权利要求8所述的冻干制剂或根据权利要求9-12任一项所述的液体制剂，其中所述多元醇选自山梨醇、甘露醇或其组合，所述氨基酸包括精氨酸、盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸或其组合；
    优选地，所述稳定剂包括山梨醇和精氨酸；更优选地，所述山梨醇以约10-100mg/mL，例如约10、20、30、40、50、60、70、80、90、100mg/mL的浓度存在，所述精氨酸以例如约10、20、30、40、50、60、70、80、90、100mg/mL存在；更优选地，所述山梨醇和所述精氨酸分别以约30mg/mL和约17.42mg/mL的浓度存在；
    优选地，所述稳定剂包括山梨醇和盐酸精氨酸；更优选地，所述山梨醇和所述盐酸精氨酸分别以约10-100mg/mL，例如约10、20、30、40、50、60、70、80、90、100mg/mL的浓度存在；更优选地，所述山梨醇和所述盐酸精氨酸分别以约30mg/mL和约21.07mg/mL的浓度存在。
  14. 根据权利要求8所述的冻干制剂或根据权利要求9-13任一项所述的液体制剂，其中所述稳定剂还包括甲硫氨酸；优选地，所述甲硫氨酸以约1-100mg/mL，例如约1、5、10、20、30、40、50、60、70、80、90、100mg/mL的浓度存在；更优选地，所述甲硫氨酸以约 1-50mg/mL；更优选地，所述甲硫氨酸以约7.49mg/mL的浓度存在。
  15. 根据权利要求8所述的冻干制剂或根据权利要求9-14任一项所述的液体制剂，其特征在于：
    所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约2.94mg/ml枸橼酸钠、约50mg/ml山梨醇、约0.20mg/ml聚山梨酯80，所述水溶液具有约5.5的pH；
    或所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约2.94mg/ml枸橼酸钠、约50mg/ml山梨醇、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH；
    或所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约2.94mg/ml枸橼酸钠、约50mg/ml山梨醇、约2.94mg/ml甲硫氨酸、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH；
    或所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约2.94mg/ml枸橼酸钠、约50mg/ml山梨醇、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.5的pH；
    或所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约2.94mg/ml枸橼酸钠、约50mg/ml山梨醇、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH；
    或所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约1.55mg/ml组氨酸、约50mg/ml山梨醇、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH；
    或所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约1.55mg/ml组氨酸、约50mg/ml山梨醇、约7.49mg/ml甲硫氨酸、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH；
    更优选地，所述水溶液包含：20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约1.55mg/ml组氨酸、约30mg/ml山梨醇、约7.49mg/ml甲硫氨酸、约17.42mg/ml精氨酸、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH；
    更优选地，所述水溶液包含：约20-170mg/ml重组全人源抗程序性死亡受体1(PD-1)和抗程序性死亡配体1(PD-L1)双特异性抗体、约0.85mg/ml组氨酸、约1.00mg/ml盐酸组氨酸、约30.00mg/ml山梨醇、约7.49mg/ml甲硫氨酸、约21.07mg/ml盐酸精氨酸、约0.20mg/ml聚山梨酯80，所述水溶液具有约6.0的pH。
  16. 根据权利要求1-8任一项所述的冻干制剂或根据权利要求9-15任一项的液体制剂，其中所述抗体或其抗原结合片段结合人类PD-L1(SEQ ID NO:1)及人类PD-1(SEQ ID NO:2)；
    优选地，所述抗体或其抗原结合片段包含第一重链(HCl)，其包含第一重链可变区(HCVR1)和恒定区；第一轻链(LC1)，其包含第一轻链可变区(LCVR1)和恒定区；第二重链(HC2)，其包含第二重链可变区(HCVR2)和恒定区；以及第二轻链(LC2)，其包含第二轻链可变区(LCVR2)和恒定区，其中：
    1)所述HCVR1包含SEQ ID NO:3所示的重链可变区所含的三个互补决定区域HCDR1、HCDR2和HCDR3，并且所述LCVR1包含SEQ ID NO:4所示的轻链可变区所含的LCDR1、LCDR2和LCDR3；以及
    2)所述HCVR2包含SEQ ID NO:5所示的重链可变区中所含的三个互补决定区域(CDR)HCDR1、HCDR2和HCDR3，并且所述LCVR2包含SEQ ID NO:8所示的轻链可变区所含的三个互补决定区域LCDR1、LCDR2和LCDR3。
  17. 根据权利要求1-8任一项所述的冻干制剂或根据权利要求9-15任一项的液体制剂，其中所述抗体或其抗原结合片段包含第一重链(HCl)，其包含第一重链可变区(HCVR1)和恒定区；第一轻链(LC1)，其包含第一轻链可变区(LCVR1)和恒定区；第二重链(HC2)，其包含第二重链可变区(HCVR2)和恒定区；以及第二轻链(LC2)，其包含第二轻链可变区(LCVR2)和恒定区，其中：
    a)所述HCVR1包含具有SEQ ID NO:16所示的氨基酸序列的互补决定区域CDR1、具有SEQ ID NO:17所示的氨基酸序列的互补决定区域CDR2及具有SEQ ID NO:18所示的氨基酸序列的互补决定区域CDR3；
    b)所述LCVR1包含具有SEQ ID NO:19所示的氨基酸序列的互补决定区域CDR1、具有SEQ ID NO:20所示的氨基酸序列的互补决定区域CDR2及具有SEQ ID NO:21所示的氨基酸序列的互补决定区域CDR3；
    c)所述HCVR2包含具有SEQ ID NO:22所示的氨基酸序列的互补决定区域CDR1、具有SEQ ID NO:23或SEQ ID NO:24所示的氨基酸序列的互补决定区域CDR2及具有SEQ ID NO:25所示的氨基酸序列的互补决定区域CDR3；以及
    d)所述LCVR2包含具有SEQ ID NO:26所示的氨基酸序列的互补决定区域CDR1、具有SEQ ID NO:27所示的氨基酸序列的互补决定区域CDR2及具有SEQ ID NO:28所示的氨基酸序列的互补决定区域CDR3。
  18. 根据权利要求1-8任一项所述的冻干制剂或根据权利要求9-15任一项的液体制剂，其中所述抗体或其抗原结合片段包含第一重链(HCl)，其包含第一重链可变区(HCVR1)和恒定区；第一轻链(LC1)，其包含第一轻链可变区(LCVR1)和恒定区；第二重链(HC2)，其包含第二重链可变区(HCVR2)和恒定区；以及第二轻链(LC2)，其包含第二轻链可变区(LCVR2)和恒定区，其中：
    所述抗体或其抗原结合片段包含与SEQ ID NO:3具有至少90％,95％,98％或99％或更高 同一性的HCVR1；和/或与SEQ ID NO:4具有至少90％,95％,98％或99％或更高同一性的LCVR1；和/或与SEQ ID NO:5具有至少90％,95％,98％或99％或更高同一性的HCVR2；和/或与SEQ ID NO:8具有至少90％,95％,98％或99％或更高同一性的LCVR2；
    优选地，a)所述HCVR1包含具有SEQ ID NO:3所示的氨基酸序列；b)所述LCVR1包含具有SEQ ID NO:4所示的氨基酸序列；c)所述HCVR2包含具有SEQ ID NO:5所示的氨基酸序列；及d)所述LCVR2包含具有SEQ ID NO:8所示的氨基酸序列。
  19. 根据权利要求1-8任一项所述的冻干制剂或根据权利要求9-15任一项的液体制剂，其中所述抗体或其抗原结合片段包含第一重链(HCl)，其包含第一重链可变区(HCVR1)和恒定区；第一轻链(LC1)，其包含第一轻链可变区(LCVR1)和恒定区；第二重链(HC2)，其包含第二重链可变区(HCVR2)和恒定区；以及第二轻链(LC2)，其包含第二轻链可变区(LCVR2)和恒定区，其中：
    所述抗体或其抗原结合片段包含与SEQ ID NO:49或SEQ ID NO:29具有至少90％,95％,98％或99％或更高同一性的HC1；和/或与SEQ ID NO:30具有至少90％,95％,98％或99％或更高同一性的LC1；和/或与SEQ ID NO:33或SEQ ID NO:31或SEQ ID NO:32具有至少90％,95％,98％或99％或更高同一性的HC2；和/或与SEQ ID NO:34具有至少90％,95％,98％或99％或更高同一性的LC2；
    优选地，所述HC1、所述LC1、所述HC2及所述LC2分别包含SEQ ID NO:49、SEQ ID NO:30、SEQ ID NO:31及SEQ ID NO:34所示的氨基酸序列；或者分别包含SEQ ID NO:49、SEQ ID NO:30、SEQ ID NO:32及SEQ ID NO:34所示的氨基酸序列；或者分别包含SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:33及SEQ ID NO:34所示的氨基酸序列；
    更优选地，所述抗体或其抗原结合片段含有衍生自人IgG1的Fc部分变体。
  20. 根据权利要求1-8任一项所述的冻干制剂或根据权利要求9-15任一项的液体制剂，其中所述抗体选自：PD-1/PD-L1双特异性Ab v13844、Ab v3.0或Ab v3.2；优选地，所述抗体为PD-1/PD-L1双特异性Ab v3.2。
  21. 根据权利要求1-8任一项所述的冻干制剂或根据权利要求9-15任一项的液体制剂，其中所述抗体或其抗原结合片段在至少约12个月内表现出稳定性，优选地，所述抗体或其抗原结合片段在至少约24个月内表现出稳定性，更优选地，所述抗体或其抗原结合片段在至少约36个月内表现出稳定性。
  22. 根据权利要求1-8任一项所述的冻干制剂或根据权利要求9-15任一项的液体制剂，其中所述制剂在储存后，例如在2-8℃储存至少24个月后，或在室温储存至少6个月后，或在40℃±2℃储存1个月后，是稳定的，优选地具有如下特征之一或多项：
    (i)通过SEC-HPLC法测量，制剂具有大于或等于90％的纯度，优选大于或等于92％、94％、 95％、96％、98％或99％的纯度；
    (ii)通过还原型或非还原型CE-SDS法测量，制剂具有大于或等于90％的纯度，优选大于或等于92％、94％、96％、98％的纯度；
    (iii)通过iCIEF或CEX-HPLC法测量，主成分≥35％，优选大于或等于35％、40％、45％或50％。
  23. 递送装置，其包含根据权利要求1-8任一项所述的冻干制剂或根据权利要求9-15任一项的液体制剂，用于静脉内、皮下、皮内或者肌内注射，或静脉内输注。
  24. 预填装注射器，其包含根据权利要求1-8任一项所述的冻干制剂或根据权利要求9-15任一项的液体制剂，用于静脉内、皮下、皮内或者肌内注射，或静脉内输注。
  25. 根据权利要求1-8任一项所述的冻干制剂或根据权利要求9-15任一项的液体制剂的用途，用于制备在受试者中预防和/或治疗肿瘤的递送装置或预填装注射器或药物；例如，所述肿瘤是癌症，优选地，所述癌症为霍奇金或非霍奇金淋巴瘤、黑色素瘤、肾细胞癌、肾癌、肺癌、膀胱癌、胃及食道癌、结肠直肠癌、肝癌、肝细胞癌、胆管癌、胰脏癌、乳癌、三阴性乳癌、卵巢癌、子宫内膜癌、前列腺癌、小细胞肺癌(SCLC)、非小细胞肺癌(NSCLC)、间皮瘤、头颈的鳞状细胞癌(SCCHN)、软组织肉瘤或多形性神经胶母细胞瘤；更优选地，该肺癌是NSCLC、小细胞肺癌或间皮瘤；优选地，其中所述递送装置或预填装注射器或药物可与一或多种选自由以下组成的群组的抗肿瘤剂同时、分开或依序给药：顺铂(cisplatin)、卡铂(carboplatin)、达卡巴嗪(dacarbazine)、脂质体多柔比星(liposomal doxorubicin)、多西他赛(docetaxel)、环磷酰胺(cyclophosphamide)及多柔比星、诺维本(navelbine)、艾瑞布林(eribulin)、太平洋紫杉醇(paclitaxel)、用于可注射悬浮液的太平洋紫杉醇蛋白质结合颗粒、伊沙匹隆(ixabepilone)、卡培他滨(capecitabine)、FOLFOX(亚叶酸钙(leucovorin)、氟尿嘧啶(fluorouracil)及奥沙利铂(oxaliplatin))、FOLFIRI(亚叶酸钙、氟尿嘧啶及伊立替康(irinotecan))、吉西他滨(gemcitabine)、托泊替康(topotecan)、脂质体伊立替康、培美曲塞(pemetrexed)、西妥昔单抗(cetuximab)、尼沃鲁单抗(nivolumab)、伊匹单抗(ipilimumab)、匹利珠单抗(pidilizumab)、派姆单抗(pembrolizumab)、曲美木单抗(tremelimumab)、乌瑞鲁单抗(urelumab)、利丽单抗(lirilumab)、阿替珠单抗(atezolizumab)、爱帕司他(epacadostat)及德瓦鲁单抗(durvalumab)；更优选地，所述递送装置或预填装注射器或药物可与电离辐射同时、分开或依次施用。
  26. 一种预防和/或治疗肿瘤的方法，包括向受试者施用有效量的根据权利要求1-8任一项所述的冻干制剂或根据权利要求9-15任一项的液体制剂；例如，所述肿瘤是癌症，优选地，所述癌症为霍奇金或非霍奇金淋巴瘤、黑色素瘤、肾细胞癌、肾癌、肺癌、膀胱癌、胃及食道癌、结肠直肠癌、肝癌、肝细胞癌、胆管癌、胰脏癌、乳癌、三阴性乳癌、卵巢癌、子宫内膜癌、前列腺癌、小细胞肺癌(SCLC)、非小细胞肺癌(NSCLC)、间皮瘤、头颈的鳞状细胞癌 (SCCHN)、软组织肉瘤或多形性神经胶母细胞瘤；更优选地，该肺癌是NSCLC、小细胞肺癌或间皮瘤。
  27. 根据权利要求26所述的方法，还包括同时，分开或依次给予一种或多种抗肿瘤剂，所述抗肿瘤剂选自顺铂(cisplatin)、卡铂(carboplatin)、达卡巴嗪(dacarbazine)、脂质体多柔比星(liposomal doxorubicin)、多西他赛(docetaxel)、环磷酰胺(cyclophosphamide)及多柔比星、诺维本(navelbine)、艾瑞布林(eribulin)、太平洋紫杉醇(paclitaxel)、用于可注射悬浮液的太平洋紫杉醇蛋白质结合颗粒、伊沙匹隆(ixabepilone)、卡培他滨(capecitabine)、FOLFOX(亚叶酸钙(leucovorin)、氟尿嘧啶(fluorouracil)及奥沙利铂(oxaliplatin))、FOLFIRI(亚叶酸钙、氟尿嘧啶及伊立替康(irinotecan))、吉西他滨(gemcitabine)、托泊替康(topotecan)、脂质体伊立替康、培美曲塞(pemetrexed)、西妥昔单抗(cetuximab)、尼沃鲁单抗(nivolumab)、伊匹单抗(ipilimumab)、匹利珠单抗(pidilizumab)、派姆单抗(pembrolizumab)、曲美木单抗(tremelimumab)、乌瑞鲁单抗(urelumab)、利丽单抗(lirilumab)、阿替珠单抗(atezolizumab)、爱帕司他(epacadostat)及德瓦鲁单抗(durvalumab)。
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