WO2020259605A1 - 包含抗cd47/pd-l1双特异性抗体的制剂及其制备方法和用途 - Google Patents

包含抗cd47/pd-l1双特异性抗体的制剂及其制备方法和用途 Download PDF

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WO2020259605A1
WO2020259605A1 PCT/CN2020/098172 CN2020098172W WO2020259605A1 WO 2020259605 A1 WO2020259605 A1 WO 2020259605A1 CN 2020098172 W CN2020098172 W CN 2020098172W WO 2020259605 A1 WO2020259605 A1 WO 2020259605A1
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liquid
preparation
antibody
protein
seq
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PCT/CN2020/098172
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English (en)
French (fr)
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朱兴贵
马一冬
汪音爵
周凯松
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信达生物制药(苏州)有限公司
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Priority to CA3144244A priority Critical patent/CA3144244A1/en
Priority to JP2021577124A priority patent/JP2022540781A/ja
Priority to KR1020227002361A priority patent/KR20220036998A/ko
Priority to BR112021026414A priority patent/BR112021026414A2/pt
Priority to US17/621,946 priority patent/US20220251210A1/en
Priority to CN202080046515.9A priority patent/CN114040777A/zh
Priority to AU2020304112A priority patent/AU2020304112A1/en
Priority to EP20833335.1A priority patent/EP3991745A1/en
Publication of WO2020259605A1 publication Critical patent/WO2020259605A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to the field of antibody preparations. More specifically, the present invention relates to a bispecific antibody comprising recombinant anti-differentiation antigen cluster 47 (CD47) and anti-programmed death ligand 1 (PD-L1) (also known as anti-CD47/PD-L1 bispecific antibody) )
  • a bispecific antibody comprising recombinant anti-differentiation antigen cluster 47 (CD47) and anti-programmed death ligand 1 (PD-L1) (also known as anti-CD47/PD-L1 bispecific antibody)
  • CD47 anti-differentiation antigen cluster 47
  • PD-L1 anti-programmed death ligand 1
  • Pharmaceutical preparations especially stable high-concentration antibody liquid preparations, and methods for preparing the pharmaceutical preparations, and therapeutic and/or preventive uses of the pharmaceutical preparations.
  • PD-L1 (also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)) is a 40kDa type I transmembrane protein. PD-L1 binds to its receptor PD-1 present on activated T cells, and down-regulates T cell activation (Latchman et al., 2001 Nat Immunol 2: 261-8; Carter et al. 2002 Eur J Immunol 32: 634-43 ). PD-L1 expression has been found in many cancers, including human lung cancer, ovarian cancer, colon cancer, and a variety of myeloma, and PD-L1 expression is often associated with poor prognosis of cancer (Iwai et al.
  • Other anti-PD-L1 antibodies include YW243.55.S70 (the heavy and light chain variable regions are shown in SEQ ID NOs 20 and 21 in WO2010/077634) and the anti-PD-L1 antibody disclosed in WO2007/005874.
  • CD47 Cluster of differentiation 47
  • IAP integrin-associated protein
  • CD47 interacts with SIRP ⁇ , a cell surface immunoglobulin, which is mainly expressed by macrophages and dendritic cells as its ligand, to produce a series of cascade reactions, and thereby inhibit the expression of macrophages and dendritic cells. The uptake and phagocytosis of CD47 cells. Overexpression of CD47 was observed in tumors. However, CD47 is also expressed in many normal tissues, which leads to the non-specific binding of antibodies that target only CD47 to normal blood system cells, causing the phenomenon of antigen sink.
  • the anti-CD47/PD-L1 bispecific antibody can simultaneously target CD47 and PD-L1 on tumor cells, it promotes the anti-CD47/PD-L1 bispecific antibody pair by specifically binding to PD-L1 on tumor cells The selective binding of tumor cells avoids binding to CD47 expressed in many normal tissues. Therefore, the anti-CD47/PD-L1 bispecific antibody has the advantage of enhancing the anti-tumor effect while reducing side effects.
  • the PCT application of PCT/CN2018/123886 discloses a novel antibody format, and constructs and expresses an anti-CD47/PD-L1 bispecific antibody with the novel antibody format .
  • the anti-CD47/PD-L1 bispecific antibody was administered to tumor-bearing mice produced by inoculating NOD-SCID mice with Raji-PD-L1 cells. The results showed that it was incompatible with the administration of anti-CD47 monoclonal antibodies and anti-PD-L1 monoclonal antibodies In comparison, the anti-CD47/PD-L1 bispecific antibody has significantly improved anti-tumor activity, can significantly inhibit tumor growth, and even make the tumor disappear completely.
  • anti-CD47/PD-L1 bispecific antibody also exhibits significantly reduced hemagglutination, so it will have significantly reduced side effects in clinical treatment.
  • anti-CD47/PD-L1 bispecific antibody preparations that can be used to treat, prevent or delay various diseases related to the SIRP ⁇ /CD47 signaling pathway and the PD1/PD-L1 signaling pathway.
  • the antibody preparation In addition to being formulated in a way that makes the antibody suitable for administration to a subject, the antibody preparation also needs to be formulated in a way that maintains its stability during storage and subsequent use. For example, if the antibody is not properly formulated in the liquid, the antibody in the liquid solution tends to decompose, aggregate, or undergo undesirable chemical modification.
  • the stability of the antibody in the antibody preparation depends on the buffer, stabilizer and surfactant used in the preparation.
  • anti-CD47/PD-L1 bispecific antibodies Although some anti-CD47/PD-L1 bispecific antibodies are known, there is still a need in the art for novel pharmaceutical formulations containing anti-CD47/PD-L1 bispecific antibodies that are sufficiently stable and suitable for administration to subjects . Therefore, a suitable anti-CD47/PD-L1 bispecific antibody preparation is needed to treat or prevent diseases.
  • the present invention meets the above-mentioned needs by providing a pharmaceutical preparation containing an anti-CD47/PD-L1 bispecific antibody protein that specifically binds to CD47 and PD-L1.
  • the present invention provides a liquid antibody preparation comprising (i) anti-CD47/PD-L1 bispecific antibody protein; (ii) buffer, (iii) stabilizer, and (iv) surfactant .
  • the anti-CD47/PD-L1 bispecific antibody protein contained in the antibody preparation of the present invention is a three-chain antibody comprising a first polypeptide chain and a second polypeptide chain that specifically bind to CD47.
  • the /VL pair serves as the first antigen binding site
  • the first VHH on the third polypeptide chain that specifically binds to PD-L1 serves as the single domain second antigen binding site and the second VHH serves as the single domain third antigen A binding site
  • a VH/VL pair that specifically binds PD-L1 on the first polypeptide chain and the second polypeptide chain as the first antigen binding site, and the third polypeptide chain specifically binds to CD47
  • the first VHH serves as the single domain second antigen binding site and the second VHH serves as the single domain third antigen binding site.
  • the anti-CD47/PD-L1 bispecific antibody protein can be at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or more
  • the affinity constant binds to CD47 on the surface of tumor cells, thereby blocking the binding of CD47 to SIRP ⁇ on the surface of macrophages, and promoting the phagocytosis of tumor cells by macrophages in the infiltration area of tumor tissue; and at least about 10 7 M -1 , Preferably about 10 8 M -1 and more preferably about 10 9 M -1 or stronger affinity constant binds to PD-L1 on the surface of tumor cells, thereby inhibiting PD-1 on T cells and PD on the surface of tumor cells
  • the combination of -L1 induces T cell activation and exerts anti-tumor effects.
  • the anti-CD47/PD-L1 bispecific antibody protein is the recombinant anti-CD47/PD-L1 double disclosed in the PCT application of PCT/CN2018/123886 (application date: December 26, 2018). Specific antibody protein.
  • the entire content of the PCT application is hereby incorporated by reference.
  • the anti-CD47/PD-L1 bispecific antibody protein is a three-chain antibody comprising a first polypeptide chain and a second polypeptide chain that specifically bind CD47 VH/ The VL pair serves as the first antigen binding site, and the first VHH on the third polypeptide chain that specifically binds to PD-L1 serves as the single domain second antigen binding site and the second VHH serves as the single domain third antigen binding Site, where the VH/VL pair on the first polypeptide chain and the second polypeptide chain that specifically binds to CD47 as the first antigen binding site includes GSIEHYYWS (SEQ ID NO: 3) derived from the anti-CD47 antibody ADI-29341 ) Shown in VH CDR1, YIYYSGSTNYNPSLKS (SEQ ID NO: 4) shown in VH CDR2 shown in ARGKTGSAA (SEQ ID NO: 5) shown in VH CDR3 shown in RASQGISRWLA (SEQ ID NO: 10) shown
  • the VH/VL pair that specifically binds to CD47 on the first polypeptide chain and the second polypeptide chain of the anti-CD47/PD-L1 bispecific antibody protein comprises as the first antigen binding site
  • the region sequence has a sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity
  • the second and third antigen-binding sites of the single domain that specifically bind to PD-L1 both include the amino acid sequence shown in SEQ ID NO: 15 and/or SEQ ID NO: 16, or are substantially the same (for example, At least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) sequence:
  • the anti-CD47/PD-L1 bispecific antibody protein comprises the first polypeptide chain shown in SEQ ID NO: 1, the second polypeptide chain shown in SEQ ID NO: 8, and SEQ ID NO :14 or the third polypeptide chain shown in SEQ ID NO: 22, or substantially identical to any of the sequences (for example, at least 80%, 85%, 90%, 92%, 95%, 97%, 98% %, 99% or higher identity) sequence.
  • the anti-CD47/PD-L1 bispecific antibody protein is an anti-CD47/PD-L1 bispecific antibody protein recombinantly expressed in HEK293 cells or CHO cells.
  • the concentration of the anti-CD47/PD-L1 bispecific antibody protein in the liquid antibody preparation of the present invention is about 1-200 mg/ml. In another embodiment, the concentration of the anti-CD47/PD-L1 bispecific antibody protein in the liquid antibody preparation of the present invention is about 5-150 mg/mL. In other embodiments, the concentration of the anti-CD47/PD-L1 bispecific antibody protein in the liquid antibody preparation of the present invention is about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 , 110, 120, 130, 140 or 150mg/ml.
  • the concentration of the buffer in the liquid antibody formulation of the present invention is about 1-30 mM. In one embodiment, the concentration of the buffer in the liquid antibody formulation of the present invention is about 5-25 mM, for example, about 5, 10, 15, 20, 25 mM.
  • the buffer is selected from histidine, histidine hydrochloride and combinations thereof.
  • the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 50-500 mM. In one embodiment, the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 100-400 mM, for example, about 100, 150, 200, 250, 300, 350, 400 mM.
  • the stabilizer is selected from sorbitol, sucrose, trehalose, arginine, arginine hydrochloride and any combination thereof, more preferably sucrose, arginine and/or arginine hydrochloride.
  • the liquid antibody preparation contains arginine hydrochloride as a stabilizer, preferably arginine hydrochloride at about 50-250 mM, preferably about 100-200 mM (e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM); and/or containing sucrose as a stabilizer, preferably sucrose in about 50-250 mM, preferably about 100-200 mM (e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM).
  • the concentration of the surfactant in the liquid antibody preparation of the present invention is about 0.1-1 mg/ml. In one embodiment, the concentration of the surfactant in the liquid antibody preparation of the present invention is about 0.2-0.8 mg/ml, for example about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml.
  • the surfactant is a nonionic surfactant. In one embodiment, the surfactant is selected from polysorbate surfactants. In a specific embodiment, the surfactant in the liquid antibody formulation of the present invention is polysorbate-80.
  • the liquid formulation further includes a metal chelating agent (e.g., EDTA), for example, about 0.002-0.2 mg/ml metal chelating agent (e.g., EDTA).
  • a metal chelating agent e.g., EDTA
  • the liquid formulation further contains about 0.01-0.1 mg/ml, such as about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.1 mg/ml of a metal chelating agent (for example, EDTA).
  • the pH of the liquid formulation is about 6.4-7.0. In some embodiments, the pH of the liquid formulation is any value from about 6.4 to 7.0, such as about 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0.
  • the liquid preparation is a pharmaceutical preparation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection. In one embodiment, the liquid formulation is an intravenous infusion.
  • liquid antibody formulation of the invention comprises:
  • the liquid formulation further contains 0.002-0.2 mg/ml metal chelating agent (for example, EDTA);
  • metal chelating agent for example, EDTA
  • the pH of the liquid formulation is about 6.4-7.0, preferably about 6.5.
  • liquid antibody preparation of the present invention comprises:
  • the liquid formulation further contains about 0.01-0.1 mg/ml metal chelating agent (for example, EDTA);
  • metal chelating agent for example, EDTA
  • the pH of the liquid formulation is about 6.4-7.0, preferably about 6.5.
  • liquid antibody preparation of the invention comprises
  • the liquid formulation further contains a metal chelating agent (for example, EDTA), for example, about 0.02 mg/ml EDTA;
  • EDTA metal chelating agent
  • the pH of the liquid formulation is about 6.4-7.0, preferably about 6.5.
  • liquid antibody preparation of the invention comprises
  • the liquid formulation further contains a metal chelating agent (for example, EDTA), for example, about 0.02 mg/ml EDTA;
  • EDTA metal chelating agent
  • the pH of the liquid formulation is about 6.4-7.0, preferably about 6.5.
  • liquid antibody preparation of the invention comprises
  • the liquid formulation further contains a metal chelating agent (for example, EDTA), for example, about 0.02 mg/ml EDTA;
  • EDTA metal chelating agent
  • the pH of the liquid formulation is about 6.4-7.0, preferably about 6.5.
  • the present invention provides a solid antibody preparation, which is obtained by subjecting the liquid antibody preparation of the present invention to curing treatment.
  • the curing treatment is performed by, for example, a crystallization method, a spray drying method, or a freeze drying method.
  • the solid antibody preparation is, for example, in the form of a lyophilized powder injection.
  • the solid antibody preparation can be reconstituted in a suitable solvent before use to form the reconstituted preparation of the present invention.
  • the reconstituted preparation is also a liquid antibody preparation of the present invention.
  • the appropriate solvent is selected from water for injection, organic solvent for injection, including but not limited to oil for injection, ethanol, propylene glycol, etc., or a combination thereof.
  • the liquid preparation of the present invention can be stored stably for a long time, for example, at least 24 months or more.
  • the liquid formulation of the present invention can be heated at about -80°C to about 45°C, for example -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about Store at 35°C, about 38°C, about 40°C, about 42°C or about 45°C for at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months , At least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months , At least 36 months, or longer, is stable.
  • the liquid formulation of the present invention can be stored stably for at least 24 months.
  • the liquid formulation of the invention is stable at at least 40°C.
  • the liquid formulation of the present invention is stable at about 2°C-8°C for at least 12 months, preferably at least 24 months.
  • the liquid formulation of the present invention is stable at room temperature or, for example, about 25°C for at least 3 months, preferably at least 6 months.
  • the liquid formulation of the invention remains stable at about 40°C for at least 1 month.
  • the stability of the preparation after storage can be indicated by detecting changes in the appearance, visible foreign matter, protein content, purity, and/or charge variants of the preparation. In one embodiment, it can be in a high temperature stress test, for example after storage at 40°C ⁇ 2°C for at least 1 week, 2 weeks or preferably 1 month, or at 25°C ⁇ 2°C for at least 1 month or 2 Months later, the stability of the liquid preparation of the present invention was tested.
  • the stability of the liquid formulation of the present invention is visually inspected, wherein the liquid formulation of the present invention remains clear to slightly opalescent in appearance, a colorless to pale yellow liquid, and no foreign matter. In one embodiment, by visual inspection under a clarity detector, no visible foreign matter exists in the preparation. In one embodiment, after storage, the stability of the liquid formulation of the present invention is checked by measuring the change in protein content, for example, by ultraviolet spectrophotometry (UV) method, relative to the initial value on day 0 of storage, the rate of change in protein content is Not more than 20%, preferably not more than 10%, for example 7-8%, preferably not more than 5%.
  • UV ultraviolet spectrophotometry
  • the stability of the liquid preparation of the present invention is checked by measuring the change in turbidity of the liquid preparation of the present invention, for example, by the OD 350mm method, the change value is relative to the initial value on the 0th day of storage It does not exceed 0.04, more preferably does not exceed 0.03, and more preferably does not exceed 0.02.
  • the stability of the liquid preparation of the present invention is checked by measuring the change in purity of the liquid preparation of the present invention, wherein size exclusion high performance liquid chromatography (SEC-HPLC) is used to check the stability of the liquid preparation of the present invention.
  • SEC-HPLC size exclusion high performance liquid chromatography
  • the change value of monomer purity does not exceed 10%, for example, does not exceed 5%, 4%, 3%, for example 1-2%, preferably does not exceed 1%.
  • the stability of the liquid preparation of the invention is checked by measuring the purity change of the liquid preparation of the invention, wherein the stability of the liquid preparation of the invention is checked by non-reduced and/or reduced sodium lauryl sulfate capillary electrophoresis (CE- In the SDS method, the change value of the monomer purity does not exceed 10%, for example, it does not exceed 9.5%, 8.5%, 7.5%, 6.5%.
  • the stability of the liquid preparation of the present invention is tested by imaging capillary isoelectric focusing electrophoresis (iCIEF), wherein the charge variant of the antibody (principal component, acidic) is compared to the initial value on day 0 of storage.
  • iCIEF capillary isoelectric focusing electrophoresis
  • the total change value of the component and the alkaline component does not exceed 50%, for example, it does not exceed 45%, 40%, 35%, 30%, 25%.
  • the stability of the liquid preparation of the present invention is tested by cation exchange high performance liquid chromatography (CEX-HPLC method), wherein the charge variant of the antibody (The total change value of the main component, acidic component and alkaline component) does not exceed 40%, for example, does not exceed 38%, 36%, 34%, 32%, 30%.
  • CEX-HPLC method cation exchange high performance liquid chromatography
  • the formulation is stable after storage, such as after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 3 months, or after storage at 40°C ⁇ 2°C for 1 month ,
  • the formulation is stable after storage, such as after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 3 months, or after storage at 40°C ⁇ 2°C for 1 month ,
  • the preparation has a purity greater than 90%, preferably greater than 95%, 96%, 97%, 98%, 99% purity;
  • the preparation has a purity greater than 85%, preferably greater than 86%, 87%, 88%, 89%;
  • the relative binding activity of the anti-CD47/PD-L1 bispecific antibody protein in the preparation is 70%-130%, for example, 70%, 80% , 90%, 100%, 110%, 120%, 130%.
  • the present invention provides a delivery device comprising the liquid antibody preparation or solid antibody preparation of the present invention.
  • the delivery device of the present invention is provided in the form of a pre-filled syringe containing the liquid antibody preparation or solid antibody preparation of the present invention, for example for intravenous, subcutaneous, intradermal or intramuscular injection, intravenous infusion .
  • the present invention provides a method for delivering anti-CD47/PD-L1 bispecific antibody protein to a subject, such as a mammal, comprising the step of administering the liquid antibody preparation or solid antibody preparation of the present invention to the subject
  • 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 solid antibody preparation of the present invention for preparing treatment, prevention, or delay in a subject with SIRP ⁇ /CD47 signaling pathway and PD1/PD-L1 signaling pathway Delivery devices (eg, pre-filled syringes) or drugs for related conditions, such as various blood diseases and solid tumors, including but not limited to acute myelogenous leukemia (AML), chronic myelogenous leukemia, and acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphoma (NHL), multiple myeloma (MM), lymphoma, breast cancer, gastric cancer, lung cancer, esophageal cancer, bowel cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, Bladder cancer, glioma, melanoma and other solid tumors; autoimmune diseases and inflammatory conditions, such as allergic asthma or ulcerative colitis; rejection of cell or tissue or organ transplants, including non-human tissue transplants, including non
  • Figure 1 illustrates the structure of an anti-CD47/PD-L1 bispecific antibody, in which the first polypeptide chain is paired with the second polypeptide chain to form a first antigen binding site, and the third polypeptide chain contains a single domain first Two antigen binding sites and a single domain third antigen binding site, and there is a flexible connecting peptide between the single domain second antigen binding site and the single domain third antigen binding site of the third polypeptide chain.
  • Figure 2 shows the trends of protein purity in each sample measured by SEC-HPLC after anti-CD47/PD-L1 bispecific antibody preparations are placed at pH 6.4, 6.5, 6.8 and 7.0 at 40°C ⁇ 2°C for different times .
  • T0 on the abscissa in the figure represents 0 days; 2W represents 2 weeks; 1M represents 1 month.
  • Figure 3 shows the anti-CD47/PD-L1 bispecific antibody preparations at pH 6.4, 6.5, 6.8, and 7.0 at 40°C ⁇ 2°C for different periods of time, and the protein purity in each sample determined by the non-reducing CE-SDS method Change trend graph.
  • T0 on the abscissa in the figure represents 0 days; 2W represents 2 weeks; 1M represents 1 month.
  • Figure 4 shows the changes in the principal components of charge variants in each sample measured by the iCIEF method after anti-CD47/PD-L1 bispecific antibody preparations are placed at pH 6.4, 6.5, 6.8 and 7.0 at 40°C ⁇ 2°C for different times Trend. T0 on the abscissa in the figure represents 0 days; 2W represents 2 weeks; 1M represents 1 month.
  • Figure 5 shows that the anti-CD47/PD-L1 bispecific antibody preparations (prescription 2-5) with different stabilizers were stored at 40°C ⁇ 2°C for 0 days, 1 week, 2 weeks, and 4 weeks.
  • the main peak purity determined by SEC-HPLC method changes with time.
  • T0 on the abscissa in the figure represents 0 days; 1W represents 1 week; 2W represents 2 weeks; 4W represents 4 weeks.
  • Figure 6 shows that the anti-CD47/PD-L1 bispecific antibody preparations (prescription 2-5) with different stabilizers are stored at 40°C ⁇ 2°C for 0 days, 1 week, 2 weeks, and 4 weeks.
  • T0 on the abscissa in the figure represents 0 days; 1W represents 1 week; 2W represents 2 weeks; 4W represents 4 weeks.
  • Figure 7 shows that the anti-CD47/PD-L1 bispecific antibody preparations (prescription 2-5) with different stabilizers are stored at 40°C ⁇ 2°C for 0 days, 1 week, 2 weeks, and 4 weeks.
  • the main component of the charge variant measured by iCIEF method changes with time.
  • T0 on the abscissa in the figure represents 0 days; 1W represents 1 week; 2W represents 2 weeks; 4W represents 4 weeks.
  • the term “comprising” or “including” means to include 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 covers 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.
  • antibody is used in the broadest sense, and refers to a protein containing an antigen binding site, covering natural antibodies and artificial antibodies of various structures, including but not limited to tri-chain antibodies, 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 consists 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 terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant region does not directly participate in the binding of the antibody to the antigen, but exhibits multiple effector functions.
  • 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 the mammal under test 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-CD47/PD-L1 bispecific antibody preparation is also abbreviated as "antibody preparation of the present invention” herein, meaning that it contains anti-CD47/PD-L1 bispecific antibody protein as an active ingredient and contains pharmaceutically acceptable Preparation of excipients. After combining the anti-CD47/PD-L1 bispecific antibody protein with pharmaceutically acceptable excipients, the anti-CD47/PD-L1 bispecific antibody protein as the active ingredient is suitable for therapeutic or prophylactic administration to humans or non-humans animal.
  • 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-CD47/PD-L1 bispecific 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.
  • the antibody preparation is considered “stable.”
  • the anti-CD47/PD-L1 bispecific antibody protein preparation of the present invention exhibits low to undetectable antibody aggregation or degradation or chemical modification during manufacturing, preparation, transportation and long-term storage, As a result, there is little or no loss of biological activity of the anti-CD47/PD-L1 bispecific antibody protein, showing a high degree of stability.
  • the anti-CD47/PD-L1 bispecific antibody protein preparation 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 or at 40°C for at least 1 month, and/or at 2-8°C 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, stability testing is performed by performing various stress tests on antibody preparations.
  • the formulated anti-CD47/PD-L1 bispecific antibody protein preparation can be filled into glass vials to test the antibody stability under high temperature stress.
  • the preparation After a period of storage, 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.
  • the accumulation of antibodies in the preparation can potentially lead to an increased immune response in the patient, leading to safety issues. Therefore, there is a need to minimize or prevent aggregation of antibodies in the formulation.
  • Light scattering methods can be used to determine visible aggregates in the formulation.
  • 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, wherein 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 92% of the anti-CD47/PD-L1 bispecific 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 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-CD47/PD-L1 bispecific antibody protein list body.
  • the specific temperature at which the pharmaceutical preparation is stored 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, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C.
  • the pharmaceutical preparation is considered stable.
  • the anti-CD47/PD-L1 bispecific antibody protein monomer is considered stable. If stored at about 5°C for 9 months, at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% are detected The anti-CD47/PD-L1 bispecific antibody protein monomer is considered stable. If stored at about 5°C for 9 months, at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% are detected The anti-CD47/PD-L1 bispecific antibody protein monomer is considered stable.
  • the antibody in the preparation After a period of storage, if the antibody in the preparation does not show significant chemical changes, 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 modified 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 variant of the antibody in
  • the "acceptable degree" of chemical stability can mean that the percentage change of the charge variant (such as the main component or acidic component or alkaline component) in the preparation after storage at a certain temperature for a certain period of time does not exceed 30%, such as 20% .
  • the temperature for storing 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 percentage change value of the acid component charge variant is less than about 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% Or 0.1%, the pharmaceutical preparation can be considered stable.
  • the pharmaceutical preparation can also be regarded as stable.
  • the pharmaceutical preparation can also be considered stable.
  • 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.
  • High temperature stress refers to storing the antibody preparation at room temperature or even 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-CD47/PD-L1 bispecific antibody protein formulation of the present invention is administered to a subject parenterally.
  • the anti-CD47/PD-L1 bispecific antibody protein formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular or intravenous injection.
  • the present invention provides a stable liquid antibody preparation comprising (i) an anti-CD47/PD-L1 bispecific antibody protein, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, optionally, (V) other excipients are also included, and the pH of the antibody preparation is about 6.4-7.0.
  • the liquid antibody preparation of the present invention is in the form of an injection preparation.
  • the "anti-CD47/PD-L1 bispecific antibody protein" in the antibody preparation of the present invention is a three-chain antibody comprising a first polypeptide chain and a second polypeptide chain that specifically bind to CD47
  • the VH/VL pair serves as the first antigen binding site
  • the first VHH on the third polypeptide chain that specifically binds to PD-L1 serves as the single domain second antigen binding site and the second VHH serves as the single domain third Antigen binding site
  • a VH/VL pair that specifically binds PD-L1 on the first polypeptide chain and the second polypeptide chain as the first antigen binding site, and the specific binding on the third polypeptide chain
  • the first VHH of CD47 serves as the single domain second antigen binding site and the second VHH as the single domain third antigen binding site.
  • the anti-CD47/PD-L1 bispecific antibody protein is capable of binding to CD47 with an affinity constant of at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or stronger , And can bind to PD-L1 with an affinity constant of at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or more, so that the antibody can be used as a dual A therapeutic and/or preventive agent that specifically targets CD47 molecules and PD-L1 molecules.
  • VH/VL pair that specifically binds PD-L1 or CD47, it includes 6 anti-PD-L1 antibodies reported in the prior art and 6 anti-PD-L1 antibody VH/VL pairs developed in the future A CDR or a sequence having one, two, three, four, five, six or more amino acid changes (for example, amino acid substitutions or deletions) with one or more of the 6 CDRs; or Contains 6 CDRs derived from anti-CD47 antibodies reported in the prior art and anti-CD47 antibody VH/VL pairs developed in the future or has one, two, three CDRs with one or more of the 6 CDRs A sequence of one, four, five, six or more amino acid changes (e.g., amino acid substitutions or deletions).
  • first VHH and the second VHH that specifically bind PD-L1 or CD47, they are both derived from the heavy chain variable domains of antibodies that naturally lack light chains (such as the naturally occurring heavy chain in Camelidae species). Chain antibody heavy chain variable domain).
  • the first VHH and the second VHH may be the same or different.
  • the first VHH and the second VHH may be derived from antibodies produced in camelid species such as camels, alpacas, dromedaries, llamas, and guanacos. Other species besides Camelidae can also produce heavy chain antibodies that naturally lack light chains, and such VHHs are also within the scope of the antibody protein of the present invention.
  • the first VHH and the second VHH comprise 3 CDRs derived from any anti-PD-L1 antibody reported in the prior art and an anti-PD-L1 antibody VHH developed in the future, or one or one of the 3 CDRs.
  • Multiple CDRs have a sequence of one, two, three, four, five, six or more amino acid changes (for example, amino acid substitutions or deletions); or include anti-CD47 derived from any of the prior art reports
  • the 3 CDRs of the antibody and the anti-CD47 antibody VHH developed in the future have one, two, three, four, five, six or more amino acids with one or more of the three CDRs
  • the sequence of changes e.g., amino acid substitutions or deletions).
  • the VH/VL pair that specifically binds to CD47 on the first polypeptide chain and the second polypeptide chain of the anti-CD47/PD-L1 bispecific antibody protein comprises a VH/VL pair derived from Chinese Patent Application No.
  • VH CDR1 shown in GSIEHYYWS (SEQ ID NO: 3) of the reported anti-CD47 antibody ADI-29341
  • VH CDR1 shown in YIYYSGSTNYNPSLKS (SEQ ID NO: 4) shown in ARGKTGSAA (SEQ ID NO: 5)
  • One or more of the CDRs have a sequence of one, two, three, four, five, six or more amino acid changes (for example, amino acid substitutions or deletions).
  • the first VHH and the second VHH on the third polypeptide chain of the anti-CD47/PD-L1 bispecific antibody protein that specifically bind to PD-L1 both comprise AYTISRNSMG (SEQ ID NO: 17 ) Shown in CDR1, CDR2 shown in IESDGST (SEQ ID NO: 18) and CDR3 shown in AAPKVGLGPRTALGHLAFMTLPALNY (SEQ ID NO: 19), or have one or two CDRs with one or more of the three CDRs A sequence of one, three, four, five, six or more amino acid changes (e.g., amino acid substitutions or deletions).
  • CDR or “complementarity determining region” or “CDR region” (used interchangeably with hypervariable region “HVR” herein) is an amino acid region in the variable region of an antibody that is mainly responsible for binding to an epitope.
  • the CDRs of the heavy and light chains are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • CDR1, CDR2, and CDR3 are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • Various schemes for determining the CDR sequence of a given VH or VL or VHH amino acid sequence are known in the art. For example, the Kabat complementarity determining region (CDR) is determined based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
  • amino acid changes for example, amino acid substitutions are preferably conservative amino acid substitutions.
  • conservative amino acid substitution refers to an amino acid change that results in the substitution of a certain amino acid with a chemically similar amino acid. It is well known in the art to provide conservative substitution tables of functionally similar amino acids.
  • the conservatively substituted residues are from conservative substitution table A below, preferably the preferred substituted residues shown in Table A.
  • 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; Norleucine Leu
  • the VH/VL pair that specifically binds CD47 on the first polypeptide chain and the second polypeptide chain of the anti-CD47/PD-L1 bispecific antibody protein comprises an anti-CD47 antibody ADI-
  • the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 2/9 of 29341, or the paired heavy chain variable region sequence/light chain variable region sequence has at least 90%, A sequence of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • the first VHH and the second VHH on the third polypeptide chain of the anti-CD47/PD-L1 bispecific antibody protein that specifically bind to PD-L1 comprise SEQ ID NO: 15 and/or The amino acid sequence shown in SEQ ID NO: 16, or substantially identical to it (for example, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) sequence.
  • the type of the heavy chain constant region of the immunoglobulin in the first polypeptide chain and the third polypeptide chain in the anti-CD47/PD-L1 bispecific antibody protein is not particularly limited, and is preferably an IgG1, IgG2 or IgG4 immunoglobulin Or a sequence that is substantially identical to (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to the constant region of the heavy chain. More preferably, the heavy chain constant region is the heavy chain constant region of human IgG1 immunoglobulin, or is substantially the same (for example, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) sequence.
  • the anti-CD47/PD-L1 bispecific antibody protein comprises the heavy chain constant region used in IgG4 (e.g., human IgG4). In yet another embodiment, the anti-CD47/PD-L1 bispecific antibody protein comprises a heavy chain constant region for IgG1 (e.g., human IgG1).
  • the Fc domains of the first polypeptide chain and the third polypeptide chain of the tri-chain antibody contain hinge regions with "CPPC" amino acid residues, and/or Y349C and S354C (according to Kabat's "EU Number "), thus, the first polypeptide chain and the third polypeptide chain form an interchain disulfide bond in the Fc region, thereby stabilizing the correct pairing of the first polypeptide chain and the third polypeptide chain.
  • the first polypeptide chain and/or the third polypeptide chain of the anti-CD47/PD-L1 bispecific antibody protein contains amino acid mutations in the Fc domain that affect the effector function of the antibody.
  • the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the amino acid mutation is present in the CH2 domain of the Fc region, for example, the anti-CD47/PD-L1 bispecific antibody protein is contained in the first polypeptide chain and/or the third polypeptide chain.
  • Amino acid substitutions at positions 234 and 235 EU numbering
  • the amino acid substitutions are L234A and L235A (also referred to as "LALA mutations").
  • the second polypeptide chain of the anti-CD47/PD-L1 bispecific antibody protein comprises a kappa light chain constant region or a lambda light chain constant region, for example, a human kappa light chain constant region or a human lambda light chain Constant region.
  • the Fc domains of the first polypeptide chain and the third polypeptide chain of the anti-CD47/PD-L1 bispecific antibody protein respectively contain bumps ("knobs") or holes ("Hole"), and the protrusions or holes in the Fc domain of the first polypeptide chain can be respectively placed in the holes or protrusions in the Fc domain of the third polypeptide chain,
  • the first polypeptide chain and the third polypeptide chain form a "knob-in-hole" stable association with each other.
  • the amino acid substitution T366W is included in one of the first polypeptide chain and the third polypeptide chain, and the amino acid substitution T366W is included in the other of the first polypeptide chain and the third polypeptide chain Amino acid substitutions T366S, L368A and Y407V (EU numbering).
  • the bulge in one chain can be placed in the cavity in the other chain to promote the correct pairing of the first polypeptide chain and the third polypeptide chain.
  • the immunoglobulin CH1 domain and CL domain of the first polypeptide chain and the second polypeptide chain of the anti-CD47/PD-L1 bispecific antibody protein contain bulges or holes, respectively, and The protrusions or holes in the CH1 domain can be placed in the holes or protrusions in the CL domain, respectively, so that the first polypeptide chain and the second polypeptide chain also form a "knot-in" with each other. Stable association of “buckle”.
  • the anti-CD47/PD-L1 bispecific antibody protein comprises the first polypeptide chain shown in SEQ ID NO: 1, the second polypeptide chain shown in SEQ ID NO: 8, and SEQ ID NO The third polypeptide chain shown in :14, or substantially identical to any of the sequences (for example, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more Highly identical) sequence.
  • the anti-CD47/PD-L1 bispecific antibody protein comprises the first polypeptide chain shown in SEQ ID NO: 1, the second polypeptide chain shown in SEQ ID NO: 8, and SEQ ID
  • 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 “percentage of sequence identity” can be calculated in the following way: the two best aligned sequences are compared in the comparison window, and it is determined that the same nucleic acid base (for example, A, T, C, G, I) exists 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 algorithm needed to achieve the maximum alignment within the full-length sequence being compared or within the target sequence region.
  • the anti-CD47/PD-L1 bispecific antibody protein in the antibody preparation of the present invention can bind to PD-L1 and CD47 proteins at the same time, and maintains the affinity constant of each parent antibody, thereby blocking the SIRP ⁇ /CD47 signal transduction pathway And block the PD1/PD-L1 signaling pathway, thereby being used to treat, prevent or delay various diseases or disorders related to the SIRP ⁇ /CD47 signaling pathway and/or the PD1/PD-L1 signaling pathway.
  • the anti-CD47/PD-L1 bispecific antibody protein of the present invention is the recombinant anti-CD47/PD disclosed in the PCT application of PCT/CN2018/123886 (application date: December 26, 2018) -L1 bispecific antibody protein, which has the first polypeptide chain shown in SEQ ID NO: 1, the second polypeptide chain shown in SEQ ID NO: 8, and the third polypeptide chain shown in SEQ ID NO: 14 Peptide chain.
  • the anti-CD47/PD-L1 bispecific antibody protein is recombinantly expressed and purified by HEK293 cells or CHO cells.
  • the antibody in the liquid formulation of the present invention exhibits significant anti-tumor activity.
  • the anti-CD47/PD-L1 bispecific antibody was administered to tumor-bearing mice produced by inoculating NOD-SCID mice with Raji-PD-L1 cells.
  • the results showed that it was incompatible with the administration of anti-CD47 monoclonal antibodies and anti-PD-L1 monoclonal antibodies
  • the administration of anti-CD47/PD-L1 bispecific antibodies has significantly improved anti-tumor activity, which can lead to a tumor growth inhibition rate of about 90% or higher, such as 100%; and/or a tumor disappearance rate of more than 50% .
  • the anti-CD47/PD-L1 bispecific antibody also exhibits significantly reduced hemagglutination, so it will have significantly reduced side effects in clinical treatment.
  • the amount of the anti-CD47/PD-L1 bispecific antibody protein contained in the antibody preparation of the present invention can be changed according to the specific purpose characteristics of the preparation, the specific environment, and the specific purpose of using the preparation.
  • the antibody formulation is a liquid formulation, which may contain about 5-150 mg/mL, such as about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 mg/ml of anti-CD47/PD-L1 bispecific antibody protein.
  • the buffer is an agent that can maintain the pH of the solution within an acceptable range.
  • the buffer 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 6.4-7.0, for example, a pH of about 6.5.
  • the antibody preparation of the invention has a pH of about 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0.
  • the buffer used in the formulation of the present invention is selected from histidine, histidine hydrochloride, and combinations thereof.
  • the concentration of the buffer in the liquid antibody formulation of the present invention is about 1-30 mM. In one embodiment, the concentration of the buffer in the liquid antibody formulation of the present invention is about 5-25 mM, for example, about 5, 10, 15, 20, 25 mM.
  • the buffer used in the formulation of the invention is a combination of about 16.3 mM histidine and about 3.77 mM histidine hydrochloride.
  • Suitable stabilizers for use in the present invention may be selected from sugars, polyols and amino acids and combinations thereof.
  • Sugars used as stabilizers include, but are not limited to, sucrose and trehalose.
  • the polyol used as a stabilizer includes but is not limited to sorbitol.
  • the amino acids used as stabilizers include but are not limited to arginine and arginine hydrochloride.
  • the stabilizer is present in the liquid formulation of the present invention at a concentration of about 50-500 mM, more preferably about 100-400 mM, for example, about 100, 150, 200, 250, 300, 350, 400 mM .
  • the liquid formulation of the present invention contains sucrose as a stabilizer.
  • the amount of sucrose in the liquid formulation of the present invention may be about 50-250 mM, preferably about 100-200 mM (for example, about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM).
  • the liquid formulation of the present invention contains arginine and/or arginine hydrochloride as a stabilizer.
  • the amount of arginine and/or arginine hydrochloride in the liquid formulation of the present invention may be about 50-250 mM, preferably about 100-200 mM (for example, about 100, 110, 120, 130, 140, 150, 160, 170 , 180, 190, 200mM).
  • the liquid formulation of the present invention contains a combination of sucrose, arginine and/or arginine hydrochloride as a stabilizer.
  • sucrose may be present in an amount of about 50-250 mM, preferably about 100-200 mM (e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM).
  • arginine and/or arginine hydrochloride may be at about 50-250mM, preferably about 100-200mM (e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200mM).
  • surfactant refers to an organic substance having 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.
  • the surfactant in the liquid formulation of the present invention is a nonionic surfactant, for example, alkyl poly(ethylene oxide).
  • specific nonionic surfactants that can be included in the formulation of the present invention include, for example, polysorbates such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; Nick waits.
  • the liquid 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 can be changed according to the specific purpose characteristics of the preparation, the specific environment, and the specific purpose of using the preparation.
  • the formulation may contain about 0.1-1 mg/ml, preferably about 0.2-0.8 mg/ml, for example about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml of polysorbate Class surfactants (for example, polysorbate-80).
  • the antibody liquid preparation of the present invention may or may not contain other excipients.
  • the antibody liquid formulation of the present invention contains a metal chelator (for example, EDTA or a salt thereof) as an excipient. In another embodiment, the antibody liquid formulation of the present invention does not contain a metal chelator (for example, EDTA or a salt thereof). In one embodiment, the liquid antibody formulation of the present invention to which a metal chelating agent (e.g., EDTA or its salt) is added has higher stability than a corresponding formulation without adding a metal chelating agent (e.g., EDTA or its salt) .
  • a metal chelating agent e.g., EDTA or its salt
  • excipients can also be used in the formulation of the present invention.
  • the excipients include, for example, flavoring agents, antimicrobial agents, sweeteners, antistatic agents, antioxidants, 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 present invention provides a stable formulation containing anti-CD47/PD-L1 bispecific antibody protein.
  • the anti-CD47/PD-L1 bispecific antibody protein used in the formulation of the present invention can be prepared using techniques known in the art for antibody production.
  • the anti-CD47/PD-L1 bispecific antibody protein can be recombinantly prepared.
  • the anti-CD47/PD-L1 bispecific antibody protein of the present invention is prepared by recombinant expression in HEK293 cells or CHO cells, for example, as described in PCT/CN2018/123886, recombinantly prepared antibodies CD47/PD-L1 bispecific antibody protein.
  • recombinantly produced 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. After obtaining antibodies of sufficient purity, preparations containing antibodies can be prepared according to methods known in the art.
  • the following steps can be used for preparation: (1) After 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 capture antibody
  • 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 for measuring the purity of monoclonal antibodies using capillary as the 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 the 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 for analysis.
  • a UV detector is used to detect the migration of the protein and obtain an electrophoretic 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 monoclonal antibody standards and quality control. This method is mainly based on the molecular size or hydrodynamic radius difference to separate 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 of the sum of all peak areas.
  • the SEC-HPLC method the percentage of antibody monomers in the preparation product can be measured, and the content information of soluble aggregates and shears can be given.
  • Imaging capillary isoelectric focusing electrophoresis can be used to analyze the charge heterogeneity of monoclonal antibodies. This method can provide 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 methylcellulose, pi molecular weight standards and ampholytes.
  • 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 peak eluted before the main peak ie, the main component
  • the protein-related peak eluted after the main peak is classified as the basic component.
  • the relative amounts of the main component, acidic component and basic component 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.
  • the formulation sample 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 efficacy or biological activity of the antibody can be tested.
  • the binding ability of antibodies and their antigen molecules (CD47 molecules and PD-L1 molecules) in the preparation can be tested.
  • Those skilled in the art know that a variety of methods can be used to quantify the specific binding of antibodies to antigens, such as immunoassay tests, ELISA, and the like.
  • the anti-CD47/PD-L1 bispecific antibody protein preparation of the present invention is stable. In one embodiment, after storage at about 25°C, 37°C, 40°C, or 45°C for at least 1 month or 2 months, for example, after storage at 40°C ⁇ 2°C for 1 month, the antibody preparation of the present invention
  • the protein purity of the anti-CD47/PD-L1 bispecific antibody in is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more , As determined by size exclusion chromatography or by non-reduced CS-SDS.
  • the antibody preparation of the present invention At least 50%, preferably at least 55% of the intermediate anti-CD47/PD-L1 bispecific antibody protein is in non-basic and non-acidic form (ie, main peak or main charge form), as determined by the CEX-HPLC method.
  • the antibody preparation of the present invention comprising the anti-CD47/PD-L1 bispecific antibody protein of the present invention can be used to treat, prevent or delay various signal transduction pathways with SIRP ⁇ /CD47 and/or PD1/PD-L1 signal transduction pathways Related diseases or conditions.
  • Disease or disorder related to the SIRP ⁇ /CD47 signaling pathway and/or “Disease or disorder related to the PD1/PD-L1 signaling pathway” herein means that the anti-CD47/PD-L1 bispecific antibody of the present invention can be used
  • the preparation of the present invention containing the anti-CD47/PD-L1 bispecific antibody protein can be used to prevent or treat various blood diseases and solid tumors in subjects, including but not limited to acute myelogenous leukemia (AML), Chronic myelogenous leukemia, acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL), multiple myeloma (MM), lymphoma, breast cancer, gastric cancer, lung cancer, esophageal cancer, bowel cancer, ovarian cancer, Cervical cancer, kidney cancer, pancreatic cancer, bladder cancer, glioma, melanoma and other solid tumors.
  • AML acute myelogenous leukemia
  • ALL acute lymphocytic leukemia
  • NHL non-Hodgkin lymphoma
  • MM multiple myeloma
  • lymphoma breast cancer
  • gastric cancer lung cancer
  • lung cancer esophageal cancer
  • bowel cancer ovarian cancer
  • the preparation of the present invention containing the anti-CD47/PD-L1 bispecific antibody protein also has Potential benefits for use in human stem cell transplantation.
  • the preparation of the present invention comprising the anti-CD47/PD-L1 bispecific antibody protein can be used to treat, prevent or diagnose autoimmune diseases and inflammatory disorders mediated by SIRP ⁇ + cells in a subject, for example, Allergic asthma or ulcerative colitis.
  • autoimmune diseases and inflammatory disorders mediated by SIRP ⁇ + cells for example, Allergic asthma or ulcerative colitis.
  • These conditions include acute and chronic inflammatory conditions, allergic and allergic diseases, autoimmune diseases, ischemic conditions, severe infections, and rejection of cell or tissue or organ transplants, including non-human tissue transplants (xenografts) ) Repulsion, etc.
  • the present invention also provides the use of the preparation of the present invention in the preparation of a medicament, wherein the medicament is used to deliver anti-CD47/PD-L1 bispecific antibody protein to mammals, or to treat, prevent or ameliorate the aforementioned diseases and disorders One or more of.
  • the mammal is a human.
  • the antibody formulation of the present invention can be administered to a subject or patient in various ways.
  • administration can be by infusion or by syringe.
  • the present invention provides a delivery device (e.g., a syringe) comprising the antibody formulation of the present invention (e.g., a pre-filled syringe).
  • the patient will receive an effective amount of anti-CD47/PD-L1 bispecific antibody protein as the main active ingredient, that is, an amount sufficient to treat, ameliorate or prevent the target disease or condition.
  • 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 patient’s age, weight, health and/or gender, the nature and extent of the disease, the activity of the particular antibody, and the body’s 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 effective dose may be about 0.005 mg/kg body weight to about 50 mg/kg body weight, or about 0.1 mg/kg body weight to about 20 mg/kg body weight.
  • the application of known antibody-based drugs can provide some guidance.
  • the dosage can be a single dose schedule or a multiple dose schedule.
  • CE-SDS Sodium Lauryl Sulfate Capillary Gel Electrophoresis
  • FLD-HPLC fluorescence detection-high performance liquid chromatography
  • iCIEF Imaging Capillary Isoelectric Focusing Electrophoresis
  • N/A means "Not applicable”.
  • 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. Dilute the sample to be tested with ultrapure water to 2mg/ml as the test solution. Take the preparation buffer solution and dilute it with the same treatment method as the 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, adjust the pH to 6.8 with hydro
  • 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.
  • iCIEF method imaging capillary isoelectric focusing electrophoresis (iCIEF method) detection.
  • the inner diameter of the capillary is 100 ⁇ m and the total length is 5cm.
  • MC solution 0.5% methylcellulose solution
  • ultrapure water should be used to rinse the capillary column.
  • the vacuum injection method is used to inject for 55 seconds, the pre-focusing voltage and time are 1.5kV and 1 minute, the focusing voltage and time are 3kV and 8 minutes, and the sampling time is 55 seconds.
  • the wavelength is 280nm.
  • Cathodic Stabilizer is 500mmol/L arginine solution
  • Anodic Stabilizer is 200mmol/L iminooxalic acid
  • 3mol/L urea improves protein solubility
  • 0.5% MC solution reduces protein and capillary The adhesion between. Dilute the test product to 0.5 mg/ml with water, take 20 ⁇ l of the diluted test product solution, add 83 ⁇ l of the premix solution to it and mix well to prepare the test sample solution. Use the preparation buffer to operate in the same way to prepare a blank solution.
  • Dilute streptavidin (Thermo, catalog number: 21125) with 1 ⁇ PBS to 1 ⁇ g/ml, 100 ⁇ l/well, and coat on 96-well microtiter plate at 37°C for 2 hours. After washing the plate, add blocking solution (5% FBS, 300 ⁇ l/well) at 37°C for 2h.
  • the anti-CD47/PD-L1 bispecific antibody was diluted with 2% FBS to 40 ⁇ g/ml in 100 ⁇ l/well, and diluted 4-fold to the 12th concentration (0.01 ⁇ 10000ng/ml).
  • the anti-CD47/PD-L1 bispecific antibody Kh2NF-PC was recombinantly expressed and purified in HEK293 cells (purchased from INVITROGEN).
  • the anti-CD47/PD-L1 bispecific antibody Kh2NF-PC antibody is composed of 3 polypeptide chains, and each polypeptide chain has the following amino acid sequences from N-terminus to C-terminus:
  • the peptide chain #1 contains the following VH amino acid sequence derived from the anti-CD47 antibody ADI29341:
  • amino acid sequence of the Fc region derived from human IgG1 at the C-terminus of the CH1 amino acid sequence is as follows:
  • the peptide chain #2 contains the following VL amino acid sequence derived from the anti-CD47 antibody ADI29341:
  • the peptide chain #3 contains the following first and second anti-PD-L1 VHH amino acid sequences:
  • amino acid sequence of the connecting peptide between the first and second anti-PD-L1VHH amino acid sequences GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 20);
  • amino acid sequence of the Fc region derived from human IgG1 at the C-terminus of the second anti-PD-L1VHH amino acid sequence is as follows:
  • Example 2 One of the test of the influence of pH on the stability of the formulation
  • This example examines the stability of formulations containing anti-CD47/PD-L1 bispecific antibodies at pH 5.0 to 6.5.
  • a total of 4 pH values were designed, namely 5.0, 5.5, 6.0 and 6.5.
  • x means sampling at this point in time.
  • the obtained samples are first put in an ultra-low temperature refrigerator and stored for inspection, and defrosted and sent for inspection as needed.
  • the standard for determining whether the quality of the sample has not changed compared with the initial value is set to determine whether the sample has changed. See Table 2 for details.
  • N/A means that the appearance of the sample is unqualified and has not been tested.
  • N/A means that the appearance of the sample is unqualified and has not been tested.
  • the protein purity of each sample was determined by the non-reduced CE-SDS method and the reduced CE-SDS method. The results are shown in Table 5 and Table 6. The results showed that the protein purity of the samples with pH 6.5 value was decreased by 7.7% and 3.7% respectively compared with the samples of 0 days under the condition of 40°C ⁇ 2°C for 1 month.
  • N/A means that the appearance of the sample is unqualified and has not been tested.
  • N/A means that the appearance of the sample is unqualified and has not been tested.
  • N/A means that the appearance of the sample is unqualified and has not been tested.
  • N/A 1 means that the sample is unqualified and has not been tested
  • N/A 2 means that the test item is not set.
  • N/A means that the detection item is not set.
  • N/A means that the detection item is not set.
  • N/A means that the detection item is not set.
  • N/A means that the detection item is not set.
  • Example 2 and Example 3 show that the anti-CD47/PD-L1 bispecific antibody (for example, Kh2NF-PC) protein is placed at pH 5.0 ⁇ 6.2 at 40°C ⁇ 2°C. Months, with the extension of time, the appearance of the sample will appear turbid or milky white precipitation; and when placed at pH 6.4-7.0 at 40 °C ⁇ 2 °C for one month, the appearance of the sample and visible foreign matter are both qualified, and the protein content has not changed significantly. The relative binding activity of CD47 and PD-L1 did not change significantly. Therefore, in the subsequent examples, pH 6.5 is selected from pH 6.4-7.0 for subsequent experiments.
  • Kh2NF-PC bispecific antibody
  • a total of 5 prescriptions were designed, and the detailed prescription information is shown in Table 14.
  • adjust the protein content of each prescription to about 100.0mg/ml; add polysorbate 80 to make the final concentration of polysorbate 80 0.20mg/ml; filter and dispense into vials, stopper and cap.
  • the stability of each sample was investigated under the condition of 40°C ⁇ 2°C.
  • the specific scheme is shown in Table 15.
  • the detection indicators are appearance, visible foreign matter, protein content, purity (SEC-HPLC method) and charge variants (iCIEF method).
  • Prescription 1 20mM histidine, 5% sorbitol, 0.02% polysorbate 80, pH 6.0
  • Prescription 3 20mM histidine, 8% trehalose, 0.02% polysorbate 80, pH 6.5
  • Prescription 4 20mM histidine, 180mM arginine hydrochloride, 0.02% polysorbate 80, pH 6.5
  • Prescription 5 20mM histidine, 4% sucrose, 100mM arginine hydrochloride, 0.02% polysorbate 80, pH 6.5
  • prescription 1 has turbidity or precipitation after one week of inspection; the appearance and visible foreign matter of other prescription samples are qualified.
  • N/A means that the appearance of the sample is unqualified and has not been tested.
  • N/A means that the appearance of the sample is unqualified and has not been tested.
  • N/A means that the appearance of the sample is unqualified and has not been tested
  • N/A means that the appearance of the sample is unqualified and has not been tested
  • the osmotic pressure was measured on the 0 o'clock (T0) samples of prescription 4 and prescription 5. Each group of samples was measured twice, and the results were averaged.
  • Table 20 shows the results of osmotic pressure measurement on the samples of prescription 4 and prescription 5 at time 0 (T0).
  • the osmolality of human plasma is about 285-310 mOsmol/kg
  • the osmolarity of prescription 4 and prescription 5 are within the acceptable range of osmolality of pharmaceutical preparations.
  • the osmotic pressure of prescription 4 is closer to that of human plasma, it is preferable to use the preparation of prescription 4.
  • EDTA is a representative metal chelator. This example investigated the effect of EDTA on the protein stability of the anti-CD47/PD-L1 bispecific antibody Kh2NF-PC.
  • prescription 6 was a control group without EDTA
  • prescription 7 was a group with a final concentration of 0.02mg/ml EDTA. See Table 21 for detailed prescription information.
  • Table 23 show that from the detection results of the charge variant (CEX-HPLC method), prescription 7 shows a better advantage than the main peak of the charge variant of prescription 6; from polysorbate 80 (FLD-HPLC) According to the test results of method), the content of polysorbate 80 in prescription 6 decreased with time; in prescription 7, the metal chelating agent EDTA was added to inhibit the degradation of polysorbate 80 caused by metal ions, so prescription 7 Better than prescription 6.
  • EDTA can bind metal ions, and it can inhibit the degradation of polysorbate 80 from at least the following two aspects.
  • metal chelating agent added in the formulation can inhibit the degradation of polysorbate 80 by binding metal ions and improve the stability of the formulation.
  • the most preferred formulation is: about 100.0 mg/ml recombinant anti-differentiation antigen cluster 47 (CD47) and anti-programmed death ligand 1 (PD-L1) bispecific antibody, about 2.52 mg/ml histamine Acid, 0.79mg/ml histidine hydrochloride, 37.92mg/ml arginine hydrochloride, 0.50mg/ml polysorbate 80, 0.02mg/ml EDTA, pH 6.5.
  • CD47 recombinant anti-differentiation antigen cluster 47
  • PD-L1 anti-programmed death ligand 1
  • Example 5 Using the preparation protocol of Example 5 (about 100.0 mg/ml recombinant anti-differentiation antigen cluster 47 (CD47) and anti-programmed death ligand 1 (PD-L1) bispecific antibody, about 2.52 mg/ml histidine, 0.79 (mg/ml histidine hydrochloride, 37.92mg/ml arginine hydrochloride, 0.50mg/ml polysorbate 80, 0.02mg/ml EDTA, pH 6.5) prepare 500L of preparation, and conduct stability investigation.
  • CD47 recombinant anti-differentiation antigen cluster 47
  • PD-L1 anti-programmed death ligand 1
  • 500L The following preparations of 500L were prepared: 101.8mg/ml recombinant anti-CD47/PD-L1 bispecific antibody protein, 2.52mg/ml histidine, 0.79mg/ml histidine hydrochloride, 37.92mg/ml arginine hydrochloride , 0.50mg/ml polysorbate 80, 0.02mg/ml EDTA, pH 6.5, used to investigate its stability.
  • the main peak + fragment 100%, as long as the main peak meets the condition of ⁇ 90%, the remaining ⁇ 10% is the amount of fragments. Therefore, the amount of fragments is given as report data. Same below.
  • the content of heavy chain and light chain + non-glycosylated heavy chain + fragment 100%, as long as the content of heavy chain and light chain meets the condition of ⁇ 90%, the remaining ⁇ 10% It is the amount of non-glycosylated heavy chains and fragments. Therefore, the respective data of the amount of non-glycosylated heavy chains and the amount of fragments are given as report data. Same below.
  • the main component + acidic component + alkaline component 100%, as long as the amount of the main component meets the condition of ⁇ 44.9%, the remaining ⁇ 55.1% is the acidic component and the alkaline component Therefore, the respective data of the amount of acidic components and the amount of alkaline components are given as report data. Same below.
  • the regulations are: the relevant regulations of the Pharmacopoeia of the People's Republic of China (2015 Edition, Part Three), the same below, for example, the general rule 0904 "Visible Foreign Body Inspection Law" stipulates the inspection of visible foreign bodies.
  • 1.N/A means no detection.
  • the corresponding index generally only detects the first and last end points. If the first and last end points are both in line with the requirements, the value of each point in the process is considered to be in line with the requirements.
  • the main peak + NGHC CD47 + fragment 100%. As long as the main peak meets the condition of ⁇ 90%, the remaining ⁇ 10% is the amount of NGHC CD47 chains and fragments. Therefore, the amount of NGHC CD47 The respective data of the amount and the amount of fragments are given as report data.
  • N/A means no detection.
  • the corresponding index generally only detects the first and last endpoints. If both the first and last endpoints meet the requirements, the value of each point in the process is considered to meet the requirements.
  • N/A means no detection.
  • the corresponding index generally only detects the first and last endpoints. If both the first and last endpoints meet the requirements, the value of each point in the process is considered to meet the requirements.

Abstract

本发明涉及包含抗CD47/PD-L1双特异性抗体的制剂，尤其涉及包含抗CD47/PD-L1双特异性抗体、缓冲剂、稳定剂和表面活性剂的药物制剂。此外，本发明还涉及这些制剂的疾病治疗或预防用途。

Description

包含抗CD47/PD-L1双特异性抗体的制剂及其制备方法和用途 技术领域
本发明涉及抗体制剂领域。更具体而言，本发明涉及包含重组的抗分化抗原簇47(CD47)和抗程序性死亡配体1(PD-L1)双特异性抗体(也称为抗CD47/PD-L1双特异性抗体)的药物制剂，尤其是稳定的高浓度抗体液体制剂，以及用于制备所述药物制剂的方法，以及所述药物制剂的治疗和/或预防用途。
背景技术
PD-L1(也称为分化抗原簇274(CD274)或B7同源物1(B7-H1))，是40kDa I型跨膜蛋白。PD-L1与活化的T细胞上存在的其受体PD-1结合，下调T细胞活化(Latchman等人，2001 Nat Immunol 2：261-8；Carter等人，2002 Eur J Immunol 32：634-43)。已经在许多癌中发现了PD-L1表达，包括人肺癌、卵巢癌、结肠癌和多种骨髓瘤等，并且PD-L1表达经常与癌的预后不良相关(Iwai等人(2002)PNAS 99:12293-7；Ohigashi等人(2005)Clin Cancer Res 11:2947-53；Okazaki等人(2007)Intern.Immun.19:813-24；Thompson等人(2006)Cancer Res.66:3381-5)。已经提出通过抑制PD1与PD-L1的局部相互作用可以在一部分肿瘤患者中逆转免疫抑制。
罗氏(Roche)研发的抗PD-L1抗体Atezolizumab、德国默克(Merck KGaA)和美国辉瑞(Pfizer)合作开发的抗PD-L1抗体Avelumab、阿斯利康研发的Durvalumab显示了对部分肿瘤患者的治疗效果。其它抗PD-L1抗体包括YW243.55.S70(重链和轻链可变区显示在WO2010/077634中的SEQ ID NOs 20和21中)和WO2007/005874中公开的抗PD-L1抗体等。
分化抗原簇47(CD47)，也被称为整联蛋白相关蛋白(IAP)，是免疫球蛋白超家族成员。CD47与主要由巨噬细胞和树突细胞表达的一种作为其配体的细胞表面免疫球蛋白SIRPα相互作用，产生一系列的级联反应，并由此抑制巨噬细胞和树突细胞对表达CD47的细胞的摄取和吞噬作用。在肿瘤中观察到存在CD47的过度表达。但是CD47在许多正常组织中也有表达，这导致仅以CD47为靶点的抗体通常与正常血液系统细胞的非特异性结合，引起了抗原沉默(antigen sink)现象。
抗CD47/PD-L1双特异性抗体由于能够同时靶向CD47和肿瘤细胞上的PD-L1，通过与肿瘤细胞上PD-L1的特异性结合促进了抗CD47/PD-L1双特异性抗体对肿瘤细胞的选择性结合，避免了与许多正常组织中表达的CD47结合，因此，抗CD47/PD-L1双特异性抗体具有在增强抗肿瘤作用的同时减少副作用的优势。
PCT/CN2018/123886的PCT申请(申请日：2018年12月26日)公开了一种新型的抗体样式，并构建和表达了具有所述新型抗体样式的抗CD47/PD-L1双特异性抗体。对使用Raji-PD-L1细胞接种NOD-SCID小鼠产生的荷瘤小鼠施用抗CD47/PD-L1双特异性抗体，结果表明，与施用抗CD47单克隆抗体和抗PD-L1单克隆抗体相比较，抗CD47/PD-L1双特异性抗体具有显著提高的抗肿瘤活性，能够显著抑制肿瘤的生长，甚至能使得肿瘤完全消失。此外，抗CD47/PD-L1双特异性抗体还表现出显著降低的血细胞凝集作用，因此在临床治疗中将具有显著降低的副作用。本领域中需要能够用来治疗、预防或延缓各种与SIRPα/CD47信号传导通路和PD1/PD-L1信号传导通路相关的疾病的抗CD47/PD-L1双特异性抗体制剂。
抗体制剂除了需要以使抗体适于施用给受试者的方式调配之外，还需要以在储存以及后续使用期间维持其稳定性的方式来调配。例如，如果抗体没有适当地在液体中得以调配，则液体溶液中的该抗体倾向于分解、聚集或发生不希望的化学修饰等。抗体在抗体制剂中的稳定性取决于制剂中所使用的缓冲剂、稳定剂和表面活性剂等。
尽管已知一些抗CD47/PD-L1双特异性抗体，但在本领域中对于含有足够稳定且适于施用给受试者的抗CD47/PD-L1双特异性抗体的新颖药物制剂仍存在需要。因此，需要合适的 抗CD47/PD-L1双特异性抗体制剂，以用来治疗或预防疾病。
发明概述
本发明通过提供含有特异结合CD47和PD-L1的抗CD47/PD-L1双特异性抗体蛋白的药物制剂来满足上述需求。
在一个方面，本发明提供了一种液体抗体制剂，其包含(i)抗CD47/PD-L1双特异性抗体蛋白；(ii)缓冲剂，(iii)稳定剂，和(iv)表面活性剂。
本发明抗体制剂中包含的抗CD47/PD-L1双特异性抗体蛋白是一种三链抗体，所述三链抗体包含第一多肽链和第二多肽链上的特异性结合CD47的VH/VL对作为第一抗原结合位点，以及第三多肽链上的特异性结合PD-L1的第一VHH作为单结构域第二抗原结合位点和第二VHH作为单结构域第三抗原结合位点；或者包含第一多肽链和第二多肽链上的特异性结合PD-L1的VH/VL对作为第一抗原结合位点，以及第三多肽链上的特异性结合CD47的第一VHH作为单结构域第二抗原结合位点和第二VHH作为单结构域第三抗原结合位点。在一些实施方案中，所述抗CD47/PD-L1双特异性抗体蛋白能够以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与肿瘤细胞表面的CD47结合，由此阻断CD47与巨噬细胞表面SIRPα的结合，促进肿瘤组织浸润区的巨噬细胞对肿瘤细胞的吞噬作用；并以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与肿瘤细胞表面的PD-L1结合，由此抑制T细胞上的PD-1与肿瘤细胞表面PD-L1的结合，诱导T细胞活化并发挥抗肿瘤作用。
在一个实施方案中，所述抗CD47/PD-L1双特异性抗体蛋白为PCT/CN2018/123886的PCT申请(申请日：2018年12月26日)中公开的重组抗CD47/PD-L1双特异性抗体蛋白。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。在一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白是一种三链抗体，所述三链抗体包含第一多肽链和第二多肽链上的特异性结合CD47的VH/VL对作为第一抗原结合位点，以及第三多肽链上的特异性结合PD-L1的第一VHH作为单结构域第二抗原结合位点和第二VHH作为单结构域第三抗原结合位点，其中第一多肽链和第二多肽链上的特异性结合CD47的VH/VL对作为第一抗原结合位点包含衍生自抗CD47抗体ADI-29341的GSIEHYYWS(SEQ ID NO:3)所示的VH CDR1、YIYYSGSTNYNPSLKS(SEQ ID NO:4)所示的VH CDR2、ARGKTGSAA(SEQ ID NO:5)所示的VH CDR3、RASQGISRWLA(SEQ ID NO:10)所示的VL CDR1、AASSLQS(SEQ ID NO:11)所示的VL CDR2和QQTVSFPIT(SEQ ID NO:12)所示的VL CDR3，或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；所述第三多肽链上的特异性结合PD-L1的单结构域第二和第三抗原结合位点均包含AYTISRNSMG(SEQ ID NO:17)所示的CDR1、IESDGST(SEQ ID NO:18)所示的CDR2和AAPKVGLGPRTALGHLAFMTLPALNY(SEQ ID NO:19)所示的CDR3，或者与所述3个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列，
在一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白的所述第一多肽链和第二多肽链上的特异性结合CD47的VH/VL对作为第一抗原结合位点包含衍生自抗CD47抗体ADI-29341的SEQ ID NO:2/9的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，所述第三多肽链上的特异性结合PD-L1的单结构域第二和第三抗原结合位点均包含SEQ ID NO:15和/或SEQ ID NO:16所示的氨基酸序列，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列：
Figure PCTCN2020098172-appb-000001
Figure PCTCN2020098172-appb-000002
Figure PCTCN2020098172-appb-000003
Figure PCTCN2020098172-appb-000004
Figure PCTCN2020098172-appb-000005
在一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白包含SEQ ID NO:1所示的第一多肽链、SEQ ID NO:8所示的第二多肽链、和SEQ ID NO:14或SEQ ID NO:22所示的第三多肽链，或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列。
Figure PCTCN2020098172-appb-000006
Figure PCTCN2020098172-appb-000007
Figure PCTCN2020098172-appb-000008
Figure PCTCN2020098172-appb-000009
在一个实施方案中，所述抗CD47/PD-L1双特异性抗体蛋白是在HEK293细胞或CHO 细胞中重组表达的抗CD47/PD-L1双特异性抗体蛋白。
在一个实施方案中，本发明的液体抗体制剂中的抗CD47/PD-L1双特异性抗体蛋白的浓度为约1-200mg/ml。在另一个实施方案中，本发明的液体抗体制剂中的抗CD47/PD-L1双特异性抗体蛋白的浓度为约5-150mg/mL。在其他实施方案中，本发明的液体抗体制剂中的抗CD47/PD-L1双特异性抗体蛋白的浓度为约5、10、20、30、40、50、60、70、80、90、100、110、120、130、140或150mg/ml。
在一个实施方案中，本发明的液体抗体制剂中的缓冲剂的浓度为约1-30mM。在一个实施方案中，本发明的液体抗体制剂中的缓冲剂的浓度为约5-25mM，例如，约5、10、15、20、25mM。
在一个实施方案中，所述缓冲剂选自组氨酸、盐酸组氨酸和它们的组合。
在一个实施方案中，本发明的液体抗体制剂中的稳定剂的浓度为约50-500mM。在一个实施方案中，本发明的液体抗体制剂中的稳定剂的浓度为约100-400mM，例如，约100、150、200、250、300、350、400mM。
在一个实施方案中，所述稳定剂选自山梨醇、蔗糖、海藻糖、精氨酸、盐酸精氨酸和它们的任意组合，更优选为蔗糖、精氨酸和/或盐酸精氨酸。在一个实施方案中，所述液体抗体制剂包含盐酸精氨酸作为稳定剂，优选地盐酸精氨酸以约50-250mM，优选地约100-200mM(例如，约100、110、120、130、140、150、160、170、180、190、200mM)的量存在；和/或包含蔗糖作为稳定剂，优选地蔗糖以约50-250mM，优选地约100-200mM(例如，约100、110、120、130、140、150、160、170、180、190、200mM)的量存在。
在一个实施方案中，本发明的液体抗体制剂中的表面活性剂的浓度为约0.1-1mg/ml。在一个实施方案中，本发明的液体抗体制剂中的表面活性剂的浓度为约0.2-0.8mg/ml，例如约0.2、0.3、0.4、0.5、0.6、0.7、0.8mg/ml。
在一个实施方案中，所述表面活性剂是非离子型表面活性剂。在一个实施方案中，所述表面活性剂选自聚山梨酯类表面活性剂。在一个具体实施方案中，本发明的液体抗体制剂中的表面活性剂是聚山梨酯-80。
在一些实施方案中，所述液体制剂还包含金属螯合剂(例如，EDTA)，例如，约0.002－0.2mg/ml的金属螯合剂(例如，EDTA)。在一个实施方案中，所述液体制剂还包含约0.01-0.1mg/ml，例如约0.01、0.02、0.03、0.04、0.05、0.06、0.08、0.1mg/ml的金属螯合剂(例如，EDTA)。
在一个实施方案中，所述液体制剂的pH值为约6.4-7.0。在一些实施方案中，所述液体制剂的pH值为约6.4-7.0中的任意值，例如约6.4、6.5、6.6、6.7、6.8、6.9、7.0。
在一个实施方案中，所述液体制剂为药物制剂，优选为注射剂，更优选为皮下注射剂或静脉内注射剂。在一个实施方案中，所述液体制剂为静脉输注剂。
在一个实施方案中，本发明的液体抗体制剂包含：
(i)约1-200mg/ml的抗CD47/PD-L1双特异性抗体蛋白；
(ii)约1-30mM的组氨酸和/或盐酸组氨酸；
(iii)约50-500mM的蔗糖、精氨酸和/或盐酸精氨酸，和
(iv)约0.1-1mg/ml聚山梨醇酯80；
任选地，所述液体制剂还包含0.002－0.2mg/ml金属螯合剂(例如，EDTA)；
其中所述液体制剂的pH为约6.4－7.0，优选地约6.5。
在一个优选的实施方案中，本发明的液体抗体制剂包含：
(i)约50-150mg/ml的抗CD47/PD-L1双特异性抗体蛋白；
(ii)约3-25mM的组氨酸和/或盐酸组氨酸；
(iii)约150-300mM的蔗糖、精氨酸和/或盐酸精氨酸，和
(iv)约0.2-0.8mg/ml聚山梨醇酯80；
任选地，所述液体制剂还包含约0.01-0.1mg/ml金属螯合剂(例如，EDTA)；
其中所述液体制剂的pH为约6.4－7.0，优选地约6.5。
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约100mg/ml的抗CD47/PD-L1双特异性抗体蛋白；
(ii)约20mM组氨酸；
(iii)约180mM盐酸精氨酸，和
(iv)约0.2mg/ml聚山梨醇酯80；
任选地，所述液体制剂还包含金属螯合剂(例如，EDTA)，例如约0.02mg/ml EDTA；
其中所述液体制剂的pH为约6.4－7.0，优选地约6.5。
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约100mg/ml的抗CD47/PD-L1双特异性抗体蛋白；
(ii)约20mM组氨酸；
(iii)约100mM盐酸精氨酸和4％蔗糖，和
(iv)约0.2mg/ml聚山梨醇酯80；
任选地，所述液体制剂还包含金属螯合剂(例如，EDTA)，例如约0.02mg/ml EDTA；
其中所述液体制剂的pH为约6.4－7.0，优选地约6.5。
在一个优选的实施方案中，本发明的液体抗体制剂包含
(i)约100mg/ml的抗CD47/PD-L1双特异性抗体蛋白；
(ii)约2.52mg/ml组氨酸和约0.79mg/ml盐酸组氨酸；
(iii)约37.92mg/ml盐酸精氨酸，和
(iv)约0.5mg/ml聚山梨醇酯80；
任选地，所述液体制剂还包含金属螯合剂(例如，EDTA)，例如约0.02mg/ml EDTA；
其中所述液体制剂的pH为约6.4－7.0，优选地约6.5。
另一方面，本发明提供了ー种固体抗体制剂，其是通过将本发明的液体抗体制剂经固化处理而获得的。所述固化处理是通过例如结晶法、喷雾干燥法、冷冻干燥法实施的。在一个优选的实施方案中，所述固体抗体制剂例如是冻干粉针剂形式。固体抗体制剂可在使用前，通过重构于适当的溶媒中，形成本发明的重构制剂。所述重构制剂也是一种本发明的液体抗体制剂。在一个实施方案中，所述适当的溶媒选自注射用水、注射用有机溶剂，包括但不限于注射用油、乙醇、丙二醇等，或其组合。
本发明的液体制剂可以长期稳定储存，例如至少24个月或更长时间。在一个实施方案中，本发明的液体制剂可以在约-80℃至约45℃，例如-80℃、约-30℃、约-20℃、约0℃、约5℃、约25℃、约35℃、约38℃、约40℃、约42℃或约45℃的条件下，储存至少10天、至少20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，至少36个月，或更长时间，是稳定的。
在一个实施方案中，本发明的液体制剂可以稳定储存至少24个月。在再一实施方案中，本发明的液体制剂在至少40℃是稳定的。在再一实施方案中，本发明的液体制剂在约2℃-8℃保持稳定至少12个月，优选至少24个月。在一个实施方案中，本发明的液体制剂在室温或例如约25℃保持稳定至少3个月，优选至少6个月。在再一实施方案中，本发明的液体制剂在约40℃保持稳定至少1个月。
在一个实施方案中，可以通过检测制剂的外观、可见异物、蛋白含量、纯度、和/或电荷变异体的变化，来指示储存后制剂的稳定性。在一个实施方案中，可以在高温胁迫试验中，例如在40℃±2℃储存至少1周、2周或优选地1个月后，或在25℃±2℃储存至少1个月或2个月后，检测本发明液体制剂的稳定性。
在一个实施方案中，在储存后，通过目视检查本发明液体制剂的稳定性，其中本发明液体制剂在外观上保持为澄明至微乳光，为无色至淡黄色液体，且无异物。在一个实施方案中， 在澄明度检测仪下目视检查，制剂中无可见异物存在。在一个实施方案中，在储存后，通过测定蛋白含量变化，检查本发明液体制剂的稳定性，其中例如通过紫外分光光度(UV)法，相对于储存第0天的初始值，蛋白含量变化率不超过20％，优选不超过10％，例如7-8％，优选不超过5％。在一个实施方案中，在储存后，通过测定本发明液体制剂的浊度变化，检查本发明液体制剂的稳定性，其中例如通过OD 350mm法检测，相对于储存第0天的初始值，变化值不超过0.04，更优选地不超过0.03，更优选地不超过0.02。在一个实施方案中，在储存后，通过测定本发明液体制剂的纯度变化，检查本发明液体制剂的稳定性，其中通过尺寸排阻高效液相色谱法(SEC-HPLC)，相对于储存第0天的初始值，单体纯度的变化值不超过10％，例如不超过5％、4％、3％、例如1-2％，优选不超过1％。在一个实施方案中，在储存后，通过测定本发明液体制剂的纯度变化，检查本发明液体制剂的稳定性，其中通过非还原型和/或还原型十二烷基硫酸钠毛细管电泳(CE-SDS)法，单体纯度的变化值下降不超过10％，例如不超过9.5％、8.5％、7.5％、6.5％。在一个实施方案中，在储存后，通过成像毛细管等电聚焦电泳(iCIEF)检测本发明液体制剂的稳定性，其中相对于储存第0天的初始值，抗体的电荷变异体(主成分、酸性组分和碱性组分)的变化值总和不超过50％，例如不超过45％、40％、35％、30％、25％。在一个实施方案中，在储存后，通过阳离子交换高效液相色谱法(CEX-HPLC法)检测本发明液体制剂的稳定性，其中相对于储存第0天的初始值，抗体的电荷变异体(主成分、酸性组分和碱性组分)的变化值总和不超过40％，例如不超过38％、36％、34％、32％、30％。
在一个实施方案中，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3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95％、96％、97％、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85％的纯度，优选大于86％、87％、88％、89％的纯度；
(iii)通过iCIEF法测量，相对于储存第0天的初始值，制剂中抗CD47/PD-L1双特异性抗体蛋白的各组分(主成分、酸性组分和碱性组分)的变化值总和不超过50％，例如不超过45％、40％、35％、30％、25％；
(iv)通过CEX-HPLC法测量，相对于储存第0天的初始值，制剂中抗CD47/PD-L1双特异性抗体蛋白的各组分(主成分、酸性组分和碱性组分)的变化值总和不超过40％，例如不超过38％、36％、34％、32％、30％；
(v)通过ELISA法测量，相对于储存第0天的初始值，制剂中抗CD47/PD-L1双特异性抗体蛋白的相对结合活性为70％-130％，例如，为70％、80％、90％、100％、110％、120％、130％。
在一个方面，本发明提供了一种递送装置，其包含本发明的液体抗体制剂或固体抗体制剂。在一个实施方案中，本发明的递送装置以包含本发明的液体抗体制剂或固体抗体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、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CD47/PD-L1双特异性抗体蛋白的方法，包括给予所述受试者本发明的液体抗体制剂或固体抗体制剂的步骤，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固体抗体制剂的用途，用于制备在受试者中治疗、预防或延缓与SIRPα/CD47信号传导通路和PD1/PD-L1信号传导通路相关的病症的递送装置(如，预填装注射器)或药物，所述病症例如各种血液病和实体瘤，包括但不限于急性骨髓性白血病(AML)，慢性骨髓性白血病，急性淋巴细胞白血病(ALL)，非霍奇金淋巴瘤(NHL)，多发性骨髓瘤(MM)、淋巴瘤、乳腺癌、胃癌、肺癌、食管癌、肠 癌、卵巢癌、宫颈癌、肾癌、胰腺癌、膀胱癌、神经胶质瘤、黑素瘤和其他实体瘤；自身免疫病和炎性病症，例如，过敏性哮喘或溃疡性结肠炎；细胞或组织或器官移植物排斥，包括非人组织移植物(异种移植物)排斥。
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例示了一种抗CD47/PD-L1双特异性抗体的结构，其中第一多肽链与第二多肽链配对形成第一抗原结合位点，第三多肽链包含单结构域第二抗原结合位点和单结构域第三抗原结合位点，且在第三多肽链的单结构域第二抗原结合位点和单结构域第三抗原结合位点之间具有柔性连接肽。
图2显示了抗CD47/PD-L1双特异性抗体制剂在pH 6.4、6.5、6.8和7.0于40℃±2℃放置不同时间后，通过SEC-HPLC法测定的各样品中蛋白纯度变化趋势图。图中横坐标上的T0表示0天；2W表示2周；1M表示1个月。
图3显示了抗CD47/PD-L1双特异性抗体制剂在pH 6.4、6.5、6.8和7.0于40℃±2℃放置不同时间后，通过非还原型CE-SDS法测定的各样品中蛋白纯度变化趋势图。图中横坐标上的T0表示0天；2W表示2周；1M表示1个月。
图4显示了抗CD47/PD-L1双特异性抗体制剂在pH 6.4、6.5、6.8和7.0于40℃±2℃放置不同时间后，通过iCIEF法测定的各样品中电荷变异体主成分的变化趋势图。图中横坐标上的T0表示0天；2W表示2周；1M表示1个月。
图5显示了将具有不同稳定剂的抗CD47/PD-L1双特异性抗体制剂(处方2-5)置于40℃±2℃储存0天、1周、2周、和4周后，通过SEC-HPLC法测定的主峰纯度随时间的变化图。图中横坐标上的T0表示0天；1W表示1周；2W表示2周；4W表示4周。
图6显示了将具有不同稳定剂的抗CD47/PD-L1双特异性抗体制剂(处方2-5)置于40℃±2℃储存0天、1周、2周、和4周后，通过非还原型CE-SDS法测定的主峰纯度随时间的变化图。图中横坐标上的T0表示0天；1W表示1周；2W表示2周；4W表示4周。
图7显示了将具有不同稳定剂的抗CD47/PD-L1双特异性抗体制剂(处方2-5)置于40℃±2℃储存0天、1周、2周、和4周后，通过iCIEF法测定的电荷变异体主成分随时间的变化图。图中横坐标上的T0表示0天；1W表示1周；2W表示2周；4W表示4周。
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。
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，包括但不限于三链抗体、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。
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CD47/PD-L1双特异性抗体制剂”在本文中也简称为“本发明的抗体制剂”，意指包含抗CD47/PD-L1双特异性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CD47/PD-L1双特异性抗体蛋白与可药用赋形剂组合后，作为活性成分的抗CD47/PD-L1双特异性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CD47/PD-L1双特异性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CD47/PD-L1双特异性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CD47/PD-L1双特异性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CD47/PD-L1双特异性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或在40℃稳定至少1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CD47/PD-L1双特异性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92％的抗CD47/PD-L1双特异性抗体蛋白单体。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88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗CD47/PD-L1双特异性抗体蛋白单体。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个月或4周之后，检测到至少约88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗CD47/PD-L1双特异性抗体蛋白单体，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88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗CD47/PD-L1双特异性抗体蛋白单体，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88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗CD47/PD-L1双特异性抗体蛋白单体，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30％，例如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酸性组分电荷变异体的百分比变化值不超过约25％、20％、15％、10％、5％、4％、3％、2％、或1％。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，酸性组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，酸性组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，酸性组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。“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CD47/PD-L1双特异性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CD47/PD-L1双特异性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
本发明提供稳定的液体抗体制剂，其包含(i)抗CD47/PD-L1双特异性抗体蛋白，(ii)缓冲剂，(iii)稳定剂，和(iv)表面活性剂，任选地，还包含(v)其它赋形剂，所述抗体制剂的pH为约6.4-7.0。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CD47/PD-L1双特异性抗体蛋白
本发明抗体制剂中的“抗CD47/PD-L1双特异性抗体蛋白”是一种三链抗体，所述三链抗体包含第一多肽链和第二多肽链上的特异性结合CD47的VH/VL对作为第一抗原结合位点，以及第三多肽链上的特异性结合PD-L1的第一VHH作为单结构域第二抗原结合位点和第二VHH作为单结构域第三抗原结合位点；或者包含第一多肽链和第二多肽链上的特异性结合PD-L1的VH/VL对作为第一抗原结合位点，以及第三多肽链上的特异性结合CD47的第一VHH作为单结构域第二抗原结合位点和第二VHH作为单结构域第三抗原结合位点。所述抗CD47/PD-L1双特异性抗体蛋白能够以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与CD47结合，且能够以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与PD-L1结合，以致所述抗体可以用作双特异性靶向CD47分子和PD-L1分子的治疗剂和/或预防剂。
对于所述特异性结合PD-L1或CD47的VH/VL对，其包含衍生自任何现有技术中报导的抗PD-L1抗体和将来研发出的抗PD-L1抗体VH/VL对的6个CDR或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；或者包含衍生自任何现有技术中报导的抗CD47抗体和将来研发出的抗CD47抗体VH/VL对的6个CDR或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。
对于所述特异性结合PD-L1或CD47的第一VHH和第二VHH，它们均衍生自天然缺乏轻链的抗体的重链可变结构域(如骆驼科(Camelidae)物种中天然存在的重链抗体的重链可变结构域)。第一VHH和第二VHH可以相同或者不同。所述第一VHH和第二VHH可以源自骆驼科物种(例如骆驼、羊驼、单峰驼、驼羊和原驼)中产生的抗体。除骆驼科之外的其他物种也可以产生天然缺乏轻链的重链抗体，这类VHH也处于本发明抗体蛋白的范围内。所 述第一VHH和第二VHH包含衍生自任何现有技术中报导的抗PD-L1抗体和将来研发出的抗PD-L1抗体VHH的3个CDR或与所述3个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；或者包含衍生自任何现有技术中报导的抗CD47抗体和将来研发出的抗CD47抗体VHH的3个CDR或与所述3个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。
在一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白的所述第一多肽链和第二多肽链上的特异性结合CD47的VH/VL对包含衍生自中国专利申请号CN201710759828.9报导的抗CD47抗体ADI-29341的GSIEHYYWS(SEQ ID NO:3)所示的VH CDR1、YIYYSGSTNYNPSLKS(SEQ ID NO:4)所示的VH CDR2、ARGKTGSAA(SEQ ID NO:5)所示的VH CDR3、RASQGISRWLA(SEQ ID NO:10)所示的VL CDR1、AASSLQS(SEQ ID NO:11)所示的VL CDR2和QQTVSFPIT(SEQ ID NO:12)所示的VL CDR3，或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。
在一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白的所述第三多肽链上的特异性结合PD-L1的第一VHH和第二VHH均包含AYTISRNSMG(SEQ ID NO:17)所示的CDR1、IESDGST(SEQ ID NO:18)所示的CDR2和AAPKVGLGPRTALGHLAFMTLPALNY(SEQ ID NO:19)所示的CDR3，或者与所述3个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。
术语“CDR”或“互补决定区”或“CDR区”(在本文中与超变区“HVR”可以互换使用)，是抗体可变区中主要负责与抗原表位结合的氨基酸区域。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。本领域公知多种用于在一个给定的VH或VL或VHH氨基酸序列中确定其CDR序列的方案。例如，Kabat互补决定区(CDR)是基于序列变异性确定的并且是最常用的(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))。而Chothia指的是结构环的位置(Chothia和Lesk，J.Mol.Biol.196:901-917(1987))。AbM HVR是Kabat HVR和Chothia结构环之间的折中，并且由Oxford Molecular的AbM抗体建模软件使用。“接触性”(Contact)HVR基于对可获得的复杂晶体结构的分析。HVR也可以基于与参考CDR序列(例如本文公开的示例性CDR)具有相同的Kabat编号位置而确定。
所述氨基酸变化，例如，氨基酸置换优选地是保守氨基酸取代。“保守氨基酸取代”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
在一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白的所述第一多肽链和第二多肽链上的特异性结合CD47的VH/VL对包含衍生自抗CD47抗体ADI-29341的SEQ ID NO:2/9的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列。
在一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白的所述第三多肽链上的特异性结合PD-L1的第一VHH和第二VHH包含SEQ ID NO:15和/或SEQ ID NO:16所示的氨基酸序列，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。
不特别地限制抗CD47/PD-L1双特异性抗体蛋白中第一多肽链和第三多肽链中免疫球蛋白的重链恒定区的类型，优选地是IgG1、IgG2或IgG4免疫球蛋白的重链恒定区，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。更优选地，所述重链恒定区是人IgG1免疫球蛋白的重链恒定区，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。
在一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白包含IgG4(例如，人IgG4)中使用的重链恒定区。在又一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白包含用于IgG1(例如，人IgG1)的重链恒定区。例如，在三链抗体的第一多肽链和第三多肽链的Fc结构域中分别包含具有“CPPC”氨基酸残基的铰链区，和/或分别包含Y349C和S354C(根据Kabat的“EU编号”)，由此，第一多肽链和第三多肽链在Fc区形成链间二硫键，由此，稳定第一多肽链和第三多肽链的正确配对。
在一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白的第一多肽链和/或第三多肽链在Fc结构域中包含影响抗体效应子功能的氨基酸突变。在一个具体实施方案中，所述效应子功能是抗体依赖的细胞介导的细胞毒性(ADCC)。在一个实施方案中，所述氨基酸突变存在于Fc区的CH2结构域，例如，所述抗CD47/PD-L1双特异性抗体蛋白包含在第一多肽链和/或第三多肽链第234和235位置(EU编号)处的氨基酸置换。在一个具体实施方案中，所述氨基酸置换是L234A和L235A(也称为“LALA突变”)。
在又一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白的第二多肽链包含κ轻链恒定区或者λ轻链恒定区，例如，人κ轻链恒定区或者人λ轻链恒定区。
在一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白的第一多肽链和第三多肽链各自的Fc结构域中分别包含凸起(“结(knob)”)或空穴(“扣(hole)”)，并且第一多肽链Fc结构域中的所述凸起或空穴可分别置于第三多肽链Fc结构域中的所述空穴或凸起中，由此所述第一多肽链和第三多肽链彼此形成“结入扣(knob-in-hole)”的稳定缔合。在一个实施方案中，在所述第一多肽链和第三多肽链之一条链中包含氨基酸置换T366W，并且在所述第一多肽链和第三多肽链之另一条链中包含氨基酸置换T366S、L368A和Y407V(EU编号)。由此一条链中的凸起能够置于另一条链中的空穴中，促进第一多肽链和第三多肽链的正确配对。
在一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白的第一多肽链和第二多肽链的免疫球蛋白CH1结构域和CL结构域中分别包含凸起或空穴，并且CH1结构域中的所述凸起或空穴可分别置于CL结构域中的所述空穴或凸起中，从而所述第一多肽链和第二多肽链彼此也形成“结入扣”的稳定缔合。
在一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白包含SEQ ID NO:1所示的第一多肽链、SEQ ID NO:8所示的第二多肽链、和SEQ ID NO:14所示的第三多肽链，或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列。
又在一个实施方案中，抗CD47/PD-L1双特异性抗体蛋白包含SEQ ID NO:1所示的第一多肽链、SEQ ID NO:8所示的第二多肽链、和SEQ ID NO:22所示的第三多肽链，或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列。
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。
本发明抗体制剂中的抗CD47/PD-L1双特异性抗体蛋白能够同时与PD-L1和CD47蛋白结合，且维持了各亲本抗体的亲和力常数，由此，能够阻断SIRPα/CD47信号传导通路和阻断PD1/PD-L1信号传导通路，从而用于治疗、预防或延缓各种与SIRPα/CD47信号传导通路和/或与PD1/PD-L1信号传导通路相关的疾病或病症。
在一个优选的实施方案中，本发明的抗CD47/PD-L1双特异性抗体蛋白是PCT/CN2018/123886的PCT申请(申请日：2018年12月26日)中公开的重组抗CD47/PD-L1双特异性抗体蛋白，其具有SEQ ID NO:1所示的第一多肽链、SEQ ID NO:8所示的第二多肽链、和SEQ ID NO:14所示的第三多肽链。在一个实施方案中，该抗CD47/PD-L1双特异性抗体蛋白由HEK293细胞或CHO细胞重组表达产生并经纯化。优选地，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。对使用Raji-PD-L1细胞接种NOD-SCID小鼠产生的荷瘤小鼠施用抗CD47/PD-L1双特异性抗体，结果表明，与施用抗CD47单克隆抗体和抗PD-L1单克隆抗体相比较，施用抗CD47/PD-L1双特异性抗体具有显著提高的抗肿瘤活性，可以导致肿瘤生长抑制率达到约90％或更高，例如100％；和/或肿瘤消失率达到50％以上。此外，抗CD47/PD-L1双特异性抗体还表现出显著降低的血细胞凝集作用，因此在临床治疗中将具有显著降低的副作用。
本发明的抗体制剂中所包含的抗CD47/PD-L1双特异性抗体蛋白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5-150mg/mL，例如约5、10、20、30、40、50、60、70、80、90、100、110、120、130、140或150mg/ml的抗CD47/PD-L1双特异性抗体蛋白。
(ii)缓冲剂
缓冲剂是可以将溶液的pH维持在可接受范围的试剂。在一些实施方案中，用于本发明制剂中的缓冲剂可以将本发明制剂的pH控制在大约6.4-7.0的pH范围，例如约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6.4、6.5、6.6、6.7、6.8、6.9、7.0的pH。
在一些实施方案中，用于本发明制剂中的缓冲剂选自组氨酸、盐酸组氨酸和它们的组合。在一个实施方案中，本发明的液体抗体制剂中的缓冲剂的浓度为约1-30mM。在一个实施方案中，本发明的液体抗体制剂中的缓冲剂的浓度为约5-25mM，例如，约5、10、15、20、25mM。
在一个实施方案中，用于本发明制剂中的缓冲剂是约16.3mM组氨酸和约3.77mM盐酸组氨酸的组合。
(iii)稳定剂
用于本发明的合适的稳定剂可以选自糖、多元醇和氨基酸及其组合。对于作为稳定剂的糖包括但不限于蔗糖和海藻糖。对于作为稳定剂的多元醇包括但不限于山梨醇。对于作为稳定剂的氨基酸包括但不限于精氨酸、盐酸精氨酸。在一些实施方案中，所述稳定剂在本发明的液体制剂中以约50-500mM，更优选地约100-400mM，例如，约100、150、200、250、300、350、400mM的浓度存在。
在一个实施方案中，本发明液体制剂包含蔗糖作为稳定剂。蔗糖在本发明液体制剂中的量可以是约50-250mM，优选地约100-200mM(例如，约100、110、120、130、140、150、160、170、180、190、200mM)。
在一个实施方案中，本发明液体制剂包含精氨酸和/或盐酸精氨酸作为稳定剂。精氨酸和/或盐酸精氨酸在本发明液体制剂中的量可以是约50-250mM，优选地约100-200mM(例如，约100、110、120、130、140、150、160、170、180、190、200mM)。
在一个实施方案中，本发明液体制剂包含蔗糖、精氨酸和/或盐酸精氨酸的组合作为稳定剂。该组合中，蔗糖可以以约50-250mM，优选地约100-200mM(例如，约100、110、120、130、140、150、160、170、180、190、200mM)的量存在。在该组合中，精氨酸和/或盐酸精氨酸可以以约50-250mM，优选地约100-200mM(例如，约100、110、120、130、140、150、160、170、180、190、200mM)的量存在。
(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。
在一个实施方案中，本发明的液体制剂中的表面活性剂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，诸如聚山梨酯-20、聚山梨酯-80、聚山梨酯-60、或聚山梨酯-40；普洛尼克等。在一个优选实施方案中，本发明的液体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。在优选的一些实施方案中，制剂可含有约0.1-1mg/ml，优选地约0.2-0.8mg/ml，例如约0.2、0.3、0.4、0.5、0.6、0.7、0.8mg/ml的聚山梨酯类表面活性剂(例如，聚山梨酯-80)。
(v)其它赋形剂
本发明的抗体液体制剂中可以包含或不包含其它赋形剂。
在一个实施方案中，本发明的抗体液体制剂包含金属螯合剂(例如，EDTA或其盐)作为一种赋形剂。在另一实施方案中，本发明的抗体液体制剂不包含金属螯合剂(例如，EDTA或其盐)。在一个实施方案中，与不添加金属螯合剂(例如，EDTA或其盐)的相应制剂相比，添加金属螯合剂(例如，EDTA或其盐)的本发明抗体液体制剂具有更高的稳定性。
出于其他考虑，也可在本发明制剂中使用其它的赋形剂。所述赋形剂包括，例如，调味剂、抗微生物剂、甜味剂、抗静电剂、抗氧化剂、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)”。
II.制剂的制备
本发明提供了包含抗CD47/PD-L1双特异性抗体蛋白的稳定制剂。在本发明制剂中使用的抗CD47/PD-L1双特异性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CD47/PD-L1双特异性抗体蛋白。在一个优选的实施方案中，本发明的抗CD47/PD-L1双特异性抗体蛋白通过在HEK293细胞或CHO细胞中重组表达而制备，例如，如PCT/CN2018/123886中所述，重组制备抗CD47/PD-L1双特异性抗体蛋白。
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。
一般地，重组产生的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单克隆抗体纯度测定方法。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，是用于单克隆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)可以用于分析单克隆抗体的电荷异质性。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ampholytes。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mAb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)。
此外，可以检测抗体的功效或生物活性。例如，可以检测制剂中抗体与其抗原分子(CD47分子和PD-L1分子)的结合能力。本领域技术人员已知多种方法可以用于定量抗体与抗原的特异性结合，例如免疫测定试验，ELISA等。
本发明的抗CD47/PD-L1双特异性抗体蛋白制剂是稳定的。在一个实施方案中，于约25℃、37℃、40℃、或45℃储存至少1个月或2个月后，例如，在40℃±2℃储存1个月后，本 发明的抗体制剂中的抗CD47/PD-L1双特异性抗体蛋白纯度是至少85％、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CS-SDS所测定。在一个实施方案中，于约25℃、37℃、40℃、或45℃储存至少1个月或2个月后，例如，在40℃±2℃储存1个月后，本发明的抗体制剂中抗CD47/PD-L1双特异性抗体蛋白的至少50％，优选至少55％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CD47/PD-L1双特异性抗体蛋白的本发明的抗体制剂可以用于治疗、预防或延缓各种与SIRPα/CD47信号传导通路和/或与PD1/PD-L1信号传导通路相关的疾病或病症。“与SIRPα/CD47信号传导通路相关的疾病或病症”和/或“与PD1/PD-L1信号传导通路相关的疾病或病症”在本文中指可以用本发明抗CD47/PD-L1双特异性抗体蛋白制剂进行治疗(例如改善)或预防的疾病或病症。任何可以得益于本发明抗体制剂治疗的疾病或病症都适用于本发明。
在一个方面，包含抗CD47/PD-L1双特异性抗体蛋白的本发明制剂能够用于预防或治疗受试者的各种血液病和实体瘤，包括但不限于急性骨髓性白血病(AML)，慢性骨髓性白血病，急性淋巴细胞白血病(ALL)，非霍奇金淋巴瘤(NHL)，多发性骨髓瘤(MM)、淋巴瘤、乳腺癌、胃癌、肺癌、食管癌、肠癌、卵巢癌、宫颈癌、肾癌、胰腺癌、膀胱癌、神经胶质瘤、黑素瘤和其他实体瘤。另外，通过阻断SIRPα/CD47信号传导通路能够增进NOD小鼠系中的人干细胞植入(WO 2009/046541)，因此，包含抗CD47/PD-L1双特异性抗体蛋白的本发明制剂还具有用于人干细胞移植中的潜在益处。
在另一方面，包含抗CD47/PD-L1双特异性抗体蛋白的本发明制剂能够用于治疗、预防或诊断受试者中由SIRPα+细胞介导的自身免疫病和炎性病症，例如，过敏性哮喘或溃疡性结肠炎。这些病症包括急性和慢性炎性病症、变态反应和过敏性疾病、自身免疫病、局部缺血性病症、严重感染、和细胞或组织或器官移植物排斥，包括非人组织移植物(异种移植物)排斥等。
本发明也提供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CD47/PD-L1双特异性抗体蛋白，或用于治疗、预防或改善上述疾病和病症中的一种或多种。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CD47/PD-L1双特异性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。
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。取决于待治疗的适应症，有效剂量可为约0.005mg/kg体重至约50mg/kg体重，或约0.1mg/kg体重至约20mg/kg体重。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。
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
CE-SDS：十二烷基硫酸钠毛细管凝胶电泳
CEX-HPLC：阳离子交换高效液相色谱法
ELISA：酶联免疫吸附测定法
FLD-HPLC：荧光检测-高效液相色谱法
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
为了开发出重组抗分化抗原簇47(CD47)和抗程序性死亡配体1(PD-L1)双特异性抗体注射液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，考察了不同辅料对抗CD47/PD-L1双特异性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 PCTCN2020098172-appb-000010
注：N/A表示“不适用”(Not applicable)。
1.2.本发明的制剂研究中使用的仪器设备
Figure PCTCN2020098172-appb-000011
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；(4)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；(5)通过成像毛细管等电聚焦电泳法(iCIEF法)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；(6)通过免疫测定法，例如，直接ELISA法测定抗体制剂中抗CD47/PD-L1双特异性抗体对CD47抗原和PD-L1抗原的相对结合活性。
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。
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法)
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。采用真空进样方法进样55秒，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℃，毛细管柱温为室温，检测波长为280nm。阴极稳定剂(Cathodic Stabilizer)为500mmol/L精氨酸溶液，阳极稳定剂(Anodic Stabilizer)为200mmol/L亚氨基乙二酸，3mol/L尿素提高蛋白溶解性，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0.5mg/ml，取上述稀释后的供试品溶液20μl，向其中加入83μl预混液充分混匀制得待测样品溶液。使用制剂缓冲液同法操作，制得空白溶液。
相对结合活性(直接ELISA法)
用1×PBS稀释链亲和素(Thermo，货号：21125)至1μg/ml，100μl/孔，37℃2h包被于96孔酶标板上。洗板后加封闭液(5％FBS，300μl/孔)37℃封闭2h。用1×PBS稀释生物素化的抗原(检测抗CD47/PD-L1双特异性抗体的抗CD47端对CD47的相对结合活性时，使用购自北京Sino biological的人CD47蛋白(His标签)，货号：12283-H08H-200；检测抗CD47/PD-L1双特异性抗体的抗PD-L1端对PD-L1的相对结合活性时，使用购自ACRO BIOSYSTEMS的重组人PDL1/CD274蛋白，货号：PD1-H5229-1MG)至0.5μg/ml，100μl/孔，37℃0.5h包被于96孔酶标板上。100μl/孔以2％FBS稀释抗CD47/PD-L1双特异性抗体至40μg/ml，4倍梯度稀释至第12个浓度(0.01～10000ng/ml)。将梯度稀释的供试品以100μl/孔加入到洗过的酶标板中，37℃恒温培养箱中孵育30min。洗板后加入以2％FBS稀释的HRP缀合山羊抗人IgG-Fc片段(美国BETHYL，货号A80-104P)作为二抗(30000倍稀释，100μl/孔)37℃反应20min。洗板后加入100μl TMB显色液，显色10min后，每孔加入100μl的1mol/L H 2SO 4终止反应。以620nm为参比波长，测450nm处的OD值。以各浓度梯度样品的浓度值作为横坐标，各梯度样品的OD450nm-OD620nm值为纵坐标，应用Prism四参数拟合计算EC 50反映抗体与各抗原的结合活性。
实施例1.制备和纯化抗CD47/PD-L1双特异性抗体
根据PCT/CN2018/123886所述，通过在HEK293细胞(购自INVITROGEN公司)中重组表达和纯化了抗CD47/PD-L1双特异性抗体Kh2NF-PC。该抗CD47/PD-L1双特异性抗体Kh2NF-PC抗体由3条多肽链组成，每条多肽链从N端至C端分别具有如下氨基酸序列：
肽链#1：
Figure PCTCN2020098172-appb-000012
其中，所述肽链#1包含衍生自抗CD47抗体ADI29341的如下VH氨基酸序列：
Figure PCTCN2020098172-appb-000013
在所述VH氨基酸序列C端的衍生自人IgG1的如下CH1氨基酸序列：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT(SEQ ID NO:6)；以及
在所述CH1氨基酸序列C端的衍生自人IgG1的如下Fc区氨基酸序列：
Figure PCTCN2020098172-appb-000014
肽链#2：
Figure PCTCN2020098172-appb-000015
其中，所述肽链#2包含衍生自抗CD47抗体ADI29341的如下VL氨基酸序列：
DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVSFPITFGGGTKVEIK(SEQ ID NO:9)；以及
在所述VL氨基酸序列C端的如下人κ轻链恒定区(CL)氨基酸序列：
Figure PCTCN2020098172-appb-000016
肽链#3
Figure PCTCN2020098172-appb-000017
其中，所述肽链#3包含如下的第一和第二抗PD-L1VHH氨基酸序列：
Figure PCTCN2020098172-appb-000018
在所述第一和第二抗PD-L1VHH氨基酸序列之间的连接肽氨基酸序列：GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:20)；以及
在所述第二抗PD-L1VHH氨基酸序列C端的衍生自人IgG1的如下Fc区氨基酸序列：
Figure PCTCN2020098172-appb-000019
实施例2.pH对制剂的稳定性影响试验之一
本实施例考察了包含抗CD47/PD-L1双特异性抗体的制剂在pH 5.0至6.5的稳定性。共设计了4个pH值，分别为5.0、5.5、6.0和6.5。
2.1实验步骤
配制10mM组氨酸，5％(w/v)山梨醇缓冲液，用稀盐酸将pH分别调节为5.0、5.5、6.0和6.5，将经纯化的抗CD47/PD-L1双特异性抗体Kh2NF-PC蛋白(7.3mg/ml)超滤置换到所述不同pH值的溶液中。置换完成后，调节样品中的双特异性抗体蛋白含量至约100.0mg/ml；然后加入聚山梨酯80，使其终浓度为0.70mg/ml；过滤分装至2R西林瓶中，加塞、轧盖。将各样品于40℃±2℃条件下进行稳定性考察，具体实验方案见表1。
表1.实验方案
Figure PCTCN2020098172-appb-000020
注：(1)x表示在该时间点取样。(2)在所述时间点取样后，均将取得的样品先放入超低温冰箱中冻存待检，按需要化冻送检。
2.2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定标准，用以判断样品是否发生了变化，具体见表2。
表2.质量未发生变化的判断标准
Figure PCTCN2020098172-appb-000021
2.3处方筛选试验之一的实验结果
(1)外观和可见异物
在40℃±2℃条件下放置一个月后，pH 5.0、pH 5.5和pH 6.0样品外观均出现不同程度的浑浊和沉淀；仅有pH 6.5样品外观和可见异物均合格。
(2)蛋白含量
在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，各样品的蛋白含量检测结果见表3。结果表明，在40℃±2℃条件下放置1个月，pH 6.5样品蛋白含量未发生显著变化。
表3.在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，各样品的蛋白含量(UV法，mg/ml)
Figure PCTCN2020098172-appb-000022
Figure PCTCN2020098172-appb-000023
注：N/A表示样品外观不合格未进行检测。
(3)纯度
在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，通过SEC-HPLC法测定各样品的蛋白纯度。结果见表4。结果表明，在40℃±2℃条件下考察1个月，pH 6.5值的样品中蛋白纯度较0天的样品相比下降了4.1％。
表4.通过SEC-HPLC法对各样品测定的蛋白纯度(％)
Figure PCTCN2020098172-appb-000024
注：N/A表示样品外观不合格未进行检测。
在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，通过非还原型CE-SDS法和还原型CE-SDS法分别测定各样品的蛋白纯度。结果见表5和表6。结果表明，在40℃±2℃条件下考察1个月，pH 6.5值的样品中蛋白纯度较0天的样品相比分别下降了7.7％和3.7％。
表5.通过非还原型CE-SDS法对各样品测定的蛋白纯度(％)
Figure PCTCN2020098172-appb-000025
注：N/A表示样品外观不合格未进行检测。
表6.通过还原型CE-SDS法对各样品测定的蛋白纯度(％)
Figure PCTCN2020098172-appb-000026
注：N/A表示样品外观不合格未进行检测。
(4)电荷变异体
在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，通过iCIEF法测定各样品的电荷变异体。结果见表7。结果表明，在40℃±2℃条件下考察1个月，pH 6.5的样品主成分和酸组分均发生明显变化。与0天样品相比，酸性组分从36.6％上升至60.1％，增加了23.5％； 主成分从62.5％下降至39.1％，降低了23.4％；碱性组分无明显变化。
表7.通过iCIEF法测定的各样品的电荷变异体(％)
Figure PCTCN2020098172-appb-000027
注：N/A表示样品外观不合格未进行检测。
(5)相对结合活性
在pH 5.0、5.5、6.0和6.5于40℃±2℃放置不同时间后，通过直接ELISA法测定各样品的相对结合活性。结果见表8。结果表明，在40℃±2℃条件下考察1个月，pH 6.5的样品中，蛋白的抗CD47端和抗PD-L1端对CD47和PD-L1的相对结合活性均未发生变化。
表8.通过直接ELISA法测定的样品的相对结合活性(％)
Figure PCTCN2020098172-appb-000028
注：N/A 1表示样品外观不合格未进行检测；N/A 2表示未设置该检测项。
上述实验结果表明，对于pH 5.0、5.5、6.0和6.5的制剂，抗CD47/PD-L1双特异性抗体蛋白(例如，Kh2NF-PC蛋白)在pH 6.5时较为稳定。为了研究制剂在pH 6.5附近的pH范围内的稳定性，进行了进一步的实验。
实施例3.pH对制剂的稳定性影响试验之二
本实施例考察了pH 6.2至7.0对抗CD47/PD-L1双特异性抗体制剂中的蛋白稳定性的影响，共设计了5个pH值，分别为6.2、6.4、6.5、6.8和7.0。
3.1实验步骤
具体操作步骤与实施例2中的“2.1实验步骤”相同，除了pH值不同之外。
3.2判断标准
参见实施例2中的表2。
3.3实验结果
(1)外观和可见异物
在40℃±2℃条件下放置一个月后，仅有pH 6.2样品外观呈乳白色；而pH 6.4、6.5、6.8和7.0样品外观和可见异物均合格。
(2)蛋白含量
在pH 6.4、6.5、6.8和7.0于40℃±2℃放置不同时间后，各样品的蛋白含量检测结果见表9。结果表明，在40℃±2℃条件下放置1个月，pH 6.4、6.5、6.8和7.0样品蛋白含量均未发生显著变化。
表9.在pH 6.4、6.5、6.8和7.0于40℃±2℃放置不同时间后，各样品的蛋白含量(UV法，mg/ml)
Figure PCTCN2020098172-appb-000029
注：N/A表示未设置该检测项。
(3)纯度
在pH 6.4、6.5、6.8和7.0于40℃±2℃放置不同时间后，通过SEC-HPLC法测定各样品的蛋白纯度。结果见表10，纯度的变化趋势见图2。结果表明，在40℃±2℃条件下考察1个月，pH 6.4、6.5、6.8和7.0样品的纯度均有所下降，与0天比较分别下降了3.0％、3.7％、4.9％和5.6％。
表10.通过SEC-HPLC法对各样品测定的蛋白纯度(％)
Figure PCTCN2020098172-appb-000030
在pH 6.4、6.5、6.8和7.0于40℃±2℃放置不同时间后，通过非还原型CE-SDS法分别测定各样品的蛋白纯度。结果见表11，纯度的变化趋势见图3。结果表明，在40℃±2℃条件下考察1个月，pH 6.4、6.5、6.8和7.0样品的纯度均下降，与0天比较分别下降了7.0％、6.1％、6.7％和7.3％。
表11.通过非还原型CE-SDS法对各样品测定的蛋白纯度(％)
Figure PCTCN2020098172-appb-000031
注：N/A表示未设置该检测项。
(4)电荷变异体
在pH 6.4、6.5、6.8和7.0于40℃±2℃放置不同时间后，通过iCIEF法测定各样品的电荷变异体。结果见表12，纯度的变化趋势见图4。结果表明，在40℃±2℃条件下考察1个月，不同pH值样品主成分和酸组分均发生变化。pH值越高其主成分下降越快，酸性组分上升越快。
表12.通过iCIEF法测定的各样品的电荷变异体(％)
Figure PCTCN2020098172-appb-000032
注：N/A表示未设置该检测项。
(5)相对结合活性
在pH 6.4、6.5、6.8和7.0于40℃±2℃放置不同时间后，通过直接ELISA法测定各样品的相对结合活性。结果见表13。结果表明，在40℃±2℃条件下考察1个月，pH 6.4、pH6.5和pH 7.0样品中蛋白的抗CD47端和抗PD-L1端对CD47和PD-L1的相对结合活性均未发生明显变化。
表13.通过直接ELISA法测定的样品的相对结合活性(％)
Figure PCTCN2020098172-appb-000033
注：N/A表示未设置该检测项。
实施例2和实施例3的pH对制剂的稳定性影响试验结果表明，抗CD47/PD-L1双特异性抗体(例如，Kh2NF-PC)蛋白在pH 5.0～6.2于40℃±2℃放置一个月，随着时间的延长，样 品外观会出现浑浊或乳白色沉淀；而在pH 6.4－7.0于40℃±2℃放置一个月，样品外观和可见异物均合格，蛋白含量未发生显著变化，且对CD47和PD-L1的相对结合活性也未发生明显变化。因此，在随后的实施例中，从pH 6.4－7.0中选择pH 6.5进行后续实验。
实施例4.处方筛选试验
4.1稳定剂筛选试验
考察了不同稳定剂：山梨醇、蔗糖、海藻糖、盐酸精氨酸等对包含抗CD47/PD-L1双特异性抗体制剂稳定性的影响。
4.1.1稳定剂筛选试验步骤
共设计了5个处方，详细处方信息见表14。按照表14配制各个处方的缓冲液，将抗CD47/PD-L1双特异性抗体Kh2NF-PC蛋白(3.6mg/ml)超滤置换至各自的处方溶液中。置换完成后，调节各处方的蛋白含量至约100.0mg/ml；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，加塞、轧盖。将各样品于40℃±2℃条件下进行稳定性考察，具体方案见表15。检测指标为外观、可见异物、蛋白含量、纯度(SEC-HPLC法)和电荷变异体(iCIEF法)。
表14.稳定剂筛选试验备选处方信息表
序号 处方信息
处方1 20mM组氨酸，5％山梨醇，0.02％聚山梨酯80，pH 6.0
处方2 20mM组氨酸，5％山梨醇，0.02％聚山梨酯80，pH 6.5
处方3 20mM组氨酸，8％海藻糖，0.02％聚山梨酯80，pH 6.5
处方4 20mM组氨酸，180mM盐酸精氨酸，0.02％聚山梨酯80，pH 6.5
处方5 20mM组氨酸，4％蔗糖，100mM盐酸精氨酸，0.02％聚山梨酯80，pH 6.5
注：表中％是指％w/v，下同。
表15.稳定性考察方案
Figure PCTCN2020098172-appb-000034
注：(1)x表示该点取样。(2)上述时间点取样后均先将取得的样品放入超低温冰箱中冻存待检，按需要化冻送检。
4.1.2判定标准
判定标准具体参见实施例2中的表2。
4.1.3稳定剂筛选试验
(1)外观、可见异物
在40℃±2℃条件下观察至4周，处方1考察一周后有浑浊或沉淀；其他处方样品外观、可见异物均合格。
(2)蛋白含量
在40℃±2℃条件下观察至4周，蛋白含量结果见表16。从表16可见，在40℃±2℃条件下放置4周，处方2、处方3、处方4和处方5的蛋白含量均未发生变化。
表16.稳定剂筛选试验蛋白含量结果(UV法，mg/ml)
Figure PCTCN2020098172-appb-000035
注：N/A表示样品外观不合格未进行检测。
(3)纯度
纯度(SEC-HPLC法)：在40℃±2℃条件下观察4周的结果见表17，纯度变化趋势见图5。结果表明，在40℃±2℃条件下考察4周，处方2、处方3、处方4和处方5样品纯度均有下降，与0天样品纯度相比，分别下降了2.6％、3.0％、0.7％和0.7％。
表17.稳定剂筛选试验的纯度结果(SEC-HPLC法，％)
Figure PCTCN2020098172-appb-000036
注：N/A表示样品外观不合格未进行检测。
纯度(非还原型CE-SDS法)：在40℃±2℃条件下观察4周的结果见表18，纯度变化趋势见图6。结果表明，在40℃±2℃条件下考察4周，各处方样品纯度均有下降；与0天样品纯度相比，处方2、处方3、处方4和处方5分别下降了6.8％、7.2％、9.1％和9.2％。
表18.稳定剂筛选试验的纯度结果(非还原型CE-SDS法，％)
Figure PCTCN2020098172-appb-000037
注：N/A表示样品外观不合格未进行检测
(4)电荷变异体(iCIEF法)
在40℃±2℃条件下观察4周的电荷变异体结果见表19，电荷变异体主成分变化趋势见图7。
结果表明，40℃±2℃条件下考察4周，各处方电荷变异体主成分及酸性组分均发生明显变化，主成分下降，酸性组分上升，其变化趋势基本一致。
表19.稳定剂筛选试验的电荷变异体结果(iCIEF法，％)
Figure PCTCN2020098172-appb-000038
Figure PCTCN2020098172-appb-000039
注：N/A表示样品外观不合格未进行检测
稳定剂筛选试验结果表明，处方2、处方3、处方4和处方5在40℃±2℃条件下放置4周，样品外观、可见异物均合格，蛋白含量均未发生变化，样品纯度略有下降，其中处方4和处方5在纯度(SEC-HPLC法)和电荷变异体(iCIEF法)方面表现出明显的优势。
4.2渗透压试验
4.2.1渗透压试验步骤
利用多通道渗透压计对处方4和处方5的0时(T0)样品测定了渗透压。每组样品测量2次，结果取平均值。
4.2.2渗透压试验结果
对处方4和处方5的0时(T0)样品进行渗透压测定的结果见表20。
表20.渗透压测定结果
Figure PCTCN2020098172-appb-000040
由于人类血浆渗透压约为285－310mOsmol/kg，因此，处方4和处方5的渗透压在药物制剂渗透压的可接受范围内。另外，考虑到处方4的渗透压更接近人类血浆渗透压，优选使用处方4的制剂。
实施例5：金属螯合剂对制剂稳定性的影响
EDTA是一种代表性的金属螯合剂。本实施例研究了EDTA对抗CD47/PD-L1双特异性抗体Kh2NF-PC蛋白稳定性的影响。
5.1 EDTA对制剂稳定性的考察方案：
共设计了2个处方，其中处方6是未添加EDTA的对照组；处方7是终浓度为0.02mg/ml EDTA的组。详细处方信息见表21。按照表21配制各个处方的缓冲液，将抗CD47/PD-L1双特异性抗体Kh2NF-PC蛋白超滤置换至各自的处方溶液中。置换完成后，调节各处方的蛋 白含量至约100.0mg/ml。
表21.EDTA对制剂稳定性的处方信息表
Figure PCTCN2020098172-appb-000041
EDTA对制剂稳定性影响的详细实验条件及取样计划见表22。
表22.EDTA对制剂稳定性的考察方案
Figure PCTCN2020098172-appb-000042
注：(1)x表示该点取样。(2)上述时间点取样后均先将取得的样品放入超低温冰箱中冻存待检，按需要化冻送检。
5.2实验结果
在40℃±2℃的条件下观察至4周，结果见表23。
表23 EDTA对制剂稳定性的实验结果
Figure PCTCN2020098172-appb-000043
表23的结果显示，从电荷变异体(CEX-HPLC法)的检测结果来看，处方7相比于处方6的电荷变异体主峰表现出更好的优势；从聚山梨酯80(FLD-HPLC法)的检测结果来看，处方6的聚山梨酯80含量随时间而降低；处方7中由于加入了金属螯合剂EDTA，从而抑制了因金属离子引起的聚山梨酯80的降解，故处方7要优于处方6。EDTA作为金属螯合剂的一个例子，能够结合金属离子，其至少可从以下两个方面抑制聚山梨酯80的降解。其一是，在蛋白的整个生产过程中，包括细胞培养、纯化等相关操作步骤中，可能会引入一些金属离子，由此在有氧和金属离子共同存在的条件下，引起聚山梨酯80发生氧化降解；其二是，处方中的蛋白可能会残留一些宿主细胞蛋白，这些杂蛋白中可能存在使聚山梨酯80降解的相关酶，而酶需要金属离子作为辅因子来发挥催化功能，因此，处方中加入的金属螯合剂能够通过结合金属离子来抑制聚山梨酯80的降解，并提高处方的稳定性。
由此，确定最优选的制剂方案为：约100.0mg/ml重组抗分化抗原簇47(CD47)和抗程序性死亡配体1(PD-L1)双特异性抗体、约2.52mg/ml组氨酸、0.79mg/ml盐酸组氨酸、37.92mg/ml盐酸精氨酸、0.50mg/ml聚山梨酯80，0.02mg/ml EDTA，pH 6.5。
实施例6:500L制备物的稳定性考察
采用实施例5的制剂方案(约100.0mg/ml重组抗分化抗原簇47(CD47)和抗程序性死亡配体1(PD-L1)双特异性抗体、约2.52mg/ml组氨酸、0.79mg/ml盐酸组氨酸、37.92mg/ml盐酸精氨酸、0.50mg/ml聚山梨酯80，0.02mg/ml EDTA，pH 6.5)制备500L制备物，进行稳定性考察。
6.1待考察稳定性的制备物及考察方案
配制了500L的如下制备物：101.8mg/ml重组抗CD47/PD-L1双特异性抗体蛋白、2.52mg/ml组氨酸、0.79mg/ml盐酸组氨酸、37.92mg/ml盐酸精氨酸、0.50mg/ml聚山梨酯80，0.02mg/ml EDTA，pH 6.5，用于考察其稳定性。
表24.考察制备物的稳定性的方案
Figure PCTCN2020098172-appb-000044
表25.质量标准
Figure PCTCN2020098172-appb-000045
Figure PCTCN2020098172-appb-000046
注：1.报告数据是指对该项检测未设定接受范围，将实际检测出来的数据直接汇报即可。下同。
2.在SEC-HPLC法中，主峰+聚合体+片段＝100％，只要主峰满足≥95％的条件，剩下的≤5％就是片段和聚合体的量，因此，片段和聚合体的量这两者分别的数据以报告数据给出。下同。
3.在非还原型CE-SDS法中，主峰+片段＝100％，只要主峰满足≥90％的条件，剩下的≤10％就是片段的量，因此，片段的量以报告数据给出。下同。
4.在还原型CE-SDS法中，重链和轻链含量+非糖基化重链+片段＝100％，只要重链和轻链含量满足≥90％的条件，剩下的≤10％就是非糖基化重链和片段的量，因此，非糖基化重链的量和片段的量这两者分别的数据以报告数据给出。下同。
5.在CEX-HPLC法中，主成分+酸性组分+碱性组分＝100％，只要主成分的量满足≥44.9％的条件，剩下的≤55.1％就是酸性组分和碱性组分的量，因此，酸性组分的量和碱性组分的量这两者分别的数据以报告数据给出。下同。
6.所述规定是：《中华人民共和国药典》(2015年版，三部)的有关规定，下同，例如，通则0904“可见异物检查法”规定了可见异物的检查。
6.2稳定性考察结果
对500L制备物的稳定性考察结果如下表中所示。
表26.长期稳定性研究结果(5℃±3℃)
Figure PCTCN2020098172-appb-000047
Figure PCTCN2020098172-appb-000048
注：1.N/A表示没有检测，对应的指标一般只检测首尾两个端点，若首尾两个端点值都符合要求，则认为过程中每个点的值也是符合要求的。
2.在还原型CE-SDS法中，主峰+NGHC CD47+片段＝100％，只要主峰满足≥90％的条件，剩下的≤10％就是NGHC CD47链和片段的量，因此，NGHC CD47的量和片段的量这两者分别的数据以报告数据给出。
由表26中的检测结果可见，制备物在6个月后，满足质量标准。
表27.加速稳定性研究结果(25℃±2℃/60％RH±5％RH)
Figure PCTCN2020098172-appb-000049
注：N/A表示没有检测，对应的指标一般只检测首尾两个端点，若首尾两个端点值都符合要求，则认为过程中每个点的值也是符合要求的。
由表27中的检测结果可见，在加速稳定性实验中，制备物在6个月后，各指标均符合要 求。
表28.强制条件试验结果(40℃±2℃/75％RH±5％RH)
Figure PCTCN2020098172-appb-000050
注：N/A表示没有检测，对应的指标一般只检测首尾两个端点，若首尾两个端点值都符合要求，则认为过程中每个点的值也是符合要求的。
由表28中的检测结果可见，在强制稳定性实验中，制备物在8周后，各指标均符合要求。
总之，通过上述实验得出，本发明的制剂方案在放大生产中是满足对制剂的稳定性要求的。
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 (17)

  1. 一种液体抗体制剂，包含
    (i)抗CD47/PD-L1双特异性抗体蛋白；
    (ii)缓冲剂，
    (iii)稳定剂，
    (iv)表面活性剂，和
    任选地，(v)金属螯合剂(例如，EDTA)
    其中所述抗CD47/PD-L1双特异性抗体蛋白是一种三链抗体，所述三链抗体包含第一多肽链和第二多肽链上的特异性结合CD47的VH/VL对作为第一抗原结合位点，以及第三多肽链上的特异性结合PD-L1的第一VHH作为单结构域第二抗原结合位点和第二VHH作为单结构域第三抗原结合位点；或者包含第一多肽链和第二多肽链上的特异性结合PD-L1的VH/VL对作为第一抗原结合位点，以及第三多肽链上的特异性结合CD47的第一VHH作为单结构域第二抗原结合位点和第二VHH作为单结构域第三抗原结合位点，
    优选地，所述液体抗体制剂的pH约为6.4-7.0，例如，pH约为6.4、6.5、6.8、7.0。
  2. 权利要求1的液体抗体制剂，特征在于所述液体抗体制剂中的抗CD47/PD-L1双特异性抗体蛋白的浓度为约1-200mg/ml，优选地为约5-150mg/mL，例如约5、10、20、30、40、50、60、70、80、90、100、110、120、130、140或150mg/ml。
  3. 根据权利要求1或2所述的液体抗体制剂，特征在于所述液体抗体制剂中的缓冲剂选自组氨酸、盐酸组氨酸和它们的组合，优选地，所述缓冲剂的浓度为约1-30mM，优选地为约5-25mM，例如，约5、10、15、20、25mM。
  4. 根据权利要求1-3中任何一项所述的液体抗体制剂，特征在于所述稳定剂选自山梨醇、蔗糖、海藻糖、精氨酸、盐酸精氨酸和它们的任意组合，更优选为蔗糖、精氨酸和/或盐酸精氨酸；优选地，所述稳定剂的浓度为约50-500mM，更优选地为约100-400mM，例如，约100、150、200、250、300、350、400mM。
  5. 根据权利要求1-4中任何一项所述的液体抗体制剂，特征在于所述液体抗体制剂包含盐酸精氨酸作为稳定剂，优选地盐酸精氨酸以约50-250mM，优选地约100-200mM(例如，约100、110、120、130、140、150、160、170、180、190、200mM)的量存在；和/或包含蔗糖作为稳定剂，优选地蔗糖以约50-250mM，优选地约100-200mM(例如，约100、110、120、130、140、150、160、170、180、190、200mM)的量存在。
  6. 根据权利要求1-5中任何一项所述的液体抗体制剂，特征在于所述液体抗体制剂中的表面活性剂选自聚山梨酯类表面活性剂，优选为聚山梨酯-80。
  7. 根据权利要求1-6中任何一项所述的液体抗体制剂，特征在于所述表面活性剂的浓度为约0.1-1mg/ml，优选地为约0.2-0.8mg/ml，例如约0.2、0.3、0.4、0.5、0.6、0.7、0.8mg/ml。
  8. 根据权利要求1-7中任何一项所述的液体抗体制剂，特征在于所述液体制剂还包含金属螯合剂(例如，EDTA)，例如，约0.002－0.2mg/ml金属螯合剂(例如，EDTA)，优选地约0.01-0.1mg/ml，例如约0.01、0.02、0.03、0.04、0.05、0.06、0.08、0.1mg/ml金属螯合剂(例如，EDTA)。
  9. 根据权利要求1所述的液体抗体制剂，特征在于所述抗CD47/PD-L1双特异性抗体蛋白的第一多肽链和第二多肽链上的特异性结合CD47的VH/VL对作为第一抗原结合位点包含衍生自抗CD47抗体ADI-29341的GSIEHYYWS(SEQ ID NO:3)所示的VH CDR1、YIYYSGSTNYNPSLKS(SEQ ID NO:4)所示的VH CDR2、ARGKTGSAA(SEQ ID NO:5)所示的VH CDR3、RASQGISRWLA(SEQ ID NO:10)所示的VL CDR1、AASSLQS(SEQ ID NO:11)所示的VL CDR2和QQTVSFPIT(SEQ ID NO:12)所示的VL CDR3，或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；所述第三多肽链上的特异性结合PD-L1的单结构域第二和第三抗原 结合位点均包含AYTISRNSMG(SEQ ID NO:17)所示的CDR1、IESDGST(SEQ ID NO:18)所示的CDR2和AAPKVGLGPRTALGHLAFMTLPALNY(SEQ ID NO:19)所示的CDR3，或者与所述3个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列，
    更优选地，所述第一多肽链和第二多肽链上的特异性结合CD47的VH/VL对作为第一抗原结合位点包含衍生自抗CD47抗体ADI-29341的SEQ ID NO:2/9的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，所述第三多肽链上的特异性结合PD-L1的单结构域第二和第三抗原结合位点均包含SEQ ID NO:15和/或SEQ ID NO:16所示的氨基酸序列，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列，
    最优选地，所述三链抗体包含SEQ ID NO:1所示的第一多肽链、SEQ ID NO:8所示的第二多肽链、和SEQ ID NO:14或SEQ ID NO:22所示的第三多肽链，或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列。
  10. 根据权利要求1-9中任一项所述的液体抗体制剂，特征在于所述抗CD47/PD-L1双特异性抗体蛋白在HEK293细胞或CHO细胞中重组表达。
  11. 根据权利要求1-10中任一项所述的液体抗体制剂，特征在于所述液体制剂为注射剂，优选用于皮下注射或静脉内注射，或者为输注剂，例如用于静脉内输注。
  12. 根据权利要求1-11中任一项所述的液体抗体制剂，其包含：
    (i)约1-200mg/ml的抗CD47/PD-L1双特异性抗体蛋白；
    (ii)约1-30mM的组氨酸和/或盐酸组氨酸；
    (iii)约50-500mM的蔗糖、精氨酸和/或盐酸精氨酸，和
    (iv)约0.1-1mg/ml聚山梨醇酯80；
    任选地，所述液体制剂还包含0.002－0.2mg/ml金属螯合剂(例如，EDTA)；
    其中所述液体制剂的pH为约6.4－7.0，优选地约6.5；
    例如，所述液体抗体制剂包含
    (i)约50-150mg/ml的抗CD47/PD-L1双特异性抗体蛋白；
    (ii)约3-25mM的组氨酸和/或盐酸组氨酸；
    (iii)约150-300mM的蔗糖、精氨酸和/或盐酸精氨酸，和
    (iv)约0.2-0.8mg/ml聚山梨醇酯80；
    任选地，所述液体制剂还包含约0.01-0.1mg/ml金属螯合剂(例如，EDTA)；
    其中所述液体制剂的pH为约6.4－7.0，优选地约6.5；
    或者，所述液体抗体制剂包含
    (i)约100mg/ml的抗CD47/PD-L1双特异性抗体蛋白；
    (ii)约20mM组氨酸；
    (iii)约180mM盐酸精氨酸，和
    (iv)约0.2mg/ml聚山梨醇酯80；
    任选地，所述液体制剂还包含金属螯合剂(例如，EDTA)，例如约0.02mg/ml EDTA；
    其中所述液体制剂的pH为约6.4－7.0，优选地约6.5；
    或者，所述液体抗体制剂包含
    (i)约100mg/ml的抗CD47/PD-L1双特异性抗体蛋白；
    (ii)约20mM组氨酸；
    (iii)约100mM盐酸精氨酸和4％蔗糖，和
    (iv)约0.2mg/ml聚山梨醇酯80；
    任选地，所述液体制剂还包含金属螯合剂(例如，EDTA)，例如约0.02mg/ml EDTA；
    其中所述液体制剂的pH为约6.4－7.0，优选地约6.5；
    或者，所述液体抗体制剂包含
    (i)约100mg/ml的抗CD47/PD-L1双特异性抗体蛋白；
    (ii)约2.52mg/ml组氨酸和约0.79mg/ml盐酸组氨酸；
    (iii)约37.92mg/ml盐酸精氨酸，和
    (iv)约0.5mg/ml聚山梨醇酯80；
    任选地，所述液体制剂还包含金属螯合剂(例如，EDTA)，例如约0.02mg/ml EDTA；
    其中所述液体制剂的pH为约6.4－7.0，优选地约6.5。
  13. 根据权利要求1-12中任何一项所述的液体抗体制剂，其特征在于，该制剂在储存后，例如在2-8℃储存至少24个月后，或在室温储存至少3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95％、96％、97％、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85％的纯度，优选大于86％、87％、88％、89％、90％、91％、92％的纯度；
    (iii)通过iCIEF法测量，相对于储存第0天的初始值，制剂中抗CD47/PD-L1双特异性抗体蛋白的各组分(主成分、酸性组分和碱性组分)的变化值总和不超过50％，例如不超过48％、46％、44％、42％、40％；
    (iv)通过CEX-HPLC法测量，相对于储存第0天的初始值，制剂中抗CD47/PD-L1双特异性抗体蛋白的各组分(主成分、酸性组分和碱性组分)的变化值总和不超过40％，例如不超过38％、36％、34％、32％、30％；
    (v)通过ELISA法测量，相对于储存第0天的初始值，制剂中抗CD47/PD-L1双特异性抗体蛋白的相对结合活性为70％-130％，例如，为70％、80％、90％、100％、110％、120％、130％。
  14. ー种固体抗体制剂，其通过固化权利要求1-13中任何一项所述的液体抗体制剂而获得，所述固体抗体制剂例如是冻干粉针剂形式。
  15. 递送装置，其包含权利要求1-13中任何一项的液体抗体制剂或权利要求14的固体抗体制剂。
  16. 预填装注射器，其包含权利要求1-13中任何一项的液体抗体制剂或权利要求14的固体抗体制剂，用于静脉内注射或者肌内注射。
  17. 根据权利要求1-13中任何一项的液体抗体制剂或权利要求14的固体抗体制剂的用途，用于制备治疗、预防或延缓与SIRPα/CD47信号传导通路和PD1/PD-L1信号传导通路相关的病症的药物，所述病症例如各种实体瘤和血液病(例如，白血病、淋巴瘤、骨髓瘤，例如，多发性骨髓瘤)；自身免疫病、急性和慢性炎性病症、感染性疾病和转移性病灶。
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Cited By (2)

* Cited by examiner, † Cited by third party
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US11702474B2 (en) 2019-12-17 2023-07-18 Pfizer Inc. Antibodies specific for CD47, PD-L1, and uses thereof
WO2023011644A1 (zh) * 2021-08-06 2023-02-09 百奥泰生物制药股份有限公司 抗pd-l1/cd47双特异抗体在治疗疾病中的应用

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