WO2022222945A1 - 一种包含抗体融合蛋白的药物组合物及其用途 - Google Patents

一种包含抗体融合蛋白的药物组合物及其用途 Download PDF

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WO2022222945A1
WO2022222945A1 PCT/CN2022/087850 CN2022087850W WO2022222945A1 WO 2022222945 A1 WO2022222945 A1 WO 2022222945A1 CN 2022087850 W CN2022087850 W CN 2022087850W WO 2022222945 A1 WO2022222945 A1 WO 2022222945A1
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Prior art keywords
cancer
pharmaceutical composition
buffer
fusion protein
sirpγ
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PCT/CN2022/087850
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English (en)
French (fr)
Inventor
杨健健
田晨敏
刘洵
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Publication of WO2022222945A1 publication Critical patent/WO2022222945A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present disclosure belongs to the field of pharmaceutical preparations, and in particular relates to a pharmaceutical composition comprising an antibody fusion protein and its use as a medicine.
  • PD-1 Programmed death-1
  • PD-1 has two ligands, PD-L1 and PD-L2.
  • PD-L1 is mainly expressed on T cells, B cells, macrophages and dendritic cells (DC).
  • the expression of PD-L2 is relatively limited, mainly expressed on antigen-presenting cells, such as activated macrophages and dendritic cells.
  • PD-L1 has been detected in human tumor tissues such as breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, and melanoma, and the expression level of PD-L1 is closely related to the clinical and prognosis of patients. Since PD-L1 acts as a second signaling pathway to inhibit T cell proliferation, blocking the combination of PD-L1/PD-1 has become a very potential emerging target in the field of tumor immunotherapy.
  • CD47 is expressed or overexpressed on many tumor types, including acute myeloid leukemia, various subtypes of B-cell non-Hodgkin lymphoma, and many human solid tumor cells (Cell. 2009 Jul 23; 138(2): 286-99 ; Res. 2011;71:1374-84.). CD47 binds to signal regulatory protein alpha (SIRP ⁇ ) on macrophages, resulting in a "don't eat me” signal that prevents host cells from engulfing tumor cells, allowing them to be removed by the innate immune system (Mol Ther. 2017 Feb 1;25(2) ): 523-533).
  • SIRP ⁇ signal regulatory protein alpha
  • SIRP signal regulatory protein family
  • This family contains three type I transmembrane glycoproteins: SIRP ⁇ , SIRP ⁇ and SIRP ⁇ . Among them, both SIRP ⁇ and SIRP ⁇ can bind to CD47, but the binding capacity of SIRP ⁇ and CD47 is about 10 times weaker than that of SIRP ⁇ and CD47.
  • SIRP ⁇ is expressed on T cells and activated NK cells, and the CD47-SIRP ⁇ interaction is involved in the contact between antigen-presenting cells and T cells, co-stimulates T cell activation and promotes T cell proliferation (Piccio et al., Blood 2005, 105, 2421 -2427; BMC Struct Biol. 2013 Jul 4;13:13.).
  • the present disclosure provides a pharmaceutical composition comprising a PD-L1-SIRP ⁇ fusion protein, the composition has high stability, and after being placed at 40° C. for 28 days, the increase in aggregates does not exceed 8% (detected by SEC); in addition, , the appearance of the pharmaceutical composition in the present disclosure is still clear after shaking (25°C, 300rpm) for 8 days.
  • compositions of the present disclosure comprise a PD-L1-SIRP ⁇ fusion protein and a buffer, wherein:
  • the PD-L1-SIRP ⁇ fusion protein comprises the first chain of SEQ ID NO: 1 and the second chain of SEQ ID NO: 2;
  • the buffer is an acetate buffer or a histidine buffer, and the pH of the pharmaceutical composition is from about 5.0 to about 6.0.
  • the pH of the pharmaceutical composition is from about 5.0 to about 6.0; including but not limited to about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8 , about 5.9, about 6.0, and any range between these point values.
  • the buffer of the pharmaceutical composition is an acetate buffer.
  • the buffer of the pharmaceutical composition is an acetate buffer, and the pH of the pharmaceutical composition is from about 5.0 to about 5.5. In some embodiments, the pH of the pharmaceutical composition is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, and any range between these points. In some embodiments, the pH of the pharmaceutical composition is from about 5.2 to about 5.4. In some embodiments, the pH of the pharmaceutical composition is about 5.2.
  • the buffer of the pharmaceutical composition is a histidine buffer.
  • the buffer of the pharmaceutical composition is a histidine buffer
  • the pH of the pharmaceutical composition is from about 5.0 to about 6.0.
  • the pH of the pharmaceutical composition is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, and the like Any range between values.
  • the buffer of the pharmaceutical composition is a histidine buffer
  • the pH of the pharmaceutical composition is from about 5.5 to about 6.0.
  • the pH of the pharmaceutical composition is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, and any range between these points.
  • the pH of the pharmaceutical composition is from about 5.5 to about 5.7. In some embodiments, the pH of the pharmaceutical composition is about 5.5.
  • the acetate buffer is an acetic acid-sodium acetate buffer.
  • the histidine buffer is a histidine-hydrochloride buffer.
  • the final pH of the pharmaceutical compositions in the present disclosure is nearly identical to the buffer pH. However, it is well known to those skilled in the art that during the preparation of pharmaceutical formulations, there may be pH drift, and the drift of the final pH of the pharmaceutical composition in the present disclosure is within the range of ⁇ 0.3.
  • the buffer is a histidine-acetate buffer.
  • the PD-L1-SIRP ⁇ fusion protein concentration is about 1 mg/mL to about 100 mg/mL. In some embodiments, the concentration of the PD-L1-SIRP ⁇ fusion protein is about 30 mg/mL to about 70 mg/mL, including but not limited to about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL mL, about 70 mg/mL, and any range between these point values.
  • the concentration of PD-L1-SIRP ⁇ fusion protein in the pharmaceutical composition is from about 45 mg/mL to about 55 mg/mL, including but not limited to 45 mg/mL, 46 mg/mL, 47 mg/mL, 48 mg /mL, 49 mg/mL, 50 mg/mL, 51 mg/mL, 52 mg/mL, 53 mg/mL, 54 mg/mL, 55 mg/mL, and any range between these point values. In some embodiments, the concentration of the PD-L1-SIRP ⁇ fusion protein is 50 mg/mL.
  • the pharmaceutical composition further comprises a surfactant.
  • the surfactant is a nonionic surfactant.
  • the surfactant is selected from the group consisting of polysorbate, polysorbate 20, polysorbate 80, polyhydroxyalkene, poloxamer 188, Triton, sodium lauryl sulfonate, lauryl Sodium Sulfonate, Sodium Octyl Glycoside, Lauryl-Sulfobetaine, Myristyl-Sulfobetaine, Linole-Sulfobetaine, Stearyl-Sulfobetaine, Lauryl-Carcosine acid, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauryl amido Propyl-Betaine, Cocamidoprop
  • the surfactant is a polysorbate or a poloxamer. In some embodiments, the surfactant is polysorbate 80, polysorbate 20, or poloxamer 188. In some embodiments, the surfactant is poloxamer 188 or polysorbate 80.
  • the surfactant concentration is about 0.05 mg/mL to about 1.2 mg/mL; including but not limited to about 0.05 mg/mL, about 0.1 mg/mL mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, About 1.0 mg/mL, about 1.1 mg/mL, about 1.2 mg/mL, and any range between these point values.
  • the surfactant concentration is from about 0.2 mg/mL to about 0.8 mg/mL. In some embodiments, the surfactant concentration is from about 0.2 mg/mL to about 0.6 mg/mL. In some embodiments, the surfactant concentration is from about 0.4 mg/mL to about 0.6 mg/mL. In some embodiments, the surfactant concentration is about 0.4 mg/mL, about 0.5 mg/mL, or about 0.6 mg/mL, and any range between these point values.
  • the surfactant is polysorbate 80 at a concentration of about 0.2 mg/mL to about 0.8 mg/mL.
  • the surfactant is polysorbate 80 at a concentration of about 0.2 mg/mL to about 0.6 mg/mL.
  • the surfactant is poloxamer 188 at a concentration of about 0.4 mg/mL to about 0.8 mg/mL.
  • the surfactant is poloxamer 188 at a concentration of about 0.4 mg/mL to about 0.6 mg/mL.
  • the pharmaceutical composition further comprises a sugar.
  • the sugar is selected from the group consisting of conventional compositions (CH2O ) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like .
  • the sugar can be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, mili Disaccharide, melezitose, raffinose, mannose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, etc.
  • the sugar is selected from one or more of sucrose, mannitol, and trehalose.
  • the sugar is sucrose.
  • the pharmaceutical composition of any of the above, wherein the concentration of the sugar is from about 20 mg/mL to about 100 mg/mL; including but not limited to 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, and any range between these point values.
  • the concentration of the sugar is from about 70 mg/mL to about 90 mg/mL. In some embodiments, the concentration of the sugar is about 70 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL, or about 90 mg/mL. In some embodiments, the concentration of the sugar is 80 mg/mL.
  • the concentration of the buffer is from about 5 mM to about 50 mM; including but not limited to about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50mM, and any range between these point values.
  • the concentration of the buffer is from about 10 mM to about 30 mM. In some embodiments, the concentration of the buffer is about 10 mM, about 15 mM, about 20 mM, about 25 mM, or about 30 mM. In some embodiments, the concentration of the buffer is about 10 mM.
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pH of the pharmaceutical composition is from about 5.2 to about 6.0.
  • the pharmaceutical composition of any of the above comprises the following components:
  • PD-L1-SIRP ⁇ fusion protein at about 50 mg/mL
  • polysorbate 80 at about 0.4 mg/mL
  • sucrose at about 80 mg/mL
  • histidine at about 10 mM - a hydrochloride buffer
  • the pH of the pharmaceutical composition is from about 5.5 to about 6.0.
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • PD-L1-SIRP ⁇ fusion protein at about 50 mg/mL
  • polysorbate 80 at about 0.4 mg/mL
  • sucrose at about 80 mg/mL
  • histidine at about 10 mM - a hydrochloride buffer
  • the pH of the pharmaceutical composition is from about 5.0 to about 5.5.
  • the pharmaceutical composition of any of the above comprises the following components:
  • PD-L1-SIRP ⁇ fusion protein at about 50 mg/mL
  • polysorbate 80 at about 0.4 mg/mL
  • sucrose at about 80 mg/mL
  • histidine at about 10 mM - acetate buffer
  • the pH of the pharmaceutical composition is from about 5.0 to about 6.0.
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above comprises the following components:
  • the pharmaceutical composition of any of the above is a liquid formulation.
  • the solvent of the liquid formulation is water.
  • the present disclosure provides a lyophilized formulation that is a lyophilized form of the pharmaceutical composition of any of the foregoing.
  • the present disclosure also provides a freeze-dried preparation, characterized in that the freeze-dried preparation can form the pharmaceutical composition described in any one of the above after reconstitution.
  • the present disclosure also provides a method of preparing a lyophilized formulation, comprising the step of lyophilizing the pharmaceutical composition as described in any of the above.
  • the present disclosure also provides a lyophilized formulation obtained by lyophilizing the pharmaceutical composition according to any of the above.
  • the freeze-drying comprises the steps of pre-freezing, primary drying, and secondary drying in sequence.
  • the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.
  • the present disclosure also provides a reconstituted solution, characterized in that the reconstituted solution is prepared by reconstituting the lyophilized preparation described in any of the above.
  • the reconstitution solution as described above comprises the following components:
  • the reconstitution solution as described above comprises the following components:
  • the reconstitution solution as described above comprises the following components:
  • the reconstitution solution as described above comprises the following components:
  • the reconstitution solution as described above comprises the following components:
  • the reconstitution solution as described above comprises the following components:
  • the reconstitution solution as described above comprises the following components:
  • the reconstitution solution as described above comprises the following components:
  • the present disclosure also provides an article of manufacture comprising a container containing the pharmaceutical composition of any of the above, the lyophilized formulation of any of the above, or the reconstitution solution of any of the above.
  • the present disclosure also provides that the pharmaceutical composition of any of the above, the lyophilized formulation of any of the above, or the reconstituted solution of any of the above are prepared for use in eliminating an immunosuppression-related disease in a subject use in medicines.
  • the present disclosure also provides a method of eliminating an immunosuppression-related disease in a subject, the method comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of any of the above, the lyophilized formulation of any of the above, or any of the above item reconstitution solution.
  • the present disclosure also provides the pharmaceutical composition of any of the above, the lyophilized formulation of any of the above, or the reconstituted solution of any of the above, for use as a medicament.
  • the present disclosure also provides the pharmaceutical composition of any of the above, the lyophilized formulation of any of the above, or the reconstituted solution of any of the above, for use in eliminating an immunosuppression-related disease in a subject medicine.
  • the present disclosure also provides the pharmaceutical composition of any of the above, the lyophilized formulation of any of the above, or the reconstituted solution of any of the above in preparation for the treatment of PD-L1 or CD47-related diseases use in medicines.
  • the present disclosure also provides a method of treating a PD-L1 or CD47-related disease, the method comprising administering to a patient an effective amount of the pharmaceutical composition according to any of the above, the lyophilized according to any of the above A formulation or a reconstituted solution as in any of the above.
  • the present disclosure also provides the pharmaceutical composition of any of the above, the lyophilized formulation of any of the above, or the reconstituted solution of any of the above, for use as a treatment for PD-L1 or CD47-related diseases drug.
  • the PD-L1 or CD47-related disease is cancer, a bacterial infection, or a viral infection.
  • the PD-L1 or CD47-related disease is a cancer that expresses PD-L1 or CD47.
  • the immunosuppression-related disease comprises cancer, bacterial or viral infection.
  • the cancer includes, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, glial stromal tumor, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, Chronic myeloid leukemia, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/histiocyte-rich large B-cell lymphoma, multiple myeloma, Myeloid cell leukemia-1 protein, myelodysplastic syndrome, gastrointestinal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leuk
  • Anti-PD-L1 antibody includes full-length antibodies that can specifically bind to PD-L1, and also includes antigen-binding fragments comprising the light chain variable region and heavy chain variable region of the full-length antibody, including but not limited to the Single chain antibodies (scFv), Fab fragments or other antigen-binding fragments comprising scFv or Fab of the light chain variable region and heavy chain variable region of a full-length antibody.
  • scFv Single chain antibodies
  • Fab fragments or other antigen-binding fragments comprising scFv or Fab of the light chain variable region and heavy chain variable region of a full-length antibody.
  • SIRP ⁇ peptide refers to a human SIRP ⁇ -D1 domain peptide, which has human CD47 binding activity.
  • Linking in the linking of the SIRP ⁇ peptide to the polypeptide chain of the anti-human PD-L1 antibody refers to an operative linkage between the polypeptides, including, for example, linkage via peptide bonds, or linkage using a linker. The linkage does not result in loss of function of the SIRP ⁇ peptide and the anti-human PD-L1 antibody, respectively.
  • fusion protein generally refers to a protein resulting from the fusion of two or more proteins or polypeptides.
  • the genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be linked to each other to form a fusion gene or fused nucleic acid molecule, which can encode the fusion protein.
  • Translation of the fusion gene produces a single polypeptide having the properties of at least one, if not each, of the two or more proteins or polypeptides prior to fusion.
  • the terms fusion protein and recombinant fusion protein are used herein with the same meaning.
  • the fusion proteins described herein generally comprise at least two domains (A and B), and optionally a third component, a linker between the two domains.
  • recombinant fusion proteins typically involves removal of stop codons from the cDNA sequence encoding a first protein or polypeptide, followed by ligation or overlap extension PCR to in-frame attachment of the cDNA for the second protein sequence. This DNA sequence is then expressed by the cell as a single protein.
  • the protein can be engineered to include the entire sequence of both original proteins or polypeptides, or only a portion of either.
  • the fusion protein in the present disclosure refers to a PD-L1-SIRP ⁇ variant fusion protein, which is a tetrapeptide structure comprising an anti-PD-L1 antibody and a SIRP ⁇ variant, wherein the N-terminus of the SIRP ⁇ variant is connected by a linker to the C-terminus of the anti-PD-L1 antibody heavy chain.
  • antibody in this disclosure is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), full length antibodies or antigen binding thereof Fragments (also referred to as "antigen binding portions"), murine antibodies, chimeric antibodies or humanized antibodies, affinity matured antibodies so long as they exhibit the desired antigen binding activity.
  • Natural antibodies refer to naturally occurring immunoglobulin molecules.
  • native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains joined by disulfide bonds.
  • each heavy chain has a variable domain (VH), also known as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3).
  • VH variable domain
  • VL variable region
  • VH variable heavy domain
  • CL constant light domain
  • the full-length antibodies of the present disclosure comprise a light chain variable region linked to a light chain constant region and a heavy chain variable region and a heavy chain variable region combined from the light and heavy chain variable region combinations in Table 1, Table 2, and Table 3 A full-length antibody formed by ligation of chain constant regions.
  • Those skilled in the art can select light chain constant regions and heavy chain constant regions derived from different antibodies according to actual needs, such as light chain constant regions and heavy chain constant regions derived from human antibodies.
  • Buffer refers to a buffer that resists changes in pH through the action of its acid-base conjugated component.
  • buffers to control pH in the appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumaric acid Salt, glycylglycine and other organic acid buffers.
  • a "histidine buffer” is a buffer containing histidine ions.
  • histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, and the like.
  • the histidine buffer is a histidine-hydrochloride buffer or a histidine-acetate buffer. Histidine-acetate buffer is prepared from histidine and acetic acid, histidine-hydrochloride buffer is prepared from histidine and histidine hydrochloride, or histidine and hydrochloric acid. made.
  • citrate buffer is a buffer that includes citrate ions.
  • citrate buffers include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate, and the like.
  • a preferred citrate buffer is citrate-sodium citrate.
  • succinate buffer is a buffer that includes succinate ions.
  • succinate buffers include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate, and the like.
  • a preferred succinate buffer is succinate-sodium succinate.
  • the succinic acid-sodium succinate can be prepared from succiplatinic acid and sodium hydroxide, or can be prepared from succiplatinic acid and sodium succinate.
  • a “phosphate buffer” is a buffer that includes phosphate ions.
  • phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like.
  • the preferred phosphate buffer is disodium hydrogen phosphate-sodium hydrogen phosphate.
  • acetate buffer is a buffer that includes acetate ions.
  • acetate buffers include acetate-sodium acetate, acetate-histidine, acetate-potassium acetate, calcium acetate, acetate-magnesium acetate, and the like.
  • the acetate buffer is acetic acid-sodium acetate buffer.
  • Stabilizer refers to a component that helps maintain the structural integrity of a biopharmaceutical drug, especially during freezing and/or lyophilization and/or storage (especially when exposed to stress). This stabilizing effect can occur for a variety of reasons, usually the stabilizing agent acts as a penetrant that reduces protein denaturation. Additional stabilizers herein refer to stabilizers other than buffer systems, surfactants and sugar alcohols, eg stabilizers selected from PEG, arginine and EDTA.
  • “Pharmaceutical composition” means a mixture comprising one or more of the antibodies described herein with other chemical components, such as physiological/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to maintain the stability of the active ingredient, facilitate the administration to the organism, and facilitate the absorption of the active ingredient to exert biological activity.
  • the solvent therein is all water.
  • “Lyophilized formulation” means a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.
  • the pharmaceutical composition described in the present disclosure can achieve a stable effect: a pharmaceutical composition in which the antibody substantially retains its physical stability and/or chemical stability and/or biological activity after storage, preferably a drug
  • the composition substantially retains its physical and chemical stability and its biological activity after storage.
  • the shelf life is generally selected based on the intended shelf life of the pharmaceutical composition.
  • analytical techniques for measuring protein stability which can measure stability after storage at a selected temperature for a selected period of time.
  • a stable formulation is one in which no significant change is observed when stored at refrigerated temperature (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years .
  • stable liquid formulations include liquid formulations that exhibit desirable characteristics after storage at temperatures including 25°C for periods of time including 1 month, 3 months, and 6 months.
  • Typical example of stability Usually no more than about 10%, preferably no more than about 5% of the antibody monomers aggregate or degrade as measured by SE-HPLC. By visual analysis, the formulation was a pale yellow near colorless clear liquid or a colorless clear liquid, or clear to slightly milky white. The formulations had no more than ⁇ 10% variation in concentration, pH and osmolality. A reduction of no more than about 10%, preferably no more than about 5%, is generally observed. Typically no more than about 10% aggregates are formed, preferably no more than about 5% aggregates.
  • Antibodies show no significant increase in aggregation, precipitation and/or denaturation if after visual inspection for color and/or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS) , then the antibody "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).
  • An antibody "retains its chemical stability” in a pharmaceutical formulation if it does not exhibit significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that frequently change the chemical structure of proteins include hydrolysis or truncation (as assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (by peptide mapping such as combined with mass spectrometry or MALDI/TOF/MS, etc.) methods), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement, etc.), and isomerization (by measuring isoaspartic acid content, Peptide Mapping, etc.).
  • An antibody "retains its biological activity" in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited at the time of preparation of the pharmaceutical formulation.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions Contact with animals, humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering also mean in vitro and ex vivo treatment of, eg, cells by an agent, diagnostic, binding composition, or by another cell.
  • Treatment as it applies to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, eg, a pharmaceutical composition comprising any of the present disclosure, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to treat effect.
  • a therapeutic agent is administered in a patient or population to be treated in an amount effective to alleviate one or more symptoms of a disease, to induce regression of such symptoms or to inhibit progression of such symptoms to any clinically measured degree.
  • the amount of a therapeutic agent effective to relieve symptoms of any particular disease can vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to produce the desired effect in the patient.
  • Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (eg, methods of treatment or articles of manufacture) may be ineffective in alleviating symptoms of each target disease, the method of The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce symptoms of the target disease in a statistically significant number of patients.
  • H test Kruskal-Wallis test
  • Jonckheere-Terpstra test Jonckheere-Terpstra test
  • Wilcoxon test determine that it should reduce symptoms of the target disease in a statistically significant number of patients.
  • the disease associated with PD-L1 or CD47 is not limited in the present disclosure, as long as it is a disease associated with PD-L1 or CD47, for example, the therapeutic response induced by the fusion protein of the present disclosure can be achieved by binding human PD-L1 or CD47, The binding of PD-L1 or CD47 to its receptor/ligand is then blocked.
  • the molecules of the present disclosure are very useful to persons suffering from tumors or cancers, optionally including lymphomas, blastomas, sarcomas, leukemias , squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, glioma, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell Lymphoma, follicular lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, Small lymphocytic lymphoma, T-cell/histiocyte-rich large B-cell lymphoma, multiple myeloma, myeloid leukemia-1 protein, myelodysplastic syndrome, gastrointestinal
  • PD-L1-SIRP ⁇ fusion protein described in the present disclosure and its activity have been disclosed in WO2020177733A1, wherein the exemplified h1831K-19-S37 fusion protein has a 4-peptide structure comprising two identical first chains and two identical second strands.
  • the fusion protein sequences used in this disclosure are as follows:
  • SEC% SEC monomer content percentage
  • R-CE capillary gel electrophoresis also known as CE-SDS(R):
  • Purity percentage of reduced CE A heavy chain/A total*100%+A light chain/A total*100% (A heavy chain is the peak area of the sample heavy chain, A light chain is the peak area of the light chain in the sample, A total is the sum of all peak areas)
  • iCIEF neutral peak content percentage neutral peak area/total area*100% (total area is the sum of acidic peak, neutral peak and basic peak area).
  • Instrument used for iCIEF determination manufacturer simple protein, model muarice.
  • the freezing point method is used to measure the osmotic pressure. Based on the proportional relationship between the freezing point drop value and the molar concentration of the solution, a high-sensitivity temperature sensing element is used to measure the freezing point of the solution and convert it into osmotic pressure through electricity. Instrument manufacturer Roser Loser, model OM815.
  • Protein concentration determination instrument UV-Vis spectrophotometer, model: Nano Drop oneC, optical path is 1mm. The concentrations of the fusion proteins below were performed using a protein concentration meter.
  • the following buffers were prepared to prepare a formulation with a concentration of h1831K-19-S37 at about 50 mg/mL, and the stability of the fusion protein formulation at high temperature (40°C) was tested.
  • the buffer system is as follows:
  • His-HCl 10mM histidine-hydrochloride buffer (referred to as His-HCl), pH 5.5;
  • M stands for month
  • M0 stands for the beginning of the experiment
  • M1 stands for 1 month
  • ⁇ stands for the difference.
  • the relationship between the stability of the fusion protein and the appearance is as follows: clear > particles > obvious particles > slightly cloudy > cloudy.
  • the appearance results showed that the stability of PD-L1-SIRP ⁇ fusion protein in SA, His-HCl and AA buffer systems was better than that of CA and PB.
  • the CE results showed that when the pH was 4.5, the CE of the preparation decreased by 13.5 after being placed in AA buffer at pH 4.5 at 40°C for one month, indicating that the preparation was unstable.
  • the buffer of the formulation may be AA at pH 5.0-5.5 or His-HCl at pH 5.5-6.5.
  • D stands for days
  • D0 stands for the beginning of the experiment
  • D28 stands for 28 days
  • ⁇ stands for the difference.
  • sucrose concentration in the h1831K-19-S37 preparation can be selected from 75mg/mL to 80mg/mL.
  • D represents days
  • D0 represents the beginning of the experiment
  • D8 represents 8 days of placement.
  • D stands for days
  • D0 stands for the beginning of the experiment
  • D28 stands for 28 days
  • ⁇ stands for the difference.
  • the shaking results showed (see Table 5) that in the AA buffer, the formulations containing low concentrations of poloxamer 188 tended to produce particles; the high temperature results showed (see Table 6) that the formulations containing polysorbate 80 tended to produce aggregates , reducing the SEC purity. Therefore, the preferred surfactant is poloxamer 188, and its concentration is 0.4 mg/mL to 0.6 mg/mL.
  • Example 5 Comparison of stability of PD-L1-SIRP ⁇ fusion protein preparations with different ionic strengths
  • Freeze-drying process parameters Set temperature (°C) Setting time (min) Hold time (h) Vacuum (Pa) pre-frozen 5 10 1 N/A pre-frozen -45 50 2 N/A once dry -10 120 32 10 secondary drying 25 0 1 10 secondary drying 25 1 7 1
  • N/A means not detected.

Abstract

一种包含抗体融合蛋白的药物组合物及其用途。具体地，包含PD-L1-SIRPγ融合蛋白的药物组合物及其用途。

Description

一种包含抗体融合蛋白的药物组合物及其用途 技术领域
本披露属于药物制剂领域，具体涉及一种包含抗体融合蛋白的药物组合物，以及其作为药物的用途。
背景技术
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。
程序性死亡分子1(programmed death-l，PD-l)是1992年发现的表达在T细胞表面的一个蛋白受体，参与细胞的凋亡过程。PD-1有两个配体，分别为PD-L1和PD-L2。PD-L1的主要表达于T细胞、B细胞、巨噬细胞和树突状细胞(dendritic cell,DC)上。而PD-L2的表达相对较局限，主要表达在抗原呈递细胞上，如活化的巨噬细胞和树突状细胞。研究发现乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤等人类肿瘤组织中检测到高PD-L1的表达，且PD-L1的表达水平和患者的临床及预后紧密相关。由于PD-L1起到第二信号通路抑制T细胞增殖的作用，所以阻断PD-L1/PD-1之间结合成为了肿瘤免疫治疗领域一个非常有潜力的新兴靶点。
CD47在许多肿瘤类型上表达或过度表达，包括急性骨髓性白血病，B细胞非霍奇金淋巴瘤的多种亚型，以及许多人类实体瘤细胞(Cell.2009Jul23；138(2):286-99；Res.2011；71:1374-84.)。CD47与巨噬细胞上的信号调节蛋白α(SIRPα)结合，导致阻止宿主细胞吞噬肿瘤细胞的“不吃我”信号，从而允许它们被先天免疫系统除去(Mol Ther.2017 Feb 1；25(2):523-533)。
信号调节蛋白家族(signal regulatory protein family(SIRP))在调节免疫应答中发挥重要作用。该家族包含3种I型跨膜糖蛋白：SIRPα、SIRPβ和SIRPγ。其中SIRPα与SIRPγ均可与CD47结合，但SIRPγ与CD47的结合能力比SIRPα与CD47的结合力能力弱10倍左右。SIRPγ在T细胞和活化的NK细胞上表达，CD47-SIRPγ相互作用参与抗原呈递细胞与T细胞之间的接触，共刺激T细胞活化并促进T细胞增殖(Piccio等人，Blood 2005，105，2421-2427；BMC Struct Biol.2013 Jul 4；13:13.)。
发明内容
本披露提供一种包含PD-L1-SIRPγ融合蛋白的药物组合物，该组合物具有很高的稳定性，其在40℃放置28天后，聚体增加不超过8％(经SEC检测)；此外，本披露中的药物组合物在振摇(25℃，300rpm)8天后，外观依然澄清。
在一些实施方案中，本披露所述的药物组合物包含PD-L1-SIRPγ融合蛋白和缓冲剂，其中：
所述PD-L1-SIRPγ融合蛋白包含SEQ ID NO：1的第一链和SEQ ID NO：2 的第二链；
所述缓冲剂为醋酸盐缓冲剂或组氨酸盐缓冲剂，且所述药物组合物的pH为约5.0至约6.0。
在一些实施方案中，所述药物组合物的pH为约5.0至约6.0；包括但不限于约5.0、约5.1、约5.2、约5.3、约5.4、约5.5、约5.6、约5.7、约5.8、约5.9、约6.0，以及这些点值之间的任意范围。
在一些实施方案中，所述药物组合物的缓冲剂为醋酸盐缓冲剂。
在一些实施方案中，所述药物组合物的缓冲剂为醋酸盐缓冲剂，并且所述药物组合物的pH为约5.0至约5.5。在一些实施方案中，所述药物组合物的pH为约5.0、约5.1、约5.2、约5.3、约5.4、约5.5，以及这些点值之间的任意范围。在一些实施方案中，所述药物组合物的pH为约5.2至约5.4。在一些实施方案中，所述药物组合物的pH为约5.2。
在一些实施方案中，所述药物组合物的缓冲剂为组氨酸盐缓冲剂。
在一些实施方案中，所述药物组合物的缓冲剂为组氨酸盐缓冲剂，并且所述药物组合物的pH为约5.0至约6.0。在一些实施方案中，所述药物组合物的pH为约5.0、约5.1、约5.2、约5.3、约5.4、约5.5、约5.6、约5.7、约5.8、约5.9、约6.0，以及这些点值之间的任意范围。
在一些实施方案中，所述药物组合物的缓冲剂为组氨酸盐缓冲剂，并且所述药物组合物的pH为约5.5至约6.0。在一些实施方案中，所述药物组合物的pH为约5.5、约5.6、约5.7、约5.8、约5.9、约6.0，以及这些点值之间的任意范围。在一些实施方案中，所述药物组合物的pH为约5.5至约5.7。在一些实施方案中，所述药物组合物的pH为约5.5。
在前述药物组合物的一些实施方案中，所述的醋酸盐缓冲剂为醋酸-醋酸钠缓冲剂。
在前述药物组合物的一些实施方案中，所述的组氨酸盐缓冲剂为组氨酸-盐酸盐缓冲剂。
本披露中的药物组合物的最终pH与缓冲液pH几乎一致。但本领域技术人员公知，在制备药物制剂的过程中，可能会存在pH飘移，本披露中的药物组合物的最终pH的飘移在±0.3范围内。
在前述药物组合物的一些实施方案中，所述的缓冲剂为组氨酸-醋酸缓冲剂。
在如上任一项所述的药物组合物的一些实施方案中，所述PD-L1-SIRPγ融合蛋白浓度为约1mg/mL至约100mg/mL。在一些实施方案中，所述PD-L1-SIRPγ融合蛋白的浓度为约30mg/mL至约70mg/mL，包括但不限于约30mg/mL、约40mg/mL、约50mg/mL、约60mg/mL、约70mg/mL，以及这些点值之间的任意范围。在一些实施方案中，所述药物组合物中的PD-L1-SIRPγ融合蛋白的浓度为约45mg/mL至约55mg/mL，包括但不限于45mg/mL、46mg/mL、47mg/mL、48mg/mL、 49mg/mL、50mg/mL、51mg/mL、52mg/mL、53mg/mL、54mg/mL、55mg/mL，以及这些点值之间的任意范围。在一些实施方案中，所述PD-L1-SIRPγ融合蛋白的浓度为50mg/mL。
在如上任一项所述的药物组合物的一些实施方案中，所述药物组合物还包含表面活性剂。在一些实施方案中，所述表面活性剂是非离子表面活性剂。在一些实施方案中，所述表面活性剂选自聚山梨酯、聚山梨酯20、聚山梨酯80、聚羟亚烃、泊洛沙姆188、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基-肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇、乙烯与丙烯二醇的共聚物等。在一些实施方案中，所述表面活性剂为聚山梨酯或泊洛沙姆。在一些实施方案中，所述表面活性剂为聚山梨酯80、聚山梨酯20或泊洛沙姆188。在一些实施方案中，所述表面活性剂为泊洛沙姆188或聚山梨酯80。
在如上任一项所述的药物组合物的一些实施方案中，所述表面活性剂浓度为约0.05mg/mL至约1.2mg/mL；包括但不限于约0.05mg/mL、约0.1mg/mL、约0.2mg/mL、约0.3mg/mL、约0.4mg/mL、约0.5mg/mL、约0.6mg/mL、约0.7mg/mL、约0.8mg/mL、约0.9mg/mL、约1.0mg/mL、约1.1mg/mL、约1.2mg/mL，以及这些点值之间的任意范围。
在一些实施方案中，所述表面活性剂浓度为约0.2mg/mL至约0.8mg/mL。在一些实施方案中，所述表面活性剂浓度为约0.2mg/mL至约0.6mg/mL。在一些实施方案中，所述表面活性剂浓度为约0.4mg/mL至约0.6mg/mL。在一些实施方案中，所述表面活性剂浓度为约0.4mg/mL、约0.5mg/mL或约0.6mg/mL，以及这些点值之间的任意范围。
在一些实施方案中，所述表面活性剂为聚山梨酯80，其浓度为约0.2mg/mL至约0.8mg/mL。
在一些实施方案中，所述表面活性剂为聚山梨酯80，其浓度为约0.2mg/mL至约0.6mg/mL。
在一些实施方案中，所述表面活性剂为泊洛沙姆188，其浓度为约0.4mg/mL至约0.8mg/mL。
在一些实施方案中，所述表面活性剂为泊洛沙姆188，其浓度为约0.4mg/mL至约0.6mg/mL。
在如上任一项所述的药物组合物的一些实施方案中，所述药物组合物还包含 糖。在一些实施方案中，所述糖选自常规组合物(CH 2O) n及其衍生物、包括单糖、二糖、三糖、多糖、糖醇、还原性糖、非还原性糖等等。所述的糖可选自葡萄糖、蔗糖、海藻糖、乳糖、果糖、麦芽糖、右旋糖苷、甘油、赤藻糖醇、丙三醇、阿拉伯糖醇、xylitol、山梨糖醇、甘露醇、密里二糖、松三糖、蜜三糖、甘露三糖、水苏糖、麦芽糖、乳果糖、麦芽酮糖、山梨醇、麦芽糖醇、乳糖醇、异-麦芽酮糖等。在一些实施方案中，所述糖选自蔗糖、甘露醇和海藻糖中的一种或多种。在一些实施方案中，所述糖为蔗糖。
在一些实施方案中，如上任一项所述的药物组合物，其中所述糖的浓度为约20mg/mL至约100mg/mL；包括但不限于20mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL、100mg/mL，以及这些点值之间的任意范围。
在一些实施方案中，所述糖的浓度为约70mg/mL至约90mg/mL。在一些实施方案中，所述糖的浓度为约约70mg/mL、约75mg/mL、约80mg/mL、约85mg/mL、或约90mg/mL。在一些实施方案中，所述糖的浓度为80mg/mL。
在如上任一项所述的药物组合物的一些实施方案中，所述缓冲剂的浓度为约5mM至约50mM；包括但不限于约5mM、约10mM、约20mM、约30mM、约40mM、约50mM，以及这些点值之间的任意范围。
在一些实施方案中，所述缓冲剂的浓度为约10mM至约30mM。在一些实施方案中，所述缓冲剂的浓度为约10mM、约15mM、约20mM、约25mM或约30mM。在一些实施方案中，所述缓冲剂的浓度为约10mM。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约1mg/mL至约100mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.05mg/mL至约1.2mg/mL的泊洛沙姆188，(c)约20mg/mL至约100mg/mL的糖，和(d)约5mM至约50mM的醋酸盐缓冲剂；所述药物组合物的pH为约5.0至约6.0。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约1mg/mL至约100mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.05mg/mL至约1.2mg/mL的泊洛沙姆188，(c)约20mg/mL至约100mg/mL的糖，和(d)约5mM至约50mM的醋酸-醋酸钠缓冲剂；所述药物组合物的pH为约5.0至约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约1mg/mL至约100mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.05mg/mL至约1.2mg/mL的聚山梨酯80，(c)约20mg/mL至约100mg/mL的糖，和(d)约5mM至约50mM的组氨酸盐缓冲剂；所述药物组合物的pH为约5.0至约6.0。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约1mg/mL至约100mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.05mg/mL至约1.2mg/mL的聚山梨酯80，(c)约20mg/mL至约100mg/mL的糖，和(d)约5mM至约50mM的组氨酸-盐酸盐缓冲剂；所述药物组合物的pH为约5.5至约 6.0。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约30mg/mL至约70mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL至约0.8mg/mL的泊洛沙姆188，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约10mM至约30mM的醋酸盐缓冲剂；所述药物组合物的pH为约5.0至约6.0。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约30mg/mL至约70mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL至约0.8mg/mL的泊洛沙姆188，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约10mM至约30mM的醋酸盐缓冲剂；所述药物组合物的pH为约5.0至约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约30mg/mL至约70mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL至约0.6mg/mL的泊洛沙姆188，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约10mM至约30mM的醋酸-醋酸钠缓冲剂；所述药物组合物的pH为约5.0至约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约30mg/mL至约70mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.2mg/mL至约0.6mg/mL的聚山梨酯80，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约10mM至约30mM的组氨酸盐缓冲剂；所述药物组合物的pH为约5.0至约6.0。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约30mg/mL至约70mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.2mg/mL至约0.6mg/mL的聚山梨酯80，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约10mM至约30mM的组氨酸盐缓冲剂；所述药物组合物的pH为约5.5至约6.0。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约45mg/mL至约55mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL至约0.6mg/mL的泊洛沙姆188，(c)约70mg/mL至约90mg/mL的糖，和(d)约10mM至约30mM的醋酸-醋酸钠缓冲剂；所述药物组合物的pH为约5.0至约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约45mg/mL至约55mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.2mg/mL至约0.6mg/mL的聚山梨酯80，(c)约70mg/mL至约90mg/mL的糖，和(d)约10mM至约30mM的组氨酸-盐酸盐缓冲剂；所述药物组合物的pH为约5.5至约6.0。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约45mg/mL至约55mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL至约0.6mg/mL的泊洛沙姆188，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约10mM至约30mM的醋酸盐缓冲剂；所述药物组合物的pH为约5.0至约5.5；或
(a)约45mg/mL至约55mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.2mg/mL至约0.6mg/mL的聚山梨酯80，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约 10mM至约30mM的组氨酸盐缓冲剂；所述药物组合物的pH为约5.0至约6.0；
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约45mg/mL至约55mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL至约0.6mg/mL的泊洛沙姆188，(c)约75mg/mL至约80mg/mL的蔗糖，和(d)约10mM至约30mM的醋酸-醋酸钠缓冲剂；所述药物组合物的pH为约5.2-5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.6mg/mL的泊洛沙姆188，(c)约80mg/mL的蔗糖，和(d)约10mM的醋酸-醋酸钠缓冲剂；所述药物组合物的pH为约5.0至约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约49mg/mL至约52mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.6mg/mL的泊洛沙姆188，(c)约80mg/mL的蔗糖，和(d)约10mM的醋酸-醋酸钠缓冲剂；所述药物组合物的pH为约5.2至约5.4。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约49mg/mL至约52mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约80mg/mL的蔗糖，和(d)约10mM的组氨酸-盐酸盐缓冲剂；所述药物组合物的pH为约5.2至约6.0。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约80mg/mL的蔗糖，和(d)约10mM的组氨酸-盐酸盐缓冲剂；所述药物组合物的pH为约5.5至约6.0。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.6mg/mL的泊洛沙姆188，(c)约80mg/mL的蔗糖，和(d)约10mM的醋酸-醋酸钠缓冲剂；所述药物组合物的pH为约5.2。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约80mg/mL的蔗糖，和(d)约10mM的组氨酸-盐酸盐缓冲剂；所述药物组合物的pH为约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约10mM的醋酸-醋酸钠缓冲剂；所述药物组合物的pH为约5.0至约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约10mM的组氨酸-盐酸盐缓冲剂；所述药物组合物的pH为约5.0至约6.0。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约10mM的琥珀酸-琥珀酸钠盐缓冲剂；所述药物组合物的pH为约5.5至约6.0。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约80mg/mL的蔗糖，和(d)约10mM的组氨酸-盐酸盐缓冲剂；所述药物组合物的pH为约5.0至约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约80mg/mL的蔗糖，和(d)约10mM的组氨酸-醋酸盐缓冲剂；所述药物组合物的pH为约5.0至约6.0。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约80mg/mL的蔗糖，和(d)约10mM的琥珀酸-琥珀酸钠盐缓冲剂；所述药物组合物的pH为约5.0至约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.6mg/mL的泊洛沙姆188，(c)约80mg/mL的蔗糖，和(d)约10mM至约30mM的醋酸-醋酸钠缓冲剂；所述药物组合物的pH为约5.2至约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约75mg/mL至约80mg/mL的蔗糖，和(d)约10mM的组氨酸-盐酸盐缓冲剂；所述药物组合物的pH为约5.5-5.7。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约75mg/mL的蔗糖，和(d)约10mM的组氨酸-盐酸盐缓冲剂；所述药物组合物的pH为约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约80mg/mL的蔗糖，和(d)约10mM的组氨酸-盐酸盐缓冲剂；所述药物组合物的pH为约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.2mg/mL至约0.4mg/mL的聚山梨酯80，(c)约80mg/mL的蔗糖，和(d)约10mM的醋酸-醋酸钠盐缓冲剂；所述药物组合物的pH为约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.2mg/mL至约0.6mg/mL 的泊洛沙姆188，(c)约80mg/mL的蔗糖，和(d)约10mM的醋酸-醋酸钠盐缓冲剂；所述药物组合物的pH为约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL至约0.6mg/mL的泊洛沙姆188，(c)约80mg/mL的蔗糖，和(d)约10mM的醋酸-醋酸钠盐缓冲剂；所述药物组合物的pH为约5.5。
在一些实施方案中，如上任一项所述的药物组合物包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约80mg/mL的蔗糖，和(d)约10mM的组氨酸-盐酸盐缓冲剂；所述药物组合物的pH为约5.5。
在一些实施方案中，如上任一项所述的药物组合物是液体制剂。在一些实施方案中，所述液体制剂的溶剂是水。
本披露提供一种冻干制剂，其为前述任一项所述的药物组合物的冻干形式。
本披露还提供一种的冻干制剂，其特征在于所述冻干制剂复溶后可形成如上任一项所述的药物组合物。
本披露还提供一种制备冻干制剂的方法，其中包括将如上任一项所述的药物组合物进行冷冻干燥的步骤。
本披露还提供一种冻干制剂，所述制剂通过将如上任一项所述的药物组合物冷冻干燥获得。在一些实施方案中，所述冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。在一些实施方案中，该冻干制剂于40℃稳定至少7天，至少14天或至少28天。
本披露还提供一种复溶溶液，其特征在于所述复溶溶液是通过将如上任一项所述的冻干制剂经复溶制备获得。
在一些实施方案中，如上所述的复溶溶液包含如下组分：
(a)约30mg/mL至约70mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.2mg/mL至约0.6mg/mL的聚山梨酯80，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约10mM至约30mM的组氨酸盐缓冲剂；所述复溶溶液的pH为约5.0至约6.0。
在一些实施方案中，如上所述的复溶溶液包含如下组分：
(a)约45mg/mL至约55mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约80mg/mL的蔗糖，和(d)约10mM的组氨酸-盐酸盐缓冲剂；所述复溶溶液的pH为约5.5至约6.0。
在一些实施方案中，如上所述的复溶溶液包含如下组分：
(a)约45mg/mL至约55mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL至约0.6mg/mL的聚山梨酯80，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约10mM至约30mM的组氨酸盐缓冲剂；所述复溶溶液的pH为约5.5至约6.0。
在一些实施方案中，如上所述的复溶溶液包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80(c)约80mg/mL的蔗糖，和(d)约10mM的组氨酸-盐酸盐缓冲剂；所述复溶溶液的pH为约5.5至约6.0。
在一些实施方案中，如上所述的复溶溶液包含如下组分：
(a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约80mg/mL的蔗糖，和(d)约10mM的组氨酸-盐酸盐缓冲剂；所述复溶溶液的pH为约5.5。
在一些实施方案中，如上所述的复溶溶液包含如下组分：
(a)约45mg/mL至约55mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL至约0.6mg/mL的泊洛沙姆188，(c)约70mg/mL至约90mg/mL的糖，和(d)约10mM至约30mM的醋酸盐缓冲剂；所述复溶溶液的pH为约5.0至约6.0。
在一些实施方案中，如上所述的复溶溶液包含如下组分：
(a)约45mg/mL至约55mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL至约0.6mg/mL的泊洛沙姆188，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约10mM至约30mM的醋酸-醋酸钠缓冲剂；所述复溶溶液的pH为约5.0至约5.5。
在一些实施方案中，如上所述的复溶溶液包含如下组分：
(a)50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.6mg/mL的泊洛沙姆188，(c)约80mg/mL的蔗糖，和(d)约10mM的醋酸-醋酸钠缓冲剂；所述复溶溶液的pH为约5.2。
本披露还提供一种制品，其包括容器，该容器中装有如上任一项所述的药物组合物、如上任一项所述的冻干制剂或如上任一项所述的复溶溶液。
在一些实施方案中，本披露还提供如上任一项所述的药物组合物、如上任一项的冻干制剂或如上任一项的复溶溶液在制备用于消除受试者免疫抑制相关疾病的药物中的用途。
本披露还提供一种消除受试者免疫抑制相关疾病的方法，所述方法包括给予患者治疗有效量的如上任一项所述的药物组合物、如上任一项的冻干制剂或如上任一项的复溶溶液。
在一些实施方案中，本披露还提供如上任一项所述的药物组合物、如上任一项的冻干制剂或如上任一项的复溶溶液，用作药物。
在一些实施方案中，本披露还提供如上任一项所述的药物组合物、如上任一项的冻干制剂或如上任一项的复溶溶液，其用作消除受试者免疫抑制相关疾病的药物。
在一些实施方案中，本披露还提供如上任一项所述的药物组合物、如上任一项的冻干制剂或如上任一项的复溶溶液在制备用于治疗PD-L1或CD47相关疾病的药物中的用途。
在一些实施方案中，本披露还提供一种治疗PD-L1或CD47相关疾病的方法， 所述方法包括给予患者有效量的如上任一项所述的药物组合物、如上任一项的冻干制剂或如上任一项的复溶溶液。
在一些实施方案中，本披露还提供如上任一项所述的药物组合物、如上任一项的冻干制剂或如上任一项的复溶溶液，用作治疗PD-L1或CD47相关疾病的药物。
在一些实施方案中，所述PD-L1或CD47相关疾病为癌症、细菌感染或病毒感染。
在一些实施方案中，所述PD-L1或CD47相关疾病为表达PD-L1或CD47的癌症。
在一些实施方案中，所述免疫抑制相关疾病包括癌症、细菌或病毒感染。
在一些实施方案中，所述的癌症包括、淋巴瘤、胚细胞瘤、肉瘤、白血病、鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌、头和颈鳞状细胞癌、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、急性成淋巴细胞性白血病、急性髓细胞样白血病、慢性淋巴细胞性白血病、慢性髓细胞样白血病、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白、骨髓异常增生综合征、胃肠道癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、膀胱癌、乳腺癌、结肠癌、肝细胞癌、透明细胞肾细胞癌、头和颈癌、咽喉癌、肝胆癌、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
具体实施方式
术语
为了更容易理解本披露，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本披露所属领域的一般技术人员通常理解的含义。
本披露所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
“抗PD-L1抗体”包括能够特异性结合PD-L1的全长抗体，也包括包含该全长抗体的轻链可变区和重链可变区的抗原结合片段，包括但不限于包含该全长抗体的轻链可变区和重链可变区的单链抗体(scFv)、Fab片段或包含scFv或Fab的其他抗原结合片段。
“SIRPγ肽”指人SIRPγ-D1结构域肽，其具有结合人CD47的活性。
SIRPγ肽连接至所述抗人PD-L1抗体的多肽链中的“连接”指多肽之间的有效连接，包括例如经过肽键连接，或使用连接子连接。所述连接不会使SIRPγ肽和抗人PD-L1抗体各自的功能丧失。
术语“融合蛋白”通常指由两个或更多个蛋白或多肽融合得到的蛋白。编码所述两个或更多个蛋白或多肽的基因或核酸分子可彼此连接而形成融合基因或融合的核酸分子，该融合基因或融合的核酸分子可编码所述融合蛋白。所述融合基因的翻译产生单一多肽，其具有融合前的所述两个或更多个蛋白或多肽中至少一个、甚至每一个的性质。术语融合蛋白和重组融合蛋白在本文以相同含义使用。本文描述的融合蛋白通常包含至少两个结构域(A和B)，并且任选地包含第三组分，介于所述两个结构域之间的接头。重组融合蛋白的生成是本领域已知的，并且通常涉及自编码第一蛋白或多肽的cDNA序列去除终止密码子，然后通过连接或重叠延伸PCR以符合读框的方式附接第二蛋白的cDNA序列。该DNA序列然后会由细胞表达成为单一蛋白质。该蛋白质可以经工程化以包括两种原始蛋白质或多肽的完整序列，或仅仅任一的一部分。本披露中的融合蛋白，是指PD-L1-SIRPγ变体融合蛋白，其为包含抗PD-L1抗体和SIRPγ变体的四肽结构，其中所述的SIRPγ变体的N端通过连接子连接至抗PD-L1抗体重链的C-端。
本披露的术语“抗体”以最广义使用，其涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体，多特异性抗体(例如双特异性抗体)，全长抗体或其抗原结合片段(也称“抗原结合部分”)，鼠源抗体，嵌合抗体或人源化抗体，亲和力成熟的抗体，只要它们展现出期望的抗原结合活性。天然抗体指天然存在的免疫球蛋白分子。例如，天然IgG抗体是约150,000道尔顿的异四聚糖蛋白，由二硫键结合的两条相同轻链和两条相同重链构成。从N至C端，每条重链具有一个可变区(VH)，又称作可变重域或重链可变域，接着是三个恒定域(CH1、CH2和CH3)。类似地，从N至C端，每条轻链具有一个可变区(VL)，又称作可变轻域，或轻链可变域，接着是一个恒定轻(CL)域。“全长抗体”、“完整抗体”和“全抗体”在本文可互换使用，指具有与天然抗体结构基本类似的结构或具有含有如本文所限定的Fc区的重链的抗体。在一些实施方案中，本披露的全长抗体包括由表1、表2和表3中轻重链可变区组合中的轻链可变区与轻链恒定区连接和重链可变区与重链恒定区连接后所形成的全长抗体。本领域技术人员可以根据实际需要选择不同的抗体来源的轻链恒定区、重链恒定区，例如人抗体来源的轻链恒定区和重链恒定区。
“约”是指处于如本领域的普通技术人员所确定的特定值的可接受误差范围之内，其将部分取决于所述值是如何测量或测定的，即所述测量系统的限制。在特定测定、结果或实施方案的上下文中，除非实施例或说明书其它地方内另有明确说明，否则“约”意指给定数值±5％以内的范围。
“缓冲剂”指通过其酸-碱共轭组分的作用而耐受pH变化的缓冲剂。将pH控 制在适当范围中的缓冲剂的例子包括醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸盐、草酸盐、乳酸盐、磷酸盐、枸橼酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸和其它有机酸缓冲剂。
“组氨酸盐缓冲剂”是包含组氨酸根离子的缓冲剂。组氨酸盐缓冲剂的实例包括组氨酸-盐酸盐，组氨酸-醋酸盐，组氨酸-磷酸盐，组氨酸-硫酸盐等缓冲剂。在一些实施方案中，所述组氨酸盐缓冲剂为组氨酸-盐酸盐缓冲剂或组氨酸-醋酸盐缓冲剂。组氨酸-醋酸盐缓冲剂是组氨酸与醋酸配制而成，组氨酸-盐酸盐缓冲剂是组氨酸与组氨酸盐酸盐配制而成，或组氨酸与盐酸配制而成。
“枸橼酸盐缓冲剂”是包括枸橼酸根离子的缓冲剂。枸橼酸盐缓冲剂的实例包括枸橼酸-枸橼酸钠、枸橼酸-枸橼酸钾、枸橼酸-枸橼酸钙、枸橼酸-枸橼酸镁等。优选的枸橼酸盐缓冲剂是枸橼酸-枸橼酸钠。
“琥珀酸盐缓冲剂”是包括琥珀酸根离子的缓冲剂。琥珀酸盐缓冲剂的实例包括琥珀酸-琥珀酸钠、琥珀酸-琥珀酸钾、琥珀酸-琥珀酸钙盐等。优选的琥珀酸盐缓冲剂是琥珀酸-琥珀酸钠。示例性的，所述的琥珀酸-琥珀酸钠可由琥铂酸与氢氧化钠配制而成，或由琥铂酸与琥珀酸钠配制而成。
“磷酸盐缓冲剂”是包括磷酸根离子的缓冲剂。磷酸盐缓冲剂的实例包括磷酸氢二钠-磷酸二氢钠、磷酸氢二钠-磷酸二氢钾、磷酸氢二钠-枸橼酸等。优选的磷酸盐缓冲剂是磷酸氢二钠-磷酸二氢钠。
“醋酸盐缓冲剂”是包括醋酸根离子的缓冲剂。醋酸盐缓冲剂的实例包括醋酸-醋酸钠、醋酸组氨酸盐、醋酸-醋酸钾、醋酸醋酸钙、醋酸-醋酸镁等。在一些实施方案中，所述的醋酸盐缓冲剂是醋酸-醋酸钠缓冲剂。
“稳定剂”是指有助于维持生物制药药物的结构完整性的组分，特别是在冷冻和/或冻干和/或储存期间(特别是当暴露于应激(stress)时)。这种稳定作用可以由于多种原因而产生，通常这种稳定剂可起到减轻蛋白质变性的渗透剂的作用。本文中额外的稳定剂是指除了缓冲体系、表面活性剂和糖醇以外的稳定剂，例如选自PEG、精氨酸和EDTA的稳定剂。
“药物组合物”表示含有一种或多种本文所述抗体与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是保持活性成分的稳定性，促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
本披露中，“药物组合物”和“制剂”并不互相排斥。
本披露中所述药物组合物的溶液形式，若无特殊说明，其中的溶剂均为水。
“冻干制剂”表示液体或溶液形式的药物组合物或液体或溶液制剂经真空冷冻干燥步骤之后获得的制剂或药物组合物。
尽管本披露提供了含量范围或含量值，但本领域一般技术人员理解，所述含量范围或含量值涵盖了所测定具体值的可接受误差范围。
本披露所述的药物组合物能够达到一种稳定的效果：其中的抗体在贮藏后基 本上保留其物理稳定性和/或化学稳定性和/或生物学活性的药物组合物，优选地，药物组合物在贮藏后基本上保留其物理和化学稳定性以及其生物学活性。贮藏期一般基于药物组合物的预定保存期来选择。目前有多种测量蛋白质稳定性的分析技术，可测量在选定温度贮藏选定时间段后的稳定性。
稳定的制剂是在下述情况下没有观察到显著变化的制剂：在冷藏温度(2-8℃)保存至少3个月、优选6个月、更优选1年，且甚至更优选地多达2年。另外，稳定的液体制剂包括这样的液体制剂：其在包括25℃的温度保存包括1个月、3个月、6个月在内的时段后表现出期望的特征。稳定性的典型的例子：通过SE-HPLC测得，通常不超过约10％、优选不超过约5％的抗体单体发生聚集或降解。通过视觉分析，制剂是淡黄色近无色澄明液体或者无色澄明液体，或澄清至稍微乳白色。所述制剂的浓度、pH和重量克分子渗透压浓度具有不超过±10％变化。通常观察到不超过约10％、优选不超过约5％的减少。通常形成不超过约10％、优选不超过约5％的聚集。
如果在目检颜色和/或澄清度后，或者通过UV光散射、尺寸排阻色谱法(SEC)和动态光散射(DLS)测得，抗体没有显示出显著的聚集增加、沉淀和/或变性，那么所述抗体在药物制剂中“保留它的物理稳定性”。蛋白构象的变化可以通过荧光光谱法(其确定蛋白三级结构)和通过FTIR光谱法(其确定蛋白二级结构)来评价。
如果抗体没有显示出显著的化学改变，那么所述抗体在药物制剂中“保留它的化学稳定性”。通过检测和定量化学上改变的形式的蛋白，可以评估化学稳定性。经常改变蛋白化学结构的降解过程包括水解或截短(通过诸如尺寸排阻色谱法和CE-SDS等方法来评价)、氧化(通过诸如与质谱法或MALDI/TOF/MS结合的肽谱法等方法来评价)、脱酰胺作用(通过诸如离子交换色谱法、毛细管等电聚焦、肽谱法、异天冬氨酸测量等方法来评价)和异构化(通过测量异天冬氨酸含量、肽谱法等来评价)。
如果抗体在给定时间的生物活性是在制备药物制剂时表现出的生物活性的预定范围内，那么所述抗体在药物制剂中“保留它的生物活性”。
“施用”、“给予”和“处理”，当其应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当其应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本披露的任一种的药物 组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本披露的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
本披露中与PD-L1或CD47相关疾病没有限制，只要它是与PD-L1或CD47相关的疾病即可，例如利用本披露的融合蛋白诱导的治疗反应可通过结合人类PD-L1或CD47，然后阻遏PD-L1或CD47与其受体/配体的结合。因此，当处于适于治疗应用的制备物和制剂中时，本披露的分子对这样一些人是非常有用的，他们患有肿瘤或癌症，可选地包括淋巴瘤、胚细胞瘤、肉瘤、白血病、鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌、头和颈鳞状细胞癌、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、急性成淋巴细胞性白血病、急性髓细胞样白血病、慢性淋巴细胞性白血病、慢性髓细胞样白血病、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白、骨髓异常增生综合征、胃肠道癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、膀胱癌、乳腺癌、结肠癌、肝细胞癌、透明细胞肾细胞癌、头和颈癌、咽喉癌、肝胆癌、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
在以上说明书中提出了本披露一种或多种实施方式的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本披露，但是以下描述优选的方法和材料。通过说明书和权利要求书，本披露的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本披露所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本披露的优选实施方式。这些实施例不应以任何方式理解为限制本披露的范围，本披露的范围由权 利要求书限定。
本披露PD-L1-SIRPγ融合蛋白：
本披露中所述的PD-L1-SIRPγ融合蛋白的制备及其活性已在WO2020177733A1中公开，其中示例性的，h1831K-19-S37融合蛋白具有4肽结构，其包含两条相同的第一链和两条相同第二链。本披露中使用的融合蛋白序列如下：
h1831K-19-S37第一链：
Figure PCTCN2022087850-appb-000001
h1831K-19-S37第二链：
Figure PCTCN2022087850-appb-000002
制剂制备与检测过程中使用的设备及方法如下：
SEC分子排阻色谱法：
根据凝胶孔隙的孔径大小与高分子样品分子的线团尺寸间的相对关系而对溶质进行分离的分析的方法。
SEC％(SEC单体含量百分比)＝A单体/A总*100％(A单体为样品中主峰单体的峰面积，A总为所有峰面积之和。)
用于SEC测定的仪器：安捷伦1260；柱子：waters，XBridge
Figure PCTCN2022087850-appb-000003
SEC(300×7.8mm 3.5μm)
R-CE毛细管凝胶电泳(也称CE-SDS(R))：
将凝胶移到毛细管中作为支持介质进行的一种电泳，并在一定的电压下根据样品分子量的大小进行分离的方法。
还原CE纯度百分比＝A重链/A总*100％+A轻链/A总*100％(A重链为样品重链的峰面积，A轻链为样品中轻链的峰面积，A总为所有峰面积之和)
用于CE测定的仪器：Beckman型号plus800
iCIEF成像毛细管等点聚焦电泳：
根据蛋白质等电点pI不同进行分离的技术。
iCIEF中性峰含量百分比＝中性峰面积/总面积*100％(总面积为酸性峰、中性峰和碱性峰面积之和)。
用于iCIEF测定的仪器：厂家simple protein，型号muarice。
渗透压测定：
冰点法测定渗透压，以冰点下降值与溶液的摩尔浓度成正比例关系为基础，采用高灵敏度感温元件，测定溶液结冰点，通过电量转化为渗透压。仪器厂家罗泽Loser，型号OM815.
蛋白浓度测定：
蛋白浓度测定仪器：紫外可见分光光度计，型号：Nano Drop oneC，光程为1mm。以下融合蛋白的浓度采用蛋白浓度计。
实施例1.PD-L1-SIRPγ融合蛋白制剂pH的筛选
配制下列缓冲液，制备h1831K-19-S37浓度为约50mg/mL的制剂，并检测融合蛋白制剂在高温(40℃)下的稳定性。缓冲体系如下：
1)10mM醋酸-醋酸钠盐缓冲液(简称AA)，pH 4.5；
2)10mM AA，pH5.0；
3)10mM AA，pH5.5；
4)10mM琥珀酸-琥珀酸钠盐缓冲液(简称SA)，pH5.5；
5)10mM SA，pH6.0；
6)10mM枸橼酸-枸橼酸钠盐缓冲液(简称CA)，pH5.0；
7)10mM CA，pH5.5；
8)10mM组氨酸-盐酸盐缓冲液(简称His-HCl)，pH5.5；
9)10mM His-HCl，pH6.0；
10)10mM His-HCl，pH6.5；
11)10mM磷酸氢二钠-磷酸二氢钠盐缓冲液(简称PB)，pH6.5；
表1.pH的筛选结果
Figure PCTCN2022087850-appb-000004
Figure PCTCN2022087850-appb-000005
注：M表示月，M0表示实验开始时，M1表示放置1个月；△表示差值。
融合蛋白的稳定性与外观的关系如下：澄明>颗粒>明显颗粒>轻微浑浊>浑浊。外观结果表明，PD-L1-SIRPγ融合蛋白在SA、His-HCl和AA缓冲体系中的稳定性优于CA和PB。CE结果显示，当pH为4.5时，在pH4.5的AA缓冲液中，40℃放置一个月后，制剂的CE下降13.5，说明制剂的不稳定。因此，制剂的缓冲剂可为pH5.0-5.5的AA或pH5.5-6.5的His-HCl。
实施例2.PD-L1-SIRPγ融合蛋白制剂缓冲体系筛选
1.用下列缓冲体系，制备含约50mg/mL融合蛋白h1831K-19-S37，80mg/mL蔗糖，0.4mg/mL聚山梨酯80的制剂，检测制剂中融合蛋白的浓度，并考察制剂在40℃放置1个月的稳定性。
1)10mM AA pH5.5；
2)10mM SA pH5.5；
3)10mM His-HCl pH5.5；
表2. 40℃1个月稳定性结果
Figure PCTCN2022087850-appb-000006
注：D表示天，D0表示实验开始时；M表示月，M1表示放置1个月；△表示差值。
结果表明：当pH为5.5时，融合蛋白在AA体系中的稳定性最好。
2.分别用10mM AA pH5.0或pH5.5的缓冲液，制备含50mg/mL融合蛋白h1831K-19-S37，80mg/mL蔗糖，0.4mg/mL泊洛沙姆188的制剂，考察制剂在高温(40℃)放置28天的稳定性。
1)10mM AA pH5.0；
2)10mM AA pH5.5；
表3.不同pH 40℃28天稳定性结果
Figure PCTCN2022087850-appb-000007
注：D表示天，D0表示实验开始时，D28表示放置28天；△表示差值。
结果表明，h1831K-19-S37融合蛋白在pH 5.0-5.5之间，其制剂稳定性基本无差异。
实施例3.PD-L1-SIRPγ融合蛋白制剂中糖浓度的筛选
使用10mM His-HCl pH5.5的缓冲液，制备含50mg/mL融合蛋白h1831K-19-S37，0.4mg/mL聚山梨酯80，不同蔗糖浓度的制剂，并检测制剂的渗透压，结果见表4。
1)10mM His-HCl pH5.5，75mg/mL蔗糖；
2)10mM His-HCl pH5.5，80mg/mL蔗糖；
表4.不同蔗糖浓度渗透压结果
序号 蔗糖浓度 渗透压值(mOsm)
1 75mg/mL 291
2 80mg/mL 302
渗透压结果表明，h1831K-19-S37制剂中的蔗糖浓度可选75mg/mL-80mg/mL。
实施例4.PD-L1-SIRPγ融合蛋白制剂中表面活性剂种类和浓度的筛选
使用10mM AA pH5.5的缓冲液，制备含50mg/mL融合蛋白h1831K-19-S37，80mg/mL蔗糖和不同表面活性剂的制剂，检测制剂在振摇8天(25℃，300rpm)和高温(40℃)放置28天条件下的稳定性。表面活性剂如下：
1)0.2mg/mL泊洛沙姆188；
2)0.4mg/mL泊洛沙姆188；
3)0.6mg/mL泊洛沙姆188；
4)0.2mg/mL聚山梨酯80；
5)0.4mg/mL聚山梨酯80；
表5.表面活性剂筛选振摇8天稳定性结果
Figure PCTCN2022087850-appb-000008
注：D表示天，D0表示实验开始时，D8表示放置8天。
表6.表面活性剂筛选40℃28天稳定性结果
Figure PCTCN2022087850-appb-000009
注：D表示天，D0表示实验开始时，D28表示放置28天；△表示差值。
振摇结果表明(见表5)，在AA缓冲液中，含低浓度泊洛沙姆188的制剂易产生颗粒；高温结果表明(见表6)，含聚山梨酯80的制剂易产生聚体，使SEC纯度降低。故表面活性剂优选泊洛沙姆188，其浓度为0.4mg/mL～0.6mg/mL。
实施例5.PD-L1-SIRPγ融合蛋白制剂不同离子强度稳定性比较
用AA pH5.2不同离子强度的缓冲液，制备含50mg/mL融合蛋白h1831K-19-S37，80mg/mL蔗糖，0.6mg/mL泊洛沙姆188的制剂，检测制剂的pH和融合蛋白浓度，并考察40℃稳定性。
1)10mM AA pH5.2；
2)20mM AA pH5.2；
3)30mM AA pH5.2；
表7.不同离子强度40℃1个月稳定性结果
Figure PCTCN2022087850-appb-000010
注：D0表示实验开始时；M表示月，M1表示放置1个月；△表示差值。
结果表明：AA缓冲液的离子强度对制剂的稳定性基本无影响。
实施例6.PD-L1-SIRPγ融合蛋白制剂的冻干
制备包含10mM His-HCl pH5.5，50mg/mL融合蛋白h1831K-19-S37，80mg/mL蔗糖和0.4mg/mL聚山梨酯80的制剂，将一次干燥温度定为-10℃，冻干工艺如下表。
表8.冻干工艺
冻干工艺参数 设定温度(℃) 设定时间(min) 保持时间(h) 真空度(Pa)
预冻 5 10 1 N/A
预冻 -45 50 2 N/A
一次干燥 -10 120 32 10
二次干燥 25 0 1 10
二次干燥 25 1 7 1
注：N/A表示未检测。
结果显示，冻干制品粉饼饱满，无塌陷，稳定性良好；且1:1复溶后，复溶液的外观澄清。

Claims (17)

  1. 一种药物组合物，包含PD-L1-SIRPγ融合蛋白和缓冲剂，其中：
    所述PD-L1-SIRPγ融合蛋白包含SEQ ID NO：1的第一链和SEQ ID NO：2的第二链；
    所述缓冲剂为醋酸盐缓冲剂或组氨酸盐缓冲剂，且所述药物组合物的pH为约5.0至约6.0。
  2. 根据权利要求1所述的药物组合物，其中所述的醋酸盐缓冲剂为醋酸-醋酸钠缓冲剂；优选地，所述药物组合物的pH为约5.0至约5.5。
  3. 根据权利要求1所述的药物组合物，其中所述的组氨酸盐缓冲剂为组氨酸-盐酸盐缓冲剂；优选地，所述药物组合物的pH为约5.5至约6.0。
  4. 根据权利要求1至3中任一项所述的药物组合物，其中所述PD-L1-SIRPγ融合蛋白的浓度为约1mg/mL至约100mg/mL，优选为约45mg/mL至约55mg/mL；更优选为约50mg/mL。
  5. 根据权利要求1至4中任一项所述的药物组合物，其中所述药物组合物还包含表面活性剂，所述表面活性剂优选为聚山梨酯或泊洛沙姆，更优选为聚山梨酯80或泊洛沙姆188。
  6. 根据权利要求5所述的药物组合物，其中所述表面活性剂的浓度为约0.05mg/mL至约1.2mg/mL，优选为约0.2mg/mL至约0.8mg/mL，更优选为约0.4mg/mL至约0.6mg/mL。
  7. 根据权利要求1至6中任一项所述的药物组合物，其中所述药物组合物还包含糖，所述糖选自蔗糖、甘露醇和海藻糖中的一种或多种，优选为蔗糖。
  8. 根据权利要求7所述的药物组合物，其中所述糖的浓度为约20mg/mL至约100mg/mL，优选为约70mg/mL至约90mg/mL，更优选为约80mg/mL。
  9. 根据权利要求1至8中任一项所述的药物组合物，其中所述缓冲剂的浓度为约5mM至约50mM，优选为约10mM至约30mM，更优选为约10mM。
  10. 根据权利要求1至9中任一项所述的药物组合物，其包含如下组分：
    (a)约45mg/mL至约55mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL 至约0.6mg/mL的泊洛沙姆188，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约10mM至约30mM的醋酸盐缓冲剂；所述药物组合物的pH为约5.0至约5.5；或
    (a)约45mg/mL至约55mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.2mg/mL至约0.6mg/mL的聚山梨酯80，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约10mM至约30mM的组氨酸盐缓冲剂；所述药物组合物的pH为约5.0至约6.0；
    优选地，所述药物组合物包含如下组分：
    (a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.6mg/mL的泊洛沙姆188，(c)约80mg/mL的蔗糖，和(d)约10mM的醋酸-醋酸钠缓冲剂；所述药物组合物的pH为约5.2；或
    (a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约80mg/mL的蔗糖，和(d)约10mM的组氨酸-盐酸盐缓冲剂；所述药物组合物的pH为约5.5。
  11. 一种冻干制剂，所述冻干制剂复溶后可形成权利要求1至10中任一项所述的药物组合物。
  12. 一种冻干制剂，所述制剂通过将权利要求1至10中任一项所述的药物组合物冷冻干燥获得。
  13. 一种制备冻干制剂的方法，其包括将权利要求1至10中任一项所述的药物组合物进行冷冻干燥的步骤。
  14. 一种复溶溶液，所述复溶溶液是通过将权利要求11或12所述的冻干制剂复溶制备获得；
    优选地，所述复溶溶液包含如下组分：
    (a)约45mg/mL至约55mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.2mg/mL至约0.6mg/mL的聚山梨酯80，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约10mM至约30mM的组氨酸盐缓冲剂；所述复溶溶液的pH为约5.0至约6.0；或
    (a)约45mg/mL至约55mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL至约0.6mg/mL的泊洛沙姆188，(c)约70mg/mL至约90mg/mL的蔗糖，和(d)约10mM至约30mM的醋酸-醋酸钠缓冲剂；所述复溶溶液的pH为约5.0至约5.5；
    优选地，所述复溶溶液包含如下组分：
    (a)约50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.4mg/mL的聚山梨酯80，(c)约80mg/mL的蔗糖，和(d)约10mM的组氨酸-盐酸盐缓冲剂；所述复溶溶液的pH为约5.5；或
    (a)50mg/mL的PD-L1-SIRPγ融合蛋白，(b)约0.6mg/mL的泊洛沙姆188，(c)约80mg/mL的蔗糖，和(d)约10mM的醋酸-醋酸钠缓冲剂；所述复溶溶液的pH 为约5.2。
  15. 一种制品，其包括容器，该容器中装有如权利要求1至10中任一项所述的药物组合物，或权利要求11或12所述的冻干制剂，或权利要求14所述的复溶溶液。
  16. 一种消除受试者免疫抑制相关疾病的方法，所述方法包括给予受试者治疗有效量的权利要求1至10中任一项所述的药物组合物，或权利要求11或12所述的冻干制剂，或权利要求14所述的复溶溶液；优选地，其中所述免疫抑制相关疾病为癌症、细菌感染或病毒感染；更优选地，其中所述的癌症选自淋巴瘤、胚细胞瘤、肉瘤、白血病、鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌、头和颈鳞状细胞癌、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、急性成淋巴细胞性白血病、急性髓细胞样白血病、慢性淋巴细胞性白血病、慢性髓细胞样白血病、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白、骨髓异常增生综合征、胃肠道癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、膀胱癌、乳腺癌、结肠癌、肝细胞癌、透明细胞肾细胞癌、头和颈癌、咽喉癌、肝胆癌、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
  17. 一种治疗PD-L1或CD47相关疾病的方法，所述方法包括给予受试者治疗有效量的权利要求1至10中任一项所述的药物组合物，或权利要求11或12所述的冻干制剂，或权利要求14所述的复溶溶液；优选地，所述PD-L1或CD47相关疾病为癌症、细菌感染或病毒感染；更优选地，其中所述的癌症选自淋巴瘤、胚细胞瘤、肉瘤、白血病、鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌、头和颈鳞状细胞癌、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、急性成淋巴细胞性白血病、急性髓细胞样白血病、慢性淋巴细胞性白血病、慢性髓细胞样白血病、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白、骨髓异常增生综合征、胃肠道癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、膀胱癌、乳腺癌、结肠癌、肝细胞 癌、透明细胞肾细胞癌、头和颈癌、咽喉癌、肝胆癌、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
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