WO2021018026A1 - 一种液体制剂及其应用 - Google Patents

一种液体制剂及其应用 Download PDF

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WO2021018026A1
WO2021018026A1 PCT/CN2020/104068 CN2020104068W WO2021018026A1 WO 2021018026 A1 WO2021018026 A1 WO 2021018026A1 CN 2020104068 W CN2020104068 W CN 2020104068W WO 2021018026 A1 WO2021018026 A1 WO 2021018026A1
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polypeptide chain
seq
amino acid
acid sequence
protein
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PCT/CN2020/104068
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English (en)
French (fr)
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张晋宇
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张晋宇
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Priority to CN202080053083.4A priority Critical patent/CN114144193A/zh
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    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2046—IL-7
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2086—IL-13 to IL-16
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—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
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • A61K9/10—Dispersions; Emulsions
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons

Definitions

  • This application relates to the field of biomedicine, in particular to a liquid preparation and its application.
  • cytokine is a very important immune signal in the body, and cytokine fusion protein technology is another hot spot for tumor immunotherapy today. This method is based on the fact that these cytokines have the same or related functional activities and their respective target points are different, using genetic engineering technology to fuse two or more cytokines together.
  • cytokine fusion protein technology for tumor treatment is still not satisfactory, and there are many areas for improvement.
  • This application provides a liquid formulation, which includes an oil phase solvent system and protein.
  • the liquid preparation described in this application has an anti-tumor effect; the oil phase solvent and protein (for example, cytokine fusion protein) therein have a synergistic anti-tumor effect.
  • the application provides a liquid preparation, which includes an oil phase solvent system and a protein, wherein the oil phase solvent system includes an oil phase solvent, and the mass fraction of the oil phase solvent is about 50% or more.
  • the oil phase solvent is selected from the group consisting of glycerin, propylene glycol, polyethylene glycol, polyvinylpyrrolidone, mannitol, sorbitol, polyoxypropylene, and tromethamine.
  • the mass fraction of the oil phase solvent is about 60% or more.
  • the mass fraction of the oil phase solvent is about 70% or more.
  • the mass fraction of the protein is about 1% to about 50%.
  • the protein includes cytokines and/or antibodies.
  • the cytokine includes two or more selected from the group consisting of IL12, IL2, GMCSF, IL7, IL15, IL21, and FLT3L.
  • the cytokine is derived from a mammal.
  • the protein includes a fusion protein, and the fusion protein includes at least two cytokines selected from the group consisting of IL12, IL2, GMCSF, IL7, IL15, IL21, and FLT3L.
  • the protein includes a fusion protein, and the fusion protein includes any group of cytokines selected from:
  • the protein further includes a targeting moiety.
  • the targeting moiety can specifically recognize and/or bind to tumor-associated antigens.
  • the tumor-associated antigen is selected from the group consisting of the EDB domain of fibronectin, the EDA domain of fibronectin, and necrotic regions.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof.
  • the targeting moiety comprises an amino acid sequence shown in any one of the following groups: SEQ ID NO: 1-15.
  • the protein comprises an amino acid sequence shown in any one of the following groups: SEQ ID NO: 32-67.
  • the protein is a single chain protein.
  • the single-chain protein comprises an amino acid sequence shown in any one of the following groups: SEQ ID NO: 32-51.
  • the protein is a dimer composed of a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain is different from the second polypeptide chain.
  • the first polypeptide chain comprises IL12a and the second polypeptide chain comprises IL12b.
  • IL2 or its functional fragment is located in said first polypeptide chain or said second polypeptide chain
  • GMCSF or its functional fragment is located in said first polypeptide chain or said In the second polypeptide chain
  • the one or more targeting moieties are each independently located in the first polypeptide chain or in the second polypeptide chain.
  • the IL2 or its functional fragments, the IL12a or its functional fragments, and the GMCSF or its functional fragments are sequentially included from the N-terminus to the C-terminus. Fragment.
  • the targeting moiety, the IL12a or functional fragments thereof, the IL2 or functional fragments thereof, and the targeting moiety, the IL12a or functional fragments thereof, and the Said GMCSF or its functional fragments in the first polypeptide chain, the targeting moiety, the IL12a or functional fragments thereof, the IL2 or functional fragments thereof, and the targeting moiety, the IL12a or functional fragments thereof, and the Said GMCSF or its functional fragments.
  • the second polypeptide chain includes the IL12b or a functional fragment thereof and the targeting moiety in sequence from the N-terminus to the C-terminus.
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 53 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 52;
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 55 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 54;
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 56 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 57;
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 58 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 59;
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 60 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 61;
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 62 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 63;
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 64 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 65;
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 66 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 67.
  • this application also provides a medicine, which comprises the liquid preparation described in this application.
  • liquid formulation is formulated as an injection.
  • the medicament described in the present application further includes a diluent, wherein the diluent and the liquid preparation are not mixed with each other.
  • the application also provides the use of the liquid preparation described in the application in the preparation of drugs for treating tumors.
  • the tumor comprises lung cancer.
  • the liquid formulation described in this application is used to treat tumors.
  • this application also provides a method for treating tumors, which comprises administering the liquid formulation described in this application to a subject in need.
  • wherein the method of administration is intratumoral injection.
  • this application also provides an auxiliary material for preparing the liquid formulation described in this application, which comprises an oil phase solvent, and the mass fraction of the oil phase solvent is about 50% or more.
  • Figure 1 shows the effect of glycerol on tumor growth in mice
  • FIG. 1 shows the effect of the liquid preparation described in this application on tumor growth in mice
  • FIG. 3 shows the effect of the liquid preparation described in this application on tumor growth in mice
  • FIG. 4 shows the effect of the liquid preparation described in this application on tumor growth in mice
  • FIG. 5 shows the effect of the liquid preparation described in this application on tumor growth in mice
  • Figure 6 shows the effect of the liquid preparation described in this application on tumor growth in mice.
  • liquid preparation generally refers to a pharmaceutical composition in liquid form, which usually contains an active ingredient and a pharmaceutically acceptable carrier or excipient.
  • the liquid formulation may include an oil phase solvent system and a protein, wherein the oil phase solvent system includes an oil phase solvent, and the mass fraction of the oil phase solvent is about 50% or more.
  • the oil phase solvent may include glycerin, propylene glycol and/or polyethylene glycol.
  • the protein can be dispersed or dissolved in an oil phase solvent to prepare the liquid formulation described in this application.
  • protein can be considered as a "cytokine fusion protein", which generally refers to a fusion protein that can be obtained by fusing two or more cytokines together through gene recombination technology. It not only has the unique biological activity of its constituent factors or significantly enhances some of its activities, but it may also exert a complex biological function that is not available in a simple combination of a single cytokine through the complementary and synergistic effects of biological activities. It may also produce some new structures and biological functions.
  • cytokine fusion protein generally refers to a fusion protein that can be obtained by fusing two or more cytokines together through gene recombination technology. It not only has the unique biological activity of its constituent factors or significantly enhances some of its activities, but it may also exert a complex biological function that is not available in a simple combination of a single cytokine through the complementary and synergistic effects of biological activities. It may also produce some new structures and biological functions.
  • cytokine usually refers to the immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (such as endothelial cells, epidermal cells, fibroblasts, etc.) ) A class of small molecular proteins with extensive biological activities synthesized and secreted by stimulation. The cytokine plays an important role in regulating cell-cell interaction, cell growth and differentiation.
  • the cytokine can be selected from one or more of the following group: Interleukin (IL), FMS-related tyrosine kinase 3 ligand (FLT3L) and colony stimulating factor (Colony Stimulating) Factor, CSF).
  • the interleukin generally refers to a cytokine produced by lymphocytes, monocytes or other non-mononuclear cells.
  • the interleukin may be selected from one or more of the following group: IL12, IL2, IL7, IL15, and IL21.
  • the colony stimulating factor generally refers to a cytokine that can stimulate different hematopoietic stem cells to form cell colonies in a semi-solid medium.
  • the colony stimulating factor may be granulocyte macrophage colony stimulating factor (Granulocyte Macrophage Colony Stimulating Factor, GMCSF).
  • IL12 generally refers to interleukin-12.
  • IL12 can play an important regulatory role in cell-cell interaction, immune regulation, hematopoiesis, and inflammation.
  • the IL12 molecule is usually a heterodimer, which usually includes two subunits, the two subunits are p40 subunit (40kd) and p35 subunit (35kd), these two subunits through disulfide bond connected.
  • IL12 containing p35 subunit (35kd) can be represented as IL12a
  • IL12 containing p40 subunit (40kd) can be represented as IL12b.
  • IL-12 may refer to any natural IL-12 from any vertebrate source, which may include mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) .
  • the term “IL-12” can also include full-length or unprocessed IL-12, as well as any form of IL-12 produced by processing or artificial synthesis in cells.
  • the term also includes naturally occurring IL-12 variants, such as splice variants or allelic variants, or mutants, homologs, functional variants or functional fragments, and the like.
  • the p35 subunit in IL12 (mIL12) derived from mouse may include the amino acid sequence shown in SEQ ID NO: 16, and the p40 subunit may include the amino acid sequence shown in SEQ ID NO: 17.
  • the p35 subunit in human-derived IL12 (hIL12) may include the amino acid sequence shown in SEQ ID NO: 18, and the p40 subunit may include the amino acid sequence shown in SEQ ID NO: 19.
  • IL2 generally refers to interleukin-2, and IL2 plays an important regulatory role in cell-cell interaction, immune regulation, hematopoiesis, and inflammation.
  • the term “IL-2” can refer to any natural IL-2 from any vertebrate source, which can include mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) .
  • the term “IL-2” can also include full-length or unprocessed IL-2, as well as any form of IL-2 produced by processing or artificial synthesis in cells.
  • the term also includes naturally occurring IL-2 variants, such as splice variants or allelic variants, or mutants, homologs, functional variants or functional fragments, and the like.
  • IL2 (mIL2) derived from mouse may include the amino acid sequence shown in SEQ ID NO:20.
  • human-derived IL2 hIL2
  • IL15 generally refers to interleukin-15. IL15 plays an important regulatory role in cell-cell interaction, immune regulation, hematopoiesis, and inflammation.
  • the term “IL-15” may refer to any natural IL-15 from any vertebrate source, which may include mammals such as primates (such as humans) and rodents (such as mice and rats) .
  • the term “IL-15” can also include full-length or unprocessed IL-15, as well as any form of IL-15 produced by processing or artificial synthesis in cells.
  • the term also includes naturally-occurring IL-15 variants, such as splice variants or allelic variants, or mutants, homologues, functional variants or functional fragments, etc.
  • IL15 (mIL15) derived from mouse may include the amino acid sequence shown in SEQ ID NO:22.
  • human-derived IL15 hIL15
  • IL7 generally refers to interleukin-7.
  • IL7 plays an important regulatory role in cell-cell interaction, immune regulation, hematopoiesis, and inflammation.
  • the term “IL-7” may refer to any natural IL-7 from any vertebrate source, which may include mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) .
  • the term “IL-7” can also include full-length or unprocessed IL-7, as well as any form of IL-7 produced by processing or artificial synthesis in cells.
  • the term also includes naturally occurring IL-7 variants, such as splice variants or allelic variants, or mutants, homologues, functional variants or functional fragments, etc.
  • IL7 (mIL7) derived from mouse may include the amino acid sequence shown in SEQ ID NO:24.
  • human-derived IL7 hIL7 may include the amino acid sequence shown in SEQ ID NO:25.
  • IL21 generally refers to interleukin-21. IL21 plays an important regulatory role in cell-cell interaction, immune regulation, hematopoiesis, and inflammation.
  • the term “IL-21” can refer to any natural IL-21 from any vertebrate source, which can include mammals such as primates (such as humans) and rodents (such as mice and rats) .
  • the term “IL-21” can also include full-length or unprocessed IL-21, as well as any form of IL-21 produced by processing or artificial synthesis in cells.
  • the term also includes naturally-occurring IL-21 variants, such as splice variants or allelic variants, or mutants, homologues, functional variants or functional fragments, etc.
  • IL21 (mIL21) derived from mouse may include the amino acid sequence shown in SEQ ID NO:26.
  • human-derived IL21 hIL21
  • FLT3L generally refers to FMS-related tyrosine kinase 3 ligand, which can regulate the proliferation and differentiation of non-erythroid hematopoietic stem cells, and promote pre-B lymphocytes, dendritic cells, NK cells, and The proliferation, differentiation and maturation of toxic T lymphocytes have important anti-tumor effects.
  • FLT3L can refer to any natural FLT3L from any vertebrate source, which can include mammals such as primates (e.g., humans) and rodents (e.g., mice and rats).
  • FLT3L can also include full-length or unprocessed FLT3L, as well as any form of FLT3L produced by processing or artificial synthesis in cells.
  • the term also includes naturally occurring FLT3L variants, such as splice variants or allelic variants, or mutants, homologues, functional variants or functional fragments, etc.
  • FLT3L (mFLT3L) derived from mouse may include the amino acid sequence shown in SEQ ID NO:28.
  • human-derived FLT3L hFLT3L
  • SEQ ID NO:29 may be any form of FLT3L produced by processing or artificial synthesis in cells.
  • GMCSF generally refers to granulocyte macrophage colony stimulating factor.
  • the GMCSF may have 4 alpha-helix bundle structures.
  • the term “GMCSF” can refer to any natural GMCSF from any vertebrate source, which can include mammals such as primates (e.g., humans) and rodents (e.g., mice and rats).
  • the term “GMCSF” can also include full-length or unprocessed GMCSF, as well as any form of GMCSF produced by processing or artificial synthesis in cells.
  • the term also includes naturally occurring variants of GMCSF, such as splice variants or allelic variants, or mutants, homologues, functional variants or functional fragments, and the like.
  • GMCSF (mGMCSF) derived from mouse may include the amino acid sequence shown in SEQ ID NO:30.
  • human-derived GMCSF hGMCSF
  • antibody generally refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide that includes an antigen binding site, regardless of whether it is produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , Mutant and transplanted antibodies.
  • antibody also includes antibody fragments such as Fab, F(ab') 2 , Fv, scFv, Fd, dAbs and other antibody fragments that retain the antigen binding function (ie, specifically bind to, for example, OX40 or PD-L1). Generally, such fragments should include an antigen binding domain.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibody is composed of 5 basic heterotetramer units and another polypeptide called J chain, and contains 10 antigen binding sites, while IgA antibody includes 2-5 that can be combined with J chain to form a multivalent The basic 4-chain unit of the combination.
  • the 4-chain unit is generally about 150,000 Daltons.
  • Each L chain is connected to the H chain by a covalent disulfide bond, and two H chains are connected to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intra-chain disulfide bridges.
  • Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the ⁇ and ⁇ chains, and four CH domains for the ⁇ and ⁇ isotypes.
  • Each L chain has a variable domain (VL) at the N-terminus and a constant domain at the other end.
  • VL corresponds to VH
  • CL corresponds to the first constant domain (CH1) of the heavy chain.
  • Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • VH and VL pair together to form a single antigen binding site.
  • immunoglobulins can be divided into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains named ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the gamma and alpha classes are further divided into subclasses.
  • humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgK1.
  • targeting moiety generally refers to a type of moiety that acts on certain special tissues and cells.
  • the targeting moiety can specifically target tumor-associated antigens.
  • the targeting moiety includes an antibody or an antigen-binding fragment thereof.
  • telomere binding generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, which can be in the presence of a heterogeneous population of molecules (including biological molecules). Determine the existence of the target.
  • an antibody that specifically binds a target (which may be an epitope) is an antibody that binds to the target with greater affinity, avidity, easier, and/or longer duration than it binds to other targets.
  • the extent to which the antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA).
  • the antibody that specifically binds to the target has a dissociation constant (KD) of ⁇ 1x10 -6 M, ⁇ 1x10 -7 M, ⁇ 1x10 -8 M, ⁇ 1x10 -9 M, or ⁇ 1x10 -10 M .
  • KD dissociation constant
  • the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species.
  • specific binding may include but does not require exclusive binding.
  • tumor-associated antigen generally refers to an antigen molecule present on tumor cells or normal cells.
  • the tumor-associated antigen may include embryonic protein, glycoprotein antigen and squamous cell antigen.
  • the tumor-associated antigen may be selected from the group consisting of the EDB domain of fibronectin, the EDA domain of fibronectin, and necrotic regions.
  • the term "antigen-binding fragment” generally refers to a fragment having antigen-binding activity.
  • the antigen-binding fragment may be selected from the following group: Fab, Fab', F(ab') 2 , F(ab) 2 , dAb, isolated complementarity determining region CDR, Fv and scFv.
  • single-chain protein generally refers to a polypeptide with a primary structure composed of an uninterrupted sequence of continuous amino acid residues.
  • the single-chain protein may include the amino acid sequence shown in any one of the following groups: SEQ ID NO: 32-51.
  • the term "dimer” generally refers to a polymer complex formed by two monomer units that are usually non-covalently bonded.
  • Each monomer unit can be a macromolecule, such as a polypeptide chain or a polynucleotide.
  • the protein may be a dimer composed of a first polypeptide chain and a second polypeptide chain.
  • polypeptide chain generally refers to a macromolecule comprising two or more covalently linked peptides.
  • the peptides within the polypeptide chain can be connected to each other by a peptide bond.
  • Each polypeptide chain may include an N-terminal or amino terminal and a C-terminal or carboxy terminal.
  • a functional fragment of IL12a refers to a fragment that retains the function of IL12a.
  • the functional fragment of IL12a may be IL12a, fragment (GenBank: AIC49052.1).
  • the functional fragment of IL12b may be IL12b, fragment (GenBank: AIC54621.1).
  • injection generally refers to sterile solutions (including emulsions and suspensions) made of medicines for injection into the body, as well as sterile powders or powders that are prepared into solutions or suspensions before use. Concentrated solution.
  • injections can be injected intravenously or subcutaneously into the body.
  • the liquid preparation can be formulated as an injection.
  • the term "diluent” generally refers to a solvent used to dilute a substance, such as a solvent that reduces the concentration of a substance.
  • the medicine may further include a diluent, and the diluent and the liquid preparation are not mixed with each other.
  • tumor generally refers to neoplasms or solid lesions formed by abnormal cell growth.
  • the tumor may be a solid tumor or hematoma.
  • tumors can include lung cancer.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • the term "administration” generally refers to a method of administering a certain dose of a liquid formulation or drug to a subject (for example, a patient). Administration can be carried out by any suitable means, including parenteral, intrapulmonary and intranasal, and (if local treatment is needed) intralesional administration.
  • Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the administration may be by any suitable route, for example, by injection (such as intravenous or subcutaneous injection), depending in part on whether the administration is short-lived or long-term.
  • Various dosing schedules are covered herein, including but not limited to single administration or multiple administrations at various time points, bolus administration, and pulse infusion.
  • the administration may be intratumoral injection.
  • the "intratumoral injection” usually refers to the injection of a certain dose of liquid preparations or drugs into the tumor.
  • auxiliary material generally refers to auxiliary materials, such as materials that assist the preparation of a certain substance.
  • the auxiliary material used to prepare the liquid formulation may include an oil phase solvent, and the mass fraction of the oil phase solvent may be about 50% or more.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a liquid formulation, which may include an oil phase solvent system and protein, wherein the oil phase solvent system may include an oil phase solvent, and the mass fraction of the oil phase solvent may be about 50% or more.
  • the mass fraction of the oil phase solvent may be about 60% or more.
  • the mass fraction of the oil phase solvent may be about 70% or more.
  • the mass fraction of the oil phase solvent may be about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%. %, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 60% to about 80%, about 60% to about 90%, About 60% to about 95%, about 70% to about 90%, about 70% to about 95%, or about 80% to about 95%.
  • the oil phase solvent may be selected from the following group: glycerin, propylene glycol, polyethylene glycol, polyvinylpyrrolidone, mannitol, sorbitol, polyoxypropylene and tromethamine.
  • the oil phase solvent may be glycerin, and the mass fraction of the glycerin may be about 50% or more. In some embodiments, the mass fraction of the glycerin may be above about 60%. For example, in other embodiments, the mass fraction of the glycerin may be more than about 70%.
  • the mass fraction of the glycerin may be about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, About 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 60% to about 80%, about 60% to about 90%, about 60 % To about 95%, about 70% to about 90%, about 70% to about 95%, or about 80% to about 95%.
  • the mass fraction of the protein can be about 1% to about 50%, for example, the mass fraction of the protein can be about 1% to about 10%, about 1% to about 20%, or about 1%.
  • the liquid formulation described in the present application can be prepared by dissolving or dispersing the protein in the oil phase solvent system (for example, in an oil phase solvent).
  • oil phase solvent and the protein may be mutually soluble or immiscible with each other.
  • the liquid preparation described in this application can be used to treat tumors, wherein the oil phase solvent and protein can cooperate with each other to enhance the therapeutic effect of the tumor, for example, compared to a simple oil phase solvent or a simple Protein, the liquid preparation described in this application has a significant and enhanced effect on tumor treatment.
  • a simple protein solution usually cannot induce tumor regression, but the liquid preparation described in this application can induce tumor regression.
  • the liquid formulation can be used to inhibit tumor growth.
  • the liquid preparation of the present application can inhibit or delay the development or progression of the disease, can reduce the size of the tumor (or even substantially eliminate the tumor) by promoting the expression of cytokines, and/or can reduce and/or stabilize the disease state.
  • the liquid formulations described in this application can also include amino acids.
  • the amino acids can be glycine, glutamine, asparagine, arginine. Any one or more of acid or lysine.
  • the liquid preparation described in this application may also include an antibacterial agent or an antioxidant.
  • the antioxidant may be any one or more of ascorbic acid, sodium sulfite or sodium bisulfite.
  • the liquid preparation described in the present application may also include a buffer, and the buffer may be any one or more of borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids.
  • the liquid formulations described in this application may also include bulking agents, such as mannitol or glycine.
  • the liquid preparation described in this application may also include a chelating agent, such as ethylenediaminetetraacetic acid (EDTA.
  • EDTA ethylenediaminetetraacetic acid
  • the liquid preparation described in this application may also include a complexing agent, such as caffeine, polyvinylpyrrolidone, ⁇ -cyclodextrin or Any one or more of hydroxypropyl- ⁇ -cyclodextrin.
  • the liquid preparation described in this application may also include sugars, such as glucose, mannose or dextrin.
  • the liquid preparation described in this application may also include Preservatives, such as any of benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl paraben, propyl paraben, chlorhexidine, sorbic acid, or hydrogen peroxidekind or more.
  • Preservatives such as any of benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl paraben, propyl paraben, chlorhexidine, sorbic acid, or hydrogen peroxidekind or more.
  • the protein may include cytokines and/or antibodies.
  • the antibody can be selected from one or more of the following group: monoclonal antibody, polyclonal antibody, antibody fragment, chimeric antibody, bispecific antibody, heteroconjugate antibody, single chain (ScFv ), fusion proteins containing antibody portions (such as domain antibodies), humanized antibodies, and any other modified configurations of immunoglobulin molecules that contain antigen recognition sites of the desired specificity.
  • the antibody fragment can be selected from one or more of Fab, Fab', F(ab') 2 , Fv and Fc.
  • the fusion protein containing the antibody portion may be a domain antibody.
  • the antibody may also include glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • the antibody may be canine, rat, human, or any other origin, such as a chimeric or humanized antibody.
  • the cytokine can be any of the following: IL12, IL2, GMCSF, IL7, IL15, IL21, and FLT3L.
  • the cytokine may also include two or more selected from the group consisting of IL12, IL2, GMCSF, IL7, IL15, IL21 and FLT3L.
  • the cytokine may be derived from mammals. The mammal can be human or mouse.
  • the amino acid sequence of mouse-derived IL12a (represented by mIL12a) may be shown in SEQ ID NO.16
  • the amino acid sequence of mouse-derived IL12b (represented by mIL12b) may be shown in SEQ ID NO.17
  • the amino acid sequence of mouse-derived IL2 (represented by mIL2) can be shown in SEQ ID NO.20
  • the amino acid sequence of mouse-derived GMCSF (represented by mGMCSF) can be shown in SEQ ID NO.30, which is derived from
  • the amino acid sequence of mouse IL7 (represented by mIL7) can be shown in SEQ ID NO. 24, and the amino acid sequence of mouse-derived IL15 (represented by mIL15) can be shown in SEQ ID NO.
  • the amino acid sequence of IL21 (represented by mIL21) may be shown in SEQ ID NO.26, and the amino acid sequence of FLT3L (represented by mFLT3L) derived from mouse may be shown in SEQ ID NO.28.
  • the amino acid sequence of human-derived IL12a (represented by hIL12a) may be shown in SEQ ID NO.18
  • the amino acid sequence of human-derived IL12b (represented by hIL12b) may be shown in SEQ ID NO.19.
  • the amino acid sequence of human IL2 (represented by hIL2) may be as shown in SEQ ID NO.
  • amino acid sequence of human-derived GMCSF (represented by hGMCSF) may be as shown in SEQ ID NO. 31, which is derived from human IL7
  • the amino acid sequence (represented by hIL7) can be shown in SEQ ID NO. 25
  • the amino acid sequence of human-derived IL15 (represented by hIL15) can be shown as SEQ ID NO. 23
  • the human-derived IL21 (represented by hIL21)
  • the amino acid sequence of) may be shown in SEQ ID NO. 27, and the amino acid sequence of human-derived FLT3L (represented by hFLT3L) may be shown in SEQ ID NO. 29.
  • the protein may include a fusion protein, which fused two or more of the cytokines, that is, IL12, IL2, IL7, IL15, IL21, FLT3L and GMCSF through gene recombination technology together.
  • the protein can not only have the unique biological activities of its constituent factors, but also can exert biological functions that are not possessed by a single cytokine through the complementary and synergistic effects of biological activities, and can even produce some new structures and biological functions. Learn function.
  • the protein may include a fusion protein, and the fusion protein includes any group of cytokines selected from: a) IL12, IL2 and GMSCF; b) IL12, IL7 and GMSCF; c) IL12, IL15, and GMSCF; d) IL12, IL21, and GMSCF; e) IL12, IL2, and FLT3L; f) IL12, IL7, and FLT3L; g) IL12, IL15, and FLT3L; and, h) IL12, IL21, and FLT3L.
  • cytokines selected from: a) IL12, IL2 and GMSCF; b) IL12, IL7 and GMSCF; c) IL12, IL15, and GMSCF; d) IL12, IL21, and GMSCF; e) IL12, IL2, and FLT3L; f) IL12, IL7
  • the protein may also include a targeting moiety, and the number of the targeting moiety may be one or more.
  • the targeting moieties can be the same or different.
  • the targeting moiety can specifically recognize and/or bind to tumor-associated antigens.
  • the tumor-associated antigen may be selected from the following group: EDB domain of fibronectin, EDA domain of fibronectin, and necrotic regions.
  • the targeting moiety may include an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment may be selected from the following group: Fab, Fab', F(ab') 2 , F(ab) 2 , dAb, isolated complementarity determining region CDR, Fv and scFv.
  • the antigen-binding fragment may be a scFv.
  • the targeting moiety may include the amino acid sequence shown in any one of the following groups: SEQ ID NO: 1-15.
  • the targeting portion of the protein can be selected from the following group: L19V L (its amino acid sequence can be shown in SEQ ID NO. 10), L19V H (its amino acid sequence can be shown in SEQ ID NO. 11), F8V L (its amino acid sequence can be shown in SEQ ID NO. 12), F8V H (its amino acid sequence can be shown in SEQ ID NO. 13), NHS76V L (its amino acid sequence can be shown in SEQ ID NO. 14), and NHS76V H (its amino acid sequence can be shown in SEQ ID NO. 15).
  • the cytokine or the cytokine and the targeting moiety can be connected by a linker.
  • the linker may be a connecting peptide.
  • the linker may include the amino acid sequence shown in any one of the following groups: SEQ ID NO: 73-76.
  • the cytokines can be connected through the linker.
  • the IL12a, IL12b, IL2, IL7, IL15, IL21, FLT3L and GMCSF can be connected through the connecting peptide.
  • the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO. 73 or SEQ ID NO. 75.
  • the cytokine and the targeting moiety may be connected by the linker.
  • the targeting moiety can be connected to IL12a, IL12b, IL2, IL7, IL15, IL21, FLT3L and GMCSF through the connecting peptide.
  • the connecting peptide may include the amino acid sequence shown in any one of SEQ ID NOs: 73-76.
  • the protein may include the amino acid sequence shown in any one of the following groups: SEQ ID NO: 32-67.
  • the protein may be a single-chain protein, wherein the single-chain protein may comprise an amino acid sequence shown in any one of the following groups: SEQ ID NO: 32-51.
  • the structure of the single-chain protein may be that the C-terminus of mIL12b is fused with the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL2, and the C-terminus of mIL2 is fused to the N-terminus of mGMCSF, thereby forming mIL12b-mIL12a.
  • -mIL2-mGMCSF single-chain protein represented by mIL12bIL12aIL2GMCSF (its amino acid sequence can be shown in SEQ ID NO. 32).
  • the structure of the single-chain protein may be: the C-terminus of mIL12b is fused with the N-terminus of mIL12a, the C-terminus of mIL12a is fused with the N-terminus of mIL7, and the C-terminus of mIL7 is fused with the N-terminus of mGMCSF, thereby forming mIL12b-mIL12a.
  • -mIL7-mGMCSF single-chain protein represented by mIL12bIL12aIL7GMCSF (its amino acid sequence can be shown in SEQ ID NO. 33).
  • the structure of the single-chain protein may be that the C-terminus of mIL12b is fused with the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL15, and the C-terminus of mIL15 is fused to the N-terminus of mGMCSF, thereby forming mIL12b-mIL12a.
  • -mIL15-mGMCSF single-chain protein represented by mIL12bIL12aIL15GMCSF (its amino acid sequence can be shown in SEQ ID NO. 34).
  • the structure of the single-chain protein may be that the C-terminus of mIL12b is fused with the N-terminus of mIL12a, the C-terminus of mIL12a is fused with the N-terminus of mIL21, and the C-terminus of mIL21 is fused with the N-terminus of mGMCSF, thereby forming mIL12b-mIL12a.
  • -mIL21-mGMCSF single-chain protein represented by mIL12bIL12aIL21GMCSF (its amino acid sequence can be shown in SEQ ID NO. 35).
  • the structure of the single-chain protein may be that the C-terminus of mIL12b is fused with the N-terminus of mIL12a, the C-terminus of mIL12a is fused with the N-terminus of mIL2, and the C-terminus of mIL2 is fused with the N-terminus of mFLT3L, thereby forming mIL12b-mIL12a.
  • -mIL2-mFLT3L single-chain protein represented by mIL12bIL12aIL2FLT3L (its amino acid sequence can be shown in SEQ ID NO. 36).
  • the structure of the single-chain protein may be that the C-terminus of mIL12b is fused with the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL7, and the C-terminus of mIL7 is fused to the N-terminus of mFLT3L to form mIL12b-mIL12a.
  • -mIL7-mFLT3L single-chain protein represented by mIL12bIL12aIL7FLT3L (its amino acid sequence can be shown in SEQ ID NO. 37).
  • the structure of the single-chain protein may be that the C-terminus of mIL12b is fused with the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL15, and the C-terminus of mIL15 is fused to the N-terminus of mFLT3L, thereby forming mIL12b-mIL12a.
  • -mIL15-mFLT3L single-chain protein represented by mIL12bIL12aIL15FLT3L (its amino acid sequence can be shown in SEQ ID NO.38).
  • the structure of the single-chain protein may be that the C-terminus of mIL12b is fused with the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL21, and the C-terminus of mIL21 is fused to the N-terminus of mFLT3L, thereby forming mIL12b-mIL12a.
  • -mIL21-mFLT3L single-chain protein represented by mIL12bIL12aIL21FLT3L (its amino acid sequence can be shown in SEQ ID NO. 39).
  • the structure of the single-chain protein may be that the C-terminus of hIL12b is fused with the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL2, and the C-terminus of hIL2 is fused to the N-terminus of hGMCSF to form hIL12b-hIL12a.
  • -hIL2-hGMCSF single-chain protein represented by hIL12bIL12aIL2GMCSF (its amino acid sequence can be shown in SEQ ID NO. 40).
  • the structure of the single-chain protein may be that the C-terminus of hIL12b is fused with the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL7, and the C-terminus of hIL7 is fused to the N-terminus of hGMCSF, thereby forming hIL12b-hIL12a.
  • -hIL7-hGMCSF single-chain protein represented by hIL12bIL12aIL7GMCSF (its amino acid sequence can be shown in SEQ ID NO. 41).
  • the structure of the single-chain protein may be that the C-terminus of hIL12b is fused with the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL15, and the C-terminus of hIL15 is fused to the N-terminus of hGMCSF, thereby forming hIL12b-hIL12a.
  • -hIL15-hGMCSF single-chain protein represented by hIL12bIL12aIL15GMCSF (its amino acid sequence can be shown in SEQ ID NO. 42).
  • the structure of the single-chain protein may be that the C-terminus of hIL12b is fused with the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL21, and the C-terminus of hIL21 is fused to the N-terminus of hGMCSF, thereby forming hIL12b-hIL12a.
  • -hIL21-hGMCSF single-chain protein represented by hIL12bIL12aIL21GMCSF (its amino acid sequence can be shown in SEQ ID NO. 43).
  • the structure of the single-chain protein may be that the C-terminus of hIL12b is fused to the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL2, and the C-terminus of hIL2 is fused to the N-terminus of hFLT3L to form hIL12b-hIL12a.
  • -hIL2-hFLT3L single-chain protein represented by hIL12bIL12aIL2FLT3L (its amino acid sequence can be shown in SEQ ID NO.44).
  • the structure of the single-chain protein may be that the C-terminus of hIL12b is fused with the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL7, and the C-terminus of hIL7 is fused to the N-terminus of hFLT3L, thereby forming hIL12b-hIL12a.
  • -hIL7-hFLT3L single-chain protein represented by hIL12bIL12aIL7FLT3L (its amino acid sequence can be shown in SEQ ID NO. 45).
  • the structure of the single-chain protein may be that the C-terminus of hIL12b is fused with the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL15, and the C-terminus of hIL15 is fused to the N-terminus of hFLT3L to form hIL12b-hIL12a.
  • -hIL15-hFLT3L single-chain protein represented by hIL12bIL12aIL15FLT3L (its amino acid sequence can be shown in SEQ ID NO. 46).
  • the structure of the single-chain protein may be that the C-terminus of hIL12b is fused with the N-terminus of hIL12a, the C-terminus of hIL12a is fused to the N-terminus of hIL21, and the C-terminus of hIL21 is fused to the N-terminus of hFLT3L, thereby forming hIL12b-hIL12a.
  • -hIL21-hFLT3L single-chain protein represented by hIL12bIL12aIL21FLT3L (its amino acid sequence can be shown in SEQ ID NO. 47).
  • the single-chain protein structure may, N terminal and C-terminal mIL12b mIL12a fused, N terminal and C terminal mIL12a mIL2 fusion, N terminal and C terminal mIL2 fusion NHS76V H, C-NHS76V H end and F8V L the N-terminal fusion, the N-terminus F8V L C-terminal and F8V H fusion, the C-terminus and NHS76V L N-terminal F8V H fused to the N-terminus NHS76V L C-terminus and mGMCSF fusion, thereby forming mIL12b -mIL12a-mIL2-NHS76V H -F8V L -F8V H -NHS76V L -mGMCSF single chain protein.
  • linker of the single-chain protein contains a thrombin cleavage site
  • name of the single-chain protein is represented by mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr (its amino acid sequence may be shown in SEQ ID NO. 48).
  • the single-chain protein structure may be, hIL12b N-terminal and C-terminal fusion hIL12a, hIL12a N-terminal and C-terminal fusion of hIL2, hIL2 the N-terminus and C-terminus of the fusion NHS76V H, C-NHS76V H end and F8V L the N-terminal fusion, the N-terminus F8V L C-terminal and F8V H fusion F8V H C-terminal and NHS76V L the N-terminal fusion, NH S76V L of C and N-terminal hGMCSF fusion, to form a hIL12b-hIL12a-hIL2-NHS76V H -F8V L -F8V H -NHS76V L -hGMCSF single chain protein.
  • linker of the single-chain protein contains a thrombin cleavage site
  • name of the single-chain protein is represented by hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr (its amino acid sequence may be shown in SEQ ID NO. 49).
  • the single-chain protein structure may, N terminal and C-terminal mIL12b mIL12a fused, N terminal and C terminal mIL12a mIL2 fusion, N terminal and C terminal mIL2 fusion F8V H, C-F8V H end and F8V L the N-terminal fusion, the N-terminus F8V L C-terminal and F8V H fusion, the C-terminus and F8V L N-terminal F8V H fused to the N-terminus F8V L C-terminus and mGMCSF fusion, thereby forming mIL12b -mIL12a-mIL2-F8V H -F8V L -F8V H -F8V L -mGMCSF single-chain protein, represented by mIL12bIL12aIL2DiaF8GMCSF (its amino acid sequence can be shown in SEQ ID NO.50).
  • the single-chain protein structure may be, hIL12b N-terminal and C-terminal fusion hIL12a, hIL12a N-terminal and C-terminal fusion of hIL2, hIL2 the N-terminus and C-terminus of the fusion F8V H, C-F8V H end and F8V L the N-terminal fusion, the N-terminus F8V L C-terminal and F8V H fusion F8V H C-terminal and F8V L the N-terminal fusion, F8V L of C and N-terminal hGMCSF fused, thereby forming hIL12b -hIL12a-hIL2-F8V H -F8V L -F8V H -F8V L -hGMCSF single-chain protein, represented by hIL12bIL12aIL2DiaF8GMCSF (its amino acid sequence can be shown in SEQ ID NO. 51).
  • the protein may also be a dimer composed of a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain is different from the second polypeptide chain.
  • the first polypeptide chain may include IL12a
  • the second polypeptide chain may include IL12b.
  • IL2 or its functional fragment may be located in the first polypeptide chain or the second polypeptide chain
  • GMCSF or its functional fragment may be located in the first polypeptide chain or in the second polypeptide chain
  • the one or more targeting moieties may each independently be located in the first polypeptide chain or in the second polypeptide chain.
  • the IL2 or its functional fragments, the IL12a or its functional fragments, and the GMCSF or its functional fragments may be included in sequence from the N-terminus to the C-terminus .
  • the targeting moiety in the first polypeptide chain, the targeting moiety, the IL12a or its functional fragment, the IL2 or its functional fragment, and the GMCSF or its functional fragments.
  • the IL12b or a functional fragment thereof and the targeting moiety may be included in sequence from the N-terminus to the C-terminus.
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO: 53 and the second polypeptide chain may include SEQ ID NO. : The amino acid sequence shown in 52.
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO: 55 and the second polypeptide chain may include SEQ ID NO: : The amino acid sequence shown in 54;
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO: 56 and the second polypeptide chain may include SEQ ID NO. : The amino acid sequence shown in 57;
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO: 58 and the second polypeptide chain may include SEQ ID NO. : The amino acid sequence shown in 59;
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO: 60 and the second polypeptide chain may include SEQ ID NO. : The amino acid sequence shown in 61;
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO: 62 and the second polypeptide chain may include SEQ ID NO: : The amino acid sequence shown in 63;
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO: 64 and the second polypeptide chain may include SEQ ID NO. : The amino acid sequence shown in 65;
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO: 66 and the second polypeptide chain may include SEQ ID NO. : The amino acid sequence shown in 67.
  • the dimer, C and N terminal ends mIL12b may be fused F8V H, C terminal F8V H and L may be N-terminal fusion F8V second polypeptide chain is formed (which may be the amino acid sequence shown in SEQ ID NO.52 shown), and the C-terminus F8V H can be N-terminal fusion F8V L, F8V L N and C-terminal ends can be fused mIL12a, the C-terminus and N-terminus can mIL12a MIL2 fusion, a C MIL2
  • the terminal can be fused with the N-terminal of mGMCSF to form the first polypeptide chain (the amino acid sequence of which can be shown in SEQ ID NO.53), thereby forming mIL12b-F8V H -F8V L -F8V H -F8V L -mIL12a-mIL2-mGMCSF Dimer, represented by mIL12bscF8-scF8IL12aIL
  • the dimer, hIL12b the N-terminus and C-terminus can be fused NHS76V H, C-terminal NHS76V H and L may be N-terminal fusion NHS76V second polypeptide chain is formed (which may be the amino acid sequence shown in SEQ ID NO.54 shown), and the C-terminus and may NHS76V H NHS76V L the N-terminal fusion, a C-terminal NHS76V L may be fused and the N-terminus hIL12a, hIL12a the C-terminus and N-terminus can be fused hIL2, C-hIL2 The end can be fused with the N-terminus of hGMCSF to form the first polypeptide chain (its amino acid sequence can be shown in SEQ ID NO.
  • hIL12b-NHS76V H -N HS76V L -NHS76V H -NHS76V L -hIL12a-hIL2- hGMCSF dimer expressed as hIL12bscNHS76-scNHS76IL12aIL2GMCSF.
  • the C-terminus of mIL12a and the N-terminus of mIL2 can be fused to form the first polypeptide chain of mIL12a-mIL2 (sequence is shown in SEQ ID NO.56), and the C-terminus of mIL12b and the N-terminus of mIL12b
  • the N-terminus can be fused to form the second polypeptide chain of mIL12b-mGMCSF (sequence is shown in SEQ ID NO.57) to form a mIL12a-mIL2-mIL12b-mGMCSF dimer, which is represented by mIL12aIL2IL12bGMCSF.
  • the C-terminus of mIL12a and the N-terminus of mIL7 can be fused to form the first polypeptide chain of mIL12a-mIL7 (sequence is shown in SEQ ID NO.58), and the C-terminus of mIL12b and the N-terminus of mIL12b
  • the N-terminus can be fused to form the second polypeptide chain of mIL12b-mGMCSF (sequence shown in SEQ ID NO.59), thereby forming a mIL12a-mIL7-mIL12b-mGMCSF dimer, which is represented by mIL12aIL7IL12bGMCSF.
  • the C-terminus of mIL12a and the N-terminus of mIL21 can be fused to form the first polypeptide chain of mIL12a-mIL21 (sequence is shown in SEQ ID NO.60), and the C-terminus of mIL12b and the N-terminus of mIL12b
  • the N-terminus can be fused to form the second polypeptide chain of mIL12b-mGMCSF (sequence shown in SEQ ID NO.61) to form a mIL12a-mIL21-mIL12b-mGMCSF dimer, which is represented by mIL12aIL21IL12bGMCSF.
  • the C-terminus of hIL12a and the N-terminus of hIL2 can be fused to form the first polypeptide chain of hIL12a-hIL2 (sequence shown in SEQ ID NO.62), and the C-terminus of hIL12b and hGMCSF
  • the N-terminus can be fused to form the second polypeptide chain of hIL12b-hGMCSF (sequence shown in SEQ ID NO.63) to form a hIL12a-hIL2-hIL12b-hGMCSF dimer, which is represented by hIL12aIL2IL12bGMCSF.
  • the C-terminus of hIL12a and the N-terminus of hIL7 can be fused to form the first polypeptide chain of hIL12a-hIL7 (sequence shown in SEQ ID NO.64), and the C-terminus of hIL12b and hGMCSF
  • the N-terminus can be fused to form the second polypeptide chain of hIL12b-hGMCSF (sequence shown in SEQ ID NO.65), thereby forming a hIL12a-hIL7-hIL12b-hGMCSF dimer, which is represented by hIL12aIL7IL12bGMCSF.
  • the C-terminus of hIL12a and the N-terminus of hIL21 can be fused to form the first polypeptide chain of hIL12a-hIL21 (sequence shown in SEQ ID NO.66), and the C-terminus of hIL12b and hGMCSF
  • the N-terminus can be fused to form the second polypeptide chain of hIL12b-hGMCSF (sequence shown in SEQ ID NO. 67), thereby forming a hIL12a-hIL21-hIL12b-hGMCSF dimer, which is represented by hIL12aIL21IL12bGMCSF.
  • the protein, polypeptide, and/or amino acid sequence involved in this application should also be understood to include at least the following range: a variant or homologue that has the same or similar functions as the protein or polypeptide.
  • the variant may be a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and/or the polypeptide (for example, the protein molecule).
  • the functional variant may comprise at least one, such as 1-30, 1-20, or 1-10, and another example, 1, 2, 3, 4, or 5 amino acid substitutions.
  • the functional variant may substantially maintain the biological properties of the protein or polypeptide before the change (for example, substitution, deletion, or addition).
  • the functional variant can maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity of the protein or polypeptide before the change.
  • the homologue may be at least about 80% (for example, at least about 85%, about 90%) with the amino acid sequence of the protein and/or the polypeptide (for example, the protein molecule). %, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology protein or polypeptide .
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • the "percentage of sequence homology" can be calculated in the following way: the two sequences to be aligned are compared in the comparison window to determine the presence of the same nucleic acid base (for example, A, T, C, G) or Positions of the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) To get the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to produce the sequence homology percentage.
  • the same nucleic acid base for example, A, T, C, G
  • Positions of the same amino acid residues e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu,
  • the alignment to determine the percent sequence homology 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 homology can also be determined by the following methods: FASTA and BLAST. For a description of the FASTA algorithm, see WRPearson and DJ Lipman's "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad.
  • this application also provides a medicine, which comprises the liquid preparation described in this application.
  • the liquid preparation can be formulated as an injection, so that the liquid preparation described in the present application can be used for intravenous injection or subcutaneous injection.
  • the drugs described in this application can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, Administration is transdermal or via subcutaneous depot.
  • the medicine described in this application may also contain a diluent, wherein the diluent and the liquid preparation are not mixed with each other, so that when different concentrations of the liquid preparation described in this application are required, all the liquid preparations can be used.
  • the diluent dilutes the liquid formulation described in this application.
  • the medicament described in this application may also include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and/or non-ionics. Surfactant etc.
  • the pharmaceutically acceptable carrier may include excipients, for example, the excipients may be selected from the following group: starch, dextrin, sucrose, lactose, magnesium stearate, calcium sulfate, carboxymethyl , Talcum powder, calcium alginate gel, chitosan and nano microspheres.
  • the pharmaceutically acceptable carrier can also be selected from the following group: pH regulator, osmotic pressure regulator, solubilizer and bacteriostatic agent.
  • the application also provides the use of the liquid preparation described in the application in the preparation of drugs for treating tumors.
  • the tumor may include lung cancer.
  • this application also provides a method for treating tumors, which comprises administering the liquid formulation described in this application to a subject in need.
  • the administration method may be intratumoral injection, for example, injection of the liquid preparation described in the present application into the tumor.
  • the administration method can also be oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration
  • the medicine is administered via a subcutaneous depot.
  • the dose level of the liquid formulations described in this application to be administered may vary depending on the following conditions: the molecule being delivered, the route of administration, and the size of the patient (body weight, body surface or organ size) and / Or physical condition (age and general health condition).
  • the present application provides a liquid preparation or medicine, which is used to treat tumors.
  • the tumor may include lung cancer.
  • this application also provides an auxiliary material for preparing the liquid formulation described in this application, which comprises an oil phase solvent, the mass fraction of the oil phase solvent is about 50% or more, for example, the auxiliary material
  • the mass fraction of the oil phase solvent can be about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 60% to about 80%, about 60% to about 90%, about 60% -About 95%, about 70% to about 90%, about 70% to about 95%, or about 80% to about 95%, so that the liquid formulation described in this application can be easily prepared.
  • Standard abbreviations can be used, such as bp, base pairs; kb, kilobase pairs; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; nt, nucleotides ; Iv, intravenous injection; im, intramuscular; ip, intraperitoneal; sc, subcutaneous, etc.
  • DMEM medium, 1640 medium, and fetal bovine serum were purchased from Lifetechnologies; cell culture flasks and culture plates were purchased from Corning; Doxycycline (DOX) was purchased from Shanghai Shenggong Bioengineering Co., Ltd.; Puromycin (Puromycin) ), blasticidin were purchased from Chemicon; restriction endonucleases were purchased from Takara and NEB; ligase was purchased from NEB; DNA polymerase was purchased from Takara; plasmid extraction kits and gel recovery kits were purchased from OmegaBiotech; primers The synthesis was completed by Shanghai Shenggong Biological Engineering Co., Ltd.; the gene synthesis was completed by Nanjing GenScript Company; the ELISA kit was purchased from Boster Company.
  • the mIL12bIL12aIL2GMCSF protein has a secretion signal peptide at the front end, and 6*His is added to the back end for purification.
  • the DNA sequence corresponding to the mIL12bIL12aIL2GMCSF protein gene is synthesized. The sequence is mutated with a degenerate codon at the BamHI or XhoI site, before and after the synthesized sequence The ends have BamHI and XhoI restriction sites respectively, and the synthesized plasmid with the target gene is digested.
  • the system is as follows: 5 ⁇ g plasmid, 4 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, 37°C statically Set for 12 hours. Take out the EP tube, add 4.4 ⁇ l 10 ⁇ loading buffer, use 1% agarose gel for electrophoresis, recover the mIL12bIL12aIL2GMCSF protein gene fragment after electrophoresis, and set it aside.
  • amino acid sequence of mIL12bIL12aIL2GMCSF protein is shown in SEQ ID NO.32, and the nucleotide sequence encoding the mIL12bIL12aIL2GMCSF is shown in SEQ ID NO.68.
  • Enzyme digestion vector pLentis-CMV-MCS-IRES-PURO in EP tube the system is as follows: 2 ⁇ g pLentis-CMV-MCS-IRES-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 30 ⁇ l, Let stand at 37°C for 12 hours. Take out the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, use 1% agarose gel for electrophoresis, recover the pLentis-CMV-MCS-IRES-PURO vector fragment after electrophoresis, and set aside.
  • the system is as follows: 2 ⁇ l pLentis-CMV-MCS-IRES-PURO vector fragment, 2 ⁇ l gene fragment, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave it at room temperature to connect for 4 hours. Then the ligation system is transformed into E. coli competent.
  • the purified protein solution obtained was then used AMICON ULTRA-0.5 ultra
  • the filter tube was subjected to ultrafiltration, the buffer was replaced with PBS, and the protein concentration obtained was finally detected with IL12p70 ELISA kit.
  • the protein concentration was adjusted to 2 ⁇ g/ ⁇ l with PBS, and then aliquoted and stored at -20°C.
  • mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr protein which has a secretion signal peptide at the front end, and 6*His at the back end for purification.
  • the DNA sequence corresponding to the gene is synthesized, and the BamHI or XhoI site in the sequence is mutated with a degenerate codon, and the front and back ends of the synthesized sequence With BamHI and XhoI restriction sites respectively, the plasmid with the target gene was synthesized by restriction digestion.
  • the system is as follows: 5 ⁇ g plasmid, 4 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, and let stand at 37°C 12 hours. Take out the EP tube, add 4.4 ⁇ l 10 ⁇ loading buffer, use 1% agarose gel for electrophoresis, recover the mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr protein gene fragment after electrophoresis, and set it aside. It should be noted that the linker of mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr protein contains a thrombin cleavage site.
  • amino acid sequence of mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr protein is shown in SEQ ID NO. 48, and the nucleotide sequence encoding the mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr is shown in SEQ ID NO. 69
  • Enzyme digestion vector pLentis-CMV-MCS-IRES-PURO in EP tube the system is as follows: 2 ⁇ g pLentis-CMV-MCS-IRES-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 30 ⁇ l, Let stand at 37°C for 12 hours. Take out the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, use 1% agarose gel for electrophoresis, recover the pLentis-CMV-MCS-IRES-PURO vector fragment after electrophoresis, and set aside.
  • the system is as follows, 2 ⁇ l pLentis-CMV-MCS-IRES-PURO vector fragment, 2 ⁇ l gene fragment, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and water 4.5 ⁇ l. Leave it at room temperature to connect for 4 hours. Then the ligation system is transformed into E. coli competent.
  • the obtained purified protein solution is reused Perform ultrafiltration with AMICON ULTRA-0.5 ultrafiltration tube, replace the buffer with PBS, and use IL12p70ELISA kit to detect the protein concentration of the finally obtained protein solution. After adjusting the protein concentration to 2 ⁇ g/ ⁇ l with PBS, it is aliquoted at -20°C save.
  • the mIL12bIL12aIL7GMCSF protein has a secretion signal peptide at the front end, and 6*His is added to the back end for purification.
  • the DNA sequence corresponding to the mIL12bIL12aIL7GMCSF protein gene is synthesized. The sequence is mutated with a degenerate codon at the BamHI or XhoI site, before and after the synthesized sequence The ends have BamHI and XhoI restriction sites respectively, and the synthesized plasmid with the target gene is digested.
  • the system is as follows: 5 ⁇ g plasmid, 4 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, 37°C statically Set for 12 hours. Take out the EP tube, add 4.4 ⁇ l 10 ⁇ loading buffer, and use 1% agarose gel for electrophoresis. After electrophoresis, the mIL12bIL12aIL7GMCSF protein gene fragment is recovered and set aside.
  • amino acid sequence of mIL12bIL12aIL7GMCSF protein is shown in SEQ ID NO. 33, and the nucleotide sequence encoding the mIL12bIL12aIL7GMCSF is shown in SEQ ID NO. 70.
  • Enzyme digestion vector pLentis-CMV-MCS-IRES-PURO in EP tube the system is as follows: 2 ⁇ g pLentis-CMV-MCS-IRES-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 30 ⁇ l, Let stand at 37°C for 12 hours. Take out the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, use 1% agarose gel for electrophoresis, recover the pLentis-CMV-MCS-IRES-PURO vector fragment after electrophoresis, and set aside.
  • the system is as follows, 2 ⁇ l pLentis-CMV-MCS-IRES-PURO vector fragment, 2 ⁇ l gene fragment, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave it at room temperature to connect for 4 hours. Then the ligation system is transformed into E. coli competent.
  • the purified protein solution obtained was then used AMICON ULTRA-0.5 ultra
  • the filter tube was subjected to ultrafiltration, the buffer was replaced with PBS, and the protein concentration obtained was finally detected with IL12p70 ELISA kit.
  • the protein concentration was adjusted to 2 ⁇ g/ ⁇ l with PBS, and then aliquoted and stored at -20°C.
  • the mIL12bIL12aIL21GMCSF protein has a secretion signal peptide at the front end, and 6*His is added to the back end for purification.
  • the DNA sequence corresponding to the mIL12bIL12aIL21GMCSF protein gene is synthesized. The sequence is mutated with a degenerate codon at the BamHI or XhoI site, before and after the synthesized sequence The ends have BamHI and XhoI restriction sites respectively, and the synthesized plasmid with the target gene is digested.
  • the system is as follows: 5 ⁇ g plasmid, 4 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, 37°C statically Set for 12 hours. Take out the EP tube, add 4.4 ⁇ l 10 ⁇ loading buffer, use 1% agarose gel for electrophoresis, recover the mIL12bIL12aIL21GMCSF protein gene fragment after electrophoresis, and set it aside.
  • the amino acid sequence of mIL12bIL12aIL21GMCSF protein is shown in SEQ ID NO.35, and the nucleotide sequence encoding the mIL12bIL12aIL21GMCSF is shown in SEQ ID NO.71.
  • the vector pLentis-CMV-MCS-IRES-PURO was digested in the EP tube.
  • the system is as follows: 2 ⁇ g pLentis-CMV-MCS-IRES-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 30 ⁇ l, Let stand at 37°C for 12 hours. Take out the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, use 1% agarose gel for electrophoresis, recover the pLentis-CMV-MCS-IRES-PURO vector fragment after electrophoresis, and set aside.
  • the system is as follows, 2 ⁇ l pLentis-CMV-MCS-IRES-PURO vector fragment, 2 ⁇ l gene fragment, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave it at room temperature to connect for 4 hours. Then the ligation system is transformed into E. coli competent.
  • the purified protein solution obtained was then used AMICON ULTRA-0.5 ultra
  • the filter tube was subjected to ultrafiltration, the buffer was replaced with PBS, and the protein concentration obtained was finally detected with IL12p70 ELISA kit.
  • the protein concentration was adjusted to 2 ⁇ g/ ⁇ l with PBS, and then aliquoted and stored at -20°C.
  • the mIL12aIL2IL12bGMCSF protein is a heterodimer composed of mIL12aIL2 and mIL12bGMCSF, in which 6*His is added to the back end of mIL12aIL2 for purification, and the DNA sequence corresponding to the mIL12aIL2IL12bGMCSF protein gene is synthesized.
  • the T2A cleavage peptide is added between the two peptide chains to form a heterodimer after translation.
  • the polymer is mutated with degenerate codons at the BamHI or XhoI site in the sequence, and the front and back ends of the synthetic sequence are respectively equipped with BamHI and XhoI restriction sites.
  • the plasmid with the target gene is synthesized by restriction digestion.
  • the system is as follows: 5 ⁇ g plasmid, 4 ⁇ l enzyme digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, and let it stand at 37°C for 12 hours. Take out the EP tube, add 4.4 ⁇ l 10 ⁇ loading buffer, and use 1% agarose gel for electrophoresis. After electrophoresis, the mIL12aIL2IL12bGMCSF protein gene fragment is recovered and set aside.
  • amino acid sequence of the second polypeptide chain of mIL12aIL2IL12bGMCSF protein is shown in SEQ ID NO.57
  • amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.56
  • nucleotide sequence encoding the mIL12aIL2IL12bGMCSF is shown in SEQ ID NO.72. Shown.
  • Enzyme digestion vector pLentis-CMV-MCS-IRES-PURO in EP tube the system is as follows: 2 ⁇ g pLentis-CMV-MCS-IRES-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 30 ⁇ l, Let stand at 37°C for 12 hours. Take out the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, use 1% agarose gel for electrophoresis, recover the pLentis-CMV-MCS-IRES-PURO vector fragment after electrophoresis, and set aside.
  • the system is as follows, 2 ⁇ l pLentis-CMV-MCS-IRES-PURO vector fragment, 2 ⁇ l gene fragment, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave it at room temperature to connect for 4 hours. Then the ligation system is transformed into E. coli competent.
  • the purified protein solution obtained was then used AMICON ULTRA-0.5 ultra
  • the filter tube was subjected to ultrafiltration, the buffer was replaced with PBS, and the protein concentration finally obtained was detected with IL12p70ELISA kit.
  • the protein concentration was adjusted to 0.2 ⁇ g/ ⁇ l with PBS, and then aliquoted and stored at -20°C.
  • Example 7 The effect of mIL12bIL12aIL2GMCSF injection on tumor growth in mice
  • LLC digested and cultured mouse lung cancer cells
  • the protein solution prepared in Example 1 was used to prepare a series of liquid preparations of the application with different mass percentages of glycerol (ie, mIL12bIL12aIL2GMCSF glycerol injection).
  • the specific method is: take 50 ⁇ l of the protein solution prepared in Example 1, then add it to 50 ⁇ l glycerin, quickly mix with a pipette tip to avoid bubbles, and obtain a liquid preparation with a 50% glycerol concentration, and add 50ul protein solution to 75ul glycerol To obtain a liquid preparation with a concentration of 60% glycerol, 50ul of the protein solution is added to 117ul of glycerol to obtain a liquid preparation with a concentration of 70% glycerol.
  • the preparation method of the comparative preparation of the control group is as follows: take 50 ⁇ l of the protein solution prepared in Example 1, and then add it to 117 ⁇ l PBS, and quickly mix it with a pipette tip to avoid bubbles, and obtain the prepared comparative preparation 1. Use a 29G insulin syringe to suck the prepared liquid preparation of the application or the preparation 1 of the comparative example, and slowly inject it into the tumor. After the injection, the needle is kept for a little time to reduce the overflow of the solution. After the injection, the mice were returned to the cage, and the survival of the mice was recorded. The results are shown in Figure 2. PBS in the figure represents the control group.
  • mice survived after injection of the liquid preparation of the application (ie mIL12bIL12aIL2GMCSF glycerol injection), indicating that the glycerol solution and protein molecules have a synergistic effect. Together to inhibit tumor growth.
  • Example 8 The effect of mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr injection on tumor growth in mice
  • LLC digested and cultured mouse lung cancer cells
  • the protein solution prepared in Example 2 was used to prepare a series of liquid preparations of the application with different mass percentages of glycerol (ie, mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr glycerol injection).
  • the specific method is: take 50 ⁇ l of the protein solution prepared in Example 2, then add it to 50 ⁇ l glycerin, quickly mix with a pipette tip to avoid bubbles, and obtain a 50% glycerol concentration liquid preparation, and add 50ul protein solution to 75ul glycerol To obtain a liquid preparation with a concentration of 60% glycerol, 50ul of the protein solution is added to 117ul of glycerol to obtain a liquid preparation with a concentration of 70% glycerol.
  • the preparation method of the comparative preparation of the control group was as follows: take 50 ⁇ l of the protein solution prepared in Example 2, and then add it to 117 ⁇ l PBS, and quickly mix it with a pipette tip to avoid bubbles, and obtain the prepared comparative preparation 2. Use a 29G insulin syringe to suck the prepared liquid preparation of the application (ie, mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr glycerol injection) or comparative preparation 2, and slowly inject it into the tumor. After the injection, the needle is retained for a little time to reduce the overflow of the solution. After the injection, the mice were returned to the cage, and the survival of the mice was recorded.
  • Example 9 The effect of mIL12bIL12aIL7GMCSF injection on tumor growth in mice
  • LLC digested and cultured mouse lung cancer cells
  • the preparation method of the comparative preparation of the control group is as follows: take 50 ⁇ l of the protein solution prepared in Example 3, and then add it to 80 ⁇ l PBS, and quickly mix it with a pipette tip to avoid generating bubbles to obtain the prepared comparative preparation 3.
  • mice were returned to the cage, and the survival of the mice was recorded.
  • the results are shown in Figure 4, where PBS represents the control group. It can be seen that all mice survived after injection of the liquid preparation of the application (ie mIL12bIL12aIL7GMCSF glycerol injection), indicating that the glycerol solution and protein molecules have a synergistic effect, thereby Together to inhibit tumor growth.
  • Example 10 The effect of mIL12bIL12aIL21GMCSF injection on tumor growth in mice
  • LLC digested and cultured mouse lung cancer cells
  • the preparation method of the comparative preparation of the control group is as follows: take 50 ⁇ l of the protein solution prepared in Example 4, and then add it to 80 ⁇ l PBS, and quickly mix it with a pipette tip to avoid bubbles, and obtain the prepared comparative preparation 4.
  • mice were returned to the cage, and the survival of the mice was recorded.
  • the results are shown in Figure 5.
  • PBS in the figure represents the control group. It can be seen that all mice survived after injection of the liquid preparation of the application (ie mIL12bIL12aIL21GMCSF glycerol injection), indicating that the glycerol solution and protein molecules have a synergistic effect, thereby Together to inhibit tumor growth.
  • Example 11 The effect of mIL12aIL2IL12bGMCSF injection on tumor growth in mice
  • LLC digested and cultured mouse lung cancer cells
  • the preparation method of the comparative preparation of the control group was as follows: take 50 ⁇ l of the protein solution prepared in Example 5, and then add it to 80 ⁇ l PBS, and quickly mix it with a pipette tip to avoid bubbles to obtain the prepared comparative preparation 5.
  • mice were returned to the cage, and the survival of the mice was recorded.
  • the results are shown in Figure 6.
  • PBS in the figure represents the control group. It can be seen that all mice survived after injection of the liquid preparation of the application (ie mIL12aIL2IL12bGMCSF glycerol injection), indicating that the glycerol solution and protein molecules had a synergistic effect. Together to inhibit tumor growth.

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Abstract

一种液体制剂，其包括油相溶剂系统和蛋白质，其中所述油相溶剂系统包括油相溶剂，所述油相溶剂的质量分数为约50％以上。

Description

一种液体制剂及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种液体制剂及其应用。
背景技术
肿瘤是一种严重威胁人类健康的疾病，近年来，免疫治疗作为一种新疗法，在肿瘤治疗中显示出了巨大的潜力。细胞因子(Cytokine)是体内非常重要的免疫信号，细胞因子融合蛋白技术是当今肿瘤免疫疗法的另一个热点方向。该方法是基于这些细胞因子具有相同或相关的功能活性而各自作用靶点不同，利用基因工程技术将两种或多种细胞因子融合在一起。但是目前利用细胞因子融合蛋白技术进行肿瘤治疗的效果仍不尽人意，有较多需要改进之处。
发明内容
本申请提供了一种液体制剂，其包括油相溶剂系统和蛋白质。本申请所述液体制剂具有抗肿瘤效果；其中的油相溶剂和蛋白质(例如，细胞因子融合蛋白)具有协同的抗肿瘤效果。
本申请提供了一种液体制剂，其包括油相溶剂系统和蛋白质，其中所述油相溶剂系统包括油相溶剂，所述油相溶剂的质量分数为约50％以上。
在某些实施方式中，所述油相溶剂选自以下组：甘油、丙二醇、聚乙二醇、聚乙烯吡咯烷酮、甘露醇、山梨醇、聚氧丙烯和氨基丁三醇。
在某些实施方式中，所述油相溶剂的质量分数为约60％以上。
在某些实施方式中，所述油相溶剂的质量分数为约70％以上。
在某些实施方式中，所述蛋白质的质量分数为约1％-约50％。
在某些实施方式中，所述蛋白质包括细胞因子和/或抗体。
在某些实施方式中，所述细胞因子包括选自下组的两种或更多种：IL12、IL2、GMCSF、IL7、IL15、IL21和FLT3L。
在某些实施方式中，所述细胞因子源自哺乳动物。
在某些实施方式中，所述蛋白质包括融合蛋白，且所述融合蛋白中包含选自下组的至少两种细胞因子：IL12、IL2、GMCSF、IL7、IL15、IL21和FLT3L。
在某些实施方式中，所述蛋白质包括融合蛋白，且所述融合蛋白中包含选自以下的任一组细胞因子：
a)IL12、IL2和GMSCF；
b)IL12、IL7和GMSCF；
c)IL12、IL15和GMSCF；
d)IL12、IL21和GMSCF；
e)IL12、IL2和FLT3L；
f)IL12、IL7和FLT3L；
g)IL12、IL15和FLT3L；以及，
h)IL12、IL21和FLT3L。
在某些实施方式中，所述蛋白质还包括靶向部分。
在某些实施方式中，所述靶向部分能够特异性识别和/或结合肿瘤相关抗原。
在某些实施方式中，所述肿瘤相关抗原选自下组：纤连蛋白的EDB结构域、纤连蛋白的EDA结构域和细胞坏死区域(necrotic regions)。
在某些实施方式中，所述靶向部分包括抗体或其抗原结合片段。
在某些实施方式中，所述靶向部分包含下组中任一项所示的氨基酸序列：SEQ ID NO:1-15。
在某些实施方式中，所述蛋白质包含下组中任一项所示的氨基酸序列：SEQ ID NO:32-67。
在某些实施方式中，所述蛋白质为单链蛋白质。
在某些实施方式中，所述单链蛋白质包含下组中任一项所示的氨基酸序列：SEQ ID NO:32-51。
在某些实施方式中，所述蛋白质为由第一多肽链及第二多肽链组成的二聚体，所述第一多肽链不同于所述第二多肽链。
在某些实施方式中，所述第一多肽链包含IL12a，所述第二多肽链包含IL12b。
在某些实施方式中，IL2或其功能性片段位于所述第一多肽链中或所述第二多肽链中，GMCSF或其功能性片段位于所述第一多肽链中或所述第二多肽链中，且所述一个或多个靶向部分各自独立地位于所述第一多肽链中或所述第二多肽链中。
在某些实施方式中，在所述第一多肽链中，从N端到C端依次包含所述IL2或其功能性片段、所述IL12a或其功能性片段和所述GMCSF或其功能性片段。
在某些实施方式中，在所述第一多肽链中，从N端到C端依次包含所述靶向部分、所述IL12a或其功能性片段、所述IL2或其功能性片段以及所述GMCSF或其功能性片段。
在某些实施方式中，在所述第二多肽链中，从N端到C端依次包含所述IL12b或其功能性片段和所述靶向部分。
在某些实施方式中，其中，
a)所述第一多肽链包含SEQ ID NO:53所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:52所示的氨基酸序列；
b)所述第一多肽链包含SEQ ID NO:55所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:54所示的氨基酸序列；
c)所述第一多肽链包含SEQ ID NO:56所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:57所示的氨基酸序列；
d)所述第一多肽链包含SEQ ID NO:58所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:59所示的氨基酸序列；
e)所述第一多肽链包含SEQ ID NO:60所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:61所示的氨基酸序列；
f)所述第一多肽链包含SEQ ID NO:62所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:63所示的氨基酸序列；
g)所述第一多肽链包含SEQ ID NO:64所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:65所示的氨基酸序列；
h)所述第一多肽链包含SEQ ID NO:66所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:67所示的氨基酸序列。
另一方面，本申请还提供了一种药物，其包含本申请所述的液体制剂。
在某些实施方式中，其中所述液体制剂被配制为注射剂。
在某些实施方式中，本申请所述的药物还包含稀释剂，其中所述稀释剂与所述液体制剂彼此不混合。
另一方面，本申请还提供了本申请所述的液体制剂在制备治疗肿瘤的药物中的用途。
在某些实施方式中，其中所述肿瘤包括肺癌。
在某些实施方式中，本申请所述的液体制剂，其用于治疗肿瘤。
另一方面，本申请还提供了一种治疗肿瘤的方法，其包括向有需要的受试者施用本申请所述的液体制剂。
在某些实施方式中，其中所述施用方法为瘤内注射。
另一方面，本申请还提供了一种用于制备本申请所述的液体制剂的辅料，其包含油相溶剂，所述油相溶剂的质量分数为约50％以上。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉 及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示甘油对小鼠体内肿瘤生长的影响；
图2显示本申请所述的液体制剂对小鼠体内肿瘤生长的影响；
图3显示本申请所述的液体制剂对小鼠体内肿瘤生长的影响；
图4显示本申请所述的液体制剂对小鼠体内肿瘤生长的影响；
图5显示本申请所述的液体制剂对小鼠体内肿瘤生长的影响；
图6显示本申请所述的液体制剂对小鼠体内肿瘤生长的影响。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
以下对本申请做进一步描述：在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本申请中，术语“液体制剂”通常指一种呈液体状的药物组合物，其通常包含活性成分和可药用的载体或赋形剂。例如，在本申请中，所述液体制剂可以包括油相溶剂系统和蛋白质，其中所述油相溶剂系统包括油相溶剂，所述油相溶剂的质量分数为约50％以上。所述油相溶剂可以包括甘油、丙二醇和/或聚乙二醇。在本申请中，可以将蛋白质分散或者溶解在油相溶剂中，以制备本申请所述的液体制剂。
在本申请中，术语“蛋白质”可以被认为属于“细胞因子融合蛋白”，其通常是指能够通过基因重组技术将两种或多种细胞因子融合在一起获得的融合蛋白。其既具有其组成因子独特的生物学活性或使其某些活性显著提高，还可能会通过生物学活性的互补及协同效应发挥出较单一细胞因子简单配伍所不具备的复合生物学功能，甚至还可能会产生一些新的结构 及生物学功能。
在本申请中，术语“IL12”、“IL12a”、“IL12b”、“IL2”、“GMCSF”、“IL7”、“IL15”、“IL21”、“FLT3L”可以被认为属于“细胞因子”。所述“细胞因子”通常是指由免疫细胞(如单核、巨噬细胞、T细胞、B细胞、NK细胞等)和某些非免疫细胞(例如，内皮细胞、表皮细胞、纤维母细胞等)经刺激而合成、分泌的一类具有广泛生物学活性的小分子蛋白质。所述细胞因子对于细胞间相互作用、细胞的生长和分化有重要调节作用。在本申请中，所述细胞因子可以选自下组中的一种或多种：白细胞介素(Interleukin，IL)、FMS相关酪氨酸激酶3配体(FLT3L)和集落刺激因子(Colony Stimulating Factor，CSF)。所述白细胞介素通常是指由淋巴细胞、单核细胞或其它非单个核细胞产生的细胞因子。在本申请中，所述白细胞介素可以选自下组中的一种或多种：IL12、IL2、IL7、IL15、IL21。在本申请中，所述集落刺激因子通常是指可刺激不同的造血干细胞在半固体培养基中形成细胞集落的细胞因子。在本申请中，所述集落刺激因子可以是粒细胞巨噬细胞集落刺激因子(Granulocyte Macrophage Colony Stimulating Factor，GMCSF)。
在本申请中，术语“IL12”通常是指白细胞介素-12，IL12可以在细胞间相互作用、免疫调节、造血以及炎症过程中起重要调节作用。IL12的分子通常是一种异源二聚体，其通常包括两个亚基，两个亚基分别是p40亚基(40kd)和p35亚基(35kd)，这两个亚基通过二硫键连接在一起。在本申请中，含有p35亚基(35kd)的IL12可以以IL12a表示，含有p40亚基(40kd)的IL12可以以IL12b表示。术语“IL-12”可以指来自任何脊椎动物来源的任何天然IL-12，所述脊椎动物来源可包括哺乳动物例如灵长类动物(例如人)和啮齿类动物(例如小鼠和大鼠)。术语“IL-12”还可包含全长的或未加工的IL-12，以及由细胞中的加工或人工合成而产生的任何形式的IL-12。该术语还包含天然存在的IL-12变体，例如剪接变体或等位基因变体，或突变体、同源物、功能性变体或功能性片段等。例如，来源于小鼠的IL12(mIL12)中的p35亚基可包含如SEQ ID NO:16所示的氨基酸序列，p40亚基可包含如SEQ ID NO:17所示的氨基酸序列。又例如，来源于人的IL12(hIL12)中的p35亚基可包含如SEQ ID NO:18所示的氨基酸序列，p40亚基可包含如SEQ ID NO:19所示的氨基酸序列。
在本申请中，术语“IL2”通常是指白细胞介素-2，IL2在细胞间相互作用、免疫调节、造血以及炎症过程中起重要调节作用。术语“IL-2”可以指来自任何脊椎动物来源的任何天然IL-2，所述脊椎动物来源可包括哺乳动物例如灵长类动物(例如人)和啮齿类动物(例如小鼠和大鼠)。术语“IL-2”还可包含全长的或未加工的IL-2，以及由细胞中的加工或人工合成而产生的任何形式的IL-2。该术语还包含天然存在的IL-2变体，例如剪接变体或等位基因变体，或突变体、同源物、功能性变体或功能性片段等。例如，来源于小鼠的IL2(mIL2)可包含如SEQ  ID NO:20所示的氨基酸序列。又例如，来源于人的IL2(hIL2)可包含如SEQ ID NO:21所示的氨基酸序列。
在本申请中，术语“IL15”通常是指白细胞介素-15，IL15在细胞间相互作用、免疫调节、造血以及炎症过程中起重要调节作用。术语“IL-15”可以指来自任何脊椎动物来源的任何天然IL-15，所述脊椎动物来源可包括哺乳动物例如灵长类动物(例如人)和啮齿类动物(例如小鼠和大鼠)。术语“IL-15”还可包含全长的或未加工的IL-15，以及由细胞中的加工或人工合成而产生的任何形式的IL-15。该术语还包含天然存在的IL-15变体，例如剪接变体或等位基因变体，或突变体、同源物、功能性变体或功能性片段等。例如，来源于小鼠的IL15(mIL15)可包含如SEQ ID NO:22所示的氨基酸序列。又例如，来源于人的IL15(hIL15)可包含如SEQ ID NO:23所示的氨基酸序列。
在本申请中，术语“IL7”通常是指白细胞介素-7，IL7在细胞间相互作用、免疫调节、造血以及炎症过程中起重要调节作用。术语“IL-7”可以指来自任何脊椎动物来源的任何天然IL-7，所述脊椎动物来源可包括哺乳动物例如灵长类动物(例如人)和啮齿类动物(例如小鼠和大鼠)。术语“IL-7”还可包含全长的或未加工的IL-7，以及由细胞中的加工或人工合成而产生的任何形式的IL-7。该术语还包含天然存在的IL-7变体，例如剪接变体或等位基因变体，或突变体、同源物、功能性变体或功能性片段等。例如，来源于小鼠的IL7(mIL7)可包含如SEQ ID NO:24所示的氨基酸序列。又例如，来源于人的IL7(hIL7)可包含如SEQ ID NO:25所示的氨基酸序列。
在本申请中，术语“IL21”通常是指白细胞介素-21，IL21在细胞间相互作用、免疫调节、造血以及炎症过程中起重要调节作用。术语“IL-21”可以指来自任何脊椎动物来源的任何天然IL-21，所述脊椎动物来源可包括哺乳动物例如灵长类动物(例如人)和啮齿类动物(例如小鼠和大鼠)。术语“IL-21”还可包含全长的或未加工的IL-21，以及由细胞中的加工或人工合成而产生的任何形式的IL-21。该术语还包含天然存在的IL-21变体，例如剪接变体或等位基因变体，或突变体、同源物、功能性变体或功能性片段等。例如，来源于小鼠的IL21(mIL21)可包含如SEQ ID NO:26所示的氨基酸序列。又例如，来源于人的IL21(hIL21)可包含如SEQ ID NO:27所示的氨基酸序列。
在本申请中，术语“FLT3L”通常是指FMS相关酪氨酸激酶3配体，其可调节非红系造血干细胞的增殖和分化，促进前B淋巴细胞、树突状细胞、NK细胞、细胞毒T淋巴细胞的增殖、分化和成熟，具有重要的抗肿瘤作用。术语“FLT3L”可以指来自任何脊椎动物来源的任何天然FLT3L，所述脊椎动物来源可包括哺乳动物例如灵长类动物(例如人)和啮齿类动物(例如小鼠和大鼠)。术语“FLT3L”还可包含全长的或未加工的FLT3L，以及由细胞中的加工或 人工合成而产生的任何形式的FLT3L。该术语还包含天然存在的FLT3L变体，例如剪接变体或等位基因变体，或突变体、同源物、功能性变体或功能性片段等。例如，来源于小鼠的FLT3L(mFLT3L)可包含如SEQ ID NO:28所示的氨基酸序列。又例如，来源于人的FLT3L(hFLT3L)可包含如SEQ ID NO:29所示的氨基酸序列。
在本申请中，术语“GMCSF”通常是指粒细胞巨噬细胞集落刺激因子。所述GMCSF可带有4个α螺旋束结构。术语“GMCSF”可以指来自任何脊椎动物来源的任何天然GMCSF，所述脊椎动物来源可包括哺乳动物例如灵长类动物(例如人)和啮齿类动物(例如小鼠和大鼠)。术语“GMCSF”还可包含全长的或未加工的GMCSF，以及由细胞中的加工或人工合成而产生的任何形式的GMCSF。该术语还包含天然存在的GMCSF变体，例如剪接变体或等位基因变体，或突变体、同源物、功能性变体或功能性片段等。例如，来源于小鼠的GMCSF(mGMCSF)可包含如SEQ ID NO:30所示的氨基酸序列。又例如，来源于人的GMCSF(hGMCSF)可包含如SEQ ID NO:31所示的氨基酸序列。
在本申请中，术语“抗体”通常指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，为了本发明的目的，术语“抗体”也包括抗体片段，比如Fab、F(ab') 2、Fv、scFv、Fd、dAb和保持抗原结合功能(即，特异性结合例如OX40或PD-Ll)的其它抗体片段。通常，这样的片段应当包括抗原结合结构域。
基本的4链抗体单元是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般为约150,000道尔顿。每个L链通过一个共价二硫键与H链连接，而两个H链通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还具有规则间隔的链内二硫化桥键。每个H链在N末端具有可变结构域(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端具有可变结构域(VL)，在其另一端具有恒定结构域。VL与VH对应，且CL与重链的第一恒定结构域(CH1)相对应。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。VH和VL配对一起形成单个抗原结合位点。对于不同类别抗体的结构和性质，参见例如Basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw(eds),Appleton&Lange,Norwalk,Conn.,1994，第71页和第6章。来自任何脊椎动物物种的L链可 以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。取决于其重链(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。基于CH序列和功能方面的相对小的差异，将γ和α类进一步分成亚类，例如，人表达下述亚类：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1和IgK1。
在本申请中，术语“靶向部分”通常是指一类针对某一些特殊组织、细胞起作用的部分。例如，靶向部分能够特异性靶向肿瘤相关抗原。在本申请中，所述靶向部分包括抗体或其抗原结合片段。
本文使用的术语“特异性识别和/或结合”通常指可测量的和可再现的相互作用，比如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况可决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标的抗体。在一个实施方案中，抗体结合无关靶标的程度小于抗体对靶标的结合的约10％，如例如通过放射免疫分析(RIA)测量的。在某些实施方案中，特异性结合靶标的抗体具有<1x10 -6M、<1x10 -7M、<1x10 -8M、<1x10 -9M或<1x10 -10M的解离常数(KD)。在某些实施方案中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在另一个实施方案中，特异性结合可以包括但不要求排他性地结合。
在本申请中，术语“肿瘤相关抗原”(tumor-associated antigen，TAA)，通常是指在肿瘤细胞或正常细胞上存在的抗原分子。所述肿瘤相关抗原可以包括：胚胎性蛋白、糖蛋白抗原和鳞状细胞抗原。所述肿瘤相关抗原可以选自下组：纤连蛋白的EDB结构域、纤连蛋白的EDA结构域和细胞坏死区域(necrotic regions)。
在本申请中，术语“抗原结合片段”通常是指具有抗原结合活性的片段。在本申请中，所述抗原结合片段可以选自下组：Fab，Fab’，F(ab’) 2，F(ab) 2，dAb，分离的互补决定区CDR，Fv和scFv。
在本申请中，术语“单链蛋白质”通常是指由一个连续氨基酸残基的不间断序列组成的一级结构的多肽。例如，在本申请中，所述单链蛋白质可以包含下组中任一项所示的氨基酸序列：SEQ ID NO:32-51。
在本申请中，术语“二聚体”通常指由两个通常是非共价键合的单体单位形成的高分子复合物。每个单体单位可以是大分子，比如多肽链或多核苷酸。例如，在本申请中，所述蛋白质可以为由第一多肽链及第二多肽链组成的二聚体。
在本申请中，术语“多肽链”通常指包括两个或多个共价连接的肽的大分子。多肽链之内的肽可以通过一个肽键彼此连接。每条多肽链可以包括一个N-末端或氨基末端和一个C-末 端或羧基末端。
在本申请中，术语“功能性片段”通常是指保留某种特定功能的片段，例如，IL12a功能性片段是指保留IL12a的功能的片段。例如，IL12a功能性片段可以为IL12a，片段(GenBank:AIC49052.1)。又例如，IL12b功能性片段可以为IL12b，片段(GenBank:AIC54621.1)。
在本申请中，术语“注射剂”通常是指药物制成的供注入体内的无菌溶液(包括乳浊液和混悬液)以及供临用前配成溶液或混悬液的无菌粉末或浓溶液。例如，注射剂可以通过静脉注射或者皮下注射的方式使药物进入体内。在本申请中，所述液体制剂可以被配制为注射剂。
在本申请中，术语“稀释剂”通常指用于稀释物质的溶剂，例如使物质浓度降低的溶剂。在本申请中，所述药物还可以包含稀释剂，所述稀释剂与所述液体制剂彼此不混合。
在本申请中，术语“肿瘤”通常指由异常细胞生长形成的赘生物或实体病变。在本申请中，肿瘤可以是实体瘤或血液瘤。例如，肿瘤可包括肺癌。
在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
在本申请中，术语“施用”通常是指向受试者(例如，患者)给予一定剂量的液体制剂或药物的方法。施用可通过任何合适的方式进行，包括肠胃外、肺内和鼻内，以及(如果局部治疗需要)损伤内施用。肠胃外输注包括例如肌肉内、静脉内、动脉内、腹膜内或皮下施用。可以通过任何合适的途径，例如通过注射(诸如静脉内或皮下注射)来给药，这部分地取决于施用是短暂的或长期的。本文涵盖各种给药排程，包括但不限于单次施用或各种时间点内的多次施用、推注施用和脉冲输注。例如，在本申请中，所述施用可以为瘤内注射。所述“瘤内注射”通常是指向肿瘤内部注射一定剂量的液体制剂或者药物。
在本申请中，术语“辅料”通常是指辅助性材料，例如辅助制备某种物质的材料。例如在本申请中，用于制备所述的液体制剂的辅料可以包含油相溶剂，所述油相溶剂的质量分数可以为约50％以上。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
液体制剂
一方面，本申请提供一种液体制剂，其可以包括油相溶剂系统和蛋白质，其中所述油相溶 剂系统可以包括油相溶剂，所述油相溶剂的质量分数可以为约50％以上。
此外，在某些实施方式中，所述油相溶剂的质量分数可以为约60％以上。例如，在另一些实施方式中，所述油相溶剂的质量分数可以为约70％以上。例如，所述油相溶剂的质量分数可以为约50％-约60％、约60％-约70％、约70％-约80％、约80％-约90％、约90％-约95％、约50％-约70％、约50％-约80％、约50％-约90％、约50％-约95％、约60％-约80％、约60％-约90％、约60％-约95％、约70％-约90％、约70％-约95％、或约80％-约95％。
在本申请中，所述油相溶剂可以选自以下组：甘油、丙二醇、聚乙二醇、聚乙烯吡咯烷酮、甘露醇、山梨醇、聚氧丙烯和氨基丁三醇。
在本申请中，所述油相溶剂可以为甘油，所述甘油的质量分数可以为约50％以上。在某些实施方式中，所述甘油的质量分数可以为约60％以上。例如，在另一些实施方式中，所述甘油的质量分数可以为约70％以上。例如，所述甘油的质量分数可以为约50％-约60％、约60％-约70％、约70％-约80％、约80％-约90％、约90％-约95％、约50％-约70％、约50％-约80％、约50％-约90％、约50％-约95％、约60％-约80％、约60％-约90％、约60％-约95％、约70％-约90％、约70％-约95％、或约80％-约95％。
在本申请中，所述蛋白质的质量分数可以为约1％-约50％，例如所述蛋白质的质量分数可以为约约1％-约10％、约1％-约20％、约1％-约30％、约1％-约40％、约1％-约50％、5％-约10％、约5％-约20％、约5％-约30％、约5％-约40％、约5％-约50％、约10％-约20％、约10％-约30％、约10％-约40％、约10％-约50％、约20％-约30％、约20％-约40％、约20％-约50％、约30％-约40％、约30％-约50％、约40％-约50％、或约45％-约50％。
在某些实施方式中，可以通过将所述蛋白质溶解或者分散在所述油相溶剂系统中(例如油相溶剂中)以制备本申请所述的液体制剂。
在本申请中，所述油相溶剂和所述蛋白质可以是彼此互溶的，也可以是彼此不互溶的。
在本申请中，所述油相溶剂和所述蛋白质之间具有协同效应，从而增强本申请所述的液体制剂在治疗肿瘤方面的效果。
在本申请中，本申请所述的液体制剂可以用于治疗肿瘤，其中的油相溶剂与蛋白质可以相互协同作用，从而增强肿瘤的治疗效果，例如，相比于单纯的油相溶剂或者单纯的蛋白质，本申请所述的液体制剂在肿瘤治疗方面的效果呈现显著和增强。又例如，单纯的蛋白溶液通常无法诱导肿瘤消退，而使用本申请所述的液体制剂则可以诱导肿瘤的消退。
在本申请中，所述液体制剂可以用于抑制肿瘤生长。例如，本申请的液体制剂可以抑制或延缓疾病的发展或进展，可以通过促进细胞因子的表达减小肿瘤的大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。
此外，还需要说明的是，本申请所述的液体制剂，除了可以包括油相溶剂系统和蛋白质之外，还可以包括氨基酸，所述氨基酸可以为甘氨酸、谷氨酰胺、天冬酰胺、精氨酸或赖氨酸中的任意一种或多种。本申请所述的液体制剂还可以包括抗菌剂或者抗氧化剂。所述抗氧化剂可以为抗坏血酸、亚硫酸钠或亚硫酸氢钠中的任意一种或多种。本申请所述的液体制剂还可以包括缓冲剂，所述缓冲剂可以为硼酸盐、碳酸氢盐、Tris-HCl、柠檬酸盐、磷酸盐或其它有机酸中的任一种或多种。本申请所述的液体制剂还可以包括膨胀剂，例如甘露醇或甘氨酸。本申请所述的液体制剂还可以包括螯合剂，例如乙二胺四乙酸(EDTA。本申请所述的液体制剂还可以包括络合剂，例如咖啡因、聚乙烯吡咯烷酮、β-环糊精或羟基丙基-β-环糊精中的任一种或多种。本申请所述的液体制剂还可以包括糖类，例如葡萄糖、甘露糖或糊精。本申请所述的液体制剂还可以包括防腐剂，例如苯扎氯铵、苯甲酸、水杨酸、硫柳汞、苯乙醇、羟苯甲酸甲酯、羟苯甲酸丙酯、双氯苯双胍己烷、山梨酸或过氧化氢中的任一种或多种。
蛋白质
在本申请中，所述蛋白质可以包括细胞因子和/或抗体。
在本申请中，所述抗体可以选自下组的一种或多种：单克隆抗体、多克隆抗体、抗体片段、嵌合抗体、双特异性抗体、杂缀合物抗体、单链(ScFv)、包含抗体部分的融合蛋白(例如结构域抗体)、人源化抗体和免疫球蛋白分子的包含所需特异性的抗原识别位点的任何其它经修饰的构型。其中，抗体片段可以选自Fab、Fab′、F(ab′) 2、Fv和Fc中的一种或多种。包含抗体部分的融合蛋白可以为结构域抗体。
在本申请中，所述抗体还可以包括抗体的糖基化变体、抗体的氨基酸序列变体和共价修饰的抗体。所述抗体可以是犬的、大鼠的、人的，或任何其它来源的，例如嵌合的或人源化的抗体。
在本申请中，所述细胞因子可以为以下的任意一种：IL12、IL2、GMCSF、IL7、IL15、IL21和FLT3L。
在本申请中，所述细胞因子也可以包括选自下组的两种或更多种：IL12、IL2、GMCSF、IL7、IL15、IL21和FLT3L。其中所述细胞因子可以源自哺乳动物。所述哺乳动物可以为人或小鼠。例如，源自小鼠的IL12a(用mIL12a表示)的氨基酸序列可以如SEQ ID NO.16所示，源自小鼠的IL12b(用mIL12b表示)的氨基酸序列可以如SEQ ID NO.17所示，源自小鼠的IL2(用mIL2表示)的氨基酸序列可以如SEQ ID NO.20所示，源自小鼠的GMCSF(用mGMCSF表示)的氨基酸序列可以如SEQ ID NO.30所示，源自小鼠的IL7(用mIL7表示)的氨基酸序列可以如SEQ ID NO.24所示，源自小鼠的IL15(用mIL15表示)的氨基酸序列 可以如SEQ ID NO.22所示，源自小鼠的IL21(用mIL21表示)的氨基酸序列可以如SEQ ID NO.26所示，源自小鼠的FLT3L(用mFLT3L表示)的氨基酸序列可以如SEQ ID NO.28所示。又例如，源自人的IL12a(用hIL12a表示)的氨基酸序列可以如SEQ ID NO.18所示，源自人的IL12b(用hIL12b表示)的氨基酸序列可以如SEQ ID NO.19所示，源自人的IL2(用hIL2表示)的氨基酸序列可以如SEQ ID NO.21所示，源自人的GMCSF(用hGMCSF表示)的氨基酸序列可以如SEQ ID NO.31所示，源自人的IL7(用hIL7表示)的氨基酸序列可以如SEQ ID NO.25所示，源自人的IL15(用hIL15表示)的氨基酸序列可以如SEQ ID NO.23所示，源自人的IL21(用hIL21表示)的氨基酸序列可以如SEQ ID NO.27所示，源自人的FLT3L(用hFLT3L表示)的氨基酸序列可以如SEQ ID NO.29所示。
在本申请中，所述蛋白质可以包括融合蛋白，该融合蛋白将所述细胞因子，即IL12、IL2、IL7、IL15、IL21、FLT3L和GMCSF中的两种或多种，通过基因重组技术融合在一起。所述蛋白质可以既具有组成其因子独特的生物学活性，又可以通过生物学活性的互补及协同效应发挥出较单一细胞因子所不具备的生物学功能，甚至还可以产生一些新的结构及生物学功能。例如，在本申请中，所述蛋白质可以包括融合蛋白，且所述融合蛋白中包含选自以下的任一组细胞因子：a)IL12、IL2和GMSCF；b)IL12、IL7和GMSCF；c)IL12、IL15和GMSCF；d)IL12、IL21和GMSCF；e)IL12、IL2和FLT3L；f)IL12、IL7和FLT3L；g)IL12、IL15和FLT3L；以及，h)IL12、IL21和FLT3L。
在本申请中，所述蛋白质还可以包括靶向部分，所述靶向部分的数量可以为1个或多个。所述靶向部分可以是相同的，也可以是不同的。所述靶向部分能够特异性识别和/或结合肿瘤相关抗原。其中所述肿瘤相关抗原可以选自下组：纤连蛋白的EDB结构域、纤连蛋白的EDA结构域和细胞坏死区域(necrotic regions)。所述靶向部分可以包括抗体或其抗原结合片段。
在本申请中，所述抗原结合片段可以选自下组：Fab，Fab’，F(ab’) 2，F(ab) 2，dAb，分离的互补决定区CDR，Fv和scFv。在某些实施方式中，所述抗原结合片段可以为scFv。
在本申请中，所述靶向部分可以包含下组中任一项所示的氨基酸序列：SEQ ID NO:1-15。
例如，所述蛋白质的靶向部分可以选自下组：L19V L(其氨基酸序列可以如SEQ ID NO.10所示)、L19V H(其氨基酸序列可以如SEQ ID NO.11所示)、F8V L(其氨基酸序列可以如SEQ ID NO.12所示)、F8V H(其氨基酸序列可以如SEQ ID NO.13所示)、NHS76V L(其氨基酸序列可以如SEQ ID NO.14所示)和NHS76V H(其氨基酸序列可以如SEQ ID NO.15所示)。
需要说明的是，在本申请中，所述细胞因子之间或者所述细胞因子与所述靶向部分之间均可以通过连接子连接。所述连接子可以为连接肽。在本申请中，所述连接子可以包含下组中任一项所示的氨基酸序列：SEQ ID NO:73-76。
例如，所述细胞因子之间可以通过所述连接子连接。在本申请中，所述IL12a、IL12b、IL2、IL7、IL15、IL21、FLT3L和GMCSF之间可通过所述连接肽进行连接。例如，所述连接肽可包含如SEQ ID NO.73或SEQ ID NO.75所示的氨基酸序列。
例如，所述细胞因子和所述靶向部分可以通过所述连接子连接。在本申请中，所述靶向部分与IL12a、IL12b、IL2、IL7、IL15、IL21、FLT3L和GMCSF之间可通过所述连接肽进行连接。例如，所述连接肽可以包含SEQ ID NO:73-76中的任一项所示的氨基酸序列。
在本申请中，所述蛋白质可以包含下组中任一项所示的氨基酸序列：SEQ ID NO:32-67。
例如，在本申请中，所述蛋白质可以为单链蛋白质，其中所述单链蛋白质可以包含下组中任一项所示的氨基酸序列：SEQ ID NO:32-51。
例如，所述单链蛋白质的结构可以为，mIL12b的C端和mIL12a的N端融合，mIL12a的C端和mIL2的N端融合，mIL2的C端和mGMCSF的N端融合，从而形成mIL12b-mIL12a-mIL2-mGMCSF单链蛋白质，用mIL12bIL12aIL2GMCSF表示(其氨基酸序列可以如SEQ ID NO.32所示)。
例如，所述单链蛋白质的结构可以为，mIL12b的C端和mIL12a的N端融合，mIL12a的C端和mIL7的N端融合，mIL7的C端和mGMCSF的N端融合，从而形成mIL12b-mIL12a-mIL7-mGMCSF单链蛋白质，用mIL12bIL12aIL7GMCSF表示(其氨基酸序列可以如SEQ ID NO.33所示)。
例如，所述单链蛋白质的结构可以为，mIL12b的C端和mIL12a的N端融合，mIL12a的C端和mIL15的N端融合，mIL15的C端和mGMCSF的N端融合，从而形成mIL12b-mIL12a-mIL15-mGMCSF单链蛋白质，用mIL12bIL12aIL15GMCSF表示(其氨基酸序列可以如SEQ ID NO.34所示)。
例如，所述单链蛋白质的结构可以为，mIL12b的C端和mIL12a的N端融合，mIL12a的C端和mIL21的N端融合，mIL21的C端和mGMCSF的N端融合，从而形成mIL12b-mIL12a-mIL21-mGMCSF单链蛋白质，用mIL12bIL12aIL21GMCSF表示(其氨基酸序列可以如SEQ ID NO.35所示)。
例如，所述单链蛋白质的结构可以为，mIL12b的C端和mIL12a的N端融合，mIL12a的C端和mIL2的N端融合，mIL2的C端和mFLT3L的N端融合，从而形成mIL12b-mIL12a-mIL2-mFLT3L单链蛋白质，用mIL12bIL12aIL2FLT3L表示(其氨基酸序列可以如SEQ ID NO.36所示)。
例如，所述单链蛋白质的结构可以为，mIL12b的C端和mIL12a的N端融合，mIL12a的C端和mIL7的N端融合，mIL7的C端和mFLT3L的N端融合，从而形成mIL12b-mIL12a- mIL7-mFLT3L单链蛋白质，用mIL12bIL12aIL7FLT3L表示(其氨基酸序列可以如SEQ ID NO.37所示)。
例如，所述单链蛋白质的结构可以为，mIL12b的C端和mIL12a的N端融合，mIL12a的C端和mIL15的N端融合，mIL15的C端和mFLT3L的N端融合，从而形成mIL12b-mIL12a-mIL15-mFLT3L单链蛋白质，用mIL12bIL12aIL15FLT3L表示(其氨基酸序列可以如SEQ ID NO.38所示)。
例如，所述单链蛋白质的结构可以为，mIL12b的C端和mIL12a的N端融合，mIL12a的C端和mIL21的N端融合，mIL21的C端和mFLT3L的N端融合，从而形成mIL12b-mIL12a-mIL21-mFLT3L单链蛋白质，用mIL12bIL12aIL21FLT3L表示(其氨基酸序列可以如SEQ ID NO.39所示)。
例如，所述单链蛋白质的结构可以为，hIL12b的C端和hIL12a的N端融合，hIL12a的C端和hIL2的N端融合，hIL2的C端和hGMCSF的N端融合，从而形成hIL12b-hIL12a-hIL2-hGMCSF单链蛋白质，用hIL12bIL12aIL2GMCSF表示(其氨基酸序列可以如SEQ ID NO.40所示)。
例如，所述单链蛋白质的结构可以为，hIL12b的C端和hIL12a的N端融合，hIL12a的C端和hIL7的N端融合，hIL7的C端和hGMCSF的N端融合，从而形成hIL12b-hIL12a-hIL7-hGMCSF单链蛋白质，用hIL12bIL12aIL7GMCSF表示(其氨基酸序列可以如SEQ ID NO.41所示)。
例如，所述单链蛋白质的结构可以为，hIL12b的C端和hIL12a的N端融合，hIL12a的C端和hIL15的N端融合，hIL15的C端和hGMCSF的N端融合，从而形成hIL12b-hIL12a-hIL15-hGMCSF单链蛋白质，用hIL12bIL12aIL15GMCSF表示(其氨基酸序列可以如SEQ ID NO.42所示)。
例如，所述单链蛋白质的结构可以为，hIL12b的C端和hIL12a的N端融合，hIL12a的C端和hIL21的N端融合，hIL21的C端和hGMCSF的N端融合，从而形成hIL12b-hIL12a-hIL21-hGMCSF单链蛋白质，用hIL12bIL12aIL21GMCSF表示(其氨基酸序列可以如SEQ ID NO.43所示)。
例如，所述单链蛋白质的结构可以为，hIL12b的C端和hIL12a的N端融合，hIL12a的C端和hIL2的N端融合，hIL2的C端和hFLT3L的N端融合，从而形成hIL12b-hIL12a-hIL2-hFLT3L单链蛋白质，用hIL12bIL12aIL2FLT3L表示(其氨基酸序列可以如SEQ ID NO.44所示)。
例如，所述单链蛋白质的结构可以为，hIL12b的C端和hIL12a的N端融合，hIL12a的 C端和hIL7的N端融合，hIL7的C端和hFLT3L的N端融合，从而形成hIL12b-hIL12a-hIL7-hFLT3L单链蛋白质，用hIL12bIL12aIL7FLT3L表示(其氨基酸序列可以如SEQ ID NO.45所示)。
例如，所述单链蛋白质的结构可以为，hIL12b的C端和hIL12a的N端融合，hIL12a的C端和hIL15的N端融合，hIL15的C端和hFLT3L的N端融合，从而形成hIL12b-hIL12a-hIL15-hFLT3L单链蛋白质，用hIL12bIL12aIL15FLT3L表示(其氨基酸序列可以如SEQ ID NO.46所示)。
例如，所述单链蛋白质的结构可以为，hIL12b的C端和hIL12a的N端融合，hIL12a的C端和hIL21的N端融合，hIL21的C端和hFLT3L的N端融合，从而形成hIL12b-hIL12a-hIL21-hFLT3L单链蛋白质，用hIL12bIL12aIL21FLT3L表示(其氨基酸序列可以如SEQ ID NO.47所示)。
例如，所述单链蛋白质的结构可以为，mIL12b的C端和mIL12a的N端融合，mIL12a的C端和mIL2的N端融合，mIL2的C端和NHS76V H的N端融合，NHS76V H的C端和F8V L的N端融合，F8V L的C端和F8V H的N端融合，F8V H的C端和NHS76V L的N端融合，NHS76V L的C端和mGMCSF的N端融合，从而形成mIL12b-mIL12a-mIL2-NHS76V H-F8V L-F8V H-NHS76V L-mGMCSF单链蛋白质。需要说明的是，该单链蛋白质的连接子中含有凝血酶切割位点，该单链蛋白质的名称用mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr表示(其氨基酸序列可以如SEQ ID NO.48所示)。
例如，所述单链蛋白质的结构可以为，hIL12b的C端和hIL12a的N端融合，hIL12a的C端和hIL2的N端融合，hIL2的C端和NHS76V H的N端融合，NHS76V H的C端和F8V L的N端融合，F8V L的C端和F8V H的N端融合，F8V H的C端和NHS76V L的N端融合，NH S76V L的C端和hGMCSF的N端融合，从而形成hIL12b-hIL12a-hIL2-NHS76V H-F8V L-F8V H-NHS76V L-hGMCSF单链蛋白质。需要说明的是，该单链蛋白质的连接子中含有凝血酶切割位点，该单链蛋白质的名称用hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr表示(其氨基酸序列可以如SEQ ID NO.49所示)。
例如，所述单链蛋白质的结构可以为，mIL12b的C端和mIL12a的N端融合，mIL12a的C端和mIL2的N端融合，mIL2的C端和F8V H的N端融合，F8V H的C端和F8V L的N端融合，F8V L的C端和F8V H的N端融合，F8V H的C端和F8V L的N端融合，F8V L的C端和mGMCSF的N端融合，从而形成mIL12b-mIL12a-mIL2-F8V H-F8V L-F8V H-F8V L-mGMCSF单链蛋白质，用mIL12bIL12aIL2DiaF8GMCSF表示(其氨基酸序列可以如SEQ ID NO.50所示)。
例如，所述单链蛋白质的结构可以为，hIL12b的C端和hIL12a的N端融合，hIL12a的C端和hIL2的N端融合，hIL2的C端和F8V H的N端融合，F8V H的C端和F8V L的N端融合，F8V L的C端和F8V H的N端融合，F8V H的C端和F8V L的N端融合，F8V L的C端和hGMCSF的N端融合，从而形成hIL12b-hIL12a-hIL2-F8V H-F8V L-F8V H-F8V L-hGMCSF单链蛋白质，用hIL12bIL12aIL2DiaF8GMCSF表示(其氨基酸序列可以如SEQ ID NO.51所示)。
又例如，在本申请中，所述蛋白质还可以为由第一多肽链及第二多肽链组成的二聚体，所述第一多肽链不同于所述第二多肽链。其中所述第一多肽链可以包含IL12a，所述第二多肽链可以包含IL12b。
在本申请中，其中IL2或其功能性片段可以位于所述第一多肽链中或所述第二多肽链中，GMCSF或其功能性片段可以位于所述第一多肽链中或所述第二多肽链中，且所述一个或多个靶向部分可以各自独立地位于所述第一多肽链中或所述第二多肽链中。
在本申请中，在所述第一多肽链中，从N端到C端可以依次包含所述IL2或其功能性片段、所述IL12a或其功能性片段和所述GMCSF或其功能性片段。
在本申请中，在所述第一多肽链中，从N端到C端可以依次包含所述靶向部分、所述IL12a或其功能性片段、所述IL2或其功能性片段以及所述GMCSF或其功能性片段。
在本申请中，在所述第二多肽链中，从N端到C端可以依次包含所述IL12b或其功能性片段和所述靶向部分。
在某些实施方式中，在本申请所述的二聚体中，所述第一多肽链可以包含SEQ ID NO:53所示的氨基酸序列且所述第二多肽链可以包含SEQ ID NO:52所示的氨基酸序列。
在某些实施方式中，在本申请所述的二聚体中，所述第一多肽链可以包含SEQ ID NO:55所示的氨基酸序列且所述第二多肽链可以包含SEQ ID NO:54所示的氨基酸序列；
在某些实施方式中，在本申请所述的二聚体中，所述第一多肽链可以包含SEQ ID NO:56所示的氨基酸序列且所述第二多肽链可以包含SEQ ID NO:57所示的氨基酸序列；
在某些实施方式中，在本申请所述的二聚体中，所述第一多肽链可以包含SEQ ID NO:58所示的氨基酸序列且所述第二多肽链可以包含SEQ ID NO:59所示的氨基酸序列；
在某些实施方式中，在本申请所述的二聚体中，所述第一多肽链可以包含SEQ ID NO:60所示的氨基酸序列且所述第二多肽链可以包含SEQ ID NO:61所示的氨基酸序列；
在某些实施方式中，在本申请所述的二聚体中，所述第一多肽链可以包含SEQ ID NO:62所示的氨基酸序列且所述第二多肽链可以包含SEQ ID NO:63所示的氨基酸序列；
在某些实施方式中，在本申请所述的二聚体中，所述第一多肽链可以包含SEQ ID NO:64所示的氨基酸序列且所述第二多肽链可以包含SEQ ID NO:65所示的氨基酸序列；
在某些实施方式中，在本申请所述的二聚体中，所述第一多肽链可以包含SEQ ID NO:66所示的氨基酸序列且所述第二多肽链可以包含SEQ ID NO:67所示的氨基酸序列。
例如，在所述二聚体中，mIL12b的C端可以和F8V H的N端融合，F8V H的C端可以和F8V L的N端融合形成第二多肽链(其氨基酸序列可以如SEQ ID NO.52所示)，且F8V H的C端可以和F8V L的N端融合，F8V L的C端可以和mIL12a的N端融合，mIL12a的C端可以和mIL2的N端融合，mIL2的C端可以和mGMCSF的N端融合形成第一多肽链(其氨基酸序列可以如SEQ ID NO.53所示)，从而形成mIL12b-F8V H-F8V L-F8V H-F8V L-mIL12a-mIL2-mGMCSF二聚体，用mIL12bscF8-scF8IL12aIL2GMCSF表示。
例如，在所述二聚体中，hIL12b的C端可以和NHS76V H的N端融合，NHS76V H的C端可以和NHS76V L的N端融合形成第二多肽链(其氨基酸序列可以如SEQ ID NO.54所示)，且NHS76V H的C端可以和NHS76V L的N端融合，NHS76V L的C端可以和hIL12a的N端融合，hIL12a的C端可以和hIL2的N端融合，hIL2的C端可以和hGMCSF的N端融合形成第一多肽链(其氨基酸序列可以如SEQ ID NO.55所示)，从而形成hIL12b-NHS76V H-N HS76V L-NHS76V H-NHS76V L-hIL12a-hIL2-hGMCSF二聚体，用hIL12bscNHS76-scNHS76IL12aIL2GMCSF表示。
例如，在所述二聚体中，mIL12a的C端和mIL2的N端可以融合形成mIL12a-mIL2第一多肽链(序列如SEQ ID NO.56所示)，且mIL12b的C端和mGMCSF的N端可以融合形成mIL12b-mGMCSF第二多肽链(序列如SEQ ID NO.57所示)，从而形成mIL12a-mIL2-mIL12b-mGMCSF二聚体，用mIL12aIL2IL12bGMCSF表示。
例如，在所述二聚体中，mIL12a的C端和mIL7的N端可以融合形成mIL12a-mIL7第一多肽链(序列如SEQ ID NO.58所示)，且mIL12b的C端和mGMCSF的N端可以融合形成mIL12b-mGMCSF第二多肽链(序列如SEQ ID NO.59所示)，从而形成mIL12a-mIL7-mIL12b-mGMCSF二聚体，用mIL12aIL7IL12bGMCSF表示。
例如，在所述二聚体中，mIL12a的C端和mIL21的N端可以融合形成mIL12a-mIL21第一多肽链(序列如SEQ ID NO.60所示)，且mIL12b的C端和mGMCSF的N端可以融合形成mIL12b-mGMCSF第二多肽链(序列如SEQ ID NO.61所示)，从而形成mIL12a-mIL21-mIL12b-mGMCSF二聚体，用mIL12aIL21IL12bGMCSF表示。
例如，在所述二聚体中，hIL12a的C端和hIL2的N端可以融合形成hIL12a-hIL2第一多肽链(序列如SEQ ID NO.62所示)，且hIL12b的C端和hGMCSF的N端可以融合形成hIL12b-hGMCSF第二多肽链(序列如SEQ ID NO.63所示)，从而形成hIL12a-hIL2-hIL12b-hGMCSF二聚体，用hIL12aIL2IL12bGMCSF表示。
例如，在所述二聚体中，hIL12a的C端和hIL7的N端可以融合形成hIL12a-hIL7第一多肽链(序列如SEQ ID NO.64所示)，且hIL12b的C端和hGMCSF的N端可以融合形成hIL12b-hGMCSF第二多肽链(序列如SEQ ID NO.65所示)，从而形成hIL12a-hIL7-hIL12b-hGMCSF二聚体，用hIL12aIL7IL12bGMCSF表示。
例如，在所述二聚体中，hIL12a的C端和hIL21的N端可以融合形成hIL12a-hIL21第一多肽链(序列如SEQ ID NO.66所示)，且hIL12b的C端和hGMCSF的N端可以融合形成hIL12b-hGMCSF第二多肽链(序列如SEQ ID NO.67所示)，从而形成hIL12a-hIL21-hIL12b-hGMCSF二聚体，用hIL12aIL21IL12bGMCSF表示。
在本申请中涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中，所述变体可以为，在所述蛋白质和/或所述多肽(例如，所述蛋白质分子)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性。
在本申请中，所述同源物可以为，与所述蛋白质和/或所述多肽(例如，所述蛋白质分子)的氨基酸序列具有至少约80％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待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)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，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，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。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国 国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
药物、用途、治疗肿瘤的方法
另一方面，本申请还提供了一种药物，其包含本申请所述的液体制剂。其中所述液体制剂可以被配制为注射剂，以使本申请所述的液体制剂可以用于静脉注射或者皮下注射。
在某些实施方式中，本申请所述药物可以被配制用于口服给药，静脉内给药，肌肉内给药，在肿瘤部位的原位给药，吸入，直肠给药，阴道给药，经皮给药或通过皮下储存库给药。
在本申请中，本申请所述的药物还可以包含稀释剂，其中所述稀释剂与所述液体制剂彼此不混合，以便在需要不同浓度的本申请所述的液体制剂的时候，可以用所述稀释剂对本申请所述液体制剂进行稀释。
在某些实施方式中，本申请所述的药物还可以包含药学上可接受的载体。例如，所述药学上可接受的载体可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。例如，所述药学上可接受的载体可以包括赋形剂，例如，所述赋形剂可以选自下组：淀粉、糊精、蔗糖、乳糖、硬脂酸镁、硫酸钙、羧甲基素、滑石粉、海藻酸钙凝胶、壳聚糖和纳米微球等。例如，所述药学上可接受的载体还可以选自下组：pH调节剂、渗透压调节剂、增溶剂和抑菌剂。
另一方面，本申请还提供了本申请所述的液体制剂在制备治疗肿瘤的药物中的用途。其中所述肿瘤可以包括肺癌。
另一方面，本申请还提供了一种治疗肿瘤的方法，其包括向有需要的受试者施用本申请所述的液体制剂。
其中所述施用方法可以为瘤内注射，例如向肿瘤内部注射本申请所述的液体制剂。在某些实施方式中，所述施用方法也可以为口服给药，静脉内给药，肌肉内给药，在肿瘤部位的原位给药，吸入，直肠给药，阴道给药，经皮给药或通过皮下储存库给药。
此外，在某些实施方式中，所施用的本申请所述液体制剂的剂量水平可以视以下情况而变化：被递送的分子、给药途径和患者的大小(体重、体表面或器官大小)和/或身体状况(年龄及一般健康状况)。
另一方面，本申请提供了一种液体制剂或药物，其用于治疗肿瘤。其中所述肿瘤可以包括肺癌。
另一方面，本申请还提供了一种用于制备本申请所述的液体制剂的辅料，其包含油相溶剂，所述油相溶剂的质量分数为约50％以上，例如，所述辅料中的油相溶剂的质量分数可以为约50％-约60％、约60％-约70％、约70％-约80％、约80％-约90％、约90％-约95％、约50％-约70％、约50％-约80％、约50％-约90％、约50％-约95％、约60％-约80％、约60％-约90％、约60％-约95％、约70％-约90％、约70％-约95％、或约80％-约95％，以便本申请所述的液体制剂能够易于制备。
实施例
给出下述实施例仅仅是为了阐释本申请的液体制剂、药物和用途等，而不用于限制本申请发明的范围，也不意欲表示下述试验是进行的全部和唯一的试验。已经进行了努力以确保所用数值(例如数量、温度等)的准确性，但是应当考虑到某些实验误差和偏差。除非另有说明，否则份数为重量份，分子量为重均分子量，温度为摄氏温度，压力为常压或接近常压。可以使用标准缩写词，例如bp，碱基对；kb，千碱基对；pl，微微升；s或sec，秒；min，分钟；h或hr，小时；aa，氨基酸；nt，核苷酸；iv，静脉注射；i.m.，肌内；i.p.，腹膜内；s.c.，皮下等。
试剂：DMEM培养基、1640培养基、胎牛血清购自lifetechnologies公司；细胞培养瓶、培养板购自Corning公司；强力霉素(DOX)购自上海生工生物工程有限公司；嘌呤霉素(Puromycin)、Blasticidin购自Chemicon公司；限制性内切酶购自Takara和NEB公司；连接酶购自NEB公司；DNA聚合酶购自Takara公司；质粒提取试剂盒、胶回收试剂盒购自OmegaBiotech公司；引物合成由上海生工生物工程有限公司完成；基因合成由南京金斯瑞公司完成；ELISA试剂盒购于博士德公司。
实施例1 mIL12bIL12aIL2GMCSF蛋白质的表达
1.1构建表达载体
mIL12bIL12aIL2GMCSF蛋白质，其前端带有分泌信号肽，后端加入6*His以便纯化，合成mIL12bIL12aIL2GMCSF蛋白质基因对应的DNA序列，序列中在BamHI或XhoI位点处用简并密码子突变掉，合成序列的前后端分别带有BamHI和XhoI酶切位点，酶切合成的带有目的基因的质粒，体系如下：5μg质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12bIL12aIL2GMCSF蛋白质基因片段，待用。
mIL12bIL12aIL2GMCSF蛋白质的氨基酸序列如SEQ ID NO.32所示，编码所述 mIL12bIL12aIL2GMCSF的核苷酸序列如SEQ ID NO.68所示。
在EP管内酶切载体pLentis-CMV-MCS-IRES-PURO，体系如下：2μg pLentis-CMV-MCS-IRES-PURO载体质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-CMV-MCS-IRES-PURO载体片段，待用。
连接mIL12bIL12aIL2GMCSF和pLentis-CMV-MCS-IRES-PURO，体系如下，2μl pLentis-CMV-MCS-IRES-PURO载体片段、2μl基因片段、1μl连接酶缓冲液、0.5μl T4 DNA连接酶和水4.5μl。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37度摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定基因片段是否成功连入载体中，然后将正确的载体测序，确定构建成功。获得表达载体pLentis-CMV-mIL12bIL12aIL2GMCSF-IRES-PURO。
1.2制备表达病毒
1)消化培养的293FT细胞，计数后将3×10 6个细胞/孔铺入10cm培养皿中，培养液体积为10ml。
2)第二天晚上，观察细胞状态，如果细胞状态好，进行转染。在培养板中加入氯喹至终浓度25μM，取一只试管，加入灭菌水及以下质粒(pMD2.G 6μg+pSPAX2 15μg+上述1.1实施例获得的表达载体20μg)，总体积为1045μl，然后加入2M CaCl 2 155μl，混匀，最后再加入1200μl 2×HBS，边滴加边振荡，滴加完毕后，迅速将混合物加入到细胞培养孔中，轻轻摇晃混匀。
3)第三天早上，观察细胞状态，将培养基换为10ml新鲜DMEM培养基。
4)第五天早上，观察细胞状态，并收集培养皿中的上清，用0.45μm滤器过滤，然后置于高速离心管中，50000g离心2小时，小心弃去上清，尽量用吸水纸吸干液体，然后用200μl HBSS重悬沉淀，溶解2小时后分装成小管，-70℃保存。
1.3制备表达细胞
消化培养的293A细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml。24小时后，加入10μl表达上述目的基因的病毒(即实施例1.2获得的病毒)，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养。待细胞长满后，将其传出到培养瓶中，加入终浓度3μg/ml嘌呤霉素，继续培养，每两天更换一次培养基，并保持嘌呤霉素的浓度，筛选一周后，存活的细胞即为稳定表达所述蛋白的细胞，命名为293A-mIL12bIL12aIL2GMCSF。
1.4蛋白表达纯化
将构建的表达mIL12bIL12aIL2GMCSF的细胞293A-mIL12bIL12aIL2GMCSF，传代到15cm培养皿中，待细胞长满后，将培养基换为30ml CDM4HEK293，继续培养5天，然后收集上清，0.45μm滤器过滤，再用50kd的AMICON ULTRA-15超滤浓缩，获得的浓缩蛋白液用镍螯合磁珠(购于海狸生物科技有限公司)进行纯化，操作流程按说明书进行，获得的纯化蛋白液再用AMICON ULTRA-0.5超滤管进行超滤，将缓冲液置换为PBS，最后获得的蛋白液用IL12p70ELISA试剂盒检测蛋白浓度，将蛋白浓度用PBS调整到2μg/μl后，分装后于-20℃保存。
实施例2蛋白质分子mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr的表达
2.1构建表达载体
mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr蛋白质，其前端带有分泌信号肽，后端加入6*His以便纯化，合成基因对应的DNA序列，序列中有BamHI或XhoI位点处用简并密码子突变掉，合成序列前后端分别带有BamHI和XhoI酶切位点，酶切合成的带有目的基因的质粒，体系如下：5μg质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr蛋白质基因片段，待用。需要说明的是，mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr蛋白质的连接子中含有凝血酶切割位点。
mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr蛋白质的氨基酸序列如SEQ ID NO.48所示，编码所述mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr的核苷酸序列如SEQ ID NO.69所示
在EP管内酶切载体pLentis-CMV-MCS-IRES-PURO，体系如下：2μg pLentis-CMV-MCS-IRES-PURO载体质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-CMV-MCS-IRES-PURO载体片段，待用。
连接mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr和pLentis-CMV-MCS-IRES-PURO，体系如下，2μl pLentis-CMV-MCS-IRES-PURO载体片段、2μl基因片段、1μl连接酶缓冲液、0.5μl T4 DNA连接酶和水4.5μl。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37度摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体测序，确定构建成功。获得表达载体pLentis-CMV-mIL12bIL12aIL2DiaNHS76F8GMCSF- Thr-IRES-PURO。
2.2制备表达病毒
1)消化培养的293FT细胞，计数后将3×10 6个细胞/孔铺入10cm培养皿中，培养液体积为10ml。
2)第二天晚上，观察细胞状态，如果细胞状态好，进行转染。在培养板中加入氯喹至终浓度25μM，取一只试管，加入灭菌水及以下质粒(pMD2.G 6μg+pSPAX2 15μg+实施例2.1获得的表达载体20μg)，总体积为1045μl，然后加入2M CaCl 2 155μl，混匀，最后再加入1200μl 2×HBS，边滴加边振荡，滴加完毕后，迅速将混合物加入到细胞培养孔中，轻轻摇晃混匀。
3)第三天早上，观察细胞状态，将培养基换为10ml新鲜DMEM培养基。
4)第五天早上，观察细胞状态，并收集培养皿中的上清，用0.45μm滤器过滤，然后置于高速离心管中，50000g离心2小时，小心弃去上清，尽量用吸水纸吸干液体，然后用200μl HBSS重悬沉淀，溶解2小时后分装成小管，-70℃保存。
2.3制备表达细胞
消化培养的293A细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml。24小时后，加入10μl表达上述目的基因的病毒(即实施例2.2获得的病毒)，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养。待细胞长满后，将其传出到培养瓶中，加入终浓度3μg/ml嘌呤霉素，继续培养，每两天更换一次培养基，并保持嘌呤霉素的浓度，筛选一周后，存活的细胞即为稳定表达所述蛋白的细胞，命名为293A-mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr。
2.4蛋白表达纯化
将构建的表达mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr的细胞293A-mIL12bIL12aIL2Di aNHS76F8GMCSF-Thr，传代到15cm培养皿中，待细胞长满后，将培养基换为30ml CDM4HEK293，继续培养5天，然后收集上清，0.45μm滤器过滤，再用50kd的AMICON ULTRA-15超滤浓缩，获得的浓缩蛋白液用镍螯合磁珠(购于海狸生物科技有限公司)进行纯化，操作流程按说明书进行，获得的纯化蛋白液再用AMICON ULTRA-0.5超滤管进行超滤，将缓冲液置换为PBS，最后获得的蛋白液用IL12p70ELISA试剂盒检测蛋白浓度，将蛋白浓度用PBS调整到2μg/μl后，分装后于-20℃保存。
实施例3 mIL12bIL12aIL7GMCSF蛋白质的表达
3.1构建表达载体
mIL12bIL12aIL7GMCSF蛋白质，其前端带有分泌信号肽，后端加入6*His以便纯化，合成mIL12bIL12aIL7GMCSF蛋白质基因对应的DNA序列，序列中在BamHI或XhoI位点处用简并密码子突变掉，合成序列的前后端分别带有BamHI和XhoI酶切位点，酶切合成的带有目的基因的质粒，体系如下：5μg质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12bIL12aIL7GMCSF蛋白质基因片段，待用。
mIL12bIL12aIL7GMCSF蛋白质的氨基酸序列如SEQ ID NO.33所示，编码所述mIL12bIL12aIL7GMCSF的核苷酸序列如SEQ ID NO.70所示。
在EP管内酶切载体pLentis-CMV-MCS-IRES-PURO，体系如下：2μg pLentis-CMV-MCS-IRES-PURO载体质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-CMV-MCS-IRES-PURO载体片段，待用。
连接mIL12bIL12aIL7GMCSF和pLentis-CMV-MCS-IRES-PURO，体系如下，2μl pLentis-CMV-MCS-IRES-PURO载体片段、2μl基因片段、1μl连接酶缓冲液、0.5μl T4 DNA连接酶和水4.5μl。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37度摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定基因片段是否成功连入载体中，然后将正确的载体测序，确定构建成功。获得表达载体pLentis-CMV-mIL12bIL12aIL7GMCSF-IRES-PURO。
3.2制备表达病毒
1)消化培养的293FT细胞，计数后将3×10 6个细胞/孔铺入10cm培养皿中，培养液体积为10ml。
2)第二天晚上，观察细胞状态，如果细胞状态好，进行转染。在培养板中加入氯喹至终浓度25μM，取一只试管，加入灭菌水及以下质粒(pMD2.G 6μg+pSPAX2 15μg+上述3.1实施例获得的表达载体20μg)，总体积为1045μl，然后加入2M CaCl 2 155μl，混匀，最后再加入1200μl 2×HBS，边滴加边振荡，滴加完毕后，迅速将混合物加入到细胞培养孔中，轻轻摇晃混匀。
3)第三天早上，观察细胞状态，将培养基换为10ml新鲜DMEM培养基。
4)第五天早上，观察细胞状态，并收集培养皿中的上清，用0.45μm滤器过滤，然后置于高速离心管中，50000g离心2小时，小心弃去上清，尽量用吸水纸吸干液体，然后用200μl HBSS重悬沉淀，溶解2小时后分装成小管，-70℃保存。
3.3制备表达细胞
消化培养的293A细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml。24小时后，加入10μl表达上述目的基因的病毒(即实施例3.2获得的病毒)，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养。待细胞长满后，将其传出到培养瓶中，加入终浓度3μg/ml嘌呤霉素，继续培养，每两天更换一次培养基，并保持嘌呤霉素的浓度，筛选一周后，存活的细胞即为稳定表达所述蛋白的细胞，命名为293A-mIL12bIL12aIL7GMCSF。
3.4蛋白表达纯化
将构建的表达mIL12bIL12aIL7GMCSF的细胞293A-mIL12bIL12aIL7GMCSF，传代到15cm培养皿中，待细胞长满后，将培养基换为30ml CDM4HEK293，继续培养5天，然后收集上清，0.45μm滤器过滤，再用50kd的AMICON ULTRA-15超滤浓缩，获得的浓缩蛋白液用镍螯合磁珠(购于海狸生物科技有限公司)进行纯化，操作流程按说明书进行，获得的纯化蛋白液再用AMICON ULTRA-0.5超滤管进行超滤，将缓冲液置换为PBS，最后获得的蛋白液用IL12p70ELISA试剂盒检测蛋白浓度，将蛋白浓度用PBS调整到2μg/μl后，分装后于-20℃保存。
实施例4 mIL12bIL12aIL21GMCSF蛋白质的表达
4.1构建表达载体
mIL12bIL12aIL21GMCSF蛋白质，其前端带有分泌信号肽，后端加入6*His以便纯化，合成mIL12bIL12aIL21GMCSF蛋白质基因对应的DNA序列，序列中在BamHI或XhoI位点处用简并密码子突变掉，合成序列的前后端分别带有BamHI和XhoI酶切位点，酶切合成的带有目的基因的质粒，体系如下：5μg质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12bIL12aIL21GMCSF蛋白质基因片段，待用。
mIL12bIL12aIL21GMCSF蛋白质的氨基酸序列如SEQ ID NO.35所示，编码所述mIL12bIL12aIL21GMCSF的核苷酸序列如SEQ ID NO.71所示。
在EP管内酶切载体pLentis-CMV-MCS-IRES-PURO，体系如下：2μg pLentis-CMV-MCS- IRES-PURO载体质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-CMV-MCS-IRES-PURO载体片段，待用。
连接mIL12bIL12aIL21GMCSF和pLentis-CMV-MCS-IRES-PURO，体系如下，2μl pLentis-CMV-MCS-IRES-PURO载体片段、2μl基因片段、1μl连接酶缓冲液、0.5μl T4 DNA连接酶和水4.5μl。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37度摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定基因片段是否成功连入载体中，然后将正确的载体测序，确定构建成功。获得表达载体pLentis-CMV-mIL12bIL12aIL21GMCSF-IRES-PURO。
4.2制备表达病毒
1)消化培养的293FT细胞，计数后将3×10 6个细胞/孔铺入10cm培养皿中，培养液体积为10ml。
2)第二天晚上，观察细胞状态，如果细胞状态好，进行转染。在培养板中加入氯喹至终浓度25μM，取一只试管，加入灭菌水及以下质粒(pMD2.G 6μg+pSPAX2 15μg+上述4.1实施例获得的表达载体20μg)，总体积为1045μl，然后加入2M CaCl 2 155μl，混匀，最后再加入1200μl 2×HBS，边滴加边振荡，滴加完毕后，迅速将混合物加入到细胞培养孔中，轻轻摇晃混匀。
3)第三天早上，观察细胞状态，将培养基换为10ml新鲜DMEM培养基。
4)第五天早上，观察细胞状态，并收集培养皿中的上清，用0.45μm滤器过滤，然后置于高速离心管中，50000g离心2小时，小心弃去上清，尽量用吸水纸吸干液体，然后用200μl HBSS重悬沉淀，溶解2小时后分装成小管，-70℃保存。
4.3制备表达细胞
消化培养的293A细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml。24小时后，加入10μl表达上述目的基因的病毒(即实施例4.2获得的病毒)，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养。待细胞长满后，将其传出到培养瓶中，加入终浓度3μg/ml嘌呤霉素，继续培养，每两天更换一次培养基，并保持嘌呤霉素的浓度，筛选一周后，存活的细胞即为稳定表达所述蛋白的细胞，命名为293A-mIL12bIL12aIL21GMCSF。
4.4蛋白表达纯化
将构建的表达mIL12bIL12aIL21GMCSF的细胞293A-mIL12bIL12aIL21GMCSF，传代到15cm培养皿中，待细胞长满后，将培养基换为30ml CDM4HEK293，继续培养5天，然后收集上清，0.45μm滤器过滤，再用50kd的AMICON ULTRA-15超滤浓缩，获得的浓缩蛋白液用镍螯合磁珠(购于海狸生物科技有限公司)进行纯化，操作流程按说明书进行，获得的纯化蛋白液再用AMICON ULTRA-0.5超滤管进行超滤，将缓冲液置换为PBS，最后获得的蛋白液用IL12p70ELISA试剂盒检测蛋白浓度，将蛋白浓度用PBS调整到2μg/μl后，分装后于-20℃保存。
实施例5 mIL12aIL2IL12bGMCSF蛋白质的表达
5.1构建表达载体
mIL12aIL2IL12bGMCSF蛋白是由mIL12aIL2和mIL12bGMCSF组成的异二聚体，其中mIL12aIL2后端加入6*His以便纯化，合成mIL12aIL2IL12bGMCSF蛋白质基因对应的DNA序列，两段肽链中间加入T2A切割肽使其翻译后形成异二聚体，序列中在BamHI或XhoI位点处用简并密码子突变掉，合成序列的前后端分别带有BamHI和XhoI酶切位点，酶切合成的带有目的基因的质粒，体系如下：5μg质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12aIL2IL12bGMCSF蛋白质基因片段，待用。
mIL12aIL2IL12bGMCSF蛋白质的第二多肽链氨基酸序列如SEQ ID NO.57所示，第一多肽链氨基酸序列如SEQ ID NO.56所示，编码所述mIL12aIL2IL12bGMCSF的核苷酸序列如SEQ ID NO.72所示。
在EP管内酶切载体pLentis-CMV-MCS-IRES-PURO，体系如下：2μg pLentis-CMV-MCS-IRES-PURO载体质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-CMV-MCS-IRES-PURO载体片段，待用。
连接mIL12aIL2IL12bGMCSF和pLentis-CMV-MCS-IRES-PURO，体系如下，2μl pLentis-CMV-MCS-IRES-PURO载体片段、2μl基因片段、1μl连接酶缓冲液、0.5μl T4 DNA连接酶和水4.5μl。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37度摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定基因片段是否成功连入载体中，然后将正确的载体测序，确定构建成功。获得表达载体pLentis-CMV-mIL12aIL2IL12bGMCSF-IRES-PURO。
5.2制备表达病毒
1)消化培养的293FT细胞，计数后将3×10 6个细胞/孔铺入10cm培养皿中，培养液体积为10ml。
2)第二天晚上，观察细胞状态，如果细胞状态好，进行转染。在培养板中加入氯喹至终浓度25μM，取一只试管，加入灭菌水及以下质粒(pMD2.G 6μg+pSPAX2 15μg+上述5.1实施例获得的表达载体20μg)，总体积为1045μl，然后加入2M CaCl 2 155μl，混匀，最后再加入1200μl 2×HBS，边滴加边振荡，滴加完毕后，迅速将混合物加入到细胞培养孔中，轻轻摇晃混匀。
3)第三天早上，观察细胞状态，将培养基换为10ml新鲜DMEM培养基。
4)第五天早上，观察细胞状态，并收集培养皿中的上清，用0.45μm滤器过滤，然后置于高速离心管中，50000g离心2小时，小心弃去上清，尽量用吸水纸吸干液体，然后用200μl HBSS重悬沉淀，溶解2小时后分装成小管，-70℃保存。
5.3制备表达细胞
消化培养的293A细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml。24小时后，加入10μl表达上述目的基因的病毒(即实施例5.2获得的病毒)，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养。待细胞长满后，将其传出到培养瓶中，加入终浓度3μg/ml嘌呤霉素，继续培养，每两天更换一次培养基，并保持嘌呤霉素的浓度，筛选一周后，存活的细胞即为稳定表达所述蛋白的细胞，命名为293A-mIL12aIL2IL12bGMCSF。
5.4蛋白表达纯化
将构建的表达mIL12aIL2IL12bGMCSF的细胞293A-mIL12aIL2IL12bGMCSF，传代到15cm培养皿中，待细胞长满后，将培养基换为30ml CDM4HEK293，继续培养5天，然后收集上清，0.45μm滤器过滤，再用50kd的AMICON ULTRA-15超滤浓缩，获得的浓缩蛋白液用镍螯合磁珠(购于海狸生物科技有限公司)进行纯化，操作流程按说明书进行，获得的纯化蛋白液再用AMICON ULTRA-0.5超滤管进行超滤，将缓冲液置换为PBS，最后获得的蛋白液用IL12p70ELISA试剂盒检测蛋白浓度，将蛋白浓度用PBS调整到0.2μg/μl后，分装后于-20℃保存。
实施例6甘油注射液对小鼠肿瘤生长的影响
将5×10 5个消化培养的小鼠肺癌细胞(LLC)注射到C57BL/6小鼠身体右侧皮下，待肿 瘤的长径达到7-10mm时开始进行治疗。用PBS分别配制50％，60％和70％浓度的甘油溶液，使用29G的胰岛素注射器吸取配制的甘油溶液，缓慢注射到肿瘤内，注射体积为150μl，注射完后将针头滞留少许时间以减少溶液的溢出。注射后的小鼠放回笼内，记录小鼠的存活情况，只注射PBS的小鼠作为对照。结果如图1所示，50％浓度的甘油溶液注射后，小鼠存活率为12.5％，60％和70％浓度的甘油溶液注射后，小鼠存活率为25％，说明甘油溶液有一定的抗肿瘤效果。
实施例7 mIL12bIL12aIL2GMCSF注射液对小鼠肿瘤生长的影响
将5×10 5个消化培养的小鼠肺癌细胞(LLC)注射到C57BL/6小鼠身体右侧皮下，待肿瘤的长径达到7-10mm时开始进行治疗。
用实施例1制备的蛋白溶液配制一系列不同甘油质量百分数的本申请液体制剂(即mIL12bIL12aIL2GMCSF甘油注射液)。具体方法为：取实施例1制备的蛋白溶液50μl，然后加入到50μl甘油中，迅速用枪头吹打混匀，避免产生气泡，得到50％甘油浓度的液体制剂，蛋白溶液50ul加入到75ul甘油中，得到60％甘油浓度的液体制剂，蛋白溶液50ul加入到117ul甘油中，得到70％甘油浓度的液体制剂。对照组的对比例制剂的制备方法为：取实施例1制备的蛋白溶液50μl，然后加入到117μl PBS中，迅速用枪头吹打混匀，避免产生气泡，得到制备的对比例制剂1。使用29G的胰岛素注射器吸取配制的本申请的液体制剂或对比例制剂1，缓慢注射到肿瘤内，注射完后将针头滞留少许时间以减少溶液的溢出。注射后的小鼠放回笼内，记录小鼠的存活情况。结果如图2所示，图中PBS表示对照组，可以看出，本申请的液体制剂(即mIL12bIL12aIL2GMCSF甘油注射液)注射后，小鼠全部存活，表明甘油溶液和蛋白质分子产生了协同作用，从而共同抑制肿瘤的生长。
实施例8 mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr注射液对小鼠肿瘤生长的影响
将5×10 5个消化培养的小鼠肺癌细胞(LLC)注射到C57BL/6小鼠身体右侧皮下，待肿瘤的长径达到7-10mm时开始进行治疗。
用实施例2制备的蛋白溶液配制一系列不同甘油质量百分数的本申请液体制剂(即mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr甘油注射液)。具体方法为：取实施例2制备的蛋白溶液50μl，然后加入到50μl甘油中，迅速用枪头吹打混匀，避免产生气泡，得到50％甘油浓度的液体制剂，蛋白溶液50ul加入到75ul甘油中，得到60％甘油浓度的液体制剂，蛋白溶液50ul加入到117ul甘油中，得到70％甘油浓度的液体制剂。对照组的对比例制剂的制备方法为：取实施例2制备的蛋白溶液50μl，然后加入到117μl PBS中，迅速用枪头吹打混匀，避免产 生气泡，得到制备的对比例制剂2。使用29G的胰岛素注射器吸取配制的本申请的液体制剂(即mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr甘油注射液)或对比例制剂2，缓慢注射到肿瘤内，注射完后将针头滞留少许时间以减少溶液的溢出。注射后的小鼠放回笼内，记录小鼠的存活情况。结果如图3所示，图中PBS表示对照组，可以看出，本申请的液体制剂mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr甘油注射液注射后，小鼠全部存活，表明甘油溶液和蛋白质分子产生了协同作用，从而共同抑制肿瘤的生长。
实施例9 mIL12bIL12aIL7GMCSF注射液对小鼠肿瘤生长的影响
将5×10 5个消化培养的小鼠肺癌细胞(LLC)注射到C57BL/6小鼠身体右侧皮下，待肿瘤的长径达到7-10mm时开始进行治疗。
取实施例3制备的蛋白溶液50μl，然后加入到80μl甘油中，迅速用枪头吹打混匀，避免产生气泡，得到制备的本申请的液体制剂。对照组的对比例制剂的制备方法为：取实施例3制备的蛋白溶液50μl，然后加入到80μl PBS中，迅速用枪头吹打混匀，避免产生气泡，得到制备的对比例制剂3。使用29G的胰岛素注射器吸取配制的本申请的液体制剂或对比例制剂3，缓慢注射到肿瘤内，注射完后将针头滞留少许时间以减少溶液的溢出。注射后的小鼠放回笼内，记录小鼠的存活情况。结果如图4所示，图中PBS表示对照组，可以看出，本申请的液体制剂(即mIL12bIL12aIL7GMCSF甘油注射液)注射后，小鼠全部存活，表明甘油溶液和蛋白质分子产生了协同作用，从而共同抑制肿瘤的生长。
实施例10 mIL12bIL12aIL21GMCSF注射液对小鼠肿瘤生长的影响
将5×10 5个消化培养的小鼠肺癌细胞(LLC)注射到C57BL/6小鼠身体右侧皮下，待肿瘤的长径达到7-10mm时开始进行治疗。
取实施例4制备的蛋白溶液50μl，然后加入到80μl甘油中，迅速用枪头吹打混匀，避免产生气泡，得到制备的本申请的液体制剂。对照组的对比例制剂的制备方法为：取实施例4制备的蛋白溶液50μl，然后加入到80μl PBS中，迅速用枪头吹打混匀，避免产生气泡，得到制备的对比例制剂4。使用29G的胰岛素注射器吸取配制的本申请的液体制剂或对比例制剂4，缓慢注射到肿瘤内，注射完后将针头滞留少许时间以减少溶液的溢出。注射后的小鼠放回笼内，记录小鼠的存活情况。结果如图5所示，图中PBS表示对照组，可以看出，本申请的液体制剂(即mIL12bIL12aIL21GMCSF甘油注射液)注射后，小鼠全部存活，表明甘油溶液和蛋白质分子产生了协同作用，从而共同抑制肿瘤的生长。
实施例11 mIL12aIL2IL12bGMCSF注射液对小鼠肿瘤生长的影响
将5×10 5个消化培养的小鼠肺癌细胞(LLC)注射到C57BL/6小鼠身体右侧皮下，待肿瘤的长径达到7-10mm时开始进行治疗。
取实施例5制备的蛋白溶液50μl，然后加入到80μl甘油中，迅速用枪头吹打混匀，避免产生气泡，得到制备的本申请的液体制剂。对照组的对比例制剂的制备方法为：取实施例5制备的蛋白溶液50μl，然后加入到80μl PBS中，迅速用枪头吹打混匀，避免产生气泡，得到制备的对比例制剂5。使用29G的胰岛素注射器吸取配制的本申请的液体制剂或对比例制剂5，缓慢注射到肿瘤内，注射完后将针头滞留少许时间以减少溶液的溢出。注射后的小鼠放回笼内，记录小鼠的存活情况。结果如图6所示，图中PBS表示对照组，可以看出，本申请的液体制剂(即mIL12aIL2IL12bGMCSF甘油注射液)注射后，小鼠全部存活，表明甘油溶液和蛋白质分子产生了协同作用，从而共同抑制肿瘤的生长。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (34)

  1. 液体制剂，其包括油相溶剂系统和蛋白质，其中所述油相溶剂系统包括油相溶剂，所述油相溶剂的质量分数为约50％以上。
  2. 根据权利要求1所述的液体制剂，其中所述油相溶剂选自以下组：甘油、丙二醇、聚乙二醇、聚乙烯吡咯烷酮、甘露醇、山梨醇、聚氧丙烯和氨基丁三醇。
  3. 根据权利要求1-2中任一项所述的液体制剂，其中所述油相溶剂的质量分数为约60％以上。
  4. 根据权利要求1-3中任一项所述的液体制剂，其中所述油相溶剂的质量分数为约70％以上。
  5. 根据权利要求1-4中任一项所述的液体制剂，其中所述蛋白质的质量分数为约1％-约50％。
  6. 根据权利要求1-5中任一项所述的液体制剂，其中所述蛋白质包括细胞因子和/或抗体。
  7. 根据权利要求6所述的液体制剂，其中所述细胞因子包括选自下组的两种或更多种：IL12、IL2、GMCSF、IL7、IL15、IL21和FLT3L。
  8. 根据权利要求6-7中任一项所述的液体制剂，其中所述细胞因子源自哺乳动物。
  9. 根据权利要求1-8中任一项所述的液体制剂，其中所述蛋白质包括融合蛋白，且所述融合蛋白中包含选自下组的至少两种细胞因子：IL12、IL2、GMCSF、IL7、IL15、IL21和FLT3L。
  10. 根据权利要求1-9中任一项所述的液体制剂，其中所述蛋白质包括融合蛋白，且所述融合蛋白中包含选自以下的任一组细胞因子：
    a)IL12、IL2和GMSCF；
    b)IL12、IL7和GMSCF；
    c)IL12、IL15和GMSCF；
    d)IL12、IL21和GMSCF；
    e)IL12、IL2和FLT3L；
    f)IL12、IL7和FLT3L；
    g)IL12、IL15和FLT3L；以及，
    h)IL12、IL21和FLT3L。
  11. 根据权利要求1-10中任一项所述的液体制剂，其中所述蛋白质还包括靶向部分。
  12. 根据权利要求11所述的液体制剂，其中所述靶向部分能够特异性识别和/或结合肿瘤相关抗原。
  13. 根据权利要求12所述的液体制剂，其中所述肿瘤相关抗原选自下组：纤连蛋白的EDB结构域、纤连蛋白的EDA结构域和细胞坏死区域(necrotic regions)。
  14. 根据权利要求11-13中任一项所述的液体制剂，其中所述靶向部分包括抗体或其抗原结 合片段。
  15. 根据权利要求11-14中任一项所述的液体制剂，其中所述靶向部分包含下组中任一项所示的氨基酸序列：SEQ ID NO:1-15。
  16. 根据权利要求1-15中任一项所述的液体制剂，其中所述蛋白质包含下组中任一项所示的氨基酸序列：SEQ ID NO:32-67。
  17. 根据权利要求1-16中任一项所述的液体制剂，其中所述蛋白质为单链蛋白质。
  18. 根据权利要求17所述的液体制剂，其中所述单链蛋白质包含下组中任一项所示的氨基酸序列：SEQ ID NO:32-51。
  19. 根据权利要求1-18中任一项所述的液体制剂，其中所述蛋白质为由第一多肽链及第二多肽链组成的二聚体，所述第一多肽链不同于所述第二多肽链。
  20. 根据权利要求19所述的液体制剂，其中所述第一多肽链包含IL12a，所述第二多肽链包含IL12b。
  21. 根据权利要求19-20中任一项所述的液体制剂，其中所述IL2或其功能性片段位于所述第一多肽链中或所述第二多肽链中，所述GMCSF或其功能性片段位于所述第一多肽链中或所述第二多肽链中，且所述一个或多个靶向部分各自独立地位于所述第一多肽链中或所述第二多肽链中。
  22. 根据权利要求20-21中任一项所述的液体制剂，其中在所述第一多肽链中，从N端到C端依次包含所述IL2或其功能性片段、所述IL12a或其功能性片段和所述GMCSF或其功能性片段。
  23. 根据权利要求20-22中任一项所述的液体制剂，其中在所述第一多肽链中，从N端到C端依次包含所述靶向部分、所述IL12a或其功能性片段、所述IL2或其功能性片段以及所述GMCSF或其功能性片段。
  24. 根据权利要求20-23中任一项所述的液体制剂，其中在所述第二多肽链中，从N端到C端依次包含所述IL12b或其功能性片段和所述靶向部分。
  25. 根据权利要求19-24中任一项所述的液体制剂，其中，
    a)所述第一多肽链包含SEQ ID NO:53所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:52所示的氨基酸序列；
    b)所述第一多肽链包含SEQ ID NO:55所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:54所示的氨基酸序列；
    c)所述第一多肽链包含SEQ ID NO:56所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:57所示的氨基酸序列；
    d)所述第一多肽链包含SEQ ID NO:58所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:59所示的氨基酸序列；
    e)所述第一多肽链包含SEQ ID NO:60所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:61所示的氨基酸序列；
    f)所述第一多肽链包含SEQ ID NO:62所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:63所示的氨基酸序列；
    g)所述第一多肽链包含SEQ ID NO:64所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:65所示的氨基酸序列；
    h)所述第一多肽链包含SEQ ID NO:66所示的氨基酸序列且所述第二多肽链包含SEQ ID NO:67所示的氨基酸序列。
  26. 药物，其包含权利要求1-25中任一项所述的液体制剂。
  27. 根据权利要求26所述的药物，其中所述液体制剂被配制为注射剂。
  28. 根据权利要求26-27中任一项所述的药物，其还包含稀释剂，其中所述稀释剂与所述液体制剂彼此不混合。
  29. 权利要求1-25中任一项所述的液体制剂在制备治疗肿瘤的药物中的用途。
  30. 根据权利要求29所述的用途，其中所述肿瘤包括肺癌。
  31. 权利要求1-25中任一项所述的液体制剂，其用于治疗肿瘤。
  32. 治疗肿瘤的方法，其包括向有需要的受试者施用权利要求1-25中任一项所述的液体制剂。
  33. 根据权利要求32所述的方法，其中所述施用方法为瘤内注射。
  34. 一种用于制备权利要求1-25中任一项所述的液体制剂的辅料，其包含油相溶剂，所述油相溶剂的质量分数为约50％以上。
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