WO2021147886A1 - 一种药物组合物及其用途 - Google Patents

一种药物组合物及其用途 Download PDF

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WO2021147886A1
WO2021147886A1 PCT/CN2021/072824 CN2021072824W WO2021147886A1 WO 2021147886 A1 WO2021147886 A1 WO 2021147886A1 CN 2021072824 W CN2021072824 W CN 2021072824W WO 2021147886 A1 WO2021147886 A1 WO 2021147886A1
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polypeptide chain
seq
amino acid
acid sequence
sequence shown
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PCT/CN2021/072824
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French (fr)
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张晋宇
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张晋宇
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Priority to US17/759,114 priority Critical patent/US20230043257A1/en
Priority to CN202180009111.7A priority patent/CN114945586A/zh
Publication of WO2021147886A1 publication Critical patent/WO2021147886A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • 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]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • 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
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • 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
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • 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
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This application relates to the field of biomedicine, in particular to a pharmaceutical composition and its use.
  • 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 unsatisfactory, and there are many areas for improvement.
  • the present application provides a pharmaceutical composition comprising a protein and an immune checkpoint inhibitor, wherein the protein comprises a fusion protein, and the fusion protein comprises the cytokines IL12, IL2 and GMCSF.
  • the immune checkpoint inhibitor includes an inhibitor of PD1, PD-L1 and/or CTLA-4.
  • the cytokine is derived from a mammal.
  • 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 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. 27-52.
  • 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.
  • the IL2 or functional fragment thereof is located in the first polypeptide chain or the second polypeptide chain, and the GMCSF or functional fragment thereof is located in the first polypeptide chain Or in the second polypeptide chain.
  • the IL2 or functional fragment thereof is located in the first polypeptide chain or the second polypeptide chain
  • the GMCSF or functional fragment thereof is located in the first polypeptide chain Or in the second polypeptide chain
  • one or more of the targeting moieties are each independently located in the first polypeptide chain or in the second polypeptide chain.
  • the IL12a or its functional fragment and the IL2 or its functional fragment are included in sequence from the N-terminus to the C-terminus; or, in the first polypeptide chain, The polypeptide chain includes the IL2 or its functional fragment and the IL12a or its functional fragment in sequence from the N-terminus to the C-terminus; or, in the first polypeptide chain, from the N-terminus to the C-terminus It contains the IL12a or its functional fragment and the GMCSF or its functional fragment in sequence.
  • the second polypeptide chain includes the IL12b or its functional fragment and the GMCSF or its functional fragment in sequence from the N-terminus to the C-terminus; or, in the second polypeptide chain, The polypeptide chain includes the GMCSF or its functional fragment and the IL12b or its functional fragment in sequence from the N-terminus to the C-terminus; or, in the second polypeptide chain, from the N-terminus to the C-terminus
  • the IL12b or functional fragments thereof and the IL2 or functional fragments thereof are included in sequence.
  • 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. 57;
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO. 54 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.53 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO.58;
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO. 54 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO. 58;
  • 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. 59;
  • 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. 60.
  • the targeting moiety in the first polypeptide chain, the targeting moiety, the IL12a or functional fragments thereof, the IL2 or functional fragments thereof, and the The GMCSF or functional fragments thereof; or, in the first polypeptide chain, the IL2 or functional fragments thereof, the IL12a or functional fragments thereof, and the GMCSF or Its functional fragments.
  • the second polypeptide chain includes the IL12b or its functional fragment 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. 66 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. 66 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO. 62;
  • 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. 63;
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO. 67 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. 68 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO. 62;
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO. 69 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. 70 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO. 64;
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO. 71 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO. 64;
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO. 72 and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO. 65.
  • the present application provides a kit, which includes the pharmaceutical composition.
  • the present application also provides the use of the pharmaceutical composition or the kit in the preparation of medicines for the treatment of tumors.
  • the tumor comprises a solid tumor.
  • the tumor comprises melanoma.
  • the tumor comprises breast cancer.
  • the tumor comprises lung cancer.
  • the pharmaceutical composition or the kit described in this application is used to treat tumors.
  • this application also provides a method for treating tumors, which comprises administering the pharmaceutical composition or the kit to a subject in need.
  • the administering includes administering the protein first, and then administering the immune checkpoint inhibitor.
  • the administration includes intratumoral injection, intravenous injection, or subcutaneous injection.
  • the tumor comprises a solid tumor.
  • the tumor comprises melanoma.
  • the tumor comprises breast cancer.
  • the tumor comprises lung cancer.
  • Figures 1A-1C show the effect of induced expression of mIL12aIL2-IL12bGMCSF heterodimer on T cell PD1 expression.
  • Figure 2 shows the growth of melanoma in mice after the combined treatment of mIL12bIL12aIL2GMCSF, mIL12bIL12aIL2GMCSF and PD1 antibody described in this application.
  • Figure 3 shows the growth of melanoma in mice after combined treatment with the mIL12bIL12aIL2DiaNHS76F8GMCSF, mIL12bIL12aIL2DiaNHS76F8GMCSF and PD1 antibodies described in this application.
  • Figure 4 shows the growth of melanoma in mice after combined treatment with the mIL12aIL2-IL12bGMCSF, mIL12aIL2-IL12bGMCSF and PD1 antibodies described in this application.
  • Figure 5 shows the growth of breast cancer in mice after combined treatment with the mIL12bIL12aIL2DiaNHS76F8GMCSF, mIL12bIL12aIL2DiaNHS76F8GMCSF and PD1 antibodies described in this application.
  • Figure 6 shows the growth of lung cancer in mice after combined treatment with the mIL12bIL12aIL2DiaNHS76F8GMCSF, mIL12bIL12aIL2DiaNHS76F8GMCSF and PD1 antibody described in this application.
  • the term "pharmaceutical composition” generally refers to a composition suitable for administration to a patient.
  • the pharmaceutical composition in this application includes a protein and an immune checkpoint inhibitor, wherein the protein includes a fusion protein, and the fusion protein includes the cytokines IL12, IL2, and GMCSF.
  • the pharmaceutical composition may also include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives, etc. Suitable formulations of agents and/or adjuvants.
  • the acceptable ingredients of the composition are not toxic to the recipient at the dosage and concentration used.
  • the pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the term "immune checkpoint inhibitor” generally refers to a molecule that reduces, inhibits, interferes, or modulates one or more checkpoint proteins in whole or in part.
  • Checkpoint proteins regulate T cell activation or function.
  • a variety of checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD1 and its ligands PD-L1 and PD-L2 (Pardoll, NatureReviews Cancer 12:252 -264, 2012). These proteins are responsible for the costimulation or inhibitory interaction of T cell responses.
  • Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses.
  • Immune checkpoint inhibitors include antibodies or are derived from antibodies.
  • the immune checkpoint inhibitor may include inhibitors of PD1, PD-L1 and/or CTLA-4.
  • 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 improves 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, and even 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 improves 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, and even 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.) )
  • immune cells such as monocytes, macrophages, T cells, B cells, NK cells, etc.
  • non-immune cells such as endothelial cells, epidermal cells, fibroblasts, etc.
  • the cytokine plays an important role in regulating cell-cell interaction, cell growth and differentiation.
  • the cytokine may be selected from one or more of the following group: Interleukin (IL) and 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.
  • 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 (GMCSF).
  • IL12 generally refers to interleukin-12.
  • IL12 can play an important regulatory role in cell-cell interaction, immune regulation, hematopoiesis, and inflammation.
  • the molecule of IL12 is usually a heterodimer, which usually includes two subunits, the two subunits are p40 subunit (40kd) and p35 subunit (35kd), these two subunits are through disulfide bond connected together.
  • IL12 containing p35 subunit (35kd) can be represented as IL12a
  • IL12 containing p40 subunit (40kd) can be represented as IL12b.
  • 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.
  • murine-derived IL2 may include the amino acid sequence shown in SEQ ID NO.76
  • human-derived IL2 may include the amino acid sequence shown in SEQ ID NO.77.
  • GMCSF generally refers to granulocyte macrophage colony stimulating factor.
  • the GMCSF may have 4 alpha-helix bundle structures.
  • GMCSF (mGMCSF) derived from mouse may include the amino acid sequence shown in SEQ ID NO.20.
  • human-derived GMCSF (hGMCSF) may include the amino acid sequence shown in SEQ ID NO.21.
  • antibody generally refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide that includes an antigen binding site, 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, dAb and other antibody fragments that maintain antigen binding function. 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 heterotetrameric 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 the two H chains are connected to each other by one or more disulfide bonds depending on the isotype of the H chain.
  • 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.
  • the term "specific recognition and/or binding” generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody.
  • an antibody that specifically binds a target is an antibody that binds to the target with greater affinity, affinity, easier, and/or longer duration than it binds to other targets.
  • 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 usually 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 consisting of an uninterrupted sequence of consecutive amino acid residues.
  • the single-chain protein may include the amino acid sequence shown in any one of the following groups: SEQ ID NO. 27-52.
  • the term "dimer” generally refers to a polymer complex formed from 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-terminus or amino terminus and a C-terminus or carboxy terminus.
  • 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).
  • the term "kit” generally refers to a packaged product containing the pharmaceutical composition of the present application.
  • the kit may comprise a box or container containing the components of the kit.
  • the box or container has a label or a treatment plan approved by the Food and Drug Administration.
  • the box or container contains the components of the pharmaceutical composition of the present application, for example, the components may be contained in a plastic, polyethylene, polypropylene, ethylene or propylene container.
  • the container can be a tube or bottle with a lid.
  • the kit may also include instructions for administering the pharmaceutical composition described in this application.
  • tumor generally refers to neoplasms or solid lesions formed by abnormal cell growth.
  • the tumor may be a solid tumor or hematoma.
  • the tumor can include melanoma.
  • 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.
  • administration generally refers to a method of administering a certain dose of a liquid formulation or drug to a subject (eg, a patient).
  • Administration can be carried out by any suitable means, including parenteral, intrapulmonary and intranasal, and (if local treatment is required) 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.
  • 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.
  • the administration may also be intravenous injection or subcutaneous injection.
  • 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 pharmaceutical composition, which includes a protein and an immune checkpoint inhibitor, wherein the protein includes a fusion protein, and the fusion protein includes the cytokines IL12, IL2, and GMCSF.
  • the immune checkpoint inhibitor may include inhibitors of PD1, PD-L1 and/or CTLA-4.
  • the immune checkpoint inhibitor may be an antibody against PD1, PD-L1 and/or CTLA-4.
  • the cytokine may be derived from a mammal.
  • the mammal can be a human or a 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 GMCSF (represented by mGMCSF) may be as shown in SEQ ID NO.20.
  • the amino acid sequence of human-derived IL12a (represented by hIL12a) may be shown in SEQ ID NO.18, and 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 GMCSF (represented by hGMCSF) may be as shown in SEQ ID NO.21.
  • the amino acid sequence of IL2 derived from murine may be shown in SEQ ID NO.76, and the amino acid sequence of IL2 derived from human may be shown in SEQ ID NO.77.
  • the protein may also include a targeting moiety.
  • the number of the targeting moiety can 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 group consisting of the EDB domain of fibronectin, the EDA domain of fibronectin, and necrotic regions.
  • the targeting moiety may include an antibody or an 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 a Thrombin cleavage site.
  • the linker may include the amino acid sequence shown in any one of the following groups: SEQ ID NO. 22-26.
  • the cytokines can be connected through the linker.
  • the IL12a, IL12b, IL2 and GMCSF can be connected through the connecting peptide.
  • the connecting peptide may comprise an amino acid sequence as shown in any one of SEQ ID NOs. 22 and 24.
  • the cytokine and the targeting moiety may be connected by the linker.
  • the targeting moiety and IL12a, IL12b, IL2 and GMCSF can be connected through the connecting peptide.
  • the connecting peptide may comprise the amino acid sequence shown in any one of SEQ ID NOs. 22-26.
  • the protein may be a single-chain protein, and the single-chain protein may include an amino acid sequence shown in any one of the following groups: SEQ ID NO. 27-52.
  • the structure of the single-chain protein may be the fusion of the C-terminus of mIL12b and the N-terminus of mIL12a, the fusion of the C-terminus of mIL12a and the N-terminus of mIL2, and the fusion of the C-terminus of mIL2 and the N-terminus of mGMCSF to form mIL12b-mIL12a.
  • -mIL2-mGMCSF protein molecule (its amino acid sequence can be shown in SEQ ID NO. 27), and can be referred to as mIL12bIL12aIL2GMCSF protein for short.
  • the structure of the single-chain protein may be the fusion of the C-terminus of mIL12b and the N-terminus of mIL12a, the fusion of the C-terminus of mIL12a and the N-terminus of mGMCSF, and the fusion of the C-terminus of mGMCSF and the N-terminus of mIL2 to form mIL12b-mIL12a.
  • -mGMCSF-mIL2 protein molecule (its amino acid sequence can be shown in SEQ ID NO. 28), and can be referred to as mIL12bIL12aGMCSFIL2 protein for short.
  • the structure of the single-chain protein may be the fusion of the C-terminus of hIL12b and the N-terminus of hIL12a, the fusion of the C-terminus of hIL12a and the N-terminus of hIL2, and the fusion of the C-terminus of hIL2 and the N-terminus of hGMCSF to form hIL12b-hIL12a.
  • -hIL2-hGMCSF protein molecule (its amino acid sequence can be shown in SEQ ID NO. 29), and can be referred to as hIL12bIL12aIL2GMCSF protein for short.
  • the single-chain protein structure may be, the N-terminal and C-terminal mIL12b mIL12a fusion, the N-terminal and C-terminal mIL12a fusion mL19V H, mL19V H C-terminal and N-terminal fusion mL19V L, mL19V L C-and N-terminal mL19V H fusion mL19V H C-terminal and mL19V L the N-terminal fusion, the N-terminus mL19V L C-terminus and mIL2 fusion of the N-terminus C-terminus and mGMCSF of mIL2 fusion, thereby forming mIL12b -mIL12a-mL19V H -mL19V L -mL19V H -mL19V L -mIL2-mGMCSF protein molecule (the amino acid sequence of which may be shown in SEQ ID NO. 30), and may be referred to as
  • the single-chain protein structure may be, the N-terminal and C-terminal mIL12b mIL12a fusion, the N-terminal and C-terminal mIL12a fusion mF8V H, mF8V H C-terminal and N-terminal fusion mF8V L, mF8V L C-and N-terminal mF8V H fusion mF8V H C-terminal and mF8V L the N-terminal fusion, the N-terminus mF8V L C-terminus and mIL2 fusion of the N-terminus C-terminus and mGMCSF of mIL2 fusion, thereby forming mIL12b -mIL12a-mF8V H -mF8V L -mF8V H -mF8V L -mIL2-mGMCSF protein molecule (the amino acid sequence of which may be shown in SEQ ID NO.31), and may be referred to as
  • the single-chain protein structure may be, the N-terminal and C-terminal mIL12b mIL12a fusion, the N-terminal and C-terminal mIL12a fusion mNHS76V H, mNHS76V H C-terminal and N-terminal fusion mNHS76V L, mNHS76V L C-and N-terminal mNHS76V H fusion mNHS76V H C-terminal and mNHS76V L the N-terminal fusion, the N-terminus mNHS76V L C-terminus and mIL2 fusion of the N-terminus C-terminus and mGMCSF of mIL2 fusion, thereby forming mIL12b -mIL12a-mNHS76V H -mNHS76V L -mNHS76V H -mNHS76V L -mIL2-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.
  • the single-chain protein structure may be, the N-terminal and C-terminal mIL12b mIL12a fusion, the N-terminal and C-terminal mIL12a fusion mNHS76V H, mNHS76V H C-terminal and N-terminal fusion mF8V L, mF8V L C-and N-terminal mF8V H fusion mF8V H C-terminal and mNHS76V L the N-terminal fusion, the N-terminus mNHS76V L C-terminus and mIL2 fusion of the N-terminus C-terminus and mGMCSF of mIL2 fusion, thereby forming mIL12b -mIL12a-mNHS76V H -mF8V L -mF8V H -mNHS76V L -mIL2-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO. 33), and may be
  • the single-chain protein structure may be, the N-terminal and C-terminal mIL12b mIL12a fusion, the N-terminal and C-terminal mIL12a fusion mNHS76V H, mNHS76V H C-terminal and N-terminal fusion mL19V L, mL19V L C-and N-terminal mL19V H fusion mL19V H C-terminal and mNHS76V L the N-terminal fusion, the N-terminus mNHS76V L C-terminus and mIL2 fusion of the N-terminus C-terminus and mGMCSF of mIL2 fusion, thereby forming mIL12b -mIL12a-mNHS76V H -mL19V L -mL19V H -mNHS76V L -mIL2-mGMCSF protein molecule (the amino acid sequence of which may be shown in SEQ ID NO.34), and may
  • the single-chain protein structure may be, the N-terminal and C-terminal mIL12b mIL12a fusion, the N-terminal and C-terminal mIL12a fusion mF8V H, mF8V H C-terminal and N-terminal fusion mNHS76V L, mNHS76V L C-and N-terminal mNHS76V H fusion mNHS76V H C-terminal and mF8V L the N-terminal fusion, the N-terminus mF8V L C-terminus and mIL2 fusion of the N-terminus C-terminus and mGMCSF of mIL2 fusion, thereby forming mIL12b -mIL12a-mF8V H -mNHS76V L -mNHS76V H -mF8V L -mIL2-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.35), and may be
  • the single-chain protein structure may be, the N-terminal and C-terminal mIL12b mIL12a fusion, the N-terminal and C-terminal mIL12a fusion mF8V H, mF8V H C-terminal and N-terminal fusion mL19V L, mL19V L C-and N-terminal mL19V H fusion mL19V H C-terminal and mF8V L the N-terminal fusion, the N-terminus mF8V L C-terminus and mIL2 fusion of the N-terminus C-terminus and mGMCSF of mIL2 fusion, thereby forming mIL12b -mIL12a-mF8V H -mL19V L -mL19V H -mF8V L -mIL2-mGMCSF protein molecule (the amino acid sequence of which may be shown in SEQ ID NO. 36), and may be referred to as
  • the single-chain protein structure may be, the N-terminal and C-terminal mIL12b mIL12a fusion, the N-terminal and C-terminal mIL12a fusion mL19V H, mL19V H C-terminal and N-terminal fusion mNHS76V L, mNHS76V L N-terminal C-terminal and mNHS76V H fusion mNHS76V H C-terminal and mL19V L the N-terminal fusion, mL19V L of C and N-terminal mIL2 fused to the N-terminus C-terminus mIL2 and GMCSF fusion, thereby forming mIL12b -mIL12a-mL19V H -mNHS76V L -mNHS76V H -mL19V L -mIL2-mGMCSF protein molecule (the amino acid sequence of which may be shown in SEQ ID NO. 37), and may be referred to
  • the single-chain protein structure may be, the N-terminal and C-terminal mIL12b mIL12a fusion, the N-terminal and C-terminal mIL12a fusion mL19V H, mL19V H C-terminal and N-terminal fusion mF8V L, mF8V L C-and N-terminal mF8V H fusion mF8V H C-terminal and mL19V L the N-terminal fusion, the N-terminus mL19V L C-terminus and mIL2 fusion of the N-terminus C-terminus and mGMCSF of mIL2 fusion, thereby forming mIL12b -mIL12a-mL19V H -mF8V L -mF8V H -mL19V L -mIL2-mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO.38), and may be referred to as mIL12b
  • 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 mNHS76V H, C-mNHS76V H end and mF8V L the N-terminal fusion, mF8V L the N-terminus C-terminus and mF8V H fusion, the C-terminus and mNHS76V L N-terminal mF8V H fused to the N-terminus mNHS76V L C-terminus and mGMCSF fusion, thereby forming mIL12b -mIL12a-mIL2-mNHS76V H -mF8V L -mF8V H -mNHS76V L -mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO. 39), and may be referred to as mIL12bIL12aIL
  • 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 mF8V H, C-mF8V H end and mF8V L the N-terminal fusion, mF8V L the N-terminus C-terminus and mF8V H fusion, the C-terminus and mF8V L N-terminal mF8V H fused to the N-terminus mF8V L C-terminus and mGMCSF fusion, thereby forming mIL12b -mIL12a-mIL2-mF8V H -mF8V L -mF8V H -mF8V L -mGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO. 40), and may be referred to as mIL12bIL12aIL2DiaF8GMC
  • the structure of the single-chain protein may be: the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL2, the C-terminus of mIL2 is fused to the N-terminus of mGMCSF, and the C-terminus of mGMCSF is fused to the N-terminus of mIL2.
  • N-terminal fusion mNHS76V H, C-terminal and N-terminal mF8V L fusion mNHS76V H, mF8V L the N-terminal and C-terminal fusion mF8V H, C-terminal and N-terminal mNHS76V L fusion mF8V H, thereby forming mIL12b- mIL12a-mIL2-mGMCSF-mNHS76V H -mF8V L -mF8V H -mNHS76V L protein molecule (its amino acid sequence may be shown in SEQ ID NO. 41), and may be referred to as mIL12bIL12aIL2GMCSFDiaNHS76F8 protein.
  • the structure of the single-chain protein may be: the C-terminus of mIL12b is fused to the N-terminus of mIL12a, the C-terminus of mIL12a is fused to the N-terminus of mIL2, the C-terminus of mIL2 is fused to the N-terminus of mGMCSF, and the C-terminus of mGMCSF is fused to the N-terminus of mIL2.
  • the structure may be a single-chain protein, the N-terminal and C-terminal hIL12b hIL12a fused, N-terminal and C-terminal hIL12a fusion hL19V H, hL19V H C-terminal and N-terminal fusion hL19V L, hL19V L N-terminal C-terminal and hL19V H fusion hL19V H C-terminal and hL19V L the N-terminal fusion, hL19V L of C and N-terminal hIL2 fusion hIL2 of C and N-terminal hGMCSF fused, thereby forming hIL12b -hIL12a-hL19V H -hL19V L -hL19V H -hL19V L -hIL2-hGMCSF protein molecule (the amino acid sequence of which may be shown in SEQ ID NO. 43), and may be referred to as hIL12bIL
  • the structure may be a single-chain protein, the N-terminal and C-terminal hIL12b hIL12a fused, N-terminal and C-terminal hIL12a fusion hNHS76V H, hNHS76V H C-terminal and N-terminal fusion hNHS76V L, hNHS76V L N-terminal C-terminal and hNHS76V H fusion hNHS76V H C-terminal and hNHS76V L the N-terminal fusion, hNHS76V L of C and N-terminal hIL2 fusion hIL2 of C and N-terminal hGMCSF fused, thereby forming hIL12b -hIL12a-hNHS76V H -hNHS76V L -hNHS76V H -hNHS76V L -hIL2-hGMCSF protein molecule (the amino acid sequence of which can be shown in SEQ ID NO.44), and
  • the structure may be a single-chain protein, the N-terminal and C-terminal hIL12b hIL12a fused, N-terminal and C-terminal hIL12a fusion hNHS76V H, hNHS76V H C-terminal and N-terminal fusion hF8V L, hF8V L N-terminal C-terminal and hF8V H fusion hF8V H C-terminal and hNHS76V L the N-terminal fusion, hNHS76V L of C and N-terminal hIL2 fusion hIL2 of C and N-terminal hGMCSF fused, thereby forming hIL12b -hIL12a-hNHS76V H -hF8V L -hF8V H -hNHS76V L -hIL2-hGMCSF protein molecule (the amino acid sequence of which may be shown in SEQ ID NO.45), and may be referred to as
  • 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 hNHS76V H, C-hNHS76V H end and hF8V L the N-terminal fusion, the N-terminus hF8V L C-terminal and hF8V H fusion hF8V H C-terminal and hNHS76V L the N-terminal fusion, hNHS76V L of C and N-terminal hGMCSF fused, thereby forming hIL12b -hIL12a-hIL2-hNHS76V H -hF8V L -hF8V H -hNHS76V L -hGMCSF protein molecule (the amino acid sequence of which may be shown in SEQ ID NO. 46), and may be
  • 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 hF8V H, C-hF8V H end and hF8V L the N-terminal fusion, the N-terminus hF8V L C-terminal and hF8V H fusion hF8V H C-terminal and hF8V L the N-terminal fusion, hF8V L of C and N-terminal hGMCSF fused, thereby forming hIL12b -hIL12a-hIL2-hF8V H -hF8V L -hF8V H -hF8V L -hGMCSF protein molecule (its amino acid sequence may be shown in SEQ ID NO. 47), and may be referred to as hIL12b
  • the structure may be a single-chain protein, the N-terminal and C-terminal hIL12b hIL12a fused, N-terminal and C-terminal hIL12a fusion hL19V H, hL19V H C-terminal and N-terminal fusion hL19V L, hL19V L N-terminal C-terminal and hL19V H fusion, the C-terminus and hL19V L N-terminal hL19V H fusion hL19V L of C and N-terminal hGMCSF fused to the C-terminus hGMCSF and the N-terminus of hIL2 fused, thereby forming hIL12b -hIL12a-hL19V H -hL19V L -hL19V H -hL19V L -hGMCSF-hIL2 protein molecule (the amino acid sequence of which may be shown in SEQ ID NO. 48), and may be referred
  • the structure may be a single-chain protein, the N-terminal and C-terminal hIL12b hIL12a fused, N-terminal and C-terminal hIL12a fusion hNHS76V H, hNHS76V H C-terminal and N-terminal fusion hNHS76V L, hNHS76V L N-terminal C-terminal and hNHS76V H fusion, the C-terminus and hNHS76V L N-terminal hNHS76V H fusion hNHS76V L of C and N-terminal hGMCSF fused to the C-terminus hGMCSF and the N-terminus of hIL2 fused, thereby forming hIL12b -hIL12a-hNHS76V H -hNHS76V L -hNHS76V H -hNHS76V L -hGMCSF-hIL2 protein molecule (its amino acid sequence may be shown in SEQ.
  • the structure may be a single-chain protein, the N-terminal and C-terminal hIL12b hIL12a fused, N-terminal and C-terminal hIL12a fusion hNHS76V H, hNHS76V H C-terminal and N-terminal fusion hF8V L, hF8V L N-terminal C-terminal and hF8V H fusion, the C-terminus and hNHS76V L N-terminal hF8V H fusion hNHS76V L of C and N-terminal hGMCSF fused to the C-terminus hGMCSF and the N-terminus of hIL2 fused, thereby forming hIL12b -hIL12a-hNHS76V H -hF8V L -hF8V H -hNHS76V L -hGMCSF-hIL2 protein molecule (its amino acid sequence may be shown in SEQ ID NO.50), and
  • the single-chain protein may also be mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr (its amino acid sequence may be as shown in SEQ ID NO. 51), which has basically the same structure as the mIL12bIL12aIL2DiaNHS76F8G MCSF protein, except for the linker different.
  • the linker of mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr has a Thrombin cleavage site.
  • the single-chain protein may also be hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr (its amino acid sequence may be as shown in SEQ ID NO.52), which is basically the same in structure as hIL12bIL12aIL2DiaNHS76F8GMCSF protein, except that the linker is different. .
  • the linker of hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr has a Thrombin cleavage site.
  • 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.
  • the IL2 or its functional fragment may be located in the first polypeptide chain or the second polypeptide chain, and the GMCSF or its functional fragment may be located in the first polypeptide chain Or in the second polypeptide chain.
  • the IL12a or its functional fragment and the IL2 or its functional fragment may be included in sequence from the N-terminus to the C-terminus; or, in the first polypeptide chain, In the peptide chain, the IL2 or its functional fragment and the IL12a or its functional fragment may be included in sequence from the N-terminus to the C-terminus; or, in the first polypeptide chain, from the N-terminus to the C-terminus.
  • the IL12a or its functional fragment and the GMCSF or its functional fragment may be included in sequence.
  • the IL12b or its functional fragment and the GMCSF or its functional fragment may be included in sequence from the N-terminus to the C-terminus; or, in the second polypeptide chain, In the peptide chain, the GMCSF or its functional fragment and the IL12b or its functional fragment may be included in sequence from the N-terminus to the C-terminus; or, in the second polypeptide chain, from the N-terminus to the C-terminus.
  • the IL12b or its functional fragment and the IL2 or its functional fragment may be included in sequence.
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 53 and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 57.
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 54 and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 57; or,
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 53 and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 58; or, the first polypeptide chain may include The amino acid sequence shown in SEQ ID NO. 54 and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO.
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 55
  • Amino acid sequence and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 59; or, the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 56 and the second polypeptide chain
  • the peptide chain may include the amino acid sequence shown in SEQ ID NO.60.
  • the protein may be a dimer composed of a first polypeptide chain and a second polypeptide chain.
  • 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.53), and the C-terminus of mIL12b and mGMCSF
  • the N-terminus can be fused to form a mIL12b-mGMCSF second polypeptide chain (sequence shown in SEQ ID NO.57), which can form a mIL12a-mIL2-mIL12b-mGMCSF protein heterodimer, referred to as mIL12aIL2-IL12bGMCSF heterodimer.
  • the C-terminus of mIL2 and the N-terminus of mIL12a can be fused to form the first polypeptide chain of mIL2-mIL12a (sequence is shown in SEQ ID NO.54), and the C-terminus of mIL12b and the N-terminus of mIL12a
  • the N-terminus can be fused to form the second polypeptide chain of mIL12b-mGMCSF (sequence shown in SEQ ID NO.57), which can form a mIL2-mIL12a-mIL12b-mGMCSF protein heterodimer, referred to as mIL2IL12a-IL12bGMCSF heterodimer.
  • 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.53), and the C-terminus of mGMCSF and mIL12b
  • the N-terminus can be fused to form the second polypeptide chain of mGMCSF-mIL12b (sequence shown in SEQ ID NO.58), which can form a mIL12a-mIL2-mGMCSF-mIL12b protein heterodimer, referred to as mIL12aIL2-GMCSFIL12b heterodimer.
  • the C-terminus of mIL2 and the N-terminus of mIL12a can be fused to form the first polypeptide chain of mIL2-mIL12a (sequence is shown in SEQ ID NO.54), and the C-terminus of mGMCSF and mIL12b
  • the N-terminus can be fused to form a second polypeptide chain of mGMCSF-mIL12b (sequence shown in SEQ ID NO.58), which can form a mIL2-mIL12a-mGMCSF-mIL12b protein heterodimer, referred to as mIL2IL12a-GMCSFIL12b heterodimer.
  • the C-terminus of mIL12a and the N-terminus of mGMCSF can be fused to form the first polypeptide chain of mIL12a-mGMCSF (sequence is shown in SEQ ID NO.55), and the C-terminus of mIL12b and the N-terminus of mIL2
  • the N-terminus can be fused to form the second polypeptide chain of mIL12b-mIL2 (sequence shown in SEQ ID NO.59), which can form a mIL12a-mGMCSF-mIL12b-mIL2 protein heterodimer, referred to as mIL12aGMCSF-IL12bIL2 heterodimer.
  • 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.56), 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.60), which can form a hIL12a-hIL2-hIL12b-hGMCSF protein heterodimer, referred to as hIL12aIL2-IL12bGMCSF heterodimer.
  • the IL2 or its functional fragment may be located in the first polypeptide chain or the second polypeptide chain, and the GMCSF or its functional fragment may be located in the first polypeptide chain Or in the second polypeptide chain, and one or more of the targeting moieties may each independently be located in the first polypeptide chain or in the second polypeptide chain.
  • 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 functional fragments thereof; or, in the first polypeptide chain, the IL2 or functional fragments thereof, the IL12a or functional fragments thereof, and the GMCSF or Its functional fragments.
  • the IL12b or its functional fragment 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. 66 and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 61.
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 66 and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 62; or,
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 66 and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 63; or, the first polypeptide chain may include The amino acid sequence shown in SEQ ID NO. 67 and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO.
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 68
  • the amino acid sequence and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 62; or, the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 69 and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO.
  • the peptide chain may include the amino acid sequence shown in SEQ ID NO. 63; or, the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 70 and the second polypeptide chain may include SEQ ID NO. 64; or, the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO.
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 72 and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 65.
  • the dimer, C and N terminal ends mIL12b may be fused L19V H, C terminal L19V H and L may be N-terminal fusion L19V second polypeptide chain is formed (which may be the amino acid sequence shown in SEQ ID No.
  • mIL12b-L19V H- L19V L- mIL2-mIL12a-mGMCSF protein heterodimer referred to as mIL12bscL19-IL2IL12aGMCSF heterodimer.
  • 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.
  • mIL12bscF8-IL2IL12aGMCSF heterodimer a mIL12bscF8-IL2IL12aGMCSF heterodimer.
  • the dimer, C and N terminal ends mIL12b may 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.
  • mIL12b-NHS76V H -NHS76V L -mIL2-mIL12a-mGMCSF protein heterodimer referred to as mIL12bscNHS76-IL2IL12aGMCSF heterodimer.
  • the dimer, C and N terminal ends mIL12b may be fused L19V H, C terminal L19V H and L may be N-terminal fusion L19V second polypeptide chain is formed (which may be the amino acid sequence shown in SEQ ID NO.61 shown), and the C-terminus L19V H can be N-terminal fusion L19V L, C-terminal and N may L19V L mIL12a terminal fusion, 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.
  • mIL12b-L19V H -L19V L -L19V H -L19V L -mIL12a-mIL2-mGMCSF Protein heterodimer abbreviated as mIL12bscL19-scL19IL12aIL2GMCSF heterodimer.
  • 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.62 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 can be as shown in SEQ ID NO.
  • mIL12b-F8V H -F8V L -F8V H -F8V L -mIL12a-mIL2-mGMCSF Protein heterodimer abbreviated as mIL12bscF8-scF8IL12aIL2GMCSF heterodimer.
  • the dimer, C and N terminal ends mIL12b may 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.63 shown), and the C-terminus and may NHS76V H NHS76V L the N-terminal fusion, a C-terminal NHS76V L and N may be fused mIL12a terminal, C-terminal and N-terminal mIL12a MIL2 may be fused, C-MIL2
  • the terminal can be fused with the N-terminal of GMCSF to form the first polypeptide chain (the amino acid sequence can be shown in SEQ ID NO.
  • mIL12bscNHS76-scNHS76IL12aIL2GMCSF heterodimer mIL12bscNHS76-scNHS76IL12aIL2GMCSF heterodimer for short.
  • the dimer, hIL12b the N-terminus and C-terminus can 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.
  • hIL12bscF8-IL2IL12aGMCSF heterodimer a polypeptide chain (the amino acid sequence of which can be shown in SEQ ID NO.70 ), thereby forming hIL12b-F8V H -F8V L -hIL2-hIL12a-hGMCSF protein heterodimer, referred to as hIL12bscF8-IL2IL12aGMCSF heterodimer.
  • the dimer, hIL12b the N-terminus and C-terminus can 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.64 shown), and the C-terminus F8V H can be N-terminal fusion F8V L, F8V L N and C-terminal ends can be fused hIL12a, hIL12a the N-terminus and C-terminus of hIL2 may be fused, C-hIL2 The end can be fused with the N-terminus of hGMCSF to form the first polypeptide chain (the amino acid sequence can be as shown in SEQ ID NO.
  • hIL12b-F8V H -F8V L -F8V H -F8V L -hIL12a-hIL2-hGMCSF Protein heterodimer abbreviated as hIL12bscF8-scF8IL12aIL2GMCSF heterodimer.
  • 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.65 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 (the amino acid sequence can be as shown in SEQ ID NO.72), thereby forming hIL12b-NHS76V H -NHS76V L -NHS76V H -NHS76V L -hIL12a-hIL2-hGMCSF Protein heterodimer, ab
  • the present application provides a kit, which includes the pharmaceutical composition described in the present application.
  • the kit may comprise a box or container containing the components of the kit.
  • the box or container has a label or a treatment plan approved by the Food and Drug Administration.
  • the box or container contains the components of the pharmaceutical composition of the present application, for example, the components may be contained in a plastic, polyethylene, polypropylene, ethylene or propylene container.
  • the container can be a tube or bottle with a lid.
  • the kit may also include instructions for administering the pharmaceutical composition described in this application.
  • the application also provides the use of the pharmaceutical composition or the kit in the preparation of medicines, which can be used to treat tumors.
  • the pharmaceutical composition or the kit can be used to treat tumors.
  • this application also provides a method for treating tumors, which comprises administering the pharmaceutical composition or the kit to a subject in need.
  • the tumor may include solid tumors and non-solid tumors.
  • the tumor may include melanoma.
  • the tumor may include breast cancer.
  • the tumor may include lung cancer.
  • the administration may include the administration of the protein first, and then the administration of the immune checkpoint inhibitor.
  • the administration may include administering the single-chain protein described in this application first, and then administering the immune checkpoint inhibitor described in this application.
  • the administering may include administering the dimer described in this application first, and then administering the immune checkpoint inhibitor described in this application.
  • the administration may include intratumoral injection.
  • the pharmaceutical composition described in this application is injected 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 may be administered through a subcutaneous depot.
  • the administration may also include intravenous injection or subcutaneous injection.
  • the pharmaceutical composition 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, transdermal administration or via a subcutaneous depot.
  • the pharmaceutical composition described in the present 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.
  • DMEM medium, 1640 medium and fetal bovine serum were purchased from Lifetechnologies; cell culture flasks and culture plates were purchased from Corning; Doxycycline was purchased from Shanghai Shenggong Bioengineering Co., Ltd.; Puromycin and Blasticidin were purchased from Chemicon; Restriction endonucleases were purchased from Takara and NEB Company; Ligase was purchased from NEB Company; DNA polymerase was purchased from Takara Company; Plasmid extraction kit and gel recovery kit were purchased from OmegaBiotech Company; primer synthesis was purchased from Shanghai Shenggong Biological Engineering Co., Ltd. The company completed; gene synthesis and sequencing were completed by lifetechnologies; the flow detection antibody was purchased from Ebioscience; the PD1 blocking antibody was purchased from BioXcell; the protein magnetic bead purification kit was purchased from Beaver Bio.
  • Example 1 mIL12aIL2-IL12bGMCSF heterodimer induces the expression of PD1 in T cells
  • the digestion system is as follows: plasmid 6 ⁇ g, digestion buffer 4 ⁇ l, BamHI 1 ⁇ l, EcoRI 1 ⁇ l, add water to a total volume of 40 ⁇ l, and let 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 rtTA fragment is recovered and set aside.
  • the vector pLentis-CMV-IRES-Bsd was digested in the EP tube, and the digestion system was as follows: plasmid 2 ⁇ g, digestion buffer 3 ⁇ l, BamHI 1 ⁇ l, EcoRI 1 ⁇ l, add water to a total volume of 30 ⁇ l, and let stand at 37°C for 12 hours. Take out the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, and use 1% agarose gel for electrophoresis. After electrophoresis, the pLentis-CMV-IRES-Bsd vector fragment is recovered and set aside.
  • the system is as follows: pLentis-CMV-IRES-Bsd 2 ⁇ l, rtTA 2 ⁇ l, ligase buffer 1 ⁇ l, T4DNA ligase 0.5 ⁇ l, water 4.5 ⁇ l, put at room temperature and connect for 4 hours. Then the ligation system is transformed into E. coli competent. On the second day, colonies were picked from the transformed plate and placed in LB medium in a shaker at 37°C overnight. The plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragments were successfully attached to the vector by enzyme digestion. , And then sent the correct vector for sequencing to confirm that the first expression vector pLentis-CMV-rtTA-IRES-Bsd was successfully constructed.
  • the method is as follows:
  • B16 mouse melanoma cells After digestion culture B16 mouse melanoma cells, at 10 5 cells / well were seeded into 6-well culture volume of 1ml, 24 hours, added 10 l of the first viral expression vectors, and cultured in an incubator 24 hours , Discard the supernatant, change to a fresh medium and continue culturing. After the cells are full, transfer them to a culture flask, add blasticidin at a concentration suitable for the cells, and continue culturing. Change the medium once every two days and keep the concentration of blasticidin at 8 ⁇ g/ml. After one week of screening, the surviving cells are the cells stably expressing the regulatory protein, and this cell is named B16 (rtTA).
  • the restriction system is as follows: mIL12aIL2-IL12bGMCSF heterodimer plasmid 5 ⁇ g, Enzyme digestion buffer 4 ⁇ l, BamHI 1 ⁇ l, EcoRI 1 ⁇ l, 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, use 1% agarose gel for electrophoresis, recover the mIL12aIL2-IL12bGMCSF gene fragment after electrophoresis, and set it aside.
  • amino acid sequence of the first polypeptide chain of mIL12aIL2-IL12bGMCSF heterodimer is shown in SEQ ID NO.53, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.57; the nucleus encoding the mIL12aIL2-IL12bGMCSF The nucleotide sequence is shown in SEQ ID NO.73.
  • Enzyme digestion control expression vector pLentis-PTRE-MCS-PGK-PURO digestion system is as follows: pLentis-PTRE-MCS-PGK-PURO plasmid 2 ⁇ g, digestion buffer 3 ⁇ l, BamHI 1 ⁇ l, EcoRI 1 ⁇ l, 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-PTRE-MCS-PGK-PURO vector fragment after electrophoresis, and set aside.
  • connection system is as follows: pLentis-PTRE-MCS-PGK-PURO 2 ⁇ l, mIL12aIL2-IL12bGMCSF 2 ⁇ l, ligase buffer 1 ⁇ l, T4DNA ligase 0.5 ⁇ l, water 4.5 ⁇ l, set Connect for 4 hours at room temperature. Then the ligation system is transformed into E. coli competent. On the second day, colonies were picked from the transformed plate and placed in LB medium in a shaker at 37°C overnight. The plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragments were successfully attached to the vector by enzyme digestion. , And then sent the correct vector to sequence to confirm that the second expression vector pLentis-PTRE-mIL12aIL2-IL12bGMCSF-PGK-PURO was successfully constructed.
  • the method for preparing the virus of the mIL12aIL2-IL12bGMCSF heterodimer expression vector is the same as the method for preparing the first expression vector virus.
  • Digestion cultured B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, 24 hours, regulating the expression of viral vector (i.e. mIL12aIL2-IL12bGMCSF heterodimer expression vector Virus) 10 ⁇ l, after continuing to culture in the incubator for 24 hours, discard the supernatant and change to fresh medium to continue the culture.
  • viral vector i.e. mIL12aIL2-IL12bGMCSF heterodimer expression vector Virus
  • the surviving cells are the cells that can regulate and express mIL12aIL2-IL12bGMCSF, and this cell is named B16(rtTA)-mIL12aIL2-IL12bGMCSF.
  • mice Digest the B16(rtTA)-mIL12aIL2-IL12bGMCSF cells in the logarithmic growth phase, dilute with HBSS to 2 ⁇ 10 6 cells/ml, and inject 50 ⁇ l/mouse into 8-10 weeks old C57BL/6 female mice using a 1ml syringe There are 10 mice on the right dorsal side of the mouse. After the tumor grows, they are fed with water containing 2g/L doxycycline. The mice on the 0th day and the 3rd day after the addition of doxycycline are taken, and the spleen cells and tumor tissues are separated. After splitting the red blood cells with red blood cell lysate, filter the cells to obtain a single cell suspension.
  • FIG. 1A Figure 1A, Figure 1B and Figure 1C respectively show the expression of PD1 in mouse spleen CD4 T cells, spleen CD8 T cells and CD3 T cells in the tumor. It can be seen that CD4 T cells and CD8 T cells in the mouse spleen , The expression of PD1 in tumor-infiltrated CD3T cells was significantly increased.
  • the single-chain protein molecule mIL12bIL12aIL2GMCSF add 6*His at the end for purification, synthesize the DNA sequence corresponding to the gene, the front and back ends of the synthesized sequence have BamHI and XhoI restriction sites respectively, and the plasmid with the target gene is 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 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 aside.
  • the amino acid sequence of mIL12bIL12aIL2GMCSF single-chain protein is shown in SEQ ID NO.27, and the nucleotide sequence encoding the mIL12bIL12aIL2GMCSF is shown in SEQ ID NO.74.
  • 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 mIL12bIL12aIL2GMCSF gene fragment, 1 ⁇ l ligase buffer 4.5, 0.5 ⁇ l T4 DNA ligase and water ⁇ l, connect at room temperature 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 IL12p70ELISA kit.
  • the protein concentration was adjusted to 2 ⁇ g/ ⁇ l with PBS, and then aliquoted and stored at -20°C.
  • the single-chain protein molecule mIL12bIL12aIL2DiaNHS76F8GMCSF add 6*His to the end for purification, synthesize the DNA sequence corresponding to the gene, the front and back ends of the synthesized sequence have BamHI and XhoI restriction sites respectively, and the plasmid with the target gene is 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 for 12 hours.
  • amino acid sequence of mIL12bIL12aIL2DiaNHS76F8GMCSF single-chain protein is shown in SEQ ID NO. 39, and the nucleotide sequence of mIL12bIL12aIL2DiaNHS76F8GMCSF is shown in SEQ ID NO. 75.
  • 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 mIL12bIL12aIL2DiaNHS76F8GMCSF gene fragment, 1 ⁇ l DNA ligase 4.5 0.5 ⁇ l DNA ligase and 4.5 ⁇ l ligase buffer ⁇ l. Leave it at room temperature to connect for 4 hours. Then the ligation system is transformed into E. coli competent.
  • the constructed cells 293A-mIL12bIL12aIL2DiaNHS76F8GMCSF expressing mIL12bIL12aIL2DiaNHS76F8GMCSF were passaged to a 15cm culture dish. After the cells became full, the medium was changed to 30ml CDM4HEK293, and the culture was continued for 5 days, and then the supernatant was collected and filtered with a 0.45 ⁇ m filter. AMICON ULTRA-15 was concentrated by ultrafiltration, and the concentrated protein solution obtained was purified with nickel chelating magnetic beads (purchased from Beaver Biotechnology Co., Ltd.). The operation process was carried out according to the instructions.
  • 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 IL12p70ELISA kit.
  • the protein concentration was adjusted to 2 ⁇ g/ ⁇ l with PBS, and then aliquoted and stored at -20°C.
  • Example 4 The effect of combined treatment of mIL12bIL12aIL2GMCSF and PD1 antibody on the growth of melanoma in mice
  • Example 2 Take 25 ⁇ l of the protein solution prepared in Example 2 and add it to 50 ⁇ l of glycerol, and quickly mix it with a pipette tip to avoid air bubbles to obtain an injection preparation.
  • mice were injected with PD1 antibody (specifically BioXcell, InVivoMAb anti-mouse PD-1 (CD279), catalog number BE0146) 200 ⁇ g every 3 days One injection, 4 injections in total. Finally, compare the tumor growth of mice in each group. The results are shown in Figure 2. It can be seen that the combined treatment of mIL12bIL12aIL2GMCSF and PD1 antibody significantly inhibited tumor growth.
  • PD1 antibody specifically BioXcell, InVivoMAb anti-mouse PD-1 (CD279), catalog number BE0146
  • Example 5 The effect of combined treatment of mIL12bIL12aIL2DiaNHS76F8GMCSF and PD1 antibody on the growth of melanoma in mice
  • Example 3 Take 25 ⁇ l of the protein solution prepared in Example 3, adjust the total volume to 200 ⁇ l with PBS, and use a 29G insulin syringe to suck the protein solution into the tail vein of the mouse and inject it once a day for a total of 5 injections. After the injection, the mice were returned to the cage, and the tumor growth of the mice was recorded. The experiment was divided into 3 groups, 1. No injection group, 2. Protein solution injection group, 3. Protein solution injection and PD1 antibody treatment group (ie combined treatment group).
  • mIL12aIL2-IL12bGMCSF heterodimer gene add 6*His sequence to the C-terminus of IL2 gene for subsequent protein purification, the synthesized gene has BamHI and XhoI restriction sites at both ends, and then use BamHI and XhoI for enzyme
  • the digestion system is as follows: mIL12aIL2-IL12bGMCSF heterodimer plasmid 5 ⁇ g, digestion buffer 4 ⁇ l, BamHI 1 ⁇ l, XhoI 1 ⁇ l, add water to a total volume of 40 ⁇ l, and let stand at 37°C for 12 hours. Take out the EP tube, add 4.4 ⁇ l 10 ⁇ loading buffer, use 1% agarose gel for electrophoresis, recover the mIL12aIL2-IL12bGMCSF gene fragment after electrophoresis, and set it aside.
  • amino acid sequence of the first polypeptide chain of mIL12aIL2-IL12bGMCSF heterodimer is shown in SEQ ID NO.53, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.57; the nucleus encoding the mIL12aIL2-IL12bGMCSF The nucleotide sequence is shown in SEQ ID NO.73.
  • 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 Connect mIL12aIL2-IL12bGMCSF and pLentis-CMV-MCS-IRES-PURO, the system is as follows, 2 ⁇ l pLentis-CMV-MCS-IRES-PURO vector fragment, 2 ⁇ l mIL12aIL2-IL12bGMCSF gene fragment, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA connection Enzyme and water 4.5 ⁇ l, left at room temperature to connect for 4 hours. Then the ligation system is transformed into E. coli competent.
  • Example 7 The effect of combined treatment of mIL12aIL2-IL12bGMCSF and PD1 antibody on the growth of melanoma in mice
  • Example 6 Take 25 ⁇ l of the protein solution prepared in Example 6 and add it to 50 ⁇ l of glycerol, and quickly mix it with a pipette tip to avoid air bubbles to obtain an injection preparation.
  • mice were injected with PD1 antibody (specifically BioXcell, InVivoMAb anti-mouse PD-1 (CD279), catalog number BE0146) 200 ⁇ g every 3 days One injection, 4 injections in total. Finally, compare the tumor growth of mice in each group. The results are shown in Figure 4, it can be seen that the combined treatment of mIL12aIL2-IL12bGMCSF and PD1 antibody significantly inhibited tumor growth.
  • PD1 antibody specifically BioXcell, InVivoMAb anti-mouse PD-1 (CD279), catalog number BE0146
  • Example 8 The effect of combined treatment of mIL12bIL12aIL2DiaNHS76F8GMCSF and PD1 antibody on the growth of breast cancer in mice
  • Example 3 Take 25 ⁇ l of the protein solution prepared in Example 3, adjust the total volume to 200 ⁇ l with PBS, and use a 29G insulin syringe to suck the protein solution into the tail vein of the mouse and inject it once a day for a total of 5 injections. After the injection, the mice were returned to the cage, and the tumor growth of the mice was recorded. The experiment was divided into 3 groups, 1. No injection group, 2. Protein solution injection group, 3. Protein solution injection and PD1 antibody treatment group (ie combined treatment group).
  • PD1 antibody specifically BioXcell, InVivoMAb anti-mouse PD-1 (CD279), catalog number BE0146
  • PD1 antibody specifically BioXcell, InVivoMAb anti-mouse PD-1 (CD279), catalog number BE0146
  • Example 9 The effect of combined treatment of mIL12bIL12aIL2DiaNHS76F8GMCSF and PD1 antibody on the growth of lung cancer in mice
  • Example 3 Take 25 ⁇ l of the protein solution prepared in Example 3, adjust the total volume to 200 ⁇ l with PBS, and use a 29G insulin syringe to draw the protein solution into the tail vein of the mouse and inject it once a day for a total of 3 injections. After the injection, the mice were returned to the cage, and the tumor growth of the mice was recorded. The experiment was divided into 3 groups, 1. No injection group, 2. Protein solution injection group, 3. Protein solution injection and PD1 antibody treatment group (ie combined treatment group).
  • PD1 antibody specifically BioXcell, InVivoMAb anti-mouse PD-1 (CD279), catalog number BE0146
  • PD1 antibody specifically BioXcell, InVivoMAb anti-mouse PD-1 (CD279), catalog number BE0146

Abstract

本发明提供一种药物组合物，其包括蛋白质和免疫检查点抑制剂，其中所述蛋白质包括融合蛋白，且所述融合蛋白中包含细胞因子IL12、IL2和GMCSF。本发明还提供包括所述药物组合物的试剂盒以及所述药物组合物或所述试剂盒在制备用于***的药物中的用途。

Description

一种药物组合物及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种药物组合物及其用途。
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
一方面，本申请提供了一种药物组合物，其包括蛋白质和免疫检查点抑制剂，其中所述蛋白质包括融合蛋白，且所述融合蛋白中包含细胞因子IL12、IL2和GMCSF。
在某些实施方式中，所述免疫检查点抑制剂包括PD1、PD-L1和/或CTLA-4的抑制剂。
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.27-52。
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，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。
在某些实施方式中，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，且一个或多个所述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IL12a或其功能性片段和所述IL2或其功能性片段；或者，在所述第一多肽链中，从N端到C端依次包含所述IL2或其功能性片段和所述IL12a或其功能性片段；又或者，在所述第一多肽链中，从N端到C端依次包含所述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GMCSF或其功能性片段；或者，在所述第二多肽链中，从N端到C端依次包含所述GMCSF或其功能性片段和所述IL12b或其功能性片段；又或者，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IL2或其功能性片段。
在某些实施方式中，在所述的药物组合物中，
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.57所示的氨基酸序列；
b)所述第一多肽链包含SEQ ID NO.54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；
c)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.58所示的氨基酸序列；
d)所述第一多肽链包含SEQ ID NO.54所示的氨基酸序列且所述第二多肽链包含SEQ ID NO.58所示的氨基酸序列；
e)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.59所示的氨基酸序列；
f)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.60所示的氨基酸序列。
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；或者， 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或其功能性片段以及所述GMCSF或其功能性片段。
在某些实施方式中，在第二多肽链中，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，在所述的药物组合物中，
1)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.61所示的氨基酸序列；
2)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.62所示的氨基酸序列；
3)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.63所示的氨基酸序列；
4)所述第一多肽链包含SEQ ID NO.67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；
5)所述第一多肽链包含SEQ ID NO.68所示的氨基酸序列且所述第二多肽链包含SEQ ID NO.62所示的氨基酸序列；
6)所述第一多肽链包含SEQ ID NO.69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；
7)所述第一多肽链包含SEQ ID NO.70所示的氨基酸序列且所述第二多肽链包含SEQ ID NO.64所示的氨基酸序列；
8)所述第一多肽链包含SEQ ID NO.71所示的氨基酸序列且所述第二多肽链包含SEQ ID NO.64所示的氨基酸序列；
9)所述第一多肽链包含SEQ ID NO.72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。
另一方面，本申请提供一种试剂盒，其包括所述的药物组合物。
另一方面，本申请还提供所述的药物组合物或所述的试剂盒在制备药物中的用途，所述药物用于***。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括黑色素瘤。
在某些实施方式中，所述肿瘤包括乳腺癌。
在某些实施方式中，所述肿瘤包括肺癌。
在某些实施方式中，本申请所述的药物组合物或所述的试剂盒，其用于***。
另一方面，本申请还提供一种***的方法，其包括向有需要的受试者施用所述的药物组合物或所述的试剂盒。
在某些实施方式中，所述施用包括先施用所述蛋白质，之后施用所述免疫检查点抑制剂。
在某些实施方式中，所述施用包括瘤内注射、静脉注射或皮下注射。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括黑色素瘤。
在某些实施方式中，所述肿瘤包括乳腺癌。
在某些实施方式中，所述肿瘤包括肺癌。
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。对附图简要说明如下：
图1A-1C显示诱导表达mIL12aIL2-IL12bGMCSF异二聚体后对T细胞PD1表达的影响。
图2显示的是分别用本申请所述的mIL12bIL12aIL2GMCSF、mIL12bIL12aIL2GMCSF和PD1抗体联用治疗后小鼠体内黑色素瘤的生长情况。
图3显示的是分别用本申请所述的mIL12bIL12aIL2DiaNHS76F8GMCSF、mIL12bIL12aIL2DiaNHS76F8GMCSF和PD1抗体联用治疗后小鼠体内黑色素瘤的生长情况。
图4显示的是分别用本申请所述的mIL12aIL2-IL12bGMCSF、mIL12aIL2-IL12bGMCSF和PD1抗体联用治疗后小鼠体内黑色素瘤的生长情况。
图5显示的是分别用本申请所述的mIL12bIL12aIL2DiaNHS76F8GMCSF、mIL12bIL12aIL2DiaNHS76F8GMCSF和PD1抗体联用治疗后小鼠体内乳腺癌的生长情况。
图6显示的是分别用本申请所述的mIL12bIL12aIL2DiaNHS76F8GMCSF，mIL12bIL12aIL2DiaNHS76F8GMCSF和PD1抗体联用治疗后小鼠体内肺癌的生长情况。
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。
术语定义
在本申请中，术语“药物组合物”通常是指适合施用于患者的组合物。本申请中的药物组合物包括蛋白质和免疫检查点抑制剂，其中所述蛋白质包括融合蛋白，且所述融合蛋白中包含细胞因子IL12、IL2和GMCSF。在某些实施方式中，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂、防腐剂和/或佐剂的合适的制剂。例如，组合物的可接受成分在所用剂量和浓度下对接受者无毒。本申请的药物组合物包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“免疫检查点抑制剂”通常是指整体或部分减少、抑制、干扰或调节一种或多种检查点蛋白质的分子。检查点蛋白质调控T细胞活化或功能，已知多种检查点蛋白质，如CTLA-4以及其配体CD80和CD86；以及PD1及其配体PD-L1和PD-L2(Pardoll,NatureReviews Cancer 12:252-264,2012)。这些蛋白质负责T细胞反应的共刺激或抑制相互作用。免疫检查点蛋白调控并维持自身耐受性以及生理免疫反应的持续时间和幅度。免疫检查点抑制剂包括抗体或源自于抗体。例如，在本申请中，所述免疫检查点抑制剂可以包括PD1、PD-L1和/或CTLA-4的抑制剂。
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”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)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。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。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。例如，来源于鼠的IL2(mIL2)可包含如SEQ ID NO.76所示的氨基酸序列，来源于人的IL2(hIL2)可包含如SEQ ID NO.77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。例如，来源于小鼠的GMCSF(mGMCSF)可包含如SEQ ID NO.20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.21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。
在本申请中，术语“特异性识别和/或结合”通常是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。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.27-52。
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“试剂盒”通常是指包含本申请的药物组合物的包装产品。所述试剂盒可以包含容纳有该试剂盒组分的盒子或容器。该盒子或容器贴有标签或食品和药物管理局批准的治疗方案。该盒子或容器容纳有本申请的药物组合物的组分，例如，该组分可以容纳于塑料、聚乙烯、聚丙烯、乙烯或丙烯容器中。该容器可以是带盖的管或瓶。此外，所述试剂盒还可包含用于给予本申请所述药物组合物的使用说明。
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，肿瘤可包括黑色素瘤。
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。在某些情形中，所述施用还可以为静脉注射或皮下注射。
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。
发明详述
药物组合物和试剂盒
一方面，本申请提供一种药物组合物，其包括蛋白质和免疫检查点抑制剂，其中所述蛋白质包括融合蛋白，且所述融合蛋白中包含细胞因子IL12、IL2和GMCSF。
在本申请中，所述免疫检查点抑制剂可以包括PD1、PD-L1和/或CTLA-4的抑制剂。例如，所述免疫检查点抑制剂可以是抗PD1、PD-L1和/或CTLA-4的抗体。
在本申请中，所述细胞因子可以源自哺乳动物。例如，在某些实施方式中，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GMCSF(用mGMCSF表示)的氨基酸序列可以如SEQ ID NO.20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GMCSF(用hGMCSF表示)的氨基酸序列可以如SEQ ID NO.21所示。又例如，源自鼠的IL2的氨基酸序列可以如SEQ ID NO.76所示，源自人的IL2的氨基酸序列可以如SEQ ID NO.77所示。
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。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，所述靶向部分可以包括抗体或其抗原结合片段。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)。
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。在某些实施方式中，所述连接子可以包含凝血酶(Thrombin)切割位点。
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.22-26。
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.22和24中任一项所示的氨基酸序列。
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.22-26中的任一项所示的氨基酸序列。
在本申请中，所述蛋白质可以为单链蛋白质，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.27-52。
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蛋白质分子(其氨基酸序列可以如SEQ ID NO.27所示)，并可简称为mIL12bIL12aIL2GMCSF蛋白质。
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mGMCSF的N端融合，mGMCSF的C端和mIL2的N端融合，从而形成mIL12b-mIL12a-mGMCSF-mIL2蛋白质分子(其氨基酸序列可以如SEQ ID NO.28所示)，并可简称为mIL12bIL12aGMCSFIL2蛋白质。
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蛋白质分子(其氨基酸序列可以如SEQ ID NO.29所示)，并可简称为hIL12bIL12aIL2GMCSF蛋白质。
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mL19V H的N端融合，mL19V H的C端和mL19V L的N端融合，mL19V L的C端和mL19V H的N端融合，mL19V H的C端和mL19V L的N端融合，mL19V L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-mL19V H-mL19V L-mL19V H-mL19V L-mIL2-mGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.30所示)，并可简称为mIL12bIL12aDiaL19IL2GMCSF蛋白质。
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mF8V H的N端融合，mF8V H的C端和mF8V L的N端融合，mF8V L的C端和mF8V H的N端融合，mF8V H的C端和mF8V L的N端融合，mF8V L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-mF8V H-mF8V L-mF8V H-mF8V L-mIL2-mGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.31所示)，并可简称为mIL12bIL12aDiaF8IL2GMCSF蛋白质。
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mNHS76V H的N端融合，mNHS76V H的C端和mNHS76V L的N端融合，mNHS76V L的C端和mNHS76V H的N端融合，mNHS76V H的C端和mNHS76V L的N端融合，mNHS76V L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-mNHS76V H-mNHS76V L-mNHS76V H-mNHS76V L-mIL2-mGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.32所示)，并可简称为mIL12bIL12aDiaNHS76IL2GMCSF蛋白质。
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mNHS76V H的N端融合，mNHS76V H的C端和mF8V L的N端融合，mF8V L的C端和mF8V H的N端融合，mF8V H的C端和mNHS76V L的N端融合，mNHS76V L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-mNHS76V H-mF8V L-mF8V H-mNHS76V L-mIL2-mGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.33所示)，并可简称为mIL12bIL12aDiaNHS76F8IL2GMCSF蛋白质。
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mNHS76V H的N端融合，mNHS76V H的C端和mL19V L的N端融合，mL19V L的C端和mL19V H的N端融合，mL19V H的C端和mNHS76V L的N端融合，mNHS76V L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-mNHS76V H-mL19V L-mL19V H-mNHS76V L-mIL2-mGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.34所示)，并可简称为mIL12bIL12aDiaNHS76L19IL2GMCSF蛋白质。
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mF8V H的N端融合，mF8V H的C端和mNHS76V L的N端融合，mNHS76V L的C端和mNHS76V H的N端融合，mNHS76V H的C端和mF8V L的N端融合，mF8V L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-mF8V H-mNHS76V L-mNHS76V H-mF8V L-mIL2-mGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.35所示)，并可简称为mIL12bIL12aDiaF8NHS76IL2GMCSF蛋白质。
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mF8V H的N端融合，mF8V H的C端和mL19V L的N端融合，mL19V L的C端和 mL19V H的N端融合，mL19V H的C端和mF8V L的N端融合，mF8V L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-mF8V H-mL19V L-mL19V H-mF8V L-mIL2-mGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.36所示)，并可简称为mIL12bIL12aDiaF8L19IL2GMCSF蛋白质。
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mL19V H的N端融合，mL19V H的C端和mNHS76V L的N端融合，mNHS76V L的C端和mNHS76V H的N端融合，mNHS76V H的C端和mL19V L的N端融合，mL19V L的C端和mIL2的N端融合，mIL2的C端和GMCSF的N端融合，从而形成mIL12b-mIL12a-mL19V H-mNHS76V L-mNHS76V H-mL19V L-mIL2-mGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.37所示)，并可简称为mIL12bIL12aDiaL19NHS76IL2GMCSF蛋白质。
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mL19V H的N端融合，mL19V H的C端和mF8V L的N端融合，mF8V L的C端和mF8V H的N端融合，mF8V H的C端和mL19V L的N端融合，mL19V L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-mL19V H-mF8V L-mF8V H-mL19V L-mIL2-mGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.38所示)，并可简称为mIL12bIL12aDiaL19F8IL2GMCSF蛋白质。
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mNHS76V H的N端融合，mNHS76V H的C端和mF8V L的N端融合，mF8V L的C端和mF8V H的N端融合，mF8V H的C端和mNHS76V L的N端融合，mNHS76V L的C端和mGMCSF的N端融合，从而形成mIL12b-mIL12a-mIL2-mNHS76V H-mF8V L-mF8V H-mNHS76V L-mGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.39所示)，并可简称为mIL12bIL12aIL2DiaNHS76F8GMCSF蛋白质。
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mF8V H的N端融合，mF8V H的C端和mF8V L的N端融合，mF8V L的C端和mF8V H的N端融合，mF8V H的C端和mF8V L的N端融合，mF8V L的C端和mGMCSF的N端融合，从而形成mIL12b-mIL12a-mIL2-mF8V H-mF8V L-mF8V H-mF8V L-mGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.40所示)，并可简称为mIL12bIL12aIL2DiaF8GMCSF蛋白质。
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，mGMCSF的C端和 mNHS76V H的N端融合，mNHS76V H的C端和mF8V L的N端融合，mF8V L的C端和mF8V H的N端融合，mF8V H的C端和mNHS76V L的N端融合，从而形成mIL12b-mIL12a-mIL2-mGMCSF-mNHS76V H-mF8V L-mF8V H-mNHS76V L蛋白质分子(其氨基酸序列可以如SEQ ID NO.41所示)，并可简称为mIL12bIL12aIL2GMCSFDiaNHS76F8蛋白质。
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，mGMCSF的C端和mF8V H的N端融合，mF8V H的C端和mF8V L的N端融合，mF8V L的C端和mF8V H的N端融合，mF8V H的C端和mF8V L的N端融合，从而形成mIL12b-mIL12a-mIL2-mGMCSF-mF8V H-mF8V L-mF8V H-mF8V L蛋白质分子(其氨基酸序列可以如SEQ ID NO.42所示)，并可简称为mIL12bIL12aIL2GMCSFDiaF8蛋白质。
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hL19V H的N端融合，hL19V H的C端和hL19V L的N端融合，hL19V L的C端和hL19V H的N端融合，hL19V H的C端和hL19V L的N端融合，hL19V L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-hL19V H-hL19V L-hL19V H-hL19V L-hIL2-hGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.43所示)，并可简称为hIL12bIL12aDiaL19IL2GMCSF蛋白质。
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hNHS76V H的N端融合，hNHS76V H的C端和hNHS76V L的N端融合，hNHS76V L的C端和hNHS76V H的N端融合，hNHS76V H的C端和hNHS76V L的N端融合，hNHS76V L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-hNHS76V H-hNHS76V L-hNHS76V H-hNHS76V L-hIL2-hGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.44所示)，并可简称为hIL12bIL12aDiaNHS76IL2GMCSF蛋白质。
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hNHS76V H的N端融合，hNHS76V H的C端和hF8V L的N端融合，hF8V L的C端和hF8V H的N端融合，hF8V H的C端和hNHS76V L的N端融合，hNHS76V L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-hNHS76V H-hF8V L-hF8V H-hNHS76V L-hIL2-hGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.45所示)，并可简称为hIL12bIL12aDiaNHS76F8IL2GMCSF蛋白质。
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hNHS76V H的N端融合，hNHS76V H的C端和hF8V L 的N端融合，hF8V L的C端和hF8V H的N端融合，hF8V H的C端和hNHS76V L的N端融合，hNHS76V L的C端和hGMCSF的N端融合，从而形成hIL12b-hIL12a-hIL2-hNHS76V H-hF8V L-hF8V H-hNHS76V L-hGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.46所示)，并可简称为hIL12bIL12aIL2DiaNHS76F8GMCSF蛋白质。
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hF8V H的N端融合，hF8V H的C端和hF8V L的N端融合，hF8V L的C端和hF8V H的N端融合，hF8V H的C端和hF8V L的N端融合，hF8V L的C端和hGMCSF的N端融合，从而形成hIL12b-hIL12a-hIL2-hF8V H-hF8V L-hF8V H-hF8V L-hGMCSF蛋白质分子(其氨基酸序列可以如SEQ ID NO.47所示)，并可简称为hIL12bIL12aIL2DiaF8GMCSF蛋白质。
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hL19V H的N端融合，hL19V H的C端和hL19V L的N端融合，hL19V L的C端和hL19V H的N端融合，hL19V H的C端和hL19V L的N端融合，hL19V L的C端和hGMCSF的N端融合，hGMCSF的C端和hIL2的N端融合，从而形成hIL12b-hIL12a-hL19V H-hL19V L-hL19V H-hL19V L-hGMCSF-hIL2蛋白质分子(其氨基酸序列可以如SEQ ID NO.48所示)，并可简称为hIL12bIL12aDiaL19GMCSFIL2蛋白质。
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hNHS76V H的N端融合，hNHS76V H的C端和hNHS76V L的N端融合，hNHS76V L的C端和hNHS76V H的N端融合，hNHS76V H的C端和hNHS76V L的N端融合，hNHS76V L的C端和hGMCSF的N端融合，hGMCSF的C端和hIL2的N端融合，从而形成hIL12b-hIL12a-hNHS76V H-hNHS76V L-hNHS76V H-hNHS76V L-hGMCSF-hIL2蛋白质分子(其氨基酸序列可以如SEQ ID NO.49所示)，并可简称为hIL12bIL12aDiaNHS76GMCSFIL2蛋白质。
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hNHS76V H的N端融合，hNHS76V H的C端和hF8V L的N端融合，hF8V L的C端和hF8V H的N端融合，hF8V H的C端和hNHS76V L的N端融合，hNHS76V L的C端和hGMCSF的N端融合，hGMCSF的C端和hIL2的N端融合，从而形成hIL12b-hIL12a-hNHS76V H-hF8V L-hF8V H-hNHS76V L-hGMCSF-hIL2蛋白质分子(其氨基酸序列可以如SEQ ID NO.50所示)，并可简称为hIL12bIL12aDiaNHS76F8GMCSFIL2蛋白质。
此外，在某些实施方式中，所述单链蛋白质还可以为mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr(其氨基酸序列可以如SEQ ID NO.51所示)，其与mIL12bIL12aIL2DiaNHS76F8G MCSF蛋白质的结构基本相同，区别仅在于连接子不同。mIL12bIL12aIL2DiaNHS76F8GMCSF-Thr的连接子中带有凝血酶(Thrombin)切割位点。
此外，在某些实施方式中，所述单链蛋白质还可以为hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr(其氨基酸序列可以如SEQ ID NO.52所示)，其与hIL12bIL12aIL2DiaNHS76F8GMCSF蛋白质的结构基本相同，区别仅在于连接子不同。hIL12bIL12aIL2DiaNHS76F8GMCSF-Thr的连接子中带有凝血酶(Thrombin)切割位点。
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。
在本申请中，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，所述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，所述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。
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IL12a或其功能性片段和所述IL2或其功能性片段；或者，在所述第一多肽链中，从N端到C端可以依次包含所述IL2或其功能性片段和所述IL12a或其功能性片段；又或者，在所述第一多肽链中，从N端到C端可以依次包含所述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GMCSF或其功能性片段；或者，在所述第二多肽链中，从N端到C端可以依次包含所述GMCSF或其功能性片段和所述IL12b或其功能性片段；又或者，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IL2或其功能性片段。
在某些实施方式中，在所述药物组合物中，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.57所示的氨基酸序列；或者，所述第一多肽链可以包含SEQ ID NO.54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；或者，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.58所示的氨基酸序列；或者，所述第一多肽链可以包含SEQ ID NO.54所示的氨基酸序列且所述第二多肽链可以包含SEQ ID NO.58所示的氨基酸序列；或者，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.59所示的氨基酸序列；或者，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.60所示的氨基酸序列。
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。
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.53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)，可以形成mIL12a-mIL2-mIL12b-mGMCSF蛋白质异二聚体，简称为mIL12aIL2-IL12bGMCSF异二聚体。
例如，在所述二聚体中，mIL2的C端和mIL12a的N端可以融合形成mIL2-mIL12a第一多肽链(序列如SEQ ID NO.54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)，可以形成mIL2-mIL12a-mIL12b-mGMCSF蛋白质异二聚体，简称为mIL2IL12a-IL12bGMCSF异二聚体。
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.53所示)，且mGMCSF的C端和mIL12b的N端可以融合形成mGMCSF-mIL12b第二多肽链(序列如SEQ ID NO.58所示)，可以形成mIL12a-mIL2-mGMCSF-mIL12b蛋白质异二聚体，简称为mIL12aIL2-GMCSFIL12b异二聚体。
例如，在所述二聚体中，mIL2的C端和mIL12a的N端可以融合形成mIL2-mIL12a第一多肽链(序列如SEQ ID NO.54所示)，且mGMCSF的C端和mIL12b的N端可以融合形成mGMCSF-mIL12b第二多肽链(序列如SEQ ID NO.58所示)，可以形成mIL2-mIL12a-mGMCSF-mIL12b蛋白质异二聚体，简称为mIL2IL12a-GMCSFIL12b异二聚体。
例如，在所述二聚体中，mIL12a的C端和mGMCSF的N端可以融合形成mIL12a-mGMCSF第一多肽链(序列如SEQ ID NO.55所示)，且mIL12b的C端和mIL2的N端可以融合形成mIL12b-mIL2第二多肽链(序列如SEQ ID NO.59所示)，可以形成mIL12a-mGMCSF-mIL12b-mIL2蛋白质异二聚体，简称为mIL12aGMCSF-IL12bIL2异二聚体。
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.56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.60所示)，可以形成hIL12a-hIL2-hIL12b-hGMCSF蛋白质异二聚体，简称为hIL12aIL2-IL12bGMCSF异二聚体。
在本申请中，所述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，所述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，且一个或多个所述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；或者，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或其功能性片段以及所述GMCSF或其功能性片段。
在本申请中，在第二多肽链中，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，在所述药物组合物中，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.61所示的氨基酸序列；或者，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.62所示的氨基酸序列；或者，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.63所示的氨基酸序列；或者，所述第一多肽链可以包含SEQ ID NO.67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；或者，所述第一多肽链可以包含SEQ ID NO.68所示的氨基酸序列且所述第二多肽链可以包含SEQ ID NO.62所示的氨基酸序列；或者，所述第一多肽链可以包含SEQ ID NO.69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；或者，所述第一多肽链可以包含SEQ ID NO.70所示的氨基酸序列且所述第二多肽链可以包含SEQ ID NO.64所示的氨基酸序列；或者，所述第一多肽链可以包含SEQ ID NO.71所示的氨基酸序列且所述第二多肽链可以包含SEQ ID NO.64所示的氨基酸序列；或者，所述第一多肽链可以包含SEQ ID NO.72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。
例如，在所述二聚体中，mIL12b的C端可以和L19V H的N端融合，L19V H的C端可以和L19V 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.61所示)，且mIL2的C端可以和mIL12a的N端融合，mIL12a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.66所示)，从而形成mIL12b-L19V H-L19V L-mIL2-mIL12a-mGMCSF蛋白质异二聚体，简称为mIL12bscL19-IL2IL12aGMCSF异二聚体。
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.62所示)，且mIL2的C端可以和mIL12a的N端融合，mIL12a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.66所示)，从而形成mIL12b-F8V H-F8V L-mIL2-mIL12a-mGMCSF蛋白质异二聚体，简称为mIL12bscF8-IL2IL12aGMCSF异二聚体。
例如，在所述二聚体中，mIL12b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.63所示)，且mIL2的C端可以和mIL12a的N端融合，mIL12a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.66所示)，从而形成mIL12b-NHS76V H- NHS76V L-mIL2-mIL12a-mGMCSF蛋白质异二聚体，简称为mIL12bscNHS76-IL2IL12aGMCSF异二聚体。
例如，在所述二聚体中，mIL12b的C端可以和L19V H的N端融合，L19V H的C端可以和L19V 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.61所示)，且L19V H的C端可以和L19V L的N端融合，L19V 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.67所示)，从而形成mIL12b-L19V H-L19V L-L19V H-L19V L-mIL12a-mIL2-mGMCSF蛋白质异二聚体，简称为mIL12bscL19-scL19IL12aIL2GMCSF异二聚体。
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.62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.68所示)，从而形成mIL12b-F8V H-F8V L-F8V H-F8V L-mIL12a-mIL2-mGMCSF蛋白质异二聚体，简称为mIL12bscF8-scF8IL12aIL2GMCSF异二聚体。
例如，在所述二聚体中，mIL12b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.63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GMCSF的N端融合形成第一多肽链(其氨基酸序列可以如SEQ ID NO.69所示)，从而形成mIL12b-NHS76V H-NHS76V L-NHS76V H-NHS76V L-mIL12a-mIL2-mGMCSF蛋白质异二聚体，简称为mIL12bscNHS76-scNHS76IL12aIL2GMCSF异二聚体。
例如，在所述二聚体中，hIL12b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.64所示)，且hIL2的C端可以和hIL12a的N端融合，hIL12a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.70所示)，从而形成hIL12b-F8V H-F8V L-hIL2-hIL12a-hGMCSF蛋白质异二聚体，简称为hIL12bscF8-IL2IL12aGMCSF异二聚体。
例如，在所述二聚体中，hIL12b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.64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.71所示)，从而形成hIL12b-F8V H-F8V L-F8V H-F8V L-hIL12a-hIL2-hGMCSF蛋白质异二聚体，简称为hIL12bscF8-scF8IL12aIL2GMCSF异二聚体。
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.65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.72所示)，从而形成hIL12b-NHS76V H-NHS76V L-NHS76V H-NHS76V L-hIL12a-hIL2-hGMCSF蛋白质异二聚体，简称为hIL12bscNHS76-scNHS76IL12aIL2GMCSF异二聚体。
另一方面，本申请提供一种试剂盒，其包括本申请所述的药物组合物。所述试剂盒可以包含容纳有该试剂盒组分的盒子或容器。该盒子或容器贴有标签或食品和药物管理局批准的治疗方案。该盒子或容器容纳有本申请的药物组合物的组分，例如，该组分可以容纳于塑料、聚乙烯、聚丙烯、乙烯或丙烯容器中。该容器可以是带盖的管或瓶。此外，所述试剂盒还可包含用于给予本申请所述药物组合物的使用说明。
用途
另一方面，本申请还提供所述的药物组合物或所述的试剂盒在制备药物中的用途，所述药物可以用于***。
在本申请中，所述的药物组合物或所述的试剂盒可以用于***。
另一方面，本申请还提供一种***的方法，其包括向有需要的受试者施用所述的药物组合物或所述的试剂盒。
在本申请中，所述肿瘤可以包括实体瘤和非实体瘤。例如，所述肿瘤可以包括黑色素瘤。例如，所述肿瘤可以包括乳腺癌。例如，所述肿瘤可以包括肺癌。
在本申请中，所述施用可以包括先施用所述蛋白质，之后施用所述免疫检查点抑制剂。例如，所述施用可以包括先施用本申请所述的单链蛋白质，之后再施用本申请所述的免疫检查点抑制剂。又例如，所述施用可以包括先施用本申请所述的二聚体，之后再施用本申请所述的免疫检查点抑制剂。
在本申请中，所述施用可以包括瘤内注射。例如向肿瘤内部注射本申请所述的药物组合物。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，***给药，经皮给药或通过皮下储存库给药。在某些实施方式中，所述施用还可以包括静脉注射或皮下注射。
在某些实施方式中，本申请所述的药物组合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，***给药，经皮给药或通过皮下储存库给药。
在某些实施方式中，本申请所述的药物组合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。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的药物组合物和用途等，而不用于限制本申请发明的范围。
实施例
试剂：DMEM培养基、1640培养基和胎牛血清购自lifetechnologies公司；细胞培养瓶和培养板购自Corning公司；强力霉素购自上海生工生物工程有限公司；Puromycin和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；质粒提取试剂盒和胶回收试剂盒购自OmegaBiotech公司；引物合成由上海生工生物工程有限公司完成；基因合成和测序由lifetechnologies公司完成；流式检测抗体购于Ebioscience公司；PD1阻断抗体购于BioXcell公司；蛋白磁珠纯化试剂盒购于海狸生物。
实施例1.mIL12aIL2-IL12bGMCSF异二聚体诱导T细胞PD1的表达
1.1第一表达载体的构建
合成rtTA的DNA序列，两端带有BamHI和EcoRI位点，合成产物连接在载体pUC57内。酶切此载体，酶切体系如下：质粒6μg，酶切缓冲液4μl，BamHI 1μl，EcoRI 1μl，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rtTA片段，待用。
在EP管内酶切载体pLentis-CMV-IRES-Bsd，酶切体系如下：质粒2μg，酶切缓冲液3μl， BamHI 1μl，EcoRI 1μl，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-IRES-Bsd载体片段，待用。
连接pLentis-CMV-IRES-Bsd和rtTA，体系如下：pLentis-CMV-IRES-Bsd 2μl，rtTA 2μl，连接酶缓冲液1μl，T4DNA连接酶0.5μl，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℃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，然后将正确的载体送测序，确定第一表达载体pLentis-CMV-rtTA-IRES-Bsd构建成功。
1.2获得含第一表达载体的细胞
首先，制备第一表达载体的病毒，方法如下：
1.消化培养的293FT细胞，计数后将3×10 6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，如果细胞状态合适良好，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 5μg+pSPAX2 15μg+pLentis-CMV-rtTA-IRES-Bsd 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500μl HBSS重悬沉淀，溶解2小时后分装成小管，-70℃保存。
然后，使用第一表达载体病毒转染肿瘤细胞，方法如下：
消化培养的小鼠黑色素瘤细胞B16，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入第一表达载体病毒10μl，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，将其转出到培养瓶中，按适合此细胞的浓度加入杀稻瘟菌素(blasticidin)，继续培养，每两天更换一次培养基，并保持blasticidin的浓度为8μg/ml，筛选一周后，存活的细胞即为稳定表达调控蛋白的细胞，将此细胞命名为B16(rtTA)。
1.3编码mIL12aIL2-IL12bGMCSF异二聚体的第二表达载体的构建
合成mIL12aIL2-IL12bGMCSF异二聚体基因，合成的基因两端分别带有BamHI和EcoRI 酶切位点，然后用BamHI和EcoRI进行酶切，酶切体系如下：mIL12aIL2-IL12bGMCSF异二聚体质粒5μg，酶切缓冲液4μl，BamHI 1μl，EcoRI 1μl，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mIL12aIL2-IL12bGMCSF基因片段，待用。
mIL12aIL2-IL12bGMCSF异二聚体的第一多肽链的氨基酸序列如SEQ ID NO.53所示，第二多肽链的氨基酸序列如SEQ ID NO.57所示；编码所述mIL12aIL2-IL12bGMCSF的核苷酸序列如SEQ ID NO.73所示。
酶切调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：pLentis-PTRE-MCS-PGK-PURO质粒2μg，酶切缓冲液3μl，BamHI 1μl，EcoRI 1μl，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-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和IL2，连接体系如下：pLentis-PTRE-MCS-PGK-PURO 2μl，mIL12aIL2-IL12bGMCSF 2μl，连接酶缓冲液1μl，T4DNA连接酶0.5μl，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℃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，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-mIL12aIL2-IL12bGMCSF-PGK-PURO构建成功。
1.4调控表达mIL12aIL2-IL12bGMCSF异二聚体的细胞的制备
制备mIL12aIL2-IL12bGMCSF异二聚体表达载体的病毒，方法与第一表达载体病毒制备方法一致。消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入调控表达载体病毒(即mIL12aIL2-IL12bGMCSF异二聚体表达载体的病毒)10μl，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，按终浓度3μg/ml加入puromycin，继续培养三天后，存活的细胞即为可调控表达mIL12aIL2-IL12bGMCSF的细胞，此细胞命名为B16(rtTA)-mIL12aIL2-IL12bGMCSF。
1.5诱导表达mIL12aIL2-IL12bGMCSF异二聚体后对T细胞PD1表达的分析
将对数生长期的B16(rtTA)-mIL12aIL2-IL12bGMCSF细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，分别取加入强力霉素后第0天，第3天的小鼠，分离脾脏细胞和肿瘤组织的细胞，将细胞用红细胞裂解液裂红细胞后，筛网过滤，得到 单细胞悬液，使用此单细胞悬液进行染色，使用CD45抗体，CD3抗体，CD4抗体，CD8抗体，PD1抗体进行染色，染色后细胞用PBS清洗两次，使用流式细胞仪进行分析，确定T细胞中的PD1表达变化。结果如图1所示，其中图1A、图1B和图1C分别显示小鼠脾脏CD4T细胞、脾脏CD8T细胞和肿瘤内CD3T细胞中PD1表达情况，可以看出，小鼠脾脏内CD4T细胞、CD8T细胞，肿瘤浸润的CD3T细胞中PD1表达均显著升高。
实施例2.mIL12bIL12aIL2GMCSF单链蛋白质的表达
2.1构建表达载体
单链蛋白质分子mIL12bIL12aIL2GMCSF，末端加入6*His以便于纯化，合成基因对应的DNA序列，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mIL12bIL12aIL2GMCSF蛋白质基因片段，待用。
mIL12bIL12aIL2GMCSF单链蛋白质的氨基酸序列如SEQ ID NO.27所示，编码所述mIL12bIL12aIL2GMCSF的核苷酸序列如SEQ ID NO.74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 mIL12bIL12aIL2GMCSF的基因片段、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-mIL12bIL12aIL2GMCSF-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+上述实施例 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-mIL12bIL12aIL2GMCSF。
2.4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3.mIL12bIL12aIL2DiaNHS76F8GMCSF单链蛋白质的表达
3.1构建表达载体
单链蛋白质分子mIL12bIL12aIL2DiaNHS76F8GMCSF，末端加入6*His以便于纯化，合成基因对应的DNA序列，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蛋白质基因片段，待用。
mIL12bIL12aIL2DiaNHS76F8GMCSF单链蛋白质的氨基酸序列如SEQ ID NO.39所示，编码所述mIL12bIL12aIL2DiaNHS76F8GMCSF的核苷酸序列如SEQ ID NO.75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和pLentis-CMV-MCS-IRES-PURO，体系如下，2μl pLentis-CMV-MCS-IRES-PURO载体片段、2μl mIL12bIL12aIL2DiaNHS76F8GMCSF的基因片段、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-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+上述实施例3.1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。
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-mIL12bIL12aIL2DiaNHS76F8GMCSF。
3.4蛋白表达纯化
将构建的表达mIL12bIL12aIL2DiaNHS76F8GMCSF的细胞293A-mIL12bIL12aIL2DiaNHS76F8GMCSF，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.mIL12bIL12aIL2GMCSF和PD1抗体联合治疗对小鼠体内黑色素瘤生长的影响
将2×10 5个消化培养的小鼠黑色素瘤细胞(B16)注射到C57BL/6小鼠(6-10周龄，雌性)身体右侧皮下，待肿瘤的长径达到6-8mm时开始进行治疗。
取实施例2制备的蛋白溶液25μl，然后加入到50μl甘油中，迅速用枪头吹打混匀，避免产生气泡，得到注射制剂，使用29G的胰岛素注射器吸取配制的注射液，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，记录小鼠肿瘤的生长情况。本实验共分为3组，1.未注射组，2.注射蛋白溶液组，3.注射蛋白溶液并用PD1抗体处理组(即联合治疗组)。其中，在联合治疗组中，自蛋白溶液注射后第2天开始，在小鼠腹腔注射PD1抗体(具体为BioXcell公司，InVivoMAb anti-mouse PD-1(CD279)，货号BE0146)200μg，每3天注射一次，共注射4次。最后比较各组小鼠肿瘤的生长情况。结果如图2所示，可以看出mIL12bIL12aIL2GMCSF和PD1抗体联合治疗显著抑制了肿瘤的生长。
实施例5.mIL12bIL12aIL2DiaNHS76F8GMCSF和PD1抗体联合治疗对小鼠体内黑色素瘤生长的影响
将2×10 5个消化培养的小鼠黑色素瘤细胞(B16)注射到C57BL/6小鼠(6-10周龄，雌性)身体右侧皮下，待肿瘤的长径达到6-8mm时开始进行治疗。
取实施例3制备的蛋白溶液25μl，用PBS调节总体积到200μl，使用29G的胰岛素注射器吸取蛋白溶液注射到小鼠尾静脉中，每天注射一次，共注射5次。注射后的小鼠放回笼内，记录小鼠肿瘤的生长情况。本实验共分为3组，1.未注射组，2.注射蛋白溶液组，3.注射蛋白溶液并用PD1抗体处理组(即联合治疗组)。其中，在联合治疗组中，自小鼠完成5次蛋白溶液注射后第2天开始，在其腹腔注射PD1抗体(具体为BioXcell公司，InVivoMAb anti- mouse PD-1(CD279)，货号BE0146)200μg，每3天注射一次，共注射4次。最后比较各组小鼠肿瘤的生长情况。结果如图3所示，可以看出，mIL12bIL12aIL2DiaNHS76F8GMCSF和PD1抗体联合治疗显著抑制了肿瘤的生长。
实施例6.mIL12aIL2-IL12bGMCSF异二聚体蛋白质的表达
6.1构建表达载体
合成mIL12aIL2-IL12bGMCSF异二聚体基因，在IL2基因C端加入6*His序列用于后继的蛋白纯化，合成的基因两端分别带有BamHI和XhoI酶切位点，然后用BamHI和XhoI进行酶切，酶切体系如下：mIL12aIL2-IL12bGMCSF异二聚体质粒5μg，酶切缓冲液4μl，BamHI1μl，XhoI 1μl，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mIL12aIL2-IL12bGMCSF基因片段，待用。
mIL12aIL2-IL12bGMCSF异二聚体的第一多肽链的氨基酸序列如SEQ ID NO.53所示，第二多肽链的氨基酸序列如SEQ ID NO.57所示；编码所述mIL12aIL2-IL12bGMCSF的核苷酸序列如SEQ ID NO.73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mIL12aIL2-IL12bGMCSF和pLentis-CMV-MCS-IRES-PURO，体系如下，2μl pLentis-CMV-MCS-IRES-PURO载体片段、2μl mIL12aIL2-IL12bGMCSF的基因片段、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-mIL12aIL2-IL12bGMCSF-IRES-PURO。
6.2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。
6.3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-mIL12aIL2-IL12bGMCSF。
6.4蛋白表达纯化
将构建的表达mIL12aIL2-IL12bGMCSF的细胞293A-mIL12aIL2-IL12bGMCSF，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7.mIL12aIL2-IL12bGMCSF和PD1抗体联合治疗对小鼠体内黑色素瘤生长的影响
将2×10 5个消化培养的小鼠黑色素瘤细胞(B16)注射到C57BL/6小鼠(6-10周龄，雌性)身体右侧皮下，待肿瘤的长径达到6-8mm时开始进行治疗。
取实施例6制备的蛋白溶液25μl，然后加入到50μl甘油中，迅速用枪头吹打混匀，避免产生气泡，得到注射制剂，使用29G的胰岛素注射器吸取配制的注射液，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，记录小鼠肿瘤的生长情况。本实验共分为3组，1.未注射组，2.注射蛋白溶液组，3.注射蛋白溶液并用PD1抗体处理组(即联合治疗组)。其中，在联合治疗组中，自蛋白溶液注射后第2天开始，在小鼠腹腔注射PD1抗体(具体为BioXcell公司，InVivoMAb anti-mouse PD-1(CD279)，货号BE0146) 200μg，每3天注射一次，共注射4次。最后比较各组小鼠肿瘤的生长情况。结果如图4所示，可以看出mIL12aIL2-IL12bGMCSF和PD1抗体联合治疗显著抑制了肿瘤的生长。
实施例8.mIL12bIL12aIL2DiaNHS76F8GMCSF和PD1抗体联合治疗对小鼠体内乳腺癌生长的影响
将2×10 5个消化培养的小鼠乳腺癌细胞(4T1)注射到Balb/c小鼠(6-10周龄，雌性)身体右侧皮下，待肿瘤的长径达到6-8mm时开始进行治疗。
取实施例3制备的蛋白溶液25μl，用PBS调节总体积到200μl，使用29G的胰岛素注射器吸取蛋白溶液注射到小鼠尾静脉中，每天注射一次，共注射5次。注射后的小鼠放回笼内，记录小鼠肿瘤的生长情况。本实验共分为3组，1.未注射组，2.注射蛋白溶液组，3.注射蛋白溶液并用PD1抗体处理组(即联合治疗组)。其中，在联合治疗组中，自小鼠完成5次蛋白溶液注射后第2天开始，在其腹腔注射PD1抗体(具体为BioXcell公司，InVivoMAb anti-mouse PD-1(CD279)，货号BE0146)200μg，每3天注射一次，共注射4次。最后比较各组小鼠肿瘤的生长情况。结果如图5所示，可以看出，mIL12bIL12aIL2DiaNHS76F8GMCSF和PD1抗体联合治疗显著抑制了肿瘤的生长。
实施例9.mIL12bIL12aIL2DiaNHS76F8GMCSF和PD1抗体联合治疗对小鼠体内肺癌生长的影响
将2×10 5个消化培养的小鼠肺癌细胞(LLC)注射到C57BL/6小鼠(6-10周龄，雌性)身体右侧皮下，待肿瘤的长径达到6-8mm时开始进行治疗。
取实施例3制备的蛋白溶液25μl，用PBS调节总体积到200μl，使用29G的胰岛素注射器吸取蛋白溶液注射到小鼠尾静脉中，每天注射一次，共注射3次。注射后的小鼠放回笼内，记录小鼠肿瘤的生长情况。本实验共分为3组，1.未注射组，2.注射蛋白溶液组，3.注射蛋白溶液并用PD1抗体处理组(即联合治疗组)。其中，在联合治疗组中，自小鼠完成5次蛋白溶液注射后第2天开始，在其腹腔注射PD1抗体(具体为BioXcell公司，InVivoMAb anti-mouse PD-1(CD279)，货号BE0146)200μg，每3天注射一次，共注射4次。最后比较各组小鼠肿瘤的生长情况。结果如图6所示，可以看出，mIL12bIL12aIL2DiaNHS76F8GMCSF和PD1抗体联合治疗显著抑制了肿瘤的生长。

Claims (34)

  1. 一种药物组合物，其包括蛋白质和免疫检查点抑制剂，其中所述蛋白质包括融合蛋白，且所述融合蛋白中包含细胞因子IL12、IL2和GMCSF。
  2. 根据权利要求1所述的药物组合物，其中所述免疫检查点抑制剂包括PD1、PD-L1和/或CTLA-4的抑制剂。
  3. 根据权利要求1所述的药物组合物，其中所述细胞因子源自哺乳动物。
  4. 根据权利要求1-3中任一项所述的药物组合物，其中所述蛋白质还包括靶向部分。
  5. 根据权利要求4所述的药物组合物，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。
  6. 根据权利要求5所述的药物组合物，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)。
  7. 根据权利要求4-6中任一项所述的药物组合物，其中所述靶向部分包括抗体或其抗原结合片段。
  8. 根据权利要求4-7中任一项所述的药物组合物，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。
  9. 根据权利要求1-8中任一项所述的药物组合物，其中所述蛋白质为单链蛋白质。
  10. 根据权利要求9所述的药物组合物，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.27-52。
  11. 根据权利要求1-8中任一项所述的药物组合物，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。
  12. 根据权利要求11所述的药物组合物，其中所述第一多肽链包含IL12a，所述第二多肽链包含IL12b。
  13. 根据权利要求11-12中任一项所述的药物组合物，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。
  14. 根据权利要求11-13中任一项所述的药物组合物，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，且一个或多个所述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。
  15. 根据权利要求11-13中任一项所述的药物组合物，其中在所述第一多肽链中，从N端到C端依次包含所述IL12a或其功能性片段和所述IL2或其功能性片段；或者，在所述第一多肽链中，从N端到C端依次包含所述IL2或其功能性片段和所述IL12a或其功能性 片段；又或者，在所述第一多肽链中，从N端到C端依次包含所述IL12a或其功能性片段和所述GMCSF或其功能性片段。
  16. 根据权利要求11-13中任一项所述的药物组合物，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GMCSF或其功能性片段；或者，在所述第二多肽链中，从N端到C端依次包含所述GMCSF或其功能性片段和所述IL12b或其功能性片段；又或者，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IL2或其功能性片段。
  17. 根据权利要求11-16中任一项所述的药物组合物，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.57所示的氨基酸序列；
    b)所述第一多肽链包含SEQ ID NO.54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；
    c)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.58所示的氨基酸序列；
    d)所述第一多肽链包含SEQ ID NO.54所示的氨基酸序列且所述第二多肽链包含SEQ ID NO.58所示的氨基酸序列；
    e)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.59所示的氨基酸序列；
    f)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.60所示的氨基酸序列。
  18. 根据权利要求11-14中任一项所述的药物组合物，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；或者，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或其功能性片段以及所述GMCSF或其功能性片段。
  19. 根据权利要求11-14中任一项所述的药物组合物，其中在第二多肽链中，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。
  20. 根据权利要求11-19中任一项所述的药物组合物，其中，
    1)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.61所示的氨基酸序列；
    2)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.62所示的氨基酸序列；
    3)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.63所示的氨基酸序列；
    4)所述第一多肽链包含SEQ ID NO.67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；
    5)所述第一多肽链包含SEQ ID NO.68所示的氨基酸序列且所述第二多肽链包含SEQ ID NO.62所示的氨基酸序列；
    6)所述第一多肽链包含SEQ ID NO.69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；
    7)所述第一多肽链包含SEQ ID NO.70所示的氨基酸序列且所述第二多肽链包含SEQ ID NO.64所示的氨基酸序列；
    8)所述第一多肽链包含SEQ ID NO.71所示的氨基酸序列且所述第二多肽链包含SEQ ID NO.64所示的氨基酸序列；
    9)所述第一多肽链包含SEQ ID NO.72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。
  21. 一种试剂盒，其包括权利要求1-20中任一项所述的药物组合物。
  22. 权利要求1-20中任一项所述的药物组合物或权利要求21所述的试剂盒在制备药物中的用途，所述药物用于***。
  23. 根据权利要求22所述的用途，其中所述肿瘤包括实体瘤。
  24. 根据权利要求23所述的用途，其中所述肿瘤包括黑色素瘤。
  25. 根据权利要求23所述的用途，其中所述肿瘤包括乳腺癌。
  26. 根据权利要求23所述的用途，其中所述肿瘤包括肺癌。
  27. 权利要求1-20中任一项所述的药物组合物或权利要求21所述的试剂盒，其用于***。
  28. 一种***的方法，其包括向有需要的受试者施用权利要求1-20中任一项所述的药物组合物或权利要求21所述的试剂盒。
  29. 根据权利要求28所述的方法，其中所述施用包括先施用所述蛋白质，之后施用所述免疫检查点抑制剂。
  30. 根据权利要求28-29中任一项所述的方法，其中所述施用包括瘤内注射、静脉注射或皮下注射。
  31. 根据权利要求28-30中任一项所述的方法，其中所述肿瘤包括实体瘤。
  32. 根据权利要求28-31中任一项所述的方法，其中所述肿瘤包括黑色素瘤。
  33. 根据权利要求28-32中任一项所述的方法，其中所述肿瘤包括乳腺癌。
  34. 根据权利要求28-33中任一项所述的方法，其中所述肿瘤包括肺癌。
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