WO2018184484A1 - 细胞因子组合 - Google Patents

细胞因子组合 Download PDF

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WO2018184484A1
WO2018184484A1 PCT/CN2018/080479 CN2018080479W WO2018184484A1 WO 2018184484 A1 WO2018184484 A1 WO 2018184484A1 CN 2018080479 W CN2018080479 W CN 2018080479W WO 2018184484 A1 WO2018184484 A1 WO 2018184484A1
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functional variant
tumor
cytokine
cancer
cells
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PCT/CN2018/080479
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English (en)
French (fr)
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张晋宇
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张晋宇
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Priority to EP18781492.6A priority Critical patent/EP3607965A4/en
Priority to US16/500,429 priority patent/US11535656B2/en
Priority to CN201880023572.8A priority patent/CN110603048A/zh
Publication of WO2018184484A1 publication Critical patent/WO2018184484A1/zh
Priority to US18/056,375 priority patent/US20230203118A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2086—IL-13 to IL-16
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • 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]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • 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]
    • C07K14/5418—IL-7
    • 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]
    • C07K14/5443—IL-15

Definitions

  • This application relates to the field of cancer treatment.
  • the present application relates to a combination of cytokines comprising at least three cytokines and their use in treating tumors and/or preventing tumor recurrence or metastasis.
  • CAR-T chimeric antigen receptor T cell therapy
  • Checkpoint immune checkpoint therapy
  • the former can modify and kill T cells in a more accurate and effective way to identify and kill tumor cells in vivo.
  • Immunological checkpoints block the signaling pathways that inhibit T cell function in the tumor environment through antibody binding, allowing T cells to reactivate and kill tumor cells.
  • these two therapies are far from achieving the desired results.
  • CAR-T is not effective in solid tumors because the solid tumor is a dense, highly immunosuppressed environment, and it is difficult for CAR-T cells to enter the tumor in large quantities, and even after entering, it may be in the tumor.
  • the response rate of immunological checkpoint therapy is not high, usually only about 20-30%, indicating that only releasing T cells and re-activating them may not be enough to destroy the already formed tumors in most cases.
  • Tumor cells establish an immunosuppressive microenvironment within the tumor by secreting various proteins or direct contact.
  • a variety of immune cells, including macrophages, regulatory T cells (Treg), and bone marrow-derived inhibitory cells (MDSC) are involved in this role. Cracking the tumor immunosuppressive microenvironment is the key to the success of tumor immunotherapy. In this regard, CAR-T therapy and immune checkpoint therapy have not been done enough.
  • Cytokine is a very important immune signal in the body. Various cells secrete different cytokines, and various cytokines can act on different cells to form a complex cross-network, and the same cytokine may act on different cells in different environments, thus playing the opposite The function. None of the existing cytokines or combinations thereof have exhibited a completely satisfactory effect in antitumor.
  • the application provides cytokine combinations comprising specific cytokines and pharmaceutical compositions thereof.
  • the application provides a cytokine combination comprising at least three cytokines selected from the group consisting of IL12 or a functional variant thereof, GMCSF or a functional variant thereof, FLT3L or a functional change thereof , IL2 or a functional variant thereof, IL15 or a functional variant thereof, IL21 or a functional variant thereof and IL7 or a functional variant thereof.
  • the cytokine combination comprises any one of: i) IL12 or a functional variant thereof, GMCSF or a functional variant thereof, and IL2 or a functional variant thereof; ii) IL12 or a functional variant, GMCSF or a functional variant thereof, and IL15 or a functional variant thereof; iii) IL12 or a functional variant thereof, GMCSF or a functional variant thereof, and IL21 or a functional variant thereof; IL12 or a functional variant thereof, FLT3L or a functional variant thereof, and IL2 or a functional variant thereof; v) IL12 or a functional variant thereof, FLT3L or a functional variant thereof, and IL15 or its functionality Variant; vi) IL12 or a functional variant thereof, FLT3L or a functional variant thereof, and IL21 or a functional variant thereof; vii) IL12 or a functional variant thereof, GMCSF or a functional variant thereof, and IL7 Or a functional variant thereof; and
  • the cytokine is selected from the group consisting of a murine cytokine and a human cytokine.
  • the concentration of each cytokine in the combination of cytokines is 1-1000 ng/ ⁇ L.
  • the application provides one or more nucleic acid molecules encoding the combination of cytokines.
  • the nucleic acid molecule comprises two or more nucleic acid molecules, wherein each nucleic acid molecule encodes one or more of the cytokines, and each of the nucleic acid molecules encodes a cytokine with at least one other The nucleic acid molecules encode different cytokines.
  • the application provides a vector comprising the nucleic acid molecule.
  • the vector comprises two or more vectors, wherein each vector comprises one or more of the nucleic acid molecules, and each of the vectors comprises a cytokine encoded by the nucleic acid molecule and at least Another vector comprises nucleic acid molecules that encode different cytokines.
  • the application provides a cell comprising the nucleic acid molecule or the vector.
  • the cell comprises two or more cells, wherein each cell expresses one or more of the cytokines, and each of the cells expresses a cytokine expressed by at least another cell Different cytokines.
  • the application provides a pharmaceutical composition comprising the cytokine combination, the nucleic acid molecule, the vector or the cell, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is formulated for topical administration. In certain embodiments, the pharmaceutical composition is formulated for sustained release administration.
  • the pharmaceutical composition is formulated in a calcium alginate gel, a polylactic acid microsphere or a chitosan quaternary ammonium salt solution.
  • cytokine combination in another aspect, the use of a cytokine combination, a nucleic acid molecule, the vector, the cell, or the pharmaceutical composition described herein, in the preparation of a medicament, wherein the medicament is for treating a tumor And / or prevent tumor recurrence or metastasis.
  • the tumor is a solid tumor.
  • the tumor is selected from the group consisting of lung cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, brain and central nervous system cancer, pancreatic cancer, oral cancer, Nasopharyngeal cancer, head and neck cancer, laryngeal cancer, bone cancer, skin cancer, ovarian cancer, prostate cancer, testicular cancer, kidney cancer, bladder cancer, orbital tumor, leukemia and lymphoma.
  • the concentration of each cytokine in the combination of cytokines is 1-1000 ng/ ⁇ L.
  • the subject is a mammal.
  • the mammal is selected from the group consisting of human, mouse, rat, monkey, dog, pig, sheep, cow, and cat.
  • Figure 1 shows the therapeutic effect of mIL12+mGMCSF+mIL2 chitosan quaternary ammonium salt solution on mouse melanoma (B16F10).
  • Figure 2 shows the therapeutic effect of mIL12+mGMCSF+mIL2 chitosan quaternary ammonium salt solution on mouse colon cancer (CT26).
  • Figure 3 shows the therapeutic effect of mIL12+mGMCSF+mIL2 chitosan quaternary ammonium salt solution on mouse breast cancer (4T1).
  • Figure 4 shows the therapeutic effect of mIL12+mGMCSF+mIL2 chitosan quaternary ammonium salt solution on mouse lymphoma (EL4).
  • Figure 5 shows the therapeutic effects of different ratios of mIL12+mGMCSF+mIL2 chitosan quaternary ammonium salt solution on mouse breast cancer (4T1).
  • Figure 6 shows the therapeutic effect of mIL12+mGMCSF+mIL2 calcium alginate gel on mouse breast cancer (4T1).
  • Figure 7 shows the therapeutic effect of mIL12+mGMCSF+mIL2 polylactic acid microspheres on mouse breast cancer (4T1).
  • Figure 8 shows the therapeutic effect of cells expressing mIL12 + mGMCSF + mIL2 on mouse breast cancer (4T1).
  • Figure 9 shows the therapeutic effect of mIL12+mGMCSF+mIL15 chitosan quaternary ammonium salt solution on mouse breast cancer (4T1).
  • Figure 10 shows the therapeutic effect of mIL12+mGMCSF+mIL21 chitosan quaternary ammonium salt solution on mouse breast cancer (4T1).
  • Figure 11 shows the therapeutic effect of mIL12+mFLT3L+mIL2 chitosan quaternary ammonium salt solution on mouse breast cancer (4T1).
  • Figure 12 shows the therapeutic effect of mIL12+mFLT3L+mIL15 chitosan quaternary ammonium salt solution on mouse breast cancer (4T1).
  • Figure 13 shows the therapeutic effect of mIL12+mFLT3L+mIL21 chitosan quaternary ammonium salt solution on mouse breast cancer (4T1).
  • Figure 14 shows the therapeutic effect of hIL12+hGMCSF+hIL2 chitosan quaternary ammonium salt solution on mouse breast cancer (4T1).
  • Figure 15 shows the therapeutic effect of hIL12+hGMCSF+hIL15 chitosan quaternary ammonium salt solution on mouse breast cancer (4T1).
  • Figure 16 shows the therapeutic effect of hIL12+hGMCSF+hIL21 chitosan quaternary ammonium salt solution on mouse breast cancer (4T1).
  • Figure 17 shows the therapeutic effect of hIL12+hFLT3L+hIL2 chitosan quaternary ammonium salt solution on mouse breast cancer (4T1).
  • Figure 18 shows the therapeutic effect of hIL12+hFLT3L+hIL15 chitosan quaternary ammonium salt solution on mouse breast cancer (4T1).
  • Figure 19 shows the therapeutic effect of hIL12+hFLT3L+hIL21 chitosan quaternary ammonium salt solution on mouse breast cancer (4T1).
  • Figure 20 shows tumor growth in mouse breast cancer (4T1) untreated.
  • Figure 21 shows the resilience of cured mice to re-inoculation of tumors.
  • Figure 22 shows the therapeutic effect of mIL12+mGMCSF+mIL7 chitosan quaternary ammonium salt solution on mouse colon cancer (CT26).
  • Figure 23 shows the therapeutic effect of mIL12+mFLT3L+mIL7 chitosan quaternary ammonium salt solution on mouse colon cancer (CT26).
  • Figure 24 shows the therapeutic effect of hIL12 + hGMCSF + hIL7 chitosan quaternary ammonium salt solution on mouse colon cancer (CT26).
  • Figure 25 shows the therapeutic effect of hIL12+hFLT3L+hIL7 chitosan quaternary ammonium salt solution on mouse colon cancer (CT26).
  • Figure 26 shows the therapeutic effect of mIL12 chitosan quaternary ammonium salt solution on mouse melanoma (B16F10).
  • Figure 27 shows the therapeutic effect of mGMCSF chitosan quaternary ammonium salt solution on mouse melanoma (B16F10).
  • Figure 28 shows the therapeutic effect of mIL2 chitosan quaternary ammonium salt solution on mouse melanoma (B16F10).
  • Figure 29 shows the therapeutic effect of mIL12+mGMCSF chitosan quaternary ammonium salt solution on mouse melanoma (B16F10).
  • Figure 30 shows the therapeutic effect of mIL12+mIL2 chitosan quaternary ammonium salt solution on mouse melanoma (B16F10).
  • Figure 31 shows the therapeutic effect of mGMCSF + mIL2 chitosan quaternary ammonium salt solution on mouse melanoma (B16F10).
  • the term "functional variant” generally refers to a fragment or variant having the function of a cytokine.
  • the functional fragment or variant may have one or more substitutions, deletions or additions based on the amino acid sequence of the cytokine (eg, 1-2, 1-3, 1-4 Amino acid sequence obtained after 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more amino acids, and still retains the cytokine Features.
  • the functional fragment or variant may have at least about 80% or more (eg, at least about 80% or more, about 85% or more, about 90% or more, about 91% or more, and the amino acid sequence of the cytokine, An amino acid sequence of about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or more homologous, And still maintain the function of the cytokine.
  • the functional variant may be a natural variant (such as an allelic variant) or an artificially modified variant.
  • the homology has the art-recognized meaning, and the disclosed techniques can be used to calculate the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions, for example, along a polynucleotide or polypeptide.
  • Sequence homology is measured over the full length or along the region of the molecule (for example, see: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje , G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).
  • the functional variants described can be obtained by isolation, recombinant expression or chemical synthesis from its natural source.
  • a functional variant of the cytokine can be prepared.
  • a polyethylene glycol group (PEG) is added to the cytokine.
  • the term "cytokine” generally refers to a small molecule polypeptide that is secreted by immune cells and that modulates cellular function.
  • the cytokines have important regulatory roles for cell-cell interactions, cell growth and differentiation.
  • the cytokine can be selected from one or more of the group consisting of: interleukins and colony stimulating factors.
  • the interleukin is a type of cytokine produced by a variety of cells and acting on a variety of cells, initially referring only to cytokines produced by white blood cells and acting between white blood cells. Interleukins transmit information, activate and regulate immune cells, mediate T, B cell activation, proliferation and differentiation, and play an important role in inflammatory responses.
  • the interleukin can be selected from one or more of the group consisting of IL12, IL2, IL15, IL21, and IL7.
  • the colony stimulating factor refers to a cytokine that stimulates different hematopoietic stem cells to form cell colonies in a semi-solid medium. The colony stimulating factor plays a role in promoting proliferation and differentiation of hematopoietic stem cells at different developmental stages.
  • the colony stimulating factors may be named granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), granulocytes and macrophage colonies, respectively.
  • the colony stimulating factor can be selected from one or more of the group consisting of FMS-associated tyrosine kinase 3 ligand (FTL3L) and granulocyte macrophage colony stimulating factor (GMCSF).
  • IL2 generally refers to interleukin-2, which plays an important role in the immune response and antiviral infection of the body, and stimulates the proliferation of T cells that have been activated by specific antigens; T cells, promote cytokine production; stimulate NK cell proliferation, enhance NK killing activity and produce cytokines, induce LAK cell production; and promote B cell proliferation and secretion of antibodies; activate macrophages.
  • T cells promote cytokine production
  • B cell proliferation and secretion of antibodies activate macrophages.
  • mouse IL2 may comprise the amino acid sequence set forth in SEQ ID NO: 5.
  • the human cytokine IL2 hIL2
  • IL12 generally refers to interleukin-12, which stimulates proliferation of activated t cells, promotes differentiation of Th0 cells into Th1 cells, and induces cytotoxic activity of CTL and NK cells. Promote its secretion of cytokines such as IFN- ⁇ , TNF- ⁇ , GM-CSF; or promote the expression of NK cells and IL-2R ⁇ receptors and CD56 molecules, and enhance the ADCC effect on tumor cells.
  • the molecule of IL12 is a heterodimer, and the two subunits of the p40 subunit (40 kd) and the p35 subunit (35 kd) are linked by a disulfide bond.
  • the p35 subunit in mouse IL12 may comprise the amino acid sequence set forth in SEQ ID NO: 1
  • the p40 subunit may comprise the amino acid sequence set forth in SEQ ID NO: 2.
  • the p35 subunit in human IL12 may comprise the amino acid sequence set forth in SEQ ID NO: 8
  • the p40 subunit may comprise the amino acid sequence set forth in SEQ ID NO: 9.
  • IL15 generally refers to interleukin-15, which is produced by a variety of cells, such as activated monocytes-macrophages, epidermal cells and fibroblasts, which induce B cell proliferation and differentiation, Can play a biological activity similar to IL2.
  • mouse IL15 may comprise the amino acid sequence set forth in SEQ ID NO: 6.
  • human IL15 can comprise the amino acid sequence set forth in SEQ ID NO: 13.
  • IL7 generally refers to interleukin-7, which is secreted mainly by thymus and bone marrow stromal cells and belongs to a glycoprotein having a relative molecular mass of 25,000 to 28,000. IL7 and its receptor-mediated signal transduction pathways are mainly through the three pathways of Janus kinase, signal transduction and transcriptional activator and phosphoinositide 3-kinase.
  • mouse IL7 may comprise the amino acid sequence set forth in SEQ ID NO: 15.
  • human IL7 hIL7 can comprise the amino acid sequence set forth in SEQ ID NO: 16.
  • IL21 generally refers to interleukin-21, which is secreted by activated CD4 + T cells; IL21 is involved in the regulation of B cell proliferation, and deletion of the IL21 gene may result in the organism being more susceptible to infection by bacteria or viruses.
  • IL21 can affect the expression level of IL2 receptor protein (CD25) by regulating Bcl-6 protein.
  • mouse IL21 mIL21
  • human IL21 hIL21
  • FTL3L generally refers to a FMS-associated tyrosine kinase 3 ligand which modulates the proliferation and differentiation of non-erythroid hematopoietic stem cells and promotes pre-B lymphocytes, dendritic cells, NK cells, cells.
  • the proliferation, differentiation and maturation of toxic T lymphocytes have important anti-tumor effects.
  • mouse FTL3L may comprise the amino acid sequence set forth in SEQ ID NO:4.
  • human FTL3L hFTL3L
  • GMCSF generally refers to granulocyte macrophage colony-stimulating factor, which stimulates the proliferation, differentiation and activation of granulocytes and macrophages, increases hematopoiesis, and enhances neutrophils and neutrophils. Multiple functions of acidic cells and monocytes. GMCSF can enhance the immune activity of immune effector cells to phagocytose bacteria and kill cancer cells, and is beneficial to restore neutrophil deficiency caused by tumor chemotherapy and bone marrow transplantation.
  • mouse GMCSF may comprise the amino acid sequence set forth in SEQ ID NO:3.
  • human GMCSF hGMCSF
  • nucleic acid molecule generally refers to an isolated form of nucleotide, deoxyribonucleotide or ribonucleotide or analog thereof of any length isolated or artificially synthesized from its natural environment.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vector may include a vector mainly for inserting DNA or RNA into a cell, a vector mainly for replicating DNA or RNA, and a vector mainly for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having a plurality of the above functions.
  • the vector may be a polynucleotide that is capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.
  • the term "cell” generally refers to a plasmid or vector that may or already contains a nucleic acid molecule as described herein, or an individual cell, cell line or cell capable of expressing a cytokine/cytokine combination as described herein.
  • the cell can comprise a progeny of a single host cell. Due to natural, unexpected or deliberate mutations, the progeny cells may not necessarily be identical in morphology or genomic to the original parental cell, but are capable of expressing the cytokine/cytokine combinations described herein.
  • the cells can be obtained by transfecting the cells in vitro using the vectors described herein.
  • the term "pharmaceutically acceptable carrier” generally refers to one of the components of a pharmaceutical composition, which may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids. , sugars, chelating agents, counterions, metal complexes and/or nonionic surfactants, and the like.
  • the pharmaceutically acceptable carrier may include an excipient, for example, the excipient may be selected from the group consisting of starch, dextrin, sucrose, lactose, magnesium stearate, calcium sulfate, carboxymethyl And talcum powder.
  • the pharmaceutically acceptable carrier can also be selected from the group consisting of pH adjusters, osmotic pressure regulators, solubilizers, and bacteriostats.
  • the term "tumor” generally refers to or describes the physiological condition of a mammal, which is typically characterized by cell proliferation or a disorder of survival.
  • the tumor may be a solid tumor; for example, the solid tumor may be selected from the group consisting of lung cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, brain and central nervous system cancer.
  • metastasis generally refers to a process in which malignant tumor cells continue to grow from the primary site, through lymphatic vessels, blood vessels or direct spread, to other sites. Metastasis of malignant tumors is often the main cause of failure in cancer treatment.
  • relapse generally refers to a tumor recurrence induced by residual tumor cells in the body due to incomplete treatment.
  • the term "about” generally means a range of 0.5% to 10% above or below a specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5% above or below a specified value, Variations 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 inventors of the present application have surprisingly found that a combination of at least three cytokines selected from the group consisting of IL12, GMCSF, FLT3L, IL2, IL15, IL21 and IL7 is simultaneously administered to the inside of a tumor to locally activate the immune system in the tumor. It breaks the tumor immunosuppressive microenvironment and stimulates the immune response against the tumor, thereby effectively eliminating the tumor.
  • the application provides a cytokine combination comprising at least three (at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or more) cytokines selected from the group consisting of IL12 or a functional variant thereof, GMCSF or a functional variant thereof, FLT3L or a functional variant thereof , IL2 or a functional variant thereof, IL15 or a functional variant thereof, IL21 or a functional variant thereof and IL7 or a functional variant thereof.
  • the combination of cytokines used in the present application exhibits a strong synergistic effect, whereas administration of a single cytokine, or a combination of two cytokines, does not effectively eliminate the effects of the tumor.
  • One of the keys to the present application is to design a specific combination of proteins which do not have a significant anti-tumor effect, and to obtain an excellent anti-tumor effect by a suitable administration method.
  • an anti-tumor immune response can be stimulated in a subject in need thereof, and the activated immune system in the subject in need can recognize and destroy the tumor cells, and after the tumor is cleared, Sexual immune responses produce systemic immune memory.
  • the activated specific tumor-specific immune cells can circulate throughout the body through the blood and lymphatic system, thereby inhibiting and even clearing the lesions in other parts; on the other hand, the presence of immune memory causes the body to reappear When tumor cells have similar antigens, the immune system can quickly recognize and kill these cells, thereby avoiding tumor recurrence.
  • the cytokine in the cytokine combination of the present application acts directly on the immune system, so there is no resistance mechanism similar to a tumor-targeted drug, and it can be used repeatedly.
  • the examples of the present application show that the tumor in the mouse can be successfully cleared by administering the cytokine combination of the present application, and after inoculation of the same kind of tumor cells after a certain time, the tumor can no longer be formed.
  • cytokines since different cytokines have different mechanisms of action, a combination of various cytokines is used in the present application, so that natural immunity and acquired immunity can be simultaneously activated to achieve effective tumor treatment. For example, using a combination of three cytokines, it is possible to simultaneously activate the desired components of the immune system to achieve optimal anti-tumor immunity. If a combination of fewer cytokines is used, for example a combination comprising only two cytokines, the ability to activate certain components will be absent and the best results will not be achieved.
  • the cytokine combination can comprise three cytokines.
  • the cytokine combination can comprise any of the following groups:
  • IL12 or a functional variant thereof GMCSF or a functional variant thereof, and IL2 or a functional variant thereof;
  • IL12 or a functional variant thereof GMCSF or a functional variant thereof, and IL15 or a functional variant thereof;
  • IL12 or a functional variant thereof, GMCSF or a functional variant thereof, and IL21 or a functional variant thereof;
  • Iv IL12 or a functional variant thereof, FLT3L or a functional variant thereof, and IL2 or a functional variant thereof;
  • IL12 or a functional variant thereof FLT3L or a functional variant thereof, and IL15 or a functional variant thereof;
  • IL12 or a functional variant thereof FLT3L or a functional variant thereof, and IL21 or a functional variant thereof;
  • IL12 or a functional variant thereof GMCSF or a functional variant thereof, and IL7 or a functional variant thereof;
  • IL12 or a functional variant thereof FLT3L or a functional variant thereof, and IL7 or a functional variant thereof.
  • mouse IL2 may comprise the amino acid sequence set forth in SEQ ID NO: 5; human cytokine IL2 (hIL2) may comprise the amino acid sequence set forth in SEQ ID NO: 12.
  • the p35 subunit in mouse IL12 may comprise the amino acid sequence set forth in SEQ ID NO: 1
  • the p40 subunit may comprise the amino acid sequence set forth in SEQ ID NO: 2
  • human IL12 The p35 subunit in hIL12
  • the p40 subunit may comprise the amino acid sequence set forth in SEQ ID NO: 9.
  • mouse IL15 may comprise the amino acid sequence set forth in SEQ ID NO: 6; human IL15 (hIL15) may comprise the amino acid sequence set forth in SEQ ID NO: 13.
  • mouse IL21 may comprise the amino acid sequence set forth in SEQ ID NO: 7; human IL21 (hIL21) may comprise the amino acid sequence set forth in SEQ ID NO: 14.
  • mouse FTL3L may comprise the amino acid sequence set forth in SEQ ID NO: 4; human FTL3L (hFTL3L) may comprise the amino acid sequence set forth in SEQ ID NO: 11.
  • mouse GMCSF may comprise the amino acid sequence set forth in SEQ ID NO: 3; human GMCSF (hGMCSF) may comprise the amino acid sequence set forth in SEQ ID NO: 10.
  • mouse IL7 may comprise the amino acid sequence set forth in SEQ ID NO: 15; human IL7 (hIL7) may comprise the amino acid sequence set forth in SEQ ID NO: 16.
  • the functional variant may have substantially the same function as the cytokine (eg, IL2, IL12, IL15, IL21, FTL3L, GMCSF, or IL7) and have at least about 80 thereto % or more (eg, at least about 80% or more, about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, Amino acid sequence of about 97% or more, about 98% or more, about 99% or more or more homology.
  • cytokine eg, IL2, IL12, IL15, IL21, FTL3L, GMCSF, or IL7
  • at least about 80 thereto % or more eg, at least about 80% or more, about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more
  • the functional variant has substantially the same function as the cytokine (eg, IL2, IL12, IL15, IL21, FTL3L, GMCSF, or IL7) and comprises a Or multiple (for example, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 An amino acid sequence of addition, deletion or substitution of an amino acid.
  • cytokine eg, IL2, IL12, IL15, IL21, FTL3L, GMCSF, or IL7
  • a Or multiple for example, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10
  • An amino acid sequence of addition, deletion or substitution of an amino acid for example, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10
  • the cytokine is a mammalian cytokine, such as a murine cytokine or a human cytokine.
  • the concentration of each cytokine in the cytokine combination may be 1-1000 ng/ ⁇ L (for example, 1-1000 ng/ ⁇ L, 1-1000 ng/ ⁇ L, 2-10000 ng/ ⁇ L, 4-10000 ng). / ⁇ L, 6-100,000 ng/ ⁇ L, 8-10000 ng/ ⁇ L, 10-10000 ng/ ⁇ L, 10-1000 ng/ ⁇ L, 100-1000 ng/ ⁇ L or 10-500 ng/ ⁇ L).
  • the ratio of each cytokine in the cytokine combination may be any ratio as long as the dose of each cytokine reaches a respective therapeutically effective amount.
  • the ratio of three different cytokines in the cytokine combination can be x:y:z, where x, y, and z are independently, integers selected from 1-10 (eg, x, y, and z are independent An integer selected from the group consisting of 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10).
  • the ratio of three different cytokines in the cytokine combination can be 5:1:1, 1:5:1, 1:1:5, 4:1:1, 1:4: 1, 1:1:4, 3:1:1, 1:3:1, 1:1:3, 2:1:1, 1:2:1, 1:1:2 or 1:1:1.
  • the ratio of three different cytokines in the cytokine combination can be 1:1:1.
  • each cytokine in the cytokine combination may be administered simultaneously or at appropriate intervals (eg, may be 0.5-5 days after administration of the first and/or second cytokine) , 1-4 days, 1-3 days, 1-2 days, 11-24 hours, 10-24 hours, 9-24 hours, 8-24 hours, 7-24 hours, 6-24 hours, 5-24 hours, 4-24 hours, 3-24 hours, 2-24 hours, 1-24 hours, 55-60 minutes, 50-60 minutes, 45- 60 minutes, 40-60 minutes, 35-60 minutes, 30-60 minutes, 25-60 minutes, 20-60 minutes, 15-60 minutes, 10-60 minutes, 5-60 minutes Apply separately, at 4-60 minutes, at 3-60 minutes, at 2-60 minutes, at 1-60 minutes, at 0.5-60 minutes, or at other intervals.
  • each cytokine can be administered simultaneously.
  • the cytokines included in the cytokine combinations described herein have a strong ability to activate the immune system and thereby fight tumors, which have been used clinically for many years. However, it is precisely because these cytokines are too powerful, and low-dose systemic use can lead to more serious side effects. However, it is difficult to achieve a sufficient therapeutic concentration in the tumor. In the present application, the effect of local enrichment of cytokines in the cytokine combination in the tumor can be obtained by direct administration at the tumor site, thereby avoiding the occurrence of systemic side effects.
  • the present application has been verified by experiments that the intratumoral injection of the cytokine combination of the present application does not have a specific abnormal reaction in the mice, and it can be seen that the cytokine combination described in the present application does not bring serious problems to the subjects in need. side effect.
  • the cytokine combination can be formulated for topical administration.
  • it can be formulated for intratumoral administration.
  • the cytokine combination can be formulated for sustained release administration. This is because if cytokines are injected into the tumor site only with water or physiological saline as a carrier, these cytokines cannot stay for a long time and are quickly taken away by the circulatory system, seriously affecting their function.
  • the cytokine can be enriched for a longer period of time at the site of administration by sustained release techniques, thereby obtaining a longer residence and duration of action.
  • sustained release can be achieved by controlling the dissolution rate of the cytokine, controlling the diffusion rate of the cytokine, or using dissolution, osmosis, or using an ion exchange method or the like.
  • the cytokine in the cytokine combination can be prepared as a polylactic acid (PLA) microsphere, or in a calcium alginate gel, or by adding a chitosan quaternary ammonium salt to increase the viscosity of the cytokine solution, thereby The cytokine is retained for a longer period of time at the injection site.
  • PLA polylactic acid
  • Other sustained release techniques known in the art, such as polylactic acid-glycolic acid copolymer (PLGA) microspheres, temozolomide sustained release microspheres, block copolymer micelles, and slow release pumps, etc., can also be applied to the Cytokine combination.
  • the cytokine combinations of the present application can be administered into a tumor by any suitable means.
  • the cytokine combination can be injected directly into the tumor site.
  • intravenously administered cytokines can be enriched at the tumor site by conjugation to a tumor-targeting substance such as an antibody.
  • the application provides nucleic acids, one or more nucleic acid molecules encoding the combination of cytokines.
  • the nucleic acid molecule comprises two or more (eg, one or more, two or more, three or more, four or more, or more) nucleic acid molecules, wherein each nucleic acid molecule encodes one Or a plurality (for example, one or more, two or more, three or more, four or more or more) of the cytokines, and each of the nucleic acid molecules encodes a cytokine and at least another of the nucleic acids Molecularly encoded cytokines are different.
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by (i) amplification in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) production by clonal recombination, (iii) purification. , for example, by fractionation by gel digestion and gel electrophoresis, or (iv) synthetic, for example by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule produced by recombinant DNA techniques.
  • nucleic acids encoding the antibodies, antigen-binding fragments or variants thereof can be prepared by a variety of methods known in the art including, but not limited to, restriction fragment manipulation or synthetic oligo. Overlapping PCR of nucleotides, see Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.
  • the application provides a vector comprising the nucleic acid molecule.
  • the vector comprises two or more (eg, one or more, two or more, three or more, four or more, or more) vectors, wherein each vector comprises one or more (for example, one or more, two or more, three or more, four or more or more) of the nucleic acid molecules, and each of the vectors comprises a cytokine encoded by the nucleic acid molecule and at least another vector
  • the nucleic acid molecules contained contain different cytokines.
  • the vector may be a linear nucleic acid fragment, a circular plasmid or a viral vector (such as vaccinia virus, adenovirus, adeno-associated virus, lentivirus, etc.).
  • the nucleotide sequence encoding the cytokine can be operably linked to an expression control sequence.
  • the "regulatory sequence” may be used interchangeably with “regulatory element”, which may refer to being located upstream (5' non-coding sequence), intermediate or downstream (3' non-coding sequence) of the coding sequence, affecting the relevant coding sequence. Transcription, RNA processing or stability or translation of nucleotide sequences.
  • regulatory sequences can include, but are not limited to, a promoter, a translation leader sequence, an intron, and a polyadenylation recognition sequence.
  • "operably linked” means that a regulatory element (such as, but not limited to, a promoter sequence, a transcription termination sequence, etc.) can be ligated to a nucleic acid sequence (eg, a coding sequence or an open reading frame) such that transcription of the nucleotide sequence Controlled and regulated by the transcriptional regulatory elements.
  • a regulatory element such as, but not limited to, a promoter sequence, a transcription termination sequence, etc.
  • the application also provides cells that can be administered to a tumor site.
  • the cell comprises the nucleic acid molecule or the vector.
  • the cell comprises two or more (eg, one or more, two or more, three or more, four or more, or more) cells, wherein each cell expresses one or more (for example, one or more, two or more, three or more, four or more or more) of the cytokines, and each of the cells expresses a cytokine different from a cytokine expressed by at least another cell.
  • each cell expresses one or more (for example, one or more, two or more, three or more, four or more or more) of the cytokines, and each of the cells expresses a cytokine different from a cytokine expressed by at least another cell.
  • the cells can express all three cytokines (A, B, C); alternatively, the cells can comprise two cells, one of which Expressing one cytokine (A or B or C), another cell expressing two other cytokines (B and C; or A and C; or A and B); alternatively, the cell may comprise three types of cells, Each cell expresses a different, one cytokine.
  • the cells can continuously express the cytokines of the present application at the tumor site, thereby maintaining a certain cytokine concentration in the tumor for a long time, and exerting an anti-tumor effect.
  • methods of introducing the vector into the cell include, but are not limited to, calcium phosphate transfection, protoplast fusion, electroporation, lipofection, microinjection, and viral infection.
  • the cell may be a eukaryotic cell, such as a mammalian cell.
  • the mammal may be selected from the group consisting of human, mouse, rat, monkey, dog, pig, sheep, cow, and cat.
  • the mammal can be a human.
  • the cell can be a 293 cell.
  • the present application also provides methods of making the cytokine combinations/cytokines.
  • the method can include culturing the cell under conditions such that the cytokine combination/cytokine expression.
  • such methods can be understood by those of ordinary skill in the art by using appropriate media, appropriate temperatures and incubation times, and the like.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the cytokine combination, the nucleic acid molecule, the vector or the cell, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable adjuvant may include a solvent, a dispersion medium, a coating, an antibacterial agent and an antifungal agent, an antioxidant, an isotonic agent, an absorption delaying agent, and an excipient.
  • a solvent e.g., a solvent, a dispersion medium, a coating, an antibacterial agent and an antifungal agent, an antioxidant, an isotonic agent, an absorption delaying agent, and an excipient.
  • buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and/or nonionic surfactants, and the like can be included.
  • the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal Administration or administration via a subcutaneous reservoir.
  • the pharmaceutical composition is formulated for topical administration.
  • it can be administered intratumorally.
  • the pharmaceutical composition is formulated for sustained release administration.
  • the pharmaceutical composition is formulated in a calcium alginate gel, a polylactic acid microsphere or a chitosan quaternary ammonium salt solution.
  • the pharmaceutical composition can be used to inhibit tumor growth.
  • the pharmaceutical compositions of the present application can inhibit or delay the progression or progression of the disease, can reduce tumor size (even substantially eliminate tumors), and/or can alleviate and/or stabilize disease states.
  • the pharmaceutical compositions described herein can comprise a therapeutically effective amount of the combination of cytokines.
  • the therapeutically effective amount is a dose that is capable of preventing and/or treating (at least partially treating) a condition or disorder (eg, cancer) and/or any of its complications in a subject having or at risk of developing.
  • the "effective amount” can mean inhibiting tumor cell growth or tumor area growth by at least about 10% (eg, at least about 10%, 20%, 30%, 40) as compared to not administering the pharmaceutical composition. %, 50%, 60%, 70%, 80%, 90%, 99% or 100%) of the desired dose of the pharmaceutical composition to be administered.
  • the ability to inhibit the growth of tumor area can be evaluated by the relevant animal model of the tumor. Alternatively, it can also be evaluated by examining the ability to inhibit cell growth.
  • the pharmaceutical composition described in the present application can significantly reduce the severity of the disease, increase the frequency of the asymptomatic phase of the disease, prolong the time of the asymptomatic period of the disease, prevent damage or disability caused by the pain of the disease, and/or prevent Or treat metastasis or recurrence of the tumor.
  • the pharmaceutical composition When administered, the pharmaceutical composition may be administered at a dose of from about 0.0001 to 100 mg per kg of the body weight of the subject in need thereof (for example, may be 0.0001 to 100 mg/kg, 0.01 to 100 mg/kg, 0.05 to 100 mg). /Kg, 0.1-100 mg/Kg, 0.5-100 mg/Kg, 1-100 mg/Kg, 5-100 mg/Kg or 0.01-20 mg/Kg).
  • the dosage regimen of the pharmaceutical composition may be once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, every 3-6 Once a month, or the initial dosing interval is slightly shorter (eg, once a week to once every three weeks), the post-dosing interval is lengthened (eg, once a month to once every 3-6 months).
  • Specific dosing regimens are determined based on the disease progression of the subject in need thereof.
  • the pharmaceutical composition can be administered to a mouse when the tumor area reaches a certain threshold.
  • the application provides a method of treating a tumor and/or preventing tumor recurrence or metastasis comprising administering to a subject in need thereof the combination of a cytokine, the nucleic acid molecule, the vector, the cell, or Said pharmaceutical composition.
  • the application provides a combination of the cytokine, the nucleic acid molecule, the vector, the cell, or the pharmaceutical composition described for treating a tumor and/or preventing tumor recurrence or metastasis.
  • the present application also provides the use of the cytokine combination, the nucleic acid molecule, the vector, the cell, or the pharmaceutical composition for the preparation of a medicament, wherein the medicament is for treating a tumor and / or prevent tumor recurrence or metastasis.
  • the tumor can be a solid tumor.
  • the tumor may be selected from the group consisting of lung cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, brain and central nervous system cancer, pancreatic cancer, oral cancer, nasopharyngeal cancer, Head and neck cancer, laryngeal cancer, bone cancer, skin cancer, ovarian cancer, prostate cancer, testicular cancer, kidney cancer, bladder cancer, eyelid tumor, leukemia and lymphoma.
  • the concentration of each cytokine may be 1-1000 ng/ ⁇ L (for example, 1-100,000 ng/ ⁇ L, 1-1000 ng/ ⁇ L, 2-10000 ng). / ⁇ L, 4-10000 ng/ ⁇ L, 6-10000 ng/ ⁇ L, 8-10000 ng/ ⁇ L, 10-10000 ng/ ⁇ L, 10-1000 ng/ ⁇ L, 10-500 ng/ ⁇ L or 1-100 ng/ ⁇ L).
  • the administration can be topical.
  • it can be administered intratumorally.
  • the administration can be a single administration or multiple administrations.
  • it can be administered twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, every three to six months, or
  • the initial dosing interval is slightly shorter (eg, once a week to once every three weeks) and the post-dosing interval is lengthened (eg, once a month to once every 3-6 months).
  • the subject is a mammal.
  • the mammal is selected from the group consisting of human, mouse, rat, monkey, dog, pig, sheep, cow, and cat.
  • the subject is a human.
  • DMEM medium 1640 medium, fetal bovine serum purchased from Life Technologies
  • cell culture flasks culture plates purchased from Corning
  • Puromycin purchased from Life Technologies
  • restriction enzymes purchased from Takara and NEB
  • T4 DNA ligase was purchased from NEB
  • DNA polymerase was purchased from Takara
  • plasmid extraction kit was purchased from Omega Biotech
  • primer synthesis, gene synthesis and sequencing were performed by Life Technologies.
  • mIL12, mGMCSF, mFLT3L, mIL2, mIL15, mIL21, mIL7 represent mouse IL12, mouse GMCSF, mouse FLT3L, mouse IL2, mouse IL15, mouse IL21 and mouse IL7, respectively.
  • hIL12, hGMCSF, hFLT3L, hIL2, hIL15, hIL21, hIL7 represent human IL12, human GMCSF, human FLT3L, human IL2, human IL15, human IL21 and human IL7, respectively.
  • the above cytokines were purchased from Peprotech.
  • mice Female C57BL/6 and Balb/c mice, 6-8 weeks old, were purchased from Beijing Huakang Biotechnology Co., Ltd.
  • each fold line represents the area of the tumor in a mouse.
  • Example 1 Therapeutic effect of mIL12+mGMCSF+mIL2 chitosan quaternary ammonium salt solution on mouse melanoma
  • the cytokines mIL12, mGMCSF and mIL2 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l, and 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of mIL12+mGMCSF+mIL2, and then a pre-configured 3% shell was added. 45 ⁇ l of the glycan quaternary ammonium salt solution, carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • C26 Digested and cultured mouse colon cancer cells (CT26), 5 ⁇ 10 5 cells were injected into the right side of the body of Balb/c mice, and treatment was started when the long diameter of the tumor reached 7-9 mm.
  • the cytokines mIL12, mGMCSF and mIL2 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l, and 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of mIL12+mGMCSF+mIL2, and then a pre-configured 3% shell was added. 45 ⁇ l of the glycan quaternary ammonium salt solution, carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 3 Therapeutic effect of mIL12+mGMCSF+mIL2 chitosan quaternary ammonium salt solution on mouse breast cancer
  • the cytokines mIL12, mGMCSF and mIL2 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l, and 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of mIL12+mGMCSF+mIL2, and then a pre-configured 3% shell was added. 45 ⁇ l of the glycan quaternary ammonium salt solution, carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 4 Therapeutic effect of mIL12+mGMCSF+mIL2 chitosan quaternary ammonium salt solution on mouse lymphoma
  • Digested cultured mouse lymphoma cells EL4
  • 2 ⁇ 10 6 cells were injected into the right side of the body of C57BL/6 mice, and treatment was started when the long diameter of the tumor reached 7-9 mm.
  • the cytokines mIL12, mGMCSF and mIL2 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l, and 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of mIL12+mGMCSF+mIL2, and then a pre-configured 3% shell was added. 45 ⁇ l of the glycan quaternary ammonium salt solution, carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 5 Therapeutic effect of different ratios of mIL12+mGMCSF+mIL2 chitosan quaternary ammonium salt solution on mouse breast cancer
  • the cytokines mIL12, mGMCSF and mIL2 were solubilized in sterile water, and 200 ng/ ⁇ l and 1000 ng/ ⁇ l solutions were prepared for each cytokine, and 15 ⁇ l of 1000 ng/ ⁇ l of mIL12 and 15 ⁇ l of 200 ng/ ⁇ l of mGMCSF and 15 ⁇ l of 200 ng/ ⁇ l of mIL2 were used. After mixing, a mixture of 45 ⁇ l of mIL12+mGMCSF+mIL2 (5:1:1) was obtained, and then 45 ⁇ l of a pre-configured 3% chitosan quaternary ammonium salt solution was added, and carefully mixed with a pipette tip.
  • 15 ⁇ l of 200 ng/ ⁇ l of mIL12 and 15 ⁇ l of 1000 ng/ ⁇ l of mGMCSF and 15 ⁇ l of 200 ng/ ⁇ l of mIL2 were mixed to obtain a mixture of 45 ⁇ l of mIL12+mGMCSF+mIL2 (1:5:1), followed by a pre-configured 3% 45 ⁇ l of chitosan quaternary ammonium salt solution, carefully mixed with a pipette tip.
  • 15 ⁇ l of 200 ng/ ⁇ l of mIL12 and 15 ⁇ l of 200 ng/ ⁇ l of mGMCSF and 15 ⁇ l of 1000 ng/ ⁇ l of mIL2 were mixed to obtain a mixture of 45 ⁇ l of mIL12+mGMCSF+mIL2 (1:1:5), followed by a pre-configured 3% 45 ⁇ l of chitosan quaternary ammonium salt solution, carefully mixed with a pipette tip.
  • mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 6 Therapeutic effect of mIL12+mGMCSF+mIL2 calcium alginate gel on mouse breast cancer
  • the cytokines mIL12, mGMCSF and mIL2 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l, and 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of mIL12+mGMCSF+mIL2, and then 1.5 ⁇ l of 2M calcium chloride solution was added. , mix, and finally add 45 ⁇ l of 1% sodium alginate solution, mix with a pipette to form a gel.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine calcium alginate gel was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce the solution. overflow. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 7 Therapeutic effect of mIL12+mGMCSF+mIL2 polylactic acid microspheres on mouse breast cancer
  • Example 8 Therapeutic effect of cells expressing mIL12+mGMCSF+mIL2 on mouse breast cancer
  • the vector pLentis-CMV-MCS-IRES-PURO was digested in the EP tube, and the system was as follows: plasmid 2 ⁇ g, 3 ⁇ l of digestion buffer, 1 ⁇ l of BamHI, 1 ⁇ l of XhoI, water was added to a total volume of 30 ⁇ l, and allowed to stand at 37 ° C for 12 hours.
  • the EP tube was taken out, 3.3 ⁇ l of 10 ⁇ loading buffer was added, electrophoresis was carried out with a 1% agarose gel, and the carrier fragment was recovered after electrophoresis, and was used.
  • the DNA sequences corresponding to the coding regions of the mIL12, mGMCSF and mIL2 genes were synthesized separately, and a BamHI restriction site was added to the 5' end of the synthesis, and a XhoI restriction site was added to the 3' end.
  • the plasmid carrying the gene of interest was digested and the system was as follows: plasmid 5 ⁇ g, 4 ⁇ l of digestion buffer, 1 ⁇ l of BamHI, 1 ⁇ l of XhoI, water was added to a total volume of 40 ⁇ l, and allowed to stand at 37 ° C for 12 hours.
  • the EP tube was taken out, 4.4 ⁇ l of 10 ⁇ loading buffer was added, electrophoresis was carried out with a 1% agarose gel, and mIL12, mGMCSF and mIL2 fragments were recovered after electrophoresis, and were used.
  • the system is as follows: pLentis-CMV-MCS-IRES-PURO 2 ⁇ l, mIL12, mGMCSF or mIL2 2 ⁇ l, ligase buffer 1 ⁇ l, T4 DNA ligase 0.5 ⁇ l , water 4.5 ⁇ l. Leave at room temperature for 4 hours. The ligation system is then subjected to E. coli competent transformation. On the next day, the colonies were picked from the transformed plates, placed in a LB medium and cultured overnight in a 37-degree shaker.
  • Plasmids were extracted from the cultured bacteria using a plasmid extraction kit, and the fragments were successfully inserted into the vector by restriction enzyme digestion. Then, the correct vector was sequenced to confirm the successful construction. Viral vectors pLentis-CMV-mIL12-IRES-PURO, pLentis-CMV-mGMCSF-IRES-PURO, pLentis-CMV-mIL2-IRES-PURO expressing mIL12, mGMCSF and mIL2 were obtained.
  • the virus for the expression vector was prepared as follows: 1) The cultured 293FT cells were digested, and after counting, 3 ⁇ 10 6 cells/well were plated in a 10-cm culture dish, the volume of the culture solution was 10 ml, and a total of 5 plates were plated. 2) The next night, observe the state of the cells, and if the cells are in good condition, transfect.
  • the 293A cells were transfected with the expression virus as follows: Digested cultured 293A cells were seeded into 6-well plates at 10 5 cells/well in a culture volume of 1 ml. After 24 hours, 10 ⁇ l of the expressed virus was added, and after continuing to culture for 24 hours in the incubator, the supernatant was discarded, and the culture was continued with fresh medium. After the cells are full, transfer them out to the culture flask, add the final concentration of 3 ⁇ g/ml puromycin, continue the culture, change the medium every two days, and maintain the concentration of puromycin. After one week of screening. The surviving cells are cells stably expressing cytokines, which are 293A (mIL12), 293A (mGMCSF), and 293A (mIL2), respectively.
  • mIL12 293A
  • mGMCSF 293A
  • mIL2 293A
  • Digested cultured 293A (mIL12), 293A (mGMCSF) and 293A (mIL2) cells were collected by centrifugation, washed with medium, and then centrifuged. Resuspend the cells in the medium to 2 ⁇ 10 7 /ml, and mix 15 ⁇ l of each of the three cells to obtain 45 ⁇ l of the mixture, then add 1.5 ⁇ l of 2 M calcium chloride solution, mix, and finally add 45 ⁇ l of 1% alginic acid. The sodium solution is mixed with a pipette tip to form a gel.
  • mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then a calcium alginate gel with cytokine-expressing cells was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time. Reduce the spillage of the solution. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Six days after the first injection, the same injection procedure was performed again for tumors that did not resolve. Observe and record the growth of mouse tumors.
  • Example 9 Therapeutic effect of mIL12+mGMCSF+mIL15 chitosan quaternary ammonium salt solution on mouse breast cancer
  • the cytokines mIL12, mGMCSF and mIL15 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l. 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of mIL12+mGMCSF+mIL15, and then 45 ⁇ l of a pre-configured 3% chitosan quaternary ammonium salt solution was added, and carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 10 Therapeutic effect of mIL12+mGMCSF+mIL21 chitosan quaternary ammonium salt solution on mouse breast cancer
  • the cytokines mIL12, mGMCSF and mIL21 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l. 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of mIL12+mGMCSF+mIL21, and then 45 ⁇ l of a pre-configured 3% chitosan quaternary ammonium salt solution was added, and carefully mixed by a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 11 Therapeutic effect of mIL12+mFLT3L+mIL2 chitosan quaternary ammonium salt solution on mouse breast cancer
  • the cytokines mIL12, mFLT3L and mIL2 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l. 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of mIL12+mFLT3L+mIL2, and then 45 ⁇ l of a pre-configured 3% chitosan quaternary ammonium salt solution was added, and carefully mixed by a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 12 Therapeutic effect of mIL12+mFLT3L+mIL15 chitosan quaternary ammonium salt solution on mouse breast cancer
  • the cytokines mIL12, mFLT3L and mIL15 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l. 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of mIL12+mFLT3L+mIL15, and then 45 ⁇ l of a pre-configured 3% chitosan quaternary ammonium salt solution was added, and carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 13 Therapeutic effect of mIL12+mFLT3L+mIL21 chitosan quaternary ammonium salt solution on mouse breast cancer
  • Digested mouse breast cancer cells (4T1), 2 ⁇ 10 5 cells were injected into the right side of the body of Balb/c mice, and treatment was started when the long diameter of the tumor reached 7-9 mm.
  • the cytokines mIL12, mFLT3L and mIL21 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l. 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of mIL12+mFLT3L+mIL21, and then 45 ⁇ l of a pre-configured 3% chitosan quaternary ammonium salt solution was added, and carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 14 Therapeutic effect of hIL12+hGMCSF+hIL2 chitosan quaternary ammonium salt solution on mouse breast cancer
  • the cytokines hIL12, hGMCSF and hIL2 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l. 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of hIL12+hGMCSF+hIL2, and then 45 ⁇ l of a pre-configured 3% chitosan quaternary ammonium salt solution was added, and carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 15 Therapeutic effect of hIL12+hGMCSF+hIL15 chitosan quaternary ammonium salt solution on mouse breast cancer
  • the cytokines hIL12, hGMCSF and hIL15 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l. 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of hIL12+hGMCSF+hIL15, and then 45 ⁇ l of a pre-configured 3% chitosan quaternary ammonium salt solution was added, and carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 16 Therapeutic effect of hIL12+hGMCSF+hIL21 chitosan quaternary ammonium salt solution on mouse breast cancer
  • the cytokines hIL12, hGMCSF and hIL21 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l, and 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of hIL12+hGMCSF+hIL21, and then a pre-configured 3% shell was added. 45 ⁇ l of the glycan quaternary ammonium salt solution, carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 17 Therapeutic effect of hIL12+hFLT3L+hIL2 chitosan quaternary ammonium salt solution on mouse breast cancer
  • cytokines hIL12, hFLT3L and hIL2 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l, and 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of hIL12+hFLT3L+hIL2, and then a pre-configured 3% shell was added. 45 ⁇ l of the glycan quaternary ammonium salt solution, carefully mixed with a pipette tip.
  • mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 18 Therapeutic effect of hIL12+hFLT3L+hIL15 chitosan quaternary ammonium salt solution on mouse breast cancer
  • the cytokines hIL12, hFLT3L and hIL15 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l. 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of hIL12+hFLT3L+hIL15, and then 45 ⁇ l of a pre-configured 3% chitosan quaternary ammonium salt solution was added, and carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 19 Therapeutic effect of hIL12+hFLT3L+hIL21 chitosan quaternary ammonium salt solution on mouse breast cancer
  • the cytokines hIL12, hFLT3L and hIL21 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l. 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of hIL12+hFLT3L+hIL21, and then 45 ⁇ l of a pre-configured 3% chitosan quaternary ammonium salt solution was added, and carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 20 The effect of curing mice on re-inoculation of tumors
  • 4T1 tumor-bearing mice treated with mIL12+mGMCSF+mIL2 chitosan quaternary ammonium salt solution after 4 weeks of tumor regression, 2 ⁇ 10 5 4T1 tumor cells were injected subcutaneously in the first inoculated tumor. , observe the condition of the mice.
  • the experimental results are shown in Figure 21. No tumor growth occurred after the second injection of tumor cells, while wild-type mice injected at the same time grew tumors.
  • CT26 tumor-bearing mice treated with mIL12+mGMCSF+mIL2 chitosan quaternary ammonium salt solution were injected with 2 ⁇ 105 CT26 tumor cells subcutaneously on the contralateral side of the first tumor inoculated for 4 weeks after tumor regression.
  • Mouse condition The experimental results are shown in Figure 21.
  • A/B indicates the ratio of the number A of tumor-exposed mice to the number B of mice in which the cytokine combination is injected. For example, 2/2 means 2 injections, 2 of them have tumors; 0/5 means 5 injections, and 0 have tumors.
  • the results in Figure 21 indicate that no tumor growth occurred after the second injection of tumor cells, whereas wild-type mice injected at the same time developed tumors.
  • Example 21 Therapeutic effect of mIL12+mGMCSF+mIL7 chitosan quaternary ammonium salt solution on colon cancer in mice
  • C26 Digested and cultured mouse colon cancer cells (CT26), 5 ⁇ 10 5 cells were injected into the right side of the body of Balb/c mice, and treatment was started when the long diameter of the tumor reached 7-9 mm.
  • the cytokines mIL12, mGMCSF and mIL7 were solubilized in sterile water to a final concentration of 150 ng/ ⁇ l, and 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of mIL12+mGMCSF+mIL7, and then a pre-configured 3% shell was added. 45 ⁇ l of the glycan quaternary ammonium salt solution, carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 22 Therapeutic effect of mIL12+mFLT3L+mIL7 chitosan quaternary ammonium salt solution on colon cancer in mice
  • C26 Digested and cultured mouse colon cancer cells (CT26), 5 ⁇ 10 5 cells were injected into the right side of the body of Balb/c mice, and treatment was started when the long diameter of the tumor reached 7-9 mm.
  • cytokines mIL12, mFLT3L and mIL7 were solubilized in sterile water to a final concentration of 150 ng/ ⁇ l, and 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of mIL12+mFLT3L+mIL7, and then a pre-configured 3% shell was added. 45 ⁇ l of the glycan quaternary ammonium salt solution, carefully mixed with a pipette tip.
  • mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • C26 Digested and cultured mouse colon cancer cells (CT26), 5 ⁇ 10 5 cells were injected into the right side of the body of Balb/c mice, and treatment was started when the long diameter of the tumor reached 7-9 mm.
  • the cytokines hIL12, hGMCSF and hIL7 were solubilized in sterile water to a final concentration of 300 ng/ ⁇ l, and 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of hIL12+hGMCSF+hIL7, and then a pre-configured 3% shell was added. 45 ⁇ l of the glycan quaternary ammonium salt solution, carefully mixed with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • C26 Digested and cultured mouse colon cancer cells (CT26), 5 ⁇ 10 5 cells were injected into the right side of the body of Balb/c mice, and treatment was started when the long diameter of the tumor reached 7-9 mm.
  • cytokines hIL12, hFLT3L and hIL7 were solubilized in sterile water to a final concentration of 300 ng/ ⁇ l, and 15 ⁇ l of each was mixed to obtain a mixture of 45 ⁇ l of hIL12+hFLT3L+hIL7, and then a pre-configured 3% shell was added. 45 ⁇ l of the glycan quaternary ammonium salt solution, carefully mixed with a pipette tip.
  • mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 25 Therapeutic effect of mIL12 chitosan quaternary ammonium salt solution on mouse melanoma
  • the cytokine mIL12 was dissolved in sterile water to a final concentration of 200 ng/ ⁇ l. 15 ⁇ l was mixed with 45 ul of sterile water, and then 45 ⁇ l of a pre-configured 3% chitosan quaternary ammonium salt solution was added, carefully mixed with a pipette tip. uniform.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 26 Therapeutic effect of mGMCSF chitosan quaternary ammonium salt solution on mouse melanoma
  • the cytokine mGMCSF was solubilized in sterile water to a final concentration of 200 ng/ ⁇ l, 15 ⁇ l was mixed with 45 ul of sterile water, and then 45 ⁇ l of a pre-configured 3% chitosan quaternary ammonium salt solution was added, carefully mixed with a pipette tip. uniform.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 27 Therapeutic effect of mIL2 chitosan quaternary ammonium salt solution on mouse melanoma
  • cytokine mIL2 Dissolve the cytokine mIL2 in sterile water to a final concentration of 200 ng/ ⁇ l, mix 15 ⁇ l with 45 ul of sterile water, then add 45 ⁇ l of pre-configured 3% chitosan quaternary ammonium salt solution, carefully mix and mix with the tip of the gun. uniform.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 28 Therapeutic effect of mIL12+mGMCSF chitosan quaternary ammonium salt solution on mouse melanoma
  • the cytokines mIL12 and mGMCSF were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l, 15 ⁇ l of each was mixed, and 15 ul of sterile water was added to obtain a mixture of 45 ⁇ l of mIL12+mGMCSF, followed by pre-configured 3 45 ⁇ l of % chitosan quaternary ammonium salt solution, carefully pipet and mix with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 29 Therapeutic effect of mIL12+mIL2 chitosan quaternary ammonium salt solution on mouse melanoma
  • the cytokines mIL12 and mIL2 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l, 15 ⁇ l of each was mixed, and 15 ul of sterile water was added to obtain a mixture of 45 ⁇ l of mIL12+mIL2, followed by pre-configured 3 45 ⁇ l of % chitosan quaternary ammonium salt solution, carefully pipet and mix with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.
  • Example 30 Therapeutic effect of mGMCSF+mIL2 chitosan quaternary ammonium salt solution on mouse melanoma
  • the cytokines mGMCSF and mIL2 were solubilized in sterile water to a final concentration of 200 ng/ ⁇ l, 15 ⁇ l of each was mixed, and 15 ul of sterile water was added to obtain a mixture of 45 ⁇ l of mGMCSF+mIL2, followed by pre-configured 3 45 ⁇ l of % chitosan quaternary ammonium salt solution, carefully pipet and mix with a pipette tip.
  • the tumor-bearing mice were intraperitoneally injected with sodium pentobarbital for anesthesia, then the prepared cytokine chitosan quaternary ammonium salt solution was aspirated with a 29G insulin syringe, and slowly injected into the tumor. After the injection, the needle was retained for a little time to reduce The solution overflows. The injected mice are returned to the cage, and the temperature is kept warm until they are awakened. Observe and record the growth of mouse tumors.

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Abstract

提供用于治疗肿瘤和/或预防肿瘤的复发或转移的细胞因子组合，其包含至少3种细胞因子，所述细胞因子选自以下组：IL12或其功能性变体、GMCSF或其功能性变体、FLT3L或其功能性变体、IL2或其功能性变体、IL15或其功能性变体、IL21或其功能性变体和IL7或其功能性变体。还提供编码所述细胞因子组合的核酸分子及其载体、细胞和药物组合物，以及它们在制备用于治疗肿瘤和/或预防肿瘤复发或转移的药物中的应用。

Description

细胞因子组合
本申请要求于2017年4月6日提交的中国专利申请201710221755.8的优先权。优先权中的内容通过引用在此合并入本文。
技术领域
本申请涉及肿瘤治疗领域。具体而言，本申请涉及包含至少3种细胞因子的细胞因子组合及其在治疗肿瘤和/或预防肿瘤复发或转移方面的用途。
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。现阶段最主要的肿瘤免疫治疗方法包括嵌合抗原受体T细胞疗法(CAR-T)和免疫检查点疗法(Checkpoint)。前者通过改造T细胞，使其能够更加准确，有效地识别并杀死体内肿瘤细胞。而免疫检查点则是通过抗体结合来阻断肿瘤环境中抑制T细胞功能的信号通路，从而使得T细胞重新活化并杀伤肿瘤细胞。然而，这两种疗法还远未达到理想的效果。CAR-T在实体瘤中效果不佳，其原因在于实体瘤是一个致密的，有很强的免疫抑制的环境，CAR-T细胞难于大量的进入肿瘤内部，同时即便进入以后，也可能在肿瘤微环境中被抑制功能。免疫检查点疗法的应答率不高，通常只有20-30％左右，说明仅仅释放T细胞，使其重新活化，可能在大部分情况下并不足以消灭已经成形的肿瘤。肿瘤细胞通过分泌各种蛋白或者直接接触作用，在肿瘤内部建立起免疫抑制的微环境。多种免疫细胞，包括巨噬细胞、调节性T细胞(Treg)、骨髓来源的抑制性细胞(MDSC)等都参与了这一作用。破解肿瘤免疫抑制微环境，是肿瘤免疫疗法能否成功的关键。对此，CAR-T疗法和免疫检查点疗法都还做得不够。
细胞因子(Cytokine)是体内非常重要的免疫信号。各种细胞分泌不同的细胞因子，各种细胞因子又可以作用于不同的细胞，形成了一个复杂的交叉网络，并且，同一细胞因子在不同的环境下可能作用于不同的细胞，从而发挥完全相反的功能。现有已应用的细胞因子或者其组合中，均没有在抗肿瘤中表现出令人完全满意的效果。
发明内容
本申请提供了包含特定的细胞因子的细胞因子组合及其药物组合物。
一方面，本申请提供了细胞因子组合，其包含至少3种细胞因子，所述细胞因子选自以 下组：IL12或其功能性变体、GMCSF或其功能性变体、FLT3L或其功能性变体、IL2或其功能性变体、IL15或其功能性变体，IL21或其功能性变体和IL7或其功能性变体。
在某些实施方式中，所述细胞因子组合包含以下任一组：i)IL12或其功能性变体、GMCSF或其功能性变体，和IL2或其功能性变体；ii)IL12或其功能性变体、GMCSF或其功能性变体，和IL15或其功能性变体；iii)IL12或其功能性变体、GMCSF或其功能性变体，和IL21或其功能性变体；iv)IL12或其功能性变体、FLT3L或其功能性变体，和IL2或其功能性变体；v)IL12或其功能性变体、FLT3L或其功能性变体，和IL15或其功能性变体；vi)IL12或其功能性变体、FLT3L或其功能性变体，和IL21或其功能性变体；vii)IL12或其功能性变体、GMCSF或其功能性变体，和IL7或其功能性变体；和viii)IL12或其功能性变体、FLT3L或其功能性变体，和IL7或其功能性变体。
在某些实施方式中，所述细胞因子选自下组：鼠细胞因子和人细胞因子。
在某些实施方式中，所述细胞因子组合中的每种细胞因子的浓度为1-10000ng/μL。
另一方面，本申请提供一种或多种核酸分子，其编码所述的细胞因子组合。
在某些实施方式中，所述核酸分子包括两种以上的核酸分子，其中每种核酸分子编码一种或多种所述细胞因子，且每种所述核酸分子编码的细胞因子与至少另一种所述核酸分子编码的细胞因子不同。
另一方面，本申请提供载体，其包含所述的核酸分子。
在某些实施方式中，所述载体包括两种以上的载体，其中每种载体包含一种或多种所述核酸分子，且每种所述载体包含的所述核酸分子编码的细胞因子与至少另一种载体包含的核酸分子编码的细胞因子不同。
另一方面，本申请提供细胞，其包含所述的核酸分子或所述的载体。
在某些实施方式中，所述细胞包括两种以上的细胞，其中每种细胞表达一种或多种所述细胞因子，且每种所述细胞表达的细胞因子与至少另一种细胞表达的细胞因子不同。
另一方面，本申请提供药物组合物，其包含所述的细胞因子组合、所述的核酸分子、所述的载体或所述的细胞，以及任选地药学上可接受的载体。
在某些实施方式中，所述药物组合物被配制为适于局部施用。在某些实施方式中，所述药物组合物被配制为适于缓释施用。例如，所述药物组合物被配制于海藻酸钙凝胶、聚乳酸微球或壳聚糖季铵盐溶液中。
另一方面，本申请所述的细胞因子组合、所述的核酸分子、所述的载体、所述的细胞或所述的药物组合物在制备药物中的应用，其中所述药物用于治疗肿瘤和/或预防肿瘤复发或转 移。
在某些实施方式中，所述肿瘤是实体瘤。在某些实施方式中，所述肿瘤选自以下组：肺癌、食道癌、胃癌、结直肠癌、肝癌、乳腺癌、宫颈癌、甲状腺癌、脑及中枢神经系统癌、胰腺癌、口腔癌、鼻咽癌、头颈癌、喉癌、骨癌、皮肤癌、卵巢癌、前列腺癌、睾丸癌、肾癌、膀胱癌、眼睑肿瘤、白血病和淋巴瘤。
在某些实施方式中，所述细胞因子组合中的每种细胞因子的浓度为1-10000ng/μL。
在某些实施方式中，所述受试者是哺乳动物。例如，所述哺乳动物选自下组：人、小鼠、大鼠、猴、犬、猪、羊、牛和猫。
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公开的其它方面和优势。下文的详细描述中仅显示和描述了本公开的示例性实施方式。如本领域技术人员将认识到的，本公开的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示了mIL12+mGMCSF+mIL2壳聚糖季铵盐溶液对小鼠黑色素瘤(B16F10)的治疗效果。
图2显示了mIL12+mGMCSF+mIL2壳聚糖季铵盐溶液对小鼠结肠癌(CT26)的治疗效果。
图3显示了mIL12+mGMCSF+mIL2壳聚糖季铵盐溶液对小鼠乳腺癌(4T1)的治疗效果。
图4显示了mIL12+mGMCSF+mIL2壳聚糖季铵盐溶液对小鼠淋巴瘤(EL4)的治疗效果。
图5显示了不同比例的mIL12+mGMCSF+mIL2壳聚糖季铵盐溶液对小鼠乳腺癌(4T1)的治疗效果。
图6显示了mIL12+mGMCSF+mIL2海藻酸钙凝胶对小鼠乳腺癌(4T1)的治疗效果。
图7显示了mIL12+mGMCSF+mIL2聚乳酸微球对小鼠乳腺癌(4T1)的治疗效果。
图8显示了表达mIL12+mGMCSF+mIL2的细胞对小鼠乳腺癌(4T1)的治疗效果。
图9显示了mIL12+mGMCSF+mIL15壳聚糖季铵盐溶液对小鼠乳腺癌(4T1)的治疗效果。
图10显示了mIL12+mGMCSF+mIL21壳聚糖季铵盐溶液对小鼠乳腺癌(4T1)的治疗效果。
图11显示了mIL12+mFLT3L+mIL2壳聚糖季铵盐溶液对小鼠乳腺癌(4T1)的治疗效果。
图12显示了mIL12+mFLT3L+mIL15壳聚糖季铵盐溶液对小鼠乳腺癌(4T1)的治疗效果。
图13显示了mIL12+mFLT3L+mIL21壳聚糖季铵盐溶液对小鼠乳腺癌(4T1)的治疗效果。
图14显示了hIL12+hGMCSF+hIL2壳聚糖季铵盐溶液对小鼠乳腺癌(4T1)的治疗效果。
图15显示了hIL12+hGMCSF+hIL15壳聚糖季铵盐溶液对小鼠乳腺癌(4T1)的治疗效果。
图16显示了hIL12+hGMCSF+hIL21壳聚糖季铵盐溶液对小鼠乳腺癌(4T1)的治疗效果。
图17显示了hIL12+hFLT3L+hIL2壳聚糖季铵盐溶液对小鼠乳腺癌(4T1)的治疗效果。
图18显示了hIL12+hFLT3L+hIL15壳聚糖季铵盐溶液对小鼠乳腺癌(4T1)的治疗效果。
图19显示了hIL12+hFLT3L+hIL21壳聚糖季铵盐溶液对小鼠乳腺癌(4T1)的治疗效果。
图20显示了小鼠乳腺癌(4T1)未治疗时肿瘤生长情况。
图21显示了治愈小鼠对再次接种肿瘤的抵御效果。
图22显示了mIL12+mGMCSF+mIL7壳聚糖季铵盐溶液对小鼠结肠癌(CT26)的治疗效果。
图23显示了mIL12+mFLT3L+mIL7壳聚糖季铵盐溶液对小鼠结肠癌(CT26)的治疗效果。
图24显示了hIL12+hGMCSF+hIL7壳聚糖季铵盐溶液对小鼠结肠癌(CT26)的治疗效果。
图25显示了hIL12+hFLT3L+hIL7壳聚糖季铵盐溶液对小鼠结肠癌(CT26)的治疗效果。
图26显示了mIL12壳聚糖季铵盐溶液对小鼠黑色素瘤(B16F10)的治疗效果。
图27显示了mGMCSF壳聚糖季铵盐溶液对小鼠黑色素瘤(B16F10)的治疗效果。
图28显示了mIL2壳聚糖季铵盐溶液对小鼠黑色素瘤(B16F10)的治疗效果。
图29显示了mIL12+mGMCSF壳聚糖季铵盐溶液对小鼠黑色素瘤(B16F10)的治疗效果。
图30显示了mIL12+mIL2壳聚糖季铵盐溶液对小鼠黑色素瘤(B16F10)的治疗效果。
图31显示了mGMCSF+mIL2壳聚糖季铵盐溶液对小鼠黑色素瘤(B16F10)的治疗效果。
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。
在本申请中，术语“功能性变体”通常是指具有细胞因子的功能的片段或变体。例如，所述功能性片段或变体可以具有在所述细胞因子的氨基酸序列的基础上经过取代、缺失或添加一个或多个(例如，1-2个、1-3个、1-4个、1-5个、1-6个、1-7个、1-8个、1-9个、1-10个或更多个)氨基酸后得到的氨基酸序列，并且仍保持所述细胞因子的功能。又例如，所述功能性片段或变体可以具有与所述细胞因子的氨基酸序列具有至少约80％以上(如至少约80％以上、约85％以上、约90％以上、约91％以上、约92％以上、约93％以上、约94％以上、约95％以上、约96％以上、约97％以上、约98％以上、约99％以上或更多)同源性的氨基酸序列，并且仍保持所述细胞因子的功能。所述功能性变体可以是天然变体(如等位基因变体)，也可以是经人工修饰的变体。在本申请中，所述同源性具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比，例如，可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列同源性(例如，可参见：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988；Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993；Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994；Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987；and Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991)。测量两个多核苷酸或多肽之间的同源性水平的方法为本领域技术人员所知悉(例如，可参见：Carrillo,H.&Lipman,D.,SIAM J Applied Math 48:1073(1988))。在本申请中，在多肽的非必需区中的单个氨基酸取代(例如，合适的保守型氨基酸取代)基本上不改变所得到的多肽的生物活性(例如，可参见：Watson et al.,Molecular Biology of the Gene,4th Edition,1987,The Benjamin/Cummings Pub.co.,p.224)。在本申请中，所述的功能性变体可以通过自其天然来源分离、重组表达或化学合成而获得。例如，为了延长所述细胞因子或细胞因子组合在体内的半衰期，可以制备所述细胞因子的功能性变体。例如，在所述细胞因子上添加聚乙二醇基团(PEG)。
在本申请中，术语“细胞因子”通常是指主要由免疫细胞分泌的、能调节细胞功能的小分子多肽。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。例如，所述细胞因子可以选自下组中的一种或多种：白细胞介素和集落刺激因子。在本申请中，所述白细胞介素是由多种细胞产生并作用于多种细胞的一类细胞因子，最初仅指由白细胞产生又在白细胞间发挥作用的细胞因子。白细胞介素在传递信息，激活与调节免疫细胞，介导T、B 细胞活化、增殖与分化及在炎症反应中发挥重要的作用，目前已知的白细胞介素包括IL1-IL38。例如，所述白细胞介素可以选自下组中的一种或多种：IL12、IL2、IL15、IL21和IL7。在本申请中，所述集落刺激因子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。所述集落刺激因子对不同发育阶段的造血干细胞起促增殖、分化的作用。例如，根据集落刺激因子的作用范围，可以将所述集落刺激因子分别命名为粒细胞集落刺激因子(G-CSF)、巨噬细胞集落刺激因子(M-CSF)、粒细胞和巨噬细胞集落刺激因子(GM-CSF)和多能集落刺激因子(multi-CSF，IL3)。例如，所述集落刺激因子可以选自下组中的一种或多种：FMS相关酪氨酸激酶3配体(FTL3L)和粒细胞巨噬细胞集落刺激因子(GMCSF)。
在本申请中，术语“IL2”通常是指白细胞介素-2，IL2对机体的免疫应答和抗病毒感染等有重要作用，能刺激已被特异性抗原启动的T细胞增殖；同时IL2能活化T细胞，促进细胞因子产生；刺激NK细胞增殖，增强NK杀伤活性及产生细胞因子，诱导LAK细胞产生；并且促进B细胞增殖和分泌抗体；激活巨噬细胞。例如，小鼠IL2(mIL2)可包含如SEQ ID NO:5所示的氨基酸序列。又例如，人细胞因子IL2(hIL2)可包含如SEQ ID NO:12所示的氨基酸序列。
在本申请中，术语“IL12”通常是指白细胞介素-12，IL-12可刺激活化型t细胞增殖，促进Th0细胞细胞向Th1细胞分化；也可诱导CTL和NK细胞的细胞毒活性并促进其分泌IFN-γ、TNF-α、GM-CSF等细胞因子；或者促进NK细胞和IL-2Rα受体及CD56分子的表达，增强对肿瘤细胞的ADCC效应。IL12的分子是一种异型二聚体，p40亚基(40kd)和p35亚基(35kd)这两个亚基通过二硫键相连接。例如，小鼠IL12(mIL12)中的p35亚基可包含如SEQ ID NO:1所示的氨基酸序列，p40亚基可包含如SEQ ID NO:2所示的氨基酸序列。又例如，人IL12(hIL12)中的p35亚基可包含如SEQ ID NO:8所示的氨基酸序列，p40亚基可包含如SEQ ID NO:9所示的氨基酸序列。
在本申请中，术语“IL15”通常是指白细胞介素-15，IL15由活化的单核-巨噬细胞、表皮细胞和成纤维细胞等多种细胞产生，其可诱导B细胞增殖和分化，能够发挥类似IL2的生物学活性。例如，小鼠IL15(mIL15)可包含如SEQ ID NO:6所示的氨基酸序列。又例如，人IL15(hIL15)可包含如SEQ ID NO:13所示的氨基酸序列。
在本申请中，术语“IL7”通常是指白细胞介素-7，IL7主要由胸腺和骨髓基质细胞分泌，属于相对分子质量为25000-28000的糖蛋白。IL7及其受体介导的信号转导途径主要是通过Janus激酶、信号转导及转录活化子和磷酸肌醇3-激酶三条通路实现的。例如，小鼠IL7(mIL7)可包含如SEQ ID NO:15所示的氨基酸序列。又例如，人IL7(hIL7)可包含如SEQ ID NO:16 所示的氨基酸序列。
在本申请中，术语“IL21”通常是指白细胞介素-21，IL21由活化的CD4 +T细胞分泌；IL21参与调节B细胞增殖，缺失IL21基因可导致机体更容易受细菌或病毒的感染。研究表明，IL21可通过调控Bcl-6蛋白影响IL2受体蛋白(CD25)的表达水平。例如，小鼠IL21(mIL21)可包含如SEQ ID NO:7所示的氨基酸序列。又例如，人IL21(hIL21)可包含如SEQ ID NO:14所示的氨基酸序列。
在本申请中，术语“FTL3L”通常是指FMS相关酪氨酸激酶3配体，其可调节非红系造血干细胞的增殖和分化，促进前B淋巴细胞、树突状细胞、NK细胞、细胞毒T淋巴细胞的增殖、分化和成熟，具有重要的抗肿瘤作用。例如，小鼠FTL3L(mFTL3L)可包含如SEQ ID NO:4所示的氨基酸序列。又例如，人FTL3L(hFTL3L)可包含如SEQ ID NO:11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，其能刺激粒细胞和巨噬细胞的增殖、分化及活化，增加造血功能；亦能增强中性粒细胞、嗜酸性细胞及单核粒细胞的多种功能。GMCSF可提高免疫效应细胞吞噬细菌及杀灭癌细胞等免疫活力，并且有利于恢复因肿瘤化疗和骨髓移植引起的中性粒细胞缺乏。例如，小鼠GMCSF(mGMCSF)可包含如SEQ ID NO:3所示的氨基酸序列。又例如，人GMCSF(hGMCSF)可包含如SEQ ID NO:10所示的氨基酸序列。
在本申请中，术语“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的细胞因子/细胞因子组合的个体细胞，细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。由于天然的，意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的细胞因子/细胞因子组合即可。所述细胞可以通过使用本申请所述的载体体外转染该细胞而得到。
在本申请中，术语“药学上可接受的载体”通常是指药物组合物的组分之一，其可以包括缓冲剂、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、羧甲基素和滑石粉。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。
在本申请中，术语“肿瘤”通常是指或描述哺乳动物的生理状况，其典型特征在于细胞增殖或存活失调。例如，所述肿瘤可以为实体瘤；例如，所述实体瘤可以选自以下组：肺癌、食道癌、胃癌、结直肠癌、肝癌、乳腺癌、宫颈癌、甲状腺癌、脑及中枢神经系统癌、胰腺癌、口腔癌、鼻咽癌、头颈癌、喉癌、骨癌、皮肤癌、卵巢癌、前列腺癌、睾丸癌、肾癌、膀胱癌和淋巴瘤。
在本申请中，术语“转移”通常是指恶性肿瘤细胞从原发部位，经淋巴道、血管或直接蔓延等途径，到达其他部位继续生长的过程。恶性肿瘤的转移往往是肿瘤治疗失败的主要原因。
在本申请中，术语“复发”通常是指由于治疗的不彻底导致肿瘤细胞在体内残存而诱发的肿瘤复发。
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。
细胞因子组合
本申请的发明人令人惊奇地发现，同时将选自IL12、GMCSF、FLT3L、IL2、IL15、IL21和IL7的至少3种细胞因子的组合局部施用至肿瘤内部，能够在肿瘤内局部活化免疫系统，打破肿瘤免疫抑制微环境，激发针对肿瘤的免疫应答，从而有效清除肿瘤。
本申请提供细胞因子组合，其包含至少3种(至少1种、至少2种、至少3种、至少4种、至少5种、至少6种、至少7种、至少8种、至少9种、至少10种、至少11种、至少12种或更多种)细胞因子，所述细胞因子选自以下组：IL12或其功能性变体、GMCSF或其功能性变体、FLT3L或其功能性变体、IL2或其功能性变体、IL15或其功能性变体、IL21或其功能性变体和IL7或其功能性变体。
本申请中使用的细胞因子组合表现出很强的协同作用，而施用单个的细胞因子，或两个细胞因子的组合，均不能有效清除肿瘤的作用。本申请的关键之一在于将本身并无明显抗肿 瘤效果的蛋白，设计出特定的组合方式，并通过合适的施用方法，获得了优异的抗肿瘤的效果。
通过施用本申请的细胞因子组合，可以在有需要的受试者体内激发抗肿瘤免疫反应，有需要的受试者体内被活化的免疫系统能够识别并消灭肿瘤细胞，并且在清除肿瘤以后，获得性免疫反应能够产生全身性的免疫记忆。一方面，活化的特异性的针对肿瘤的免疫细胞能够通过血液及淋巴系统在全身循环，从而对其他部位的病灶也起到抑制乃至清除的效果；另一方面，免疫记忆的存在使得体内再出现具有相似抗原的肿瘤细胞时，免疫系统能够迅速地识别并杀灭这些细胞，从而避免肿瘤的复发。此外，本申请的细胞因子组合中的细胞因子直接作用于免疫系统，因此不存在类似于肿瘤靶向药物的耐药机制，可反复使用。例如，本申请实施例显示通过施用本申请的细胞因子组合可成功清除小鼠体内的肿瘤，在一定时间后再次接种同种肿瘤细胞，仍无法再形成肿瘤。
在本申请中，不受任何理论限制，由于不同细胞因子有不同的作用机制，本申请中使用了多种细胞因子的组合，因而可以同时活化天然免疫和获得性免疫，实现有效的肿瘤治疗。例如，使用3种细胞因子的组合，能够同时活化所需的免疫系统组分，达到最佳的激发抗肿瘤免疫效果。如果采用更少细胞因子的组合，例如仅仅包含2种细胞因子的组合，将缺少对某些组分的激活能力，无法达到最佳效果。如果采用更多种细胞因子的组合，例如4种或4种以上的细胞因子的组合，一方面可能带来更大的副作用，另一方面由于细胞因子在体内复杂的作用机理，可能会造成免疫系统紊乱，反而无法达到预期的免疫激活作用。因此，在某些实施方式中，所述的细胞因子组合可包含3种细胞因子。
例如，所述的细胞因子组合可包含以下任一组：
i)IL12或其功能性变体、GMCSF或其功能性变体，和IL2或其功能性变体；
ii)IL12或其功能性变体、GMCSF或其功能性变体，和IL15或其功能性变体；
iii)IL12或其功能性变体、GMCSF或其功能性变体，和IL21或其功能性变体；
iv)IL12或其功能性变体、FLT3L或其功能性变体，和IL2或其功能性变体；
v)IL12或其功能性变体、FLT3L或其功能性变体，和IL15或其功能性变体；
vi)IL12或其功能性变体、FLT3L或其功能性变体，和IL21或其功能性变体；
vii)IL12或其功能性变体、GMCSF或其功能性变体，和IL7或其功能性变体；和
viii)IL12或其功能性变体、FLT3L或其功能性变体，和IL7或其功能性变体。
在本申请中，小鼠IL2(mIL2)可包含如SEQ ID NO:5所示的氨基酸序列；人细胞因子IL2(hIL2)可包含如SEQ ID NO:12所示的氨基酸序列。在本申请中，小鼠IL12(mIL12) 中的p35亚基可包含如SEQ ID NO:1所示的氨基酸序列，p40亚基可包含如SEQ ID NO:2所示的氨基酸序列；人IL12(hIL12)中的p35亚基可包含如SEQ ID NO:8所示的氨基酸序列，p40亚基可包含如SEQ ID NO:9所示的氨基酸序列。在本申请中，小鼠IL15(mIL15)可包含如SEQ ID NO:6所示的氨基酸序列；人IL15(hIL15)可包含如SEQ ID NO:13所示的氨基酸序列。在本申请中，小鼠IL21(mIL21)可包含如SEQ ID NO:7所示的氨基酸序列；人IL21(hIL21)可包含如SEQ ID NO:14所示的氨基酸序列。在本申请中，小鼠FTL3L(mFTL3L)可包含如SEQ ID NO:4所示的氨基酸序列；人FTL3L(hFTL3L)可包含如SEQ ID NO:11所示的氨基酸序列。在本申请中，小鼠GMCSF(mGMCSF)可包含如SEQ ID NO:3所示的氨基酸序列；人GMCSF(hGMCSF)可包含如SEQ ID NO:10所示的氨基酸序列。在本申请中，小鼠IL7(mIL7)可包含如SEQ ID NO:15所示的氨基酸序列；人IL7(hIL7)可包含如SEQ ID NO:16所示的氨基酸序列。在某些实施方式中，所述功能性变体可以为与所述细胞因子(例如，IL2、IL12、IL15、IL21、FTL3L、GMCSF或IL7)具有基本上相同的功能，且与其具有至少约80％以上(如至少约80％以上、约85％以上、约90％以上、约91％以上、约92％以上、约93％以上、约94％以上、约95％以上、约96％以上、约97％以上、约98％以上、约99％以上或更多)同源性的氨基酸序列序列。在某些实施方式中，所述功能性变体为与所述细胞因子(例如，IL2、IL12、IL15、IL21、FTL3L、GMCSF或IL7)具有基本上相同的功能，且在其基础上包含一个或多个(例如，1-2个、1-3个、1-4个、1-5个、1-6个、1-7个、1-8个、1-9个、1-10个或更多个)氨基酸的添加、缺失或置换的氨基酸序列。
在本申请中，所述细胞因子是哺乳动物细胞因子，例如可以为鼠细胞因子或人细胞因子。
在本申请中，所述细胞因子组合中的每种细胞因子的浓度可以为1-10000ng/μL(例如，为1-100000ng/μL、1-10000ng/μL、2-10000ng/μL、4-10000ng/μL、6-10000ng/μL、8-10000ng/μL、10-10000ng/μL、10-1000ng/μL、100-1000ng/μL或10-500ng/μL)。
在本申请中，所述细胞因子组合中的每种细胞因子的比例可以是任意的比例，只要每种细胞因子的剂量都达到了各自的治疗有效量即可。例如，所述细胞因子组合中三种不同细胞因子的比例可以是x:y:z，其中x、y和z为独立地、选自1-10的整数(例如，x、y和z为独立地、选自1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10的整数)。在某些实施方式中，所述细胞因子组合中三种不同细胞因子的比例可以是5:1:1、1:5:1、1:1:5、4:1:1、1:4:1、1:1:4、3:1:1、1:3:1、1:1:3、2:1:1、1:2:1、1:1:2或1:1:1。例如，所述细胞因子组合中三种不同细胞因子的比例可以是1:1:1。
在本申请中，所述细胞因子组合中的每种细胞因子可以同时施用或在合适的时间间隔内(例如，可以为在第一种和/或第二种细胞因子施用后第0.5-5天、第1-4天、第1-3天、第1-2天、第11-24小时、第10-24小时、第9-24小时、第8-24小时、第7-24小时、第6-24小时、第5-24小时、第4-24小时、第3-24小时、第2-24小时、第1-24小时、第55-60分钟、第50-60分钟、第45-60分钟、第40-60分钟、第35-60分钟、第30-60分钟、第25-60分钟、第20-60分钟、第15-60分钟、第10-60分钟、第5-60分钟、第4-60分钟、第3-60分钟、第2-60分钟、第1-60分钟、第0.5-60分钟或其他时间间隔内)分开施用。例如，每种细胞因子可以同时施用。
本申请所述细胞因子组合中包括的细胞因子具有很强的活化免疫系统进而对抗肿瘤的能力，其已经在临床中应用多年。但正是由于这些细胞因子的功能过于强大，小剂量的全身使用，便会带来比较严重的副作用，然而其在肿瘤局部却又难以达到产生治疗效果的、足够的作用浓度。在本申请中，可以通过在肿瘤部位直接施用获得所述细胞因子组合中的细胞因子在肿瘤内局部富集的效果，从而避免了全身性的副作用的产生。本申请已经通过试验验证，瘤内注射本申请的细胞因子组合后的小鼠并未有特殊的异常反应，可见本申请所述的细胞因子组合不会给有需要的受试者带来严重的副作用。在某些实施方式中，所述细胞因子组合可被配制为适于局部施用。例如，可以被配制为瘤内施用。
在本申请中，所述细胞因子组合可被配制为缓释施用。这是因为如果仅仅以水或生理盐水为载体将细胞因子注射到肿瘤部位，这些细胞因子不能长时间停留，会快速地被循环系统带走，严重影响其发挥作用。在本申请中，可以通过缓释技术将所述细胞因子较长时间地富集在施用部位，从而获得较长的停留和作用时间。例如，可以通过控制所述细胞因子的溶出速度、控制所述细胞因子的扩散速度、或者利用溶蚀作用、渗透作用，或者采用离子交换法等达到缓释的目的。例如，可以将所述细胞因子组合中的细胞因子制备成聚乳酸(PLA)微球、或者制备于海藻酸钙凝胶中、或者通过添加壳聚糖季铵盐来增加细胞因子溶液粘度，从而将所述细胞因子较长时间地保留在注射位置。本领域已知其他的缓释技术，如聚乳酸-羟基乙酸共聚物(PLGA)微球、替莫唑胺的缓释微球、嵌段共聚物胶束和缓释泵等也均可以施用于所述的细胞因子组合。
本申请的细胞因子组合可以通过任何合适的手段施用至肿瘤内。例如所述细胞因子组合可以直接被注射至肿瘤部位。或者，可以通过与靶向肿瘤的物质(如抗体)偶联而使得静脉施用的细胞因子在肿瘤部位富集。
核酸、载体和细胞
本申请提供了核酸，一种或多种核酸分子，其编码所述的细胞因子组合。
在某些实施方式中，所述核酸分子包括两种以上(例如，1种以上、2种以上、3种以上、4种以上或更多种)的核酸分子，其中每种核酸分子编码一种或多种(例如，1种以上、2种以上、3种以上、4种以上或更多种)所述细胞因子，且每种所述核酸分子编码的细胞因子与至少另一种所述核酸分子编码的细胞因子不同。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
在本申请中，可以通过本领域已知的多种方法来制备编码所述的抗体、其抗原结合片段或变体的核酸，这些方法包括但不限于，采用限制性片段操作或采用合成性寡核苷酸的重叠延伸PCR，具体操作可参见Sambrook等人，Molecular Cloning,A Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.，1989；和Ausube等人Current Protocols in Molecular Biology,Greene Publishing and Wiley-Interscience,New York N.Y.,1993。
本申请提供了载体，其包含所述的核酸分子。
在某些实施方式中，所述载体包括两种以上(例如，1种以上、2种以上、3种以上、4种以上或更多种)的载体，其中每种载体包含一种或多种(例如，1种以上、2种以上、3种以上、4种以上或更多种)所述核酸分子，且每种所述载体包含的所述核酸分子编码的细胞因子与至少另一种载体包含的核酸分子编码的细胞因子不同。
在本申请中，所述载体可以是线性的核酸片段、环状质粒或病毒载体(如痘苗病毒、腺病毒、腺相关病毒、慢病毒等)。在所述载体中，编码所述细胞因子的核苷酸序列可以与表达调控序列可操作地连接。其中，所述“调控序列”可与“调控元件”互换使用，其可以指位于编码序列的上游(5'非编码序列)、中间或下游(3'非编码序列)，影响相关编码序列的转录、RNA加工或稳定性或者翻译的核苷酸序列。所述调控序列可包括但不限于启动子、翻译前导序列、内含子和多腺苷酸化识别序列。其中，“可操作地连接”指调控元件(例如但不限于，启动子序列、转录终止序列等)可以与核酸序列(例如，编码序列或开放读码框)连接，使得核苷酸序列的转录被所述转录调控元件控制和调节。用于将调控元件区域可操作地连接于核酸分子的技术为本领域已知。
本申请还提供了可向肿瘤部位施用的细胞。所述细胞包含所述的核酸分子或所述的载体。
在某些实施方式中，所述细胞包括两种以上(例如，1种以上、2种以上、3种以上、4种以上或更多种)的细胞，其中每种细胞表达一种或多种(例如，1种以上、2种以上、3种以上、4种以上或更多种)所述细胞因子，且每种所述细胞表达的细胞因子与至少另一种细胞表达的细胞因子不同。例如，在所述细胞因子组合包含3种细胞因子的情况下，所述细胞可以表达全部3种细胞因子(A、B、C)；或者，所述细胞可以包含两种细胞，其中一种细胞表达一种细胞因子(A或B或C)，另外一种细胞表达其它两种细胞因子(B和C；或者A和C；或者A和B)；或者，所述细胞可以包含3种细胞，每种细胞各自表达不同的、一种细胞因子。
在本申请中，所述细胞可以在肿瘤部位持续表达本申请的细胞因子，从而在能够较长时间下在肿瘤内维持一定的细胞因子浓度，发挥抗肿瘤作用。
在本申请中，将所述载体导入所述细胞的方法包括但不限于，磷酸钙转染、原生质融合、电穿孔、脂质体转染、微注射和病毒感染。
在本申请中，所述细胞可以为真核细胞，例如为哺乳动物细胞。所述哺乳动物可以选自以下组：人、小鼠、大鼠、猴、犬、猪、羊、牛和猫。例如，所述哺乳动物可以为人。例如，所述细胞可以为293细胞。
本申请还提供了制备所述细胞因子组合/细胞因子的方法。所述方法可包括，在使得所述细胞因子组合/细胞因子表达的条件下，培养所述所述细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
药物组合物和用途
本申请提供药物组合物，其包含所述的细胞因子组合、所述的核酸分子、所述的载体或所述的细胞，以及任选地药学上可接受的载体。
所述药学上可接受的佐剂可以包括溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、抗氧化剂、等渗剂、吸收延迟剂和赋形剂。例如，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。
在本申请中，所述药物组合物可被配制用于口服给药，静脉内给药，肌肉内给药，在肿瘤部位的原位给药，吸入，直肠给药，阴道给药，经皮给药或通过皮下储存库给药。
在某些实施方式中，所述药物组合物被配制为适于局部施用。例如，可以为瘤内施用。
在某些实施方式中，所述药物组合物被配制为适于缓释施用。例如，所述药物组合物被 配制于海藻酸钙凝胶、聚乳酸微球或壳聚糖季铵盐溶液中。
所述药物组合物可以用于抑制肿瘤生长。例如，本申请的药物组合物可以抑制或延缓疾病的发展或进展，可以减小肿瘤大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。
本申请所述的药物组合物可以包含治疗有效量的所述细胞因子组合。所述治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试者中的病症或病症(例如癌症)和/或其任何并发症而所需的剂量。其中，所述“有效量”可以指与不施用所述药物组合物相比，能使肿瘤细胞生长或肿瘤面积增长抑制至少约10％(例如，至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、99％或100％)所需的施用所述药物组合物的剂量。其中，抑制肿瘤面积增长的能力可以通过该肿瘤的相关动物模型来评价。或者，也可以通过检查抑制细胞生长的能力来评价。
本申请所述的药物组合物可以显著地降低疾病的严重程度，增加疾病无症状期的频率，延长疾病无症状期的时间，防治因疾病痛苦而引起的损伤或失能，和/或，预防或治疗肿瘤的转移或复发。
在施用时，所述药物组合物的给药剂量可以为约0.0001至100mg/每kg有需要的受试者的体重(例如，可以为0.0001-100mg/Kg、0.01-100mg/Kg、0.05-100mg/Kg、0.1-100mg/Kg、0.5-100mg/Kg、1-100mg/Kg、5-100mg/Kg或0.01-20mg/Kg)。
在本申请中，所述药物组合物的给药方案可以为每周给药一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次、每3-6个月一次、或起始给药间隔略短(如每周一次至每三周一次)后期给药间隔加长(如每月一次至每3-6个月一次)。具体给药方案根据有需要的受试者的疾病进展情况确定。例如，可以待小鼠的肿瘤面积达到一定阈值时，开始对其给药所述药物组合物。
本申请提供了治疗肿瘤和/或预防肿瘤复发或转移的方法，其包括向有需要的受试者施用所述的细胞因子组合、所述的核酸分子、所述的载体、所述的细胞或所述的药物组合物。
本申请提供了所述的细胞因子组合、所述的核酸分子、所述的载体、所述的细胞或所述的药物组合物，用于治疗肿瘤和/或预防肿瘤复发或转移。
本申请还提供了所述的细胞因子组合、所述的核酸分子、所述的载体、所述的细胞或所述的药物组合物在制备药物中的应用，其中所述药物用于治疗肿瘤和/或预防肿瘤复发或转移。
在本申请中，所述肿瘤可以是实体瘤。例如，所述肿瘤可以选自以下组：肺癌、食道癌、 胃癌、结直肠癌、肝癌、乳腺癌、宫颈癌、甲状腺癌、脑及中枢神经系统癌、胰腺癌、口腔癌、鼻咽癌、头颈癌、喉癌、骨癌、皮肤癌、卵巢癌、前列腺癌、睾丸癌、肾癌、膀胱癌、眼睑肿瘤、白血病和淋巴瘤。
在本申请的所述药物组合物中和/或所述方法中，每种细胞因子的浓度可以为1-10000ng/μL(例如，为1-100000ng/μL、1-10000ng/μL、2-10000ng/μL、4-10000ng/μL、6-10000ng/μL、8-10000ng/μL、10-10000ng/μL、10-1000ng/μL、10-500ng/μL或1-100ng/μL)。
其中，所述施用可以为局部施用。例如，可以为瘤内施用。所述施用可以为单次施用，也可以为多次施用。例如，可以为每周给药两次，每周给药一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次、每3-6个月一次、或起始给药间隔略短(如每周一次至每三周一次)后期给药间隔加长(如每月一次至每3-6个月一次)。
在本申请中，所述受试者是哺乳动物。例如，所述哺乳动物选自下组：人、小鼠、大鼠、猴、犬、猪、羊、牛和猫。又例如，所述受试者为人。
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装置、方法和系统的工作方式，而不用于限制本申请发明的范围。
实施例
以下将结合实施例对本申请做进一步的详细说明，但是本申请要求保护的范围并不局限于实施例表述的范围。
试剂：DMEM培养基，1640培养基，胎牛血清购自Life Technologies公司；细胞培养瓶，培养板购自Corning公司；Puromycin购自Life Technologies公司；限制性内切酶购自Takara和NEB公司；T4 DNA连接酶购自NEB公司；DNA聚合酶购自Takara公司；质粒提取试剂盒，胶回收试剂盒购自Omega Biotech公司；引物合成，基因合成和测序由Life Technologies公司完成。海藻酸钠、氯化钙、BSA、石油醚(petroleum ether)、二氯甲烷(methylene chloride)、聚乳酸(分子量20000)、聚乳酸(分子量5000)购于Sigma-Aldrich公司；壳聚糖季铵盐购于浙江金壳药业。
mIL12，mGMCSF，mFLT3L，mIL2，mIL15，mIL21,mIL7分别表示小鼠IL12、小鼠GMCSF、小鼠FLT3L、小鼠IL2、小鼠IL15，小鼠IL21和小鼠IL7。hIL12，hGMCSF，hFLT3L，hIL2，hIL15，hIL21,hIL7分别表示人IL12、人GMCSF、人FLT3L、人IL2、人IL15，人IL21和人IL7。上述细胞因子购于Peprotech公司。
6-8周龄雌性C57BL/6和Balb/c小鼠购于北京华阜康生物科技股份有限公司。
图1-20、22-31中，每条折线各代表一只小鼠体内肿瘤的面积状况。
实施例1 mIL12+mGMCSF+mIL2壳聚糖季铵盐溶液对小鼠黑色素瘤的治疗效果
消化培养的小鼠黑色素瘤细胞(B16F10)，将2×10 5个细胞注射到C57BL/6小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12、mGMCSF和mIL2，使其终浓度均为200ng/μl，每种取15μl进行混合，获得45μl mIL12+mGMCSF+mIL2的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图1。经过注射的小鼠，100％均获得肿瘤消退。但是有60％的小鼠在肿瘤消退后一至两周左右，在原肿瘤部位会复发，对于复发的肿瘤，待其长到6-8mm时，用同样的方法再次注射mIL12+mGMCSF+mIL2的壳聚糖季铵盐溶液，100％的肿瘤均完全消退。
实施例2 mIL12+mGMCSF+mIL2壳聚糖季铵盐溶液对小鼠结肠癌的治疗效果
消化培养的小鼠结肠癌细胞(CT26)，将5×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12、mGMCSF和mIL2，使其终浓度均为200ng/μl，每种取15μl进行混合，获得45μl mIL12+mGMCSF+mIL2的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图2。100％经过注射的小鼠的肿瘤完全消退。
实施例3.mIL12+mGMCSF+mIL2壳聚糖季铵盐溶液对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12、mGMCSF和mIL2，使其终浓度均为200ng/μl，每种取15μl进行混合，获得45μl mIL12+mGMCSF+mIL2的混合液，然后加入预先配置的3％的壳 聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图3。100％经过注射的小鼠的肿瘤完全消退。
实施例4 mIL12+mGMCSF+mIL2壳聚糖季铵盐溶液对小鼠淋巴瘤的治疗效果
消化培养的小鼠淋巴瘤细胞(EL4)，将2×10 6个的细胞注射到C57BL/6小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12、mGMCSF和mIL2，使其终浓度均为200ng/μl，每种取15μl进行混合，获得45μl mIL12+mGMCSF+mIL2的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图4。100％经过注射的小鼠的肿瘤完全消退。
实施例5不同比例的mIL12+mGMCSF+mIL2壳聚糖季铵盐溶液对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12、mGMCSF和mIL2，每种细胞因子配制200ng/μl和1000ng/μl的溶液，取15μl 1000ng/μl的mIL12和15μl 200ng/μl的mGMCSF和15μl 200ng/μl的mIL2进行混合，获得45μl mIL12+mGMCSF+mIL2(5:1:1)的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。
取15μl 200ng/μl的mIL12和15μl 1000ng/μl的mGMCSF和15μl 200ng/μl的mIL2进行混合，获得45μl mIL12+mGMCSF+mIL2(1:5:1)的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。
取15μl 200ng/μl的mIL12和15μl 200ng/μl的mGMCSF和15μl 1000ng/μl的mIL2进行混合，获得45μl mIL12+mGMCSF+mIL2(1:1:5)的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。
将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图5。100％经过注射的小鼠的肿瘤完全消退，说明采用含有不同比例的细胞因子的细胞因子组合也能达到良好且足够的抗肿瘤效果。
实施例6.mIL12+mGMCSF+mIL2海藻酸钙凝胶对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12、mGMCSF和mIL2，使其终浓度均为200ng/μl，每种取15μl进行混合，获得45μl mIL12+mGMCSF+mIL2的混合液，然后加入1.5μl 2M氯化钙溶液，混匀，最后再加入45μl 1％海藻酸钠溶液，用枪头吹打混匀，形成凝胶。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子海藻酸钙凝胶，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图6。100％经过注射的小鼠的肿瘤完全消退。
实施例7 mIL12+mGMCSF+mIL2聚乳酸微球对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
取2mg聚乳酸(分子量20000和分子量5000的比例1:1)，0.02mg BSA和30μg细胞因子(mIL12或mGMCSF或mIL2)，加入到二氯甲烷中，混匀，然后快速倾倒到石油醚中，待其形成微球，然后将溶液抽滤，冻干，得到包被了细胞因子的聚乳酸微球。使用时，取mIL12微球0.3mg，mGMCSF微球0.3mg，mIL2微球0.3mg，混合后用100μl DMEM重悬，混匀后用28G的注射器缓慢注射到肿瘤内，注射完后将针头滞留少许时间以减少溶液的溢出。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图7。100％经过注射的小鼠的肿瘤完全消退。
实施例8表达mIL12+mGMCSF+mIL2的细胞对小鼠乳腺癌的治疗效果
在EP管内酶切载体pLentis-CMV-MCS-IRES-PURO，体系如下：质粒2μg，酶切缓冲液3μl，BamHI 1μl，XhoI 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，电泳后回收载体片段，待用。
分别合成mIL12、mGMCSF和mIL2基因编码区对应的DNA序列，合成时在其5’端加上BamHI酶切位点，在其3’端加上XhoI酶切位点。酶切合成的带有目的基因的质粒，体系如下：质粒5μg，酶切缓冲液4μl，BamHI 1μl，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mIL12、mGMCSF和mIL2片段，待用。
连接pLentis-CMV-MCS-IRES-PURO和mIL12、mGMCSF、mIL2，体系如下：pLentis-CMV-MCS-IRES-PURO 2μl，mIL12、mGMCSF或mIL2 2μl，连接酶缓冲液1μl，T4 DNA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mIL12、mGMCSF和mIL2的病毒载体pLentis-CMV-mIL12-IRES-PURO、pLentis-CMV-mGMCSF-IRES-PURO、pLentis-CMV-mIL2-IRES-PURO。
制备表达载体的病毒，方法如下：1)消化培养的293FT细胞，计数后将3×10 6个细胞/孔铺入10-cm培养皿中，培养液体积为10ml，共铺5块板。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+pLentis-CMV-mIL12-IRES-PURO或pLentis-CMV-mGMCSF-IRES-PURO或pLentis-CMV-mIL2-IRES-PURO 20μg)，总体积为1045μl，然后加入2M CaCl2 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。
使用表达病毒转染293A细胞，方法如下：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(puromycin)，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细胞因子的细胞，分别为293A(mIL12)、 293A(mGMCSF)、293A(mIL2)。
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
消化培养的293A(mIL12)、293A(mGMCSF)和293A(mIL2)细胞，离心收集，用培养基清洗，然后再离心。用培养基重悬细胞到2×10 7个/ml，三种细胞各取15μl混合，获得45μl的混合液，然后加入1.5μl 2M氯化钙溶液，混匀，最后再加入45μl 1％海藻酸钠溶液，用枪头吹打混匀，形成凝胶。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取带有细胞因子表达细胞的海藻酸钙凝胶，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。注射完的小鼠放回笼内，注意保温，待其苏醒。第一次注射6天后，对于未消退的肿瘤，再次进行相同的注射操作。观察并记录小鼠肿瘤的生长情况。
实验结果见图8。经过注射的小鼠，60％在一次注射后获得肿瘤完全消退，另外的40％的小鼠在第一次注射的6天后进行第二次注射，第二次注射后所有小鼠的肿瘤完全消退。
实施例9 mIL12+mGMCSF+mIL15壳聚糖季铵盐溶液对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12、mGMCSF和mIL15，使其终浓度均为200ng/μl。每种取15μl进行混合，获得45μl mIL12+mGMCSF+mIL15的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图9。经过注射的小鼠，60％的小鼠获得肿瘤完全消退，40％的小鼠肿瘤生长受到抑制。
实施例10 mIL12+mGMCSF+mIL21壳聚糖季铵盐溶液对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12、mGMCSF和mIL21，使其终浓度均为200ng/μl。每种取15μl进行混合，获得45μl mIL12+mGMCSF+mIL21的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然 后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图10。经过注射的小鼠，80％的小鼠获得肿瘤完全消退，20％的小鼠肿瘤生长受到抑制。
实施例11 mIL12+mFLT3L+mIL2壳聚糖季铵盐溶液对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12、mFLT3L和mIL2，使其终浓度均为200ng/μl。每种取15μl进行混合，获得45μl mIL12+mFLT3L+mIL2的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图11。100％经过注射的小鼠的肿瘤完全消退。
实施例12 mIL12+mFLT3L+mIL15壳聚糖季铵盐溶液对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12、mFLT3L和mIL15，使其终浓度均为200ng/μl。每种取15μl进行混合，获得45μl mIL12+mFLT3L+mIL15的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图12。经过注射的小鼠，60％的小鼠获得肿瘤完全消退，40％的小鼠肿瘤生长受到抑制。
实施例13 mIL12+mFLT3L+mIL21壳聚糖季铵盐溶液对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下， 待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12、mFLT3L和mIL21，使其终浓度均为200ng/μl。每种取15μl进行混合，获得45μl mIL12+mFLT3L+mIL21的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图13。经过注射的小鼠，80％的小鼠获得肿瘤完全消退，20％的小鼠肿瘤生长受到抑制。
实施例14 hIL12+hGMCSF+hIL2壳聚糖季铵盐溶液对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子hIL12、hGMCSF和hIL2，使其终浓度均为200ng/μl。每种取15μl进行混合，获得45μl hIL12+hGMCSF+hIL2的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图14。经过注射的小鼠，20％发生肿瘤消退，其他小鼠肿瘤生长也受到了显著的抑制。未经治疗的小鼠，肿瘤会迅速生长，见图20。
实施例15 hIL12+hGMCSF+hIL15壳聚糖季铵盐溶液对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子hIL12、hGMCSF和hIL15，使其终浓度均为200ng/μl。每种取15μl进行混合，获得45μl hIL12+hGMCSF+hIL15的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图15。经过注射的小鼠，肿瘤生长均受到了显著抑制。
实施例16 hIL12+hGMCSF+hIL21壳聚糖季铵盐溶液对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子hIL12、hGMCSF和hIL21，使其终浓度均为200ng/μl，每种取15μl进行混合，获得45μl hIL12+hGMCSF+hIL21的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图16。经过注射的小鼠，肿瘤生长均受到了显著抑制。
实施例17 hIL12+hFLT3L+hIL2壳聚糖季铵盐溶液对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子hIL12、hFLT3L和hIL2，使其终浓度均为200ng/μl，每种取15μl进行混合，获得45μl hIL12+hFLT3L+hIL2的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图17。经过注射的小鼠，20％发生肿瘤消退，其他小鼠肿瘤生长也受到了显著的抑制。
实施例18 hIL12+hFLT3L+hIL15壳聚糖季铵盐溶液对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子hIL12、hFLT3L和hIL15，使其终浓度均为200ng/μl。每种取15μl进行混合，获得45μl hIL12+hFLT3L+hIL15的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G 的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图18。经过注射的小鼠，肿瘤生长均受到了显著抑制。
实施例19 hIL12+hFLT3L+hIL21壳聚糖季铵盐溶液对小鼠乳腺癌的治疗效果
消化培养的小鼠乳腺癌细胞(4T1)，将2×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子hIL12、hFLT3L和hIL21，使其终浓度均为200ng/μl。每种取15μl进行混合，获得45μl hIL12+hFLT3L+hIL21的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图19。经过注射的小鼠，肿瘤生长均受到了显著抑制。
实施例20治愈小鼠对再次接种肿瘤的抵御效果
取经过mIL12+mGMCSF+mIL2壳聚糖季铵盐溶液治疗的4T1荷瘤小鼠，待其肿瘤消退4周后，在第一次接种肿瘤的身体对侧皮下注射2×10 5个4T1肿瘤细胞，观察小鼠情况。实验结果见图21。第二次注射肿瘤细胞后未见有肿瘤长出，而同时注射的野生型小鼠均长出肿瘤。
取经过mIL12+mGMCSF+mIL2壳聚糖季铵盐溶液治疗的CT26荷瘤小鼠，待其肿瘤消退4周后，在第一次接种肿瘤的身体对侧皮下注射2×105 CT26肿瘤细胞，观察小鼠情况。实验结果见图21。图21中，“A/B”表示长出肿瘤的小鼠的数量A占注射所述细胞因子组合小鼠的数量B的比例。例如，2/2表示注射2只，有2只长出肿瘤；0/5表示注射5只，有0只长出肿瘤。图21的结果说明，第二次注射肿瘤细胞后未见有肿瘤长出，而同时注射的野生型小鼠均长出肿瘤。
实施例21 mIL12+mGMCSF+mIL7壳聚糖季铵盐溶液对小鼠结肠癌的治疗效果
消化培养的小鼠结肠癌细胞(CT26)，将5×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12、mGMCSF和mIL7，使其终浓度均为150ng/μl，每种取 15μl进行混合，获得45μl mIL12+mGMCSF+mIL7的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图22。100％经过注射的小鼠的肿瘤完全消退。
实施例22 mIL12+mFLT3L+mIL7壳聚糖季铵盐溶液对小鼠结肠癌的治疗效果
消化培养的小鼠结肠癌细胞(CT26)，将5×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12、mFLT3L和mIL7，使其终浓度均为150ng/μl，每种取15μl进行混合，获得45μl mIL12+mFLT3L+mIL7的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图23。100％经过注射的小鼠的肿瘤完全消退。
实施例23 hIL12+hGMCSF+hIL7壳聚糖季铵盐溶液对小鼠结肠癌的治疗效果
消化培养的小鼠结肠癌细胞(CT26)，将5×10 5个细胞注射到Balb/c小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子hIL12、hGMCSF和hIL7，使其终浓度均为300ng/μl，每种取15μl进行混合，获得45μl hIL12+hGMCSF+hIL7的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图24。经过注射的小鼠，肿瘤生长均受到了显著抑制。
实施例24 hIL12+hFLT3L+hIL7壳聚糖季铵盐溶液对小鼠结肠癌的治疗效果
消化培养的小鼠结肠癌细胞(CT26)，将5×10 5个细胞注射到Balb/c小鼠身体右侧皮下， 待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子hIL12、hFLT3L和hIL7，使其终浓度均为300ng/μl，每种取15μl进行混合，获得45μl hIL12+hFLT3L+hIL7的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图25。经过注射的小鼠，肿瘤生长均受到了显著抑制。
实施例25 mIL12壳聚糖季铵盐溶液对小鼠黑色素瘤的治疗效果
消化培养的小鼠黑色素瘤细胞(B16F10)，将2×10 5个细胞注射到C57BL/6小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12使其终浓度均为200ng/μl，取15μl与45ul无菌水混合，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图26。单独使用IL12不能抑制肿瘤的生长。
实施例26 mGMCSF壳聚糖季铵盐溶液对小鼠黑色素瘤的治疗效果
消化培养的小鼠黑色素瘤细胞(B16F10)，将2×10 5个细胞注射到C57BL/6小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mGMCSF使其终浓度均为200ng/μl，取15μl与45ul无菌水混合，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图27。单独使用GMCSF不能抑制肿瘤的生长。
实施例27 mIL2壳聚糖季铵盐溶液对小鼠黑色素瘤的治疗效果
消化培养的小鼠黑色素瘤细胞(B16F10)，将2×10 5个细胞注射到C57BL/6小鼠身体右 侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL2使其终浓度均为200ng/μl，取15μl与45ul无菌水混合，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图28。单独使用IL2不能抑制肿瘤的生长。
实施例28 mIL12+mGMCSF壳聚糖季铵盐溶液对小鼠黑色素瘤的治疗效果
消化培养的小鼠黑色素瘤细胞(B16F10)，将2×10 5个细胞注射到C57BL/6小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12和mGMCSF，使其终浓度均为200ng/μl，每种取15μl进行混合，再加入15ul无菌水，获得45μl mIL12+mGMCSF的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图29。IL12和GMCSF的组合不能抑制肿瘤的生长。
实施例29 mIL12+mIL2壳聚糖季铵盐溶液对小鼠黑色素瘤的治疗效果
消化培养的小鼠黑色素瘤细胞(B16F10)，将2×10 5个细胞注射到C57BL/6小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mIL12和mIL2，使其终浓度均为200ng/μl，每种取15μl进行混合，再加入15ul无菌水，获得45μl mIL12+mIL2的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图30。IL12和IL2的组合在部分小鼠上能起到一定的抑制作用，但是不能清除肿瘤。
实施例30 mGMCSF+mIL2壳聚糖季铵盐溶液对小鼠黑色素瘤的治疗效果
消化培养的小鼠黑色素瘤细胞(B16F10)，将2×10 5个细胞注射到C57BL/6小鼠身体右侧皮下，待肿瘤的长径达到7-9mm左右时开始进行治疗。
用无菌水溶解细胞因子mGMCSF和mIL2，使其终浓度均为200ng/μl，每种取15μl进行混合，再加入15ul无菌水，获得45μl mGMCSF+mIL2的混合液，然后加入预先配置的3％的壳聚糖季铵盐溶液45μl，用枪头小心吹打混匀。将荷瘤小鼠腹腔注射戊巴比妥钠进行麻醉，然后用29G的胰岛素注射器吸取配制的细胞因子壳聚糖季铵盐溶液，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。注射完的小鼠放回笼内，注意保温，待其苏醒。观察并记录小鼠肿瘤的生长情况。
实验结果见图31。GMCSF和IL2的组合不能抑制肿瘤的生长。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本文所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (20)

  1. 细胞因子组合，其包含至少3种细胞因子，所述细胞因子选自以下组：IL12或其功能性变体、GMCSF或其功能性变体、FLT3L或其功能性变体、IL2或其功能性变体、IL15或其功能性变体、IL21或其功能性变体和IL7或其功能性变体。
  2. 权利要求1的细胞因子组合，其包含以下任一组：
    i)IL12或其功能性变体、GMCSF或其功能性变体，和IL2或其功能性变体；
    ii)IL12或其功能性变体、GMCSF或其功能性变体，和IL15或其功能性变体；
    iii)IL12或其功能性变体、GMCSF或其功能性变体，和IL21或其功能性变体；
    iv)IL12或其功能性变体、FLT3L或其功能性变体，和IL2或其功能性变体；
    v)IL12或其功能性变体、FLT3L或其功能性变体，和IL15或其功能性变体；
    vi)IL12或其功能性变体、FLT3L或其功能性变体，和IL21或其功能性变体；
    vii)IL12或其功能性变体、GMCSF或其功能性变体，和IL7或其功能性变体；和
    viii)IL12或其功能性变体、FLT3L或其功能性变体，和IL7或其功能性变体。
  3. 权利要求1或2的细胞因子组合，其中所述细胞因子选自下组：鼠细胞因子和人细胞因子。
  4. 权利要求1-3中任一项所述的细胞因子组合，其中所述细胞因子组合中的每种细胞因子的浓度为1-10000ng/μL。
  5. 一种或多种核酸分子，其编码权利要求1-4中任一项所述的细胞因子组合。
  6. 权利要求5所述的核酸分子，其包括两种以上的核酸分子，其中每种核酸分子编码一种或多种所述细胞因子，且每种所述核酸分子编码的细胞因子与至少另一种所述核酸分子编码的细胞因子不同。
  7. 载体，其包含权利要求5-6中任一项所述的核酸分子。
  8. 权利要求7所述的载体，其包括两种以上的载体，其中每种载体包含一种或多种所述核酸分子，且每种所述载体包含的所述核酸分子编码的细胞因子与至少另一种载体包含的核酸分子编码的细胞因子不同。
  9. 细胞，其包含权利要求5-6中任一项所述的核酸分子或权利要求7-8中任一项所述的载体。
  10. 权利要求9所述的细胞，其包括两种以上的细胞，其中每种细胞表达一种或多种所述细胞因子，且每种所述细胞表达的细胞因子与至少另一种细胞表达的细胞因子不同。
  11. 药物组合物，其包含权利要求1-4中任一项所述的细胞因子组合、权利要求5-6中任一项所述的核酸分子、权利要求7-8中任一项所述的载体或权利要求9-10中任一项 所述的细胞，以及任选地药学上可接受的载体。
  12. 权利要求11的药物组合物，其被配制为适于局部施用。
  13. 权利要求11-12中任一项所述的药物组合物，其被配制为适于缓释施用。
  14. 如权利要求13所述的药物组合物，其被配制于海藻酸钙凝胶、聚乳酸微球或壳聚糖季铵盐溶液中。
  15. 权利要求1-4中任一项所述的细胞因子组合、权利要求5-6中任一项所述的核酸分子、权利要求7-8中任一项所述的载体、权利要求9-10中任一项所述的细胞或权利要求11-14中任一项所述的药物组合物在制备药物中的应用，其中所述药物用于治疗肿瘤和/或预防肿瘤复发或转移。
  16. 权利要求15所述的应用，其中所述肿瘤是实体瘤。
  17. 权利要求15所述的应用，其中所述肿瘤选自以下组：肺癌、食道癌、胃癌、结直肠癌、肝癌、乳腺癌、宫颈癌、甲状腺癌、脑及中枢神经系统癌、胰腺癌、口腔癌、鼻咽癌、头颈癌、喉癌、骨癌、皮肤癌、卵巢癌、前列腺癌、睾丸癌、肾癌、膀胱癌、眼睑肿瘤、白血病和淋巴瘤。
  18. 权利要求15-17中任一项的应用，所述细胞因子组合中的每种细胞因子的浓度为1-10000ng/μL。
  19. 权利要求15-18中任一项所述的应用，其中所述受试者是哺乳动物。
  20. 权利要求20所述的应用，其中所述哺乳动物选自下组：人、小鼠、大鼠、猴、犬、猪、羊、牛和猫。
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