WO2020024922A1 - 蛋白质异二聚体及其用途 - Google Patents

蛋白质异二聚体及其用途 Download PDF

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WO2020024922A1
WO2020024922A1 PCT/CN2019/098300 CN2019098300W WO2020024922A1 WO 2020024922 A1 WO2020024922 A1 WO 2020024922A1 CN 2019098300 W CN2019098300 W CN 2019098300W WO 2020024922 A1 WO2020024922 A1 WO 2020024922A1
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factor
terminus
polypeptide chain
seq
protein heterodimer
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PCT/CN2019/098300
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French (fr)
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张晋宇
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张晋宇
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Priority to US17/264,193 priority Critical patent/US11795203B2/en
Priority to EP19843948.1A priority patent/EP3831945A4/en
Priority to JP2021505757A priority patent/JP2021532796A/ja
Priority to CN201980050580.6A priority patent/CN112513276B/zh
Publication of WO2020024922A1 publication Critical patent/WO2020024922A1/zh

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    • 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
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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]
    • C07K14/5418—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]
    • C07K14/5434—IL-12
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This application relates to the field of tumor treatment, and in particular, this application relates to protein heterodimers and uses thereof.
  • cytokine is a very important immune signal in the body
  • cytokine fusion protein technology is another hot spot in tumor immunotherapy. This method is based on the fact that these cytokines have the same or related functional activities and have different targets, and genetic engineering techniques are used to fuse two or more cytokines together.
  • the effect of tumor treatment using cytokine fusion protein technology is still unsatisfactory, and there are many areas for improvement.
  • the application provides a protein heterodimer and use thereof.
  • the protein heterodimer has at least a beneficial effect of good antitumor effect.
  • the present application provides a protein heterodimer comprising a first polypeptide chain and a second polypeptide chain different from the first polypeptide chain, wherein the first polypeptide chain comprises IL12a and A first factor fused to the IL12a, the second polypeptide chain comprising IL12b and a second factor fused to the IL12b, and wherein the first factor and the second factor are each independently selected from the group : IL2, GMCSF, IL7, IL15, IL21 and FLT3L.
  • the first factor is different from the second factor.
  • the first factor and the second factor are different from each other and are selected from the group consisting of cytokine group A and cytokine group B, respectively, and the cytokine group A is selected from the group consisting of IL2, IL7, and IL15. And IL21, and cytokine group B is selected from the group consisting of GMCSF and FLT3L.
  • the C-terminus of the IL12a and the N-terminus of the first factor are fused directly or indirectly.
  • the N-terminus of the IL12a is fused directly or indirectly to the C-terminus of the first factor.
  • the C-terminus of the IL12b is fused directly or indirectly to the N-terminus of the second factor.
  • the N-terminus of the IL12b is fused directly or indirectly to the C-terminus of the second factor.
  • the first factor and the second factor are selected from the group consisting of:
  • the first factor is IL2 and the second factor is GMCSF;
  • the first factor is IL7, and the second factor is GMCSF;
  • the first factor is IL15 and the second factor is GMCSF;
  • the first factor is IL21 and the second factor is GMCSF;
  • the first factor is IL2, and the second factor is FLT3L;
  • the first factor is IL7, and the second factor is FLT3L;
  • the first factor is IL15 and the second factor is FLT3L;
  • the first factor is IL21 and the second factor is FLT3L;
  • the first factor is GMCSF, and the second factor is IL2;
  • the first factor is GMCSF, and the second factor is IL7;
  • the first factor is GMCSF, and the second factor is IL15;
  • the first factor is GMCSF, and the second factor is IL21;
  • the first factor is FLT3L
  • the second factor is IL2
  • the first factor is FLT3L, and the second factor is IL7;
  • the first factor is FLT3L and the second factor is IL15;
  • the first factor is FLT3L
  • the second factor is IL21.
  • the IL12a, the IL12b, the first factor, and the second factor are derived from a mammal.
  • the sources of the IL12a, the IL12b, the first factor, and the second factor are the same.
  • the first polypeptide chain includes a sequence as shown in any one of SEQ ID Nos. 60-65 and SEQ ID Nos. 70-73.
  • the second polypeptide chain includes a sequence as shown in any one of SEQ ID Nos. 66-69 and SEQ ID Nos. 74-75.
  • the protein heterodimer includes a sequence as set forth in any one of SEQ ID Nos. 18-37.
  • the application provides one or more isolated nucleic acid molecules that encode the protein heterodimer.
  • the nucleic acid molecule comprises a sequence as set forth in any one of SEQ ID Nos. 38-57.
  • the present application provides an expression vector comprising a nucleotide sequence encoding the protein heterodimer.
  • the present application provides a host cell including the expression vector.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the protein heterodimer, the nucleic acid molecule, the carrier, and / or the host cell, and a pharmacological agent.
  • Acceptable carrier is not limited to the following abbreviations: “X” or “X” or “X”.
  • the present application provides a method for preparing the protein heterodimer, which comprises the steps of: culturing the host cell under the condition that the protein heterodimer can be expressed.
  • the present application provides an application of the protein heterodimer and the pharmaceutical composition in the preparation of a medicament for treating tumors.
  • the tumor comprises melanoma.
  • Figure 1 shows the growth of the tumor
  • Figure 2 shows a schematic diagram of the structure of the protein heterodimer
  • FIG. 3 shows the expression of the protein heterodimer
  • FIG. 4 shows the case where the protein heterodimer induces tumor regression
  • FIG. 5 shows the case where the protein heterodimer induces tumor regression
  • FIG. 6 shows the case where the protein heterodimer induces tumor regression
  • FIG. 7 shows the case where the protein heterodimer induces tumor regression
  • FIG. 8 shows the case where the protein heterodimer induces tumor regression
  • FIG. 9 shows the case where the protein heterodimer induces tumor regression
  • FIG. 10 shows the case where the protein heterodimer induces tumor regression
  • FIG. 11 shows the case where the protein heterodimer induces tumor regression
  • FIG. 12 shows the case where the protein heterodimer induces tumor regression
  • FIG. 13 shows the case where the protein heterodimer induces tumor regression
  • FIG. 14 shows the case where the protein heterodimer induces tumor regression
  • FIG. 15 shows the case where the protein heterodimer induces tumor regression
  • FIG. 16 shows a case where the protein heterodimer induces tumor regression
  • Figure 17 shows the construction of the protein heterodimer.
  • fusion protein generally refers to a protein composed of two or more polypeptides.
  • the polypeptide may have different characteristics, which may be biological characteristics, such as in vitro or in vivo activity; or simple chemical or physical characteristics, such as binding to a target molecule, catalyzing a reaction, etc. .
  • the polypeptide may be directly bound, or may be indirectly bound through a linker (for example, a peptide linker) or a spacer.
  • the fusion protein may be expressed by a fusion gene, encoding a
  • the nucleotide sequence of one polypeptide may be enclosed in a frame with a nucleotide sequence encoding another polypeptide different from the polypeptide.
  • the fusion gene can be expressed as a single protein by a recombinant host cell.
  • cytokine fusion protein generally refers to a fusion protein comprising two or more cytokines.
  • the cytokine fusion protein can be obtained by genetic recombination technology.
  • the cytokine fusion protein can not only have the unique biological activity of the contained cytokines, but also can significantly increase some of them, and can also play a simple compatibility with a single cytokine through the complementary and synergistic effects of biological activities Compound biological functions that are not available may even produce some new structural and biological functions.
  • the cytokine fusion protein may be a protein heterodimer.
  • the term "protein heterodimer” is a dimer composed of two different polypeptide chains.
  • the protein heterodimer may include a first polypeptide chain and a second polypeptide chain, the first polypeptide chain being different from the second polypeptide chain, wherein the first polypeptide
  • the chain may include IL12a and a first factor fused to the IL12a
  • the second polypeptide chain may include IL12b and a second factor fused to the IL12b.
  • the term "cytokine” generally refers to a small molecule protein that is mainly secreted by immune cells and can regulate cell function.
  • the cytokines have important regulatory effects on cell-cell interactions, cell growth and differentiation.
  • the cytokine may be selected from the group consisting of interleukin and colony-stimulating factor.
  • the interleukin is a type of cytokine produced by and acting on a variety of cells. Interleukins play an important role in transmitting information, activating and regulating immune cells, mediating T, B cell activation, proliferation and differentiation, and in inflammatory responses.
  • the interleukin may be selected from one or more of the following group: IL12, IL2, IL15, IL21, and IL7.
  • the colony-stimulating factor may be a cytokine that can stimulate 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 can be named as granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocytes and macrophage colonies, respectively.
  • Stimulating factor (GM-CSF) and multi-energy colony stimulating factor (multi-CSF, IL3) can be selected from one or more of the following group: FMS-related tyrosine kinase 3 ligand (FTL3L) and granulocyte macrophage colony-stimulating factor (GMCSF).
  • FMS-related tyrosine kinase 3 ligand FMS-related tyrosine kinase 3 ligand
  • GMCSF granulocyte macrophage colony-stimulating factor
  • IL12 generally refers to interleukin-12.
  • IL12 and IL-12 are used interchangeably.
  • IL12 can stimulate the proliferation of activated T cells, promote the differentiation of Th0 cells to Th1 cells; also induce the cytotoxic activity of CTL and NK cells and promote their secretion of cytokines such as IFN- ⁇ , TNF- ⁇ , GM-CSF; or promote The expression of NK cells, IL-2R ⁇ receptor and CD56 molecule enhances ADCC effect on tumor cells.
  • IL12 is a heterodimer, which includes a p40 subunit (40kd) and a p35 subunit (35kd). These two subunits can be connected by a disulfide bond.
  • the p35 subunit in mouse IL12 may include the amino acid sequence shown in SEQ ID NO: 1
  • the p40 subunit may include the amino acid sequence shown in SEQ ID NO: 2.
  • the p35 subunit in human IL12 may include the amino acid sequence shown in SEQ ID NO: 9
  • the p40 subunit may include the amino acid sequence shown in SEQ ID NO: 10.
  • IL2 generally refers to interleukin-2.
  • IL2 plays an important role in the body's immune response and anti-viral infection, and can stimulate the proliferation of T cells that have been initiated by specific antigens; at the same time, IL2 can activate T cells and promote the production of cytokines; stimulate NK cell proliferation and enhance the NK killing activity and Production of cytokines, induction of LAK cell production; and promotion of B cell proliferation and secretion of antibodies; activation of macrophages.
  • mouse IL2 may include the amino acid sequence shown in SEQ ID NO: 3.
  • human cytokine IL2 may include the amino acid sequence shown in SEQ ID NO: 11.
  • IL15 generally refers to interleukin-15.
  • IL15 is produced by activated monocytes-macrophages, epidermal cells, and fibroblasts, and can induce B cell proliferation and differentiation.
  • mouse IL15 may include the amino acid sequence shown in SEQ ID NO: 4.
  • human IL15 may include the amino acid sequence shown in SEQ ID NO: 12.
  • IL7 generally refers to interleukin-7.
  • IL7 is mainly secreted by thymus and bone marrow stromal cells, and is a glycoprotein with a relative molecular mass of 25000-28000.
  • IL7 and its receptor-mediated signal transduction pathways are mainly realized through the three pathways of Janus kinase, signal transduction and transcriptional activator, and phosphoinositide 3-kinase.
  • mouse IL7 may include the amino acid sequence shown in SEQ ID NO: 5.
  • human IL7 hIL7 may include the amino acid sequence shown in SEQ ID NO: 13.
  • IL21 generally refers to interleukin-21.
  • IL21 is secreted by activated CD4 + T cells; IL21 is involved in regulating B cell proliferation. Deletion of the IL21 gene can make the body more susceptible to infection by bacteria or viruses. Studies have shown that IL21 can affect the expression level of IL2 receptor protein (CD25) by regulating Bcl-6 protein.
  • mouse IL21 may include the amino acid sequence shown in SEQ ID NO: 6.
  • human IL21 hIL21 may include the amino acid sequence shown in SEQ ID NO: 14.
  • FTL3L generally refers to FMS-related tyrosine kinase 3 ligands.
  • FTL3L can regulate the proliferation and differentiation of non-erythroid hematopoietic stem cells, promote the proliferation, differentiation and maturation of pre-B lymphocytes, dendritic cells, NK cells, and cytotoxic T lymphocytes, and has important antitumor effects.
  • mouse FTL3L may include the amino acid sequence shown in SEQ ID NO: 7.
  • human FTL3L hFTL3L
  • GMCSF generally refers to granulocyte macrophage colony-stimulating factor. GMCSF can stimulate the proliferation, differentiation and activation of granulocytes and macrophages, and increase hematopoietic function; it can also enhance the multiple functions of neutrophils, eosinophils and monocytes. GMCSF can improve the immune activity of immune effector cells phagocytosis bacteria and kill cancer cells, and is conducive to the recovery of neutrophil deficiency caused by tumor chemotherapy and bone marrow transplantation.
  • mouse GMCSF may include the amino acid sequence shown in SEQ ID NO: 8.
  • human GMCSF hGMCSF
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides, or ribonucleotides, or the like, of any length, isolated or artificially synthesized from their natural environment.
  • the nucleic acid molecule may encode the protein heterodimer, which includes the nucleotide sequence shown in any one of SEQ ID Nos. 38-57.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host.
  • the vector can transfer the inserted nucleic acid molecules into and / or between host cells.
  • the vector may include a vector mainly used to insert DNA or RNA into a cell, a vector mainly used to replicate DNA or RNA, and a vector mainly used for expression of DNA and RNA transcription and / or translation.
  • the vector also includes a vector having a plurality of the aforementioned functions.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce a desired expression product.
  • the term "host cell” generally refers to an individual cell, cell line, or cell that can or already contains a plasmid or vector comprising a nucleic acid molecule described herein, or is capable of expressing a protein heterodimer described herein.
  • the cell may include the progeny of a single host cell. Due to natural, accidental, or intentional mutations, the progeny cells may not be exactly the same morphologically or genomically as the original parental cells, but they can express the protein heterodimer combination described in this application.
  • the host cell can be obtained by transfecting the host cell in vitro using the vector described in the present application. In the present application, by transfecting B16 (rtTA) tumor cells or 293A cells with a virus expressing a vector, a host cell capable of expressing the protein heterodimer described in the present application can be obtained.
  • 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, counter ions, metal complexes and / or non-ionic 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 , Talcum powder, calcium alginate gel, chitosan, and nanospheres.
  • the pharmaceutically acceptable carrier may also be selected from the group consisting of a pH adjuster, an osmotic pressure adjuster, a solubilizer, and a bacteriostatic agent.
  • the term "tumor” generally refers to or describes the physiological condition of a mammal, which is typically characterized by dysregulated cell proliferation or survival.
  • the tumor may be selected from the group: 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, bone, skin, ovarian, prostate, testicular, renal, bladder, eyelid, leukemia and lymphoma.
  • the present application provides a protein heterodimer, which may include a first polypeptide chain and a second polypeptide chain different from the first polypeptide chain, wherein the first polypeptide chain may comprise IL12a And a first factor fused to the IL12a, the second polypeptide chain may include IL12b and a second factor fused to the IL12b, and the first factor and the second factor may be independently selected From the lower group: IL2, GMCSF, IL7, IL15, IL21 and FLT3L.
  • the first factor may be different from the second factor.
  • the protein heterodimer may be a cytokine fusion protein, which combines two or more of the cytokines, namely, IL12 and IL2, GMCSF, IL7, IL15, IL21, and FLT3L, Fusion through genetic recombination technology.
  • the protein heterodimer can not only have the unique biological activity of its constituent factors, but also can exert biological functions that are not possessed by a single cytokine through the complementary and synergistic effects of biological activities, and can even produce some new Structure and biological function.
  • the first factor and the second factor may be different from each other and selected from cytokine group A and cytokine group B, respectively, and the cytokine group A may be selected from the following group: IL2, IL7, IL15 And IL21, and cytokine group B can be selected from the group consisting of GMCSF and FLT3L.
  • the first factor can select IL2 in cytokine group A, and the second factor can select GMCSF in cytokine group B; or the first factor can select IL7 in cytokine group A, and the second factor can select cells GMCSF in factor group B; or the first factor can select IL15 in cytokine group A, and the second factor can select GMCSF in cytokine group B; or the first factor can select IL21 in cytokine group A, and
  • the second factor can select GMCSF in cytokine group B; or the first factor can select IL2 in cytokine group A, and the second factor can select FLT3L in cytokine group B; or the first factor can select cytokine group IL7 in A, and the second factor can select FLT3L in cytokine group B; or the first factor can select IL15 in cytokine group A, and the second factor can select FLT3L in cytokine group B; or the first The factor can select IL21 in cytokin
  • the C-terminus of the IL12a in the first polypeptide chain, may be directly or indirectly fused with the N-terminus of the first factor.
  • the C-terminus of the IL12a in the first polypeptide chain, may be fused directly or indirectly with the N-terminus of IL2; or in the first polypeptide chain, the C-terminus of the IL12a may be fused to the IL7
  • the N-terminus is directly or indirectly fused; or in the first polypeptide chain, the C-terminus of the IL12a may be directly or indirectly fused with the N-terminus of IL15; or in the first polypeptide chain, the IL12a
  • the C-terminus may be fused directly or indirectly to the N-terminus of IL21; or the C-terminus of the IL12a may be fused directly or indirectly to the N-terminus of GMCSF in the first polypeptide chain; or the first polypeptide chain
  • the C-terminus of the IL12a may
  • the N-terminus of the IL12a in the first polypeptide chain, may be directly or indirectly fused with the C-terminus of the first factor.
  • the N-terminus of IL12a in the first polypeptide chain, may be fused directly or indirectly with the C-terminus of IL2; or in the first polypeptide chain, the N-terminus of IL12a may be fused with IL7
  • the C-terminus is directly or indirectly fused; or in the first polypeptide chain, the N-terminus of the IL12a may be directly or indirectly fused with the C-terminus of IL15; or in the first polypeptide chain, the IL12a
  • the N-terminus may be directly or indirectly fused with the C-terminus of IL21; or in the first polypeptide chain, the N-terminus of the IL12a may be directly or indirectly fused with the C-terminus of GMCSF; or at the first polypeptide chain
  • the N-terminus of the IL12a may be
  • the C-terminus of the IL12b may be directly or indirectly fused with the N-terminus of the second factor.
  • the C-terminus of the IL12b may be fused directly or indirectly with the N-terminus of IL2; or in the second polypeptide chain, the C-terminus of the IL12b may be fused to the IL7
  • the N-terminus is directly or indirectly fused; or in the second polypeptide chain, the C-terminus of the IL12b may be directly or indirectly fused with the N-terminus of IL15; or in the second polypeptide chain, the IL12b
  • the C-terminus may be fused directly or indirectly to the N-terminus of IL21; or the C-terminus of the IL12b may be fused directly or indirectly to the N-terminus of GMCSF in the second polypeptide chain; or in the second polypeptide chain
  • the C-terminus of the IL12b may be directly or indirectly fused with the N-terminus of the second
  • the N-terminus of the IL12b may be directly or indirectly fused with the C-terminus of the second factor.
  • the N-terminus of IL12b may be fused directly or indirectly with the C-terminus of IL2; or in the second polypeptide chain, the N-terminus of IL12b may be fused with IL7
  • the C-terminus is directly or indirectly fused; or in the second polypeptide chain, the N-terminus of the IL12b may be directly or indirectly fused with the C-terminus of IL15; or in the second polypeptide chain, the IL12b
  • the N-terminus may be fused directly or indirectly to the C-terminus of IL21; or the N-terminus of the IL12b may be fused directly or indirectly to the C-terminus of GMCSF in the second polypeptide chain; or in the second polypeptide chain
  • the N-terminus of the IL12b may be fused
  • the protein heterodimer may include any permutation and combination of positional connection relationships of all the cytokines.
  • the first factor and the second factor may be selected from the following group:
  • the first factor may be IL2, and the second factor may be GMCSF;
  • the first factor may be IL7, and the second factor may be GMCSF;
  • the first factor may be IL15, and the second factor may be GMCSF;
  • the first factor may be IL21, and the second factor may be GMCSF;
  • the first factor can be IL2, and the second factor can be FLT3L;
  • the first factor can be IL7, and the second factor can be FLT3L;
  • the first factor can be IL15, and the second factor can be FLT3L;
  • the first factor can be IL21, and the second factor can be FLT3L;
  • the first factor may be GMCSF, and the second factor may be IL2;
  • the first factor may be GMCSF, and the second factor may be IL7;
  • the first factor may be GMCSF, and the second factor may be IL15;
  • the first factor may be GMCSF, and the second factor may be IL21;
  • the first factor may be FLT3L, and the second factor may be IL2;
  • the first factor may be FLT3L, and the second factor may be IL7;
  • the first factor may be FLT3L, and the second factor may be IL15; and,
  • the first factor may be FLT3L, and the second factor may be IL21.
  • the IL12a and the first factor, and / or the IL12b and the second factor may be linked by a connecting peptide.
  • the amino acid sequence of the linker peptide can be as shown in SEQ ID.17.
  • the IL12a and the IL12b may be covalently bonded through a disulfide bond.
  • the protein heterodimer may be formed between the first polypeptide chain and the second polypeptide chain through a disulfide bond between IL12a and IL12b.
  • a nucleotide sequence encoding a self-cleaving peptide may be included between the nucleotide sequences encoding the first polypeptide chain and the second polypeptide chain.
  • the position of the self-cleaving peptide is cleaved, thereby constituting the first polypeptide chain and the second polypeptide chain and passing Disulfide bonds form the heterodimer of the protein.
  • the first polypeptide chain and the second polypeptide chain may be connected by a 2A self-cleaving peptide or a flexible amino acid linker peptide.
  • amino acid sequence of the 2A self-cleaving peptide may be shown in SEQ ID NO.58, and the nucleotide sequence encoding the 2A self-cleaving peptide may be shown in SEQ ID NO.59.
  • amino acid sequence of the flexible amino acid linker peptide may be as shown in SEQ ID.17.
  • the connecting peptide may also be included between the first polypeptide chain and the second polypeptide chain.
  • the C-terminus of IL12a can be fused with the N-terminus of IL2 to form a first polypeptide chain
  • the C-terminus of IL12b can be fused with the N-terminus of GMCSF to form a second polypeptide chain, thereby The IL12a-IL2-IL12b-GMCSF protein heterodimer is formed.
  • the C-terminus of IL2 can be fused with the N-terminus of IL12a to form a first polypeptide chain
  • the C-terminus of IL12b can be fused with the N-terminus of GMCSF to form a second polypeptide chain, thereby IL2-IL12a-IL12b-GMCSF protein heterodimer is formed.
  • the C-terminus of IL12a can be fused with the N-terminus of IL2 to form a first polypeptide chain
  • the C-terminus of GMCSF can be fused with the N-terminus of IL12b to form a second polypeptide chain, thereby The IL12a-IL2-GMCSF-IL12b protein heterodimer is formed.
  • the C-terminus of IL2 can be fused with the N-terminus of IL12a to form a first polypeptide chain
  • the C-terminus of GMCSF can be fused with the N-terminus of IL12b to form a second polypeptide chain, thereby The IL2-IL12a-GMCSF-IL12b protein heterodimer is formed.
  • the C-terminus of IL12a can be fused with the N-terminus of GMCSF to form a first polypeptide chain
  • the C-terminus of IL12b can be fused with the N-terminus of IL2 to form a second polypeptide chain, thereby IL12a-GMCSF-IL12b-IL2 protein heterodimer is formed.
  • the C-terminus of IL12a can be fused with the N-terminus of IL7 to form a first polypeptide chain
  • the C-terminus of IL12b can be fused with the N-terminus of GMCSF to form a second polypeptide chain, thereby The IL12a-IL7-IL12b-GMCSF protein heterodimer is formed.
  • the C-terminus of IL12a can be fused with the N-terminus of IL15 to form a first polypeptide chain
  • the C-terminus of IL12b can be fused with the N-terminus of GMCSF to form a second polypeptide chain, thereby The IL12a-IL15-IL12b-GMCSF protein heterodimer is formed.
  • the C-terminus of IL12a can be fused with the N-terminus of IL21 to form a first polypeptide chain
  • the C-terminus of IL12b can be fused with the N-terminus of GMCSF to form a second polypeptide chain, thereby The IL12a-IL21-IL12b-GMCSF protein heterodimer is formed.
  • the C-terminus of IL12a can be fused with the N-terminus of IL2 to form a first polypeptide chain
  • the C-terminus of IL12b can be fused with the N-terminus of FLT3L to form a second polypeptide chain, thereby The IL12a-IL2-IL12b-FLT3L protein heterodimer is formed.
  • the C-terminus of IL12a can be fused with the N-terminus of IL7 to form a first polypeptide chain
  • the C-terminus of IL12b can be fused with the N-terminus of FLT3L to form a second polypeptide chain, thereby The IL12a-IL7-IL12b-FLT3L protein heterodimer is formed.
  • the C-terminus of IL12a can be fused with the N-terminus of IL15 to form a first polypeptide chain
  • the C-terminus of IL12b can be fused with the N-terminus of FLT3L to form a second polypeptide chain, thereby The IL12a-IL15-IL12b-FLT3L protein heterodimer is formed.
  • the C-terminus of IL12a can be fused with the N-terminus of IL21 to form a first polypeptide chain
  • the C-terminus of IL12b can be fused with the N-terminus of FLT3L to form a second polypeptide chain, thereby The IL12a-IL21-IL12b-FLT3L protein heterodimer is formed.
  • the IL12a, the IL12b, the first factor, and the second factor may be derived from a mammal.
  • the mammal can be selected from the group consisting of human, mouse, rat, monkey, dog, pig, sheep, cow, and cat.
  • the mammal can be selected from the group consisting of human and mouse.
  • the IL12a, IL12b, IL2, GMCSF, IL7, IL15, IL21, and FLT3L can be selected from mice, and the mouse-derived IL12a, IL12b, IL2, GMCSF, IL7, IL15, IL21, and FLT3L can be abbreviated as mIL12a , MIL12b, mIL2, mGMCSF, mIL7, mIL15, mIL21, and mFLT3L.
  • the IL12a, IL12b, IL2, GMCSF, IL7, IL15, IL21, and FLT3L can be selected from humans, and the human-derived IL12a, IL12b, IL2, GMCSF, IL7, IL15, IL21, and FLT3L can be abbreviated as hIL12a, hIL12b , HIL2, hGMCSF, hIL7, hIL15, hIL21, and hFLT3L.
  • the sources of the IL12a, the IL12b, the first factor, and the second factor may be the same.
  • the first polypeptide chain may include a sequence shown in any one of SEQ ID Nos. 60-65 and SEQ ID Nos. 70-73.
  • the second polypeptide chain may include a sequence shown in any one of SEQ ID Nos. 66-69 and SEQ ID Nos. 74-75.
  • the protein heterodimer may include a sequence as shown in any one of SEQ ID Nos. 18-37.
  • the C-terminus of mIL12a and the N-terminus of mIL2 may be fused to form the first polypeptide chain of mIL12a-mIL2 (the sequence is shown in SEQ ID NO.60), and the C-terminus of mIL12b and The N-terminus of mGMCSF can be fused to form the second polypeptide chain of mIL12b-mGMCSF (the sequence is shown in SEQ ID NO.66), and the mIL12a-mIL2-mIL12b-mGMCSF protein heterodimer can be formed (the sequence is shown in SEQ ID ID NO.18). ⁇ ).
  • the C-terminus of mIL2 and the N-terminus of mIL12a can be fused to form the first polypeptide chain of mIL2-mIL12a (the sequence is shown in SEQ ID NO. 61), and the C-terminus of mIL12b and The N-terminus of mGMCSF can be fused to form a second polypeptide chain of mIL12b-mGMCSF (sequence is shown in SEQ ID NO.66), and mIL2-mIL12a-mIL12b-mGMCSF protein heterodimer can be formed (sequence shown in SEQ ID ID NO.19 ⁇ ).
  • the C-terminus of mIL12a and the N-terminus of mIL2 can be fused to form the first polypeptide chain of mIL12a-mIL2 (the sequence is shown in SEQ ID NO. 60), and the C-terminus of mGMCSF and The N-terminus of mIL12b can be fused to form the second polypeptide chain of mGMCSF-mIL12b (the sequence is shown in SEQ ID NO.67), and the mIL12a-mIL2-mGMCSF-mIL12b protein heterodimer can be formed (the sequence is shown in SEQ ID ID NO.20). ⁇ ).
  • the C-terminus of mIL2 and the N-terminus of mIL12a can be fused to form the first polypeptide chain of mIL2-mIL12a (sequence is shown in SEQ ID NO. 61), and the C-terminus of mGMCSF and The N-terminus of mIL12b can be fused to form the second polypeptide chain of mGMCSF-mIL12b (the sequence is shown in SEQ ID NO.67), and the mIL2-mIL12a-mGMCSF-mIL12b protein heterodimer can be formed (the sequence is shown in SEQ ID ID NO.21 ⁇ ).
  • the C-terminus of mIL12a and the N-terminus of mGMCSF can be fused to form the first polypeptide chain of mIL12a-mGMCSF (the sequence is shown in SEQ ID NO. 62), and the C-terminus of mIL12b and The N-terminus of mIL2 can be fused to form the second polypeptide chain of mIL12b-mIL2 (the sequence is shown in SEQ ID NO.68), and the mIL12a-mGMCSF-mIL12b-mIL2 protein heterodimer can be formed (the sequence is shown in SEQ ID ID NO.22). ⁇ ).
  • the C-terminus of mIL12a and the N-terminus of mIL7 can be fused to form the first polypeptide chain of mIL12a-mIL7 (the sequence is shown in SEQ ID NO. 63), and the C-terminus of mIL12b and The N-terminus of mGMCSF can be fused to form the second polypeptide chain of mIL12b-mGMCSF (sequence is shown in SEQ ID NO.66), and mIL12a-mIL7-mIL12b-mGMCSF protein heterodimer can be formed (sequence shown in SEQ ID ID NO.23 ⁇ ).
  • the C-terminus of mIL12a and the N-terminus of mIL15 can be fused to form the first polypeptide chain of mIL12a-mIL15 (the sequence is shown in SEQ ID NO.64), and the C-terminus of mIL12b and The N-terminus of mGMCSF can be fused to form the second polypeptide chain of mIL12b-mGMCSF (sequence is shown in SEQ ID NO.66), and mIL12a-mIL15-mIL12b-mGMCSF protein heterodimer can be formed (sequence shown in SEQ ID ID NO.24 ⁇ ).
  • the C-terminus of mIL12a and the N-terminus of mIL21 can be fused to form the first polypeptide chain of mIL12a-mIL21 (the sequence is shown in SEQ ID NO. 65), and the C-terminus of mIL12b and The N-terminus of mGMCSF can be fused to form the second polypeptide chain of mIL12b-mGMCSF (sequence is shown in SEQ ID NO.66), and mIL12a-mIL21-mIL12b-mGMCSF protein heterodimer can be formed (sequence shown in SEQ ID ID NO.25 ⁇ ).
  • the C-terminus of mIL12a and the N-terminus of mIL2 may be fused to form the first polypeptide chain of mIL12a-mIL2 (the sequence is shown in SEQ ID NO.60), and the C-terminus of mIL12b and The N-terminus of mFLT3L can be fused to form the second polypeptide chain of mIL12b-mFLT3L (the sequence is shown in SEQ ID NO.69), and the mIL12a-mIL2-mIL12b-mFLT3L protein heterodimer can be formed (the sequence is shown in SEQ ID ID NO.26). ⁇ ).
  • the C-terminus of mIL12a and the N-terminus of mIL7 can be fused to form the first polypeptide chain of mIL12a-mIL7 (the sequence is shown in SEQ ID NO. 63), and the C-terminus of mIL12b and The N-terminus of mFLT3L can be fused to form the second polypeptide chain of mIL12b-mFLT3L (sequence is shown in SEQ ID NO.69), and mIL12a-mIL7-mIL12b-mFLT3L protein heterodimer can be formed (sequence as shown in SEQ ID ID NO.27 ⁇ ).
  • the C-terminus of mIL12a and the N-terminus of mIL15 can be fused to form the first polypeptide chain of mIL12a-mIL15 (the sequence is shown in SEQ ID NO.64), and the C-terminus of mIL12b and The N-terminus of mFLT3L can be fused to form the second polypeptide chain of mIL12b-mFLT3L (the sequence is shown in SEQ ID NO.69), and the mIL12a-mIL15-mIL12b-mFLT3L protein heterodimer can be formed (the sequence is shown in SEQ ID ID NO.28). ⁇ ).
  • the C-terminus of mIL12a and the N-terminus of mIL21 can be fused to form the first polypeptide chain of mIL12a-mIL21 (the sequence is shown in SEQ ID NO. 65), and the C-terminus of mIL12b and The N-terminus of mFLT3L can be fused to form the second polypeptide chain of mIL12b-mFLT3L (sequence is shown in SEQ ID NO.69), and mIL12a-mIL21-mIL12b-mFLT3L protein heterodimer can be formed (sequence as shown in SEQ ID ID NO.29 ⁇ ).
  • the C-terminus of hIL12a and the N-terminus of hIL2 can be fused to form the first polypeptide chain of hIL12a-hIL2 (the sequence is shown in SEQ ID NO. 70), and the C-terminus of hIL12b and The N-terminus of hGMCSF can be fused to form the second polypeptide chain of hIL12b-hGMCSF (the sequence is shown in SEQ ID NO.74), and the hIL12a-hIL2-hIL12b-hGMCSF protein heterodimer can be formed (the sequence is shown in SEQ ID ID NO.30). ⁇ ).
  • the C-terminus of hIL12a and the N-terminus of hIL7 can be fused to form the first polypeptide chain of hIL12a-hIL7 (the sequence is shown in SEQ ID NO. 71), and the C-terminus of hIL12b and The N-terminus of hGMCSF can be fused to form the second polypeptide chain of hIL12b-hGMCSF (the sequence is shown in SEQ ID NO.74), and the hIL12a-hIL7-hIL12b-hGMCSF protein heterodimer can be formed (the sequence is shown in SEQ ID ID NO.31). ⁇ ).
  • the C-terminus of hIL12a and the N-terminus of hIL15 can be fused to form a first polypeptide chain of hIL12a-hIL15 (the sequence is shown in SEQ ID NO. 72), and the C-terminus of hIL12b and The N-terminus of hGMCSF can be fused to form the second polypeptide chain of hIL12b-hGMCSF (the sequence is shown in SEQ ID NO.74), and can form the hIL12a-hIL15-hIL12b-hGMCSF protein heterodimer (the sequence is shown in SEQ ID ID NO.32). ⁇ ).
  • the C-terminus of hIL12a and the N-terminus of hIL21 can be fused to form a first polypeptide chain of hIL12a-hIL21 (the sequence is shown in SEQ ID NO. 73), and the C-terminus of hIL12b and The N-terminus of hGMCSF can be fused to form the second polypeptide chain of hIL12b-hGMCSF (the sequence is shown in SEQ ID NO.74), and the hIL12a-hIL21-hIL12b-hGMCSF protein heterodimer can be formed (the sequence is shown in SEQ ID ID NO.33). ⁇ ).
  • the C-terminus of hIL12a and the N-terminus of hIL2 can be fused to form the first polypeptide chain of hIL12a-hIL2 (the sequence is shown in SEQ ID NO. 70), and the C-terminus of hIL12b and The N-terminus of hFLT3L can be fused to form the second polypeptide chain of hIL12b-hFLT3L (sequence is shown in SEQ ID NO.75), and hIL12a-hIL2-hIL12b-hFLT3L protein heterodimer can be formed (sequence is shown in SEQ ID ID NO.34 ⁇ ).
  • the C-terminus of hIL12a and the N-terminus of hIL7 can be fused to form the first polypeptide chain of hIL12a-hIL7 (the sequence is shown in SEQ ID NO. 71), and the C-terminus of hIL12b and The N-terminus of hFLT3L can be fused to form the second polypeptide chain of hIL12b-hFLT3L (the sequence is shown in SEQ ID NO.75), and the hIL12a-hIL7-hIL12b-hFLT3L protein heterodimer can be formed (the sequence is shown in SEQ ID ID NO.35). ⁇ ).
  • the C-terminus of hIL12a and the N-terminus of hIL15 can be fused to form a first polypeptide chain of hIL12a-hIL15 (the sequence is shown in SEQ ID NO. 72), and the C-terminus of hIL12b and The N-terminus of hFLT3L can be fused to form a second polypeptide chain of hIL12b-hFLT3L (sequence is shown in SEQ ID NO.75), and hIL12a-hIL15-hIL12b-hFLT3L protein heterodimer can be formed (sequence is shown in SEQ ID ID NO.36 ⁇ ).
  • the C-terminus of hIL12a and the N-terminus of hIL21 can be fused to form a first polypeptide chain of hIL12a-hIL21 (the sequence is shown in SEQ ID NO. 73), and the C-terminus of hIL12b and The N-terminus of hFLT3L can be fused to form the second polypeptide chain of hIL12b-hFLT3L (the sequence is shown in SEQ ID NO.75), and the hIL12a-hIL21-hIL12b-hFLT3L protein heterodimer can be formed (the sequence is shown in SEQ ID ID NO.37 ⁇ ).
  • the protein heterodimer can be used for treating tumors.
  • the application provides a use of the protein heterodimer in the preparation of a medicament, which can be used to treat a tumor.
  • the tumor may be selected from the group: melanoma, 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.
  • Nucleic acid molecule expression vector, host cell, pharmaceutical composition and preparation method
  • the application provides one or more isolated nucleic acid molecules that can encode the protein heterodimer.
  • the nucleic acid molecule may include a sequence as shown in any one of SEQ ID Nos. 38-57.
  • SEQ ID NO. 47 SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO.54, SEQ ID NO.55, SEQ ID NO.56 and SEQ ID NO.57.
  • the nucleic acid molecule can be used to treat a tumor.
  • the application provides a use of the nucleic acid molecule in the preparation of a medicament, which can be used to treat a tumor.
  • the tumor may be selected from the group: melanoma, 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 present application provides an expression vector, which may include a nucleotide sequence encoding the protein heterodimer.
  • an expression vector which may include a nucleotide sequence encoding the protein heterodimer.
  • nucleotide sequence mIL12aIL7IL12bGMCSF by separately encoding mIL12aIL2IL12bGMCSF, mIL2IL12aIL12bGMCSF, mIL12aIL2GMCSFIL12b, mIL2IL12aGMCSFIL12b, mIL12aGMCSFIL12b, mIL12aGMCSFIL12b, mIL12aGMCSFIL12b, nucleotide sequence mIL12aIL7IL12bGMCSF, mIL12aIL15IL12bGMCSF, mIL12aIL21IL12bGMCSF, mIL12aIL2IL12bFLT3L, mIL12aIL7IL12bFLT3L, mIL
  • hIL12aIL2IL12bGMCSF for example, by respectively hIL12aIL2IL12bGMCSF, hIL12aIL7IL12bGMCSF, hIL12aIL15IL12bGMCSF, hIL12aIL21IL12bGMCSF, hIL12aIL2IL12bFLT3L, hIL12aIL7IL12bFLT3L, hIL12aIL15IL12bFLT3L hIL12aIL21IL12bFLT3L and inserted into pLentis-CMV-MCS-IRES-PURO thereby construct an expression vector pLentis-CMV-hIL12aIL2IL12bGMCSF-IRES-PURO, pLentis- CMV-hIL12aIL7IL12bGMCSF-IRES-PURO, pLentis-CMV-hIL12aIL15IL12bGMCSF-IRES-PURO, pLentis-CMV-hIL
  • the expression vector can be used for treating tumors.
  • the present application provides a use of the expression vector in the preparation of a medicament, which can be used to treat a tumor.
  • the tumor may be selected from the group: melanoma, 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.
  • vectors and plasmids such as a method of inserting a gene encoding a protein into a vector and a plasmid or a method of introducing a plasmid into a host cell, are well known to those of ordinary skill in the art, and in many cases They are described in publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: Laboratory, Manual, 2nd Edition, Cold Spring, Harbor, Laboratory Press.
  • the present application provides a host cell, which may include the expression vector.
  • the host cell may be prepared by transfecting B16 (rtTA) tumor cells using the expression vector.
  • rtTA B16
  • pLentis-CMV-hIL12aIL2IL12bGMCSF-IRES-PURO pLentis-CMV-hIL12aIL7IL12bGMCSF-IRES-PURO
  • pLentis-CMV-hIL12aIL15IL12bGMCIL-ILS12 -IRES-PURO pLentis-CMV-hIL12aIL7IL12bFLT3L-IRES-PURO
  • pLentis-CMV-hIL12aIL15IL12bFLT3L-IRES-PURO pLentis-CMV-hIL12aIL21IL12bFLT3L-IRES-PURO expression vector h293A, virus transfected 293AA, and transfected 293AA with virus-infected cells -hIL12aIL7IL12bGMCSF, 293A-hIL12aIL15IL12bGMCSF, 293A-hIL12aIL21IL12
  • the host cell can be used to treat a tumor.
  • the tumor may be selected from the group: melanoma, 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 application provides a use of the host cell in the preparation of a medicament, which can be used to treat a tumor.
  • the tumor may be selected from the group: melanoma, 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 application provides a protein heterodimer, a nucleic acid molecule, an expression vector, a host cell, and a pharmaceutical composition, which is used for treating tumors.
  • a method for treating a tumor comprising the steps of administering to a subject in need thereof a protein heterodimer, a nucleic acid molecule, an expression vector, a host cell, and / or a pharmaceutical composition described herein.
  • the tumor may be selected from the group: melanoma, 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 present application provides a method for preparing the protein heterodimer, which may include the steps of: culturing the host cell under the condition that the protein heterodimer can be expressed. .
  • the present application provides a pharmaceutical composition, which may include the protein heterodimer, the nucleic acid molecule, the vector, and / or the host cell, and a pharmaceutically acceptable Carrier.
  • the pharmaceutically acceptable carrier may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counter ion, a metal complex, and / or a non-ionic surfactant.
  • Agent 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 , Talcum powder, calcium alginate gel, chitosan, and nanospheres.
  • the pharmaceutically acceptable carrier may also be selected from the group consisting of a pH adjuster, an osmotic pressure adjuster, a solubilizer, and a bacteriostatic agent.
  • the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at a tumor site, inhalation, rectal administration, vaginal administration, transdermal Dosing or via subcutaneous depots.
  • the pharmaceutical composition can be used for treating tumors.
  • the tumor may be selected from the group: melanoma, 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 pharmaceutical composition can be used to inhibit tumor growth.
  • the pharmaceutical composition of the present application can inhibit or delay the development or progression of the disease, can reduce the size of the tumor (or even substantially eliminate the tumor) by promoting the expression of cytokines, and / or can reduce and / or stabilize the disease state.
  • the pharmaceutical composition may contain a therapeutically effective amount of the protein heterodimer, the nucleic acid molecule, the vector, and / or the host cell.
  • the therapeutically effective amount is a dose required to prevent and / or treat (at least partially treat) a condition or disorder (eg, cancer) and / or any complications thereof in a subject having or at risk for development.
  • the present application provides a method for relieving or treating a tumor, which method may include administering the protein heterodimer, the nucleic acid molecule, the expression vector, the host cell, and / or the Said pharmaceutical composition.
  • the method of administration may include oral administration, intravenous administration, intramuscular administration, in situ administration at a tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or Administration is by subcutaneous depot.
  • DMEM medium, 1640 medium, fetal bovine serum were purchased from lifetechnologies company; cell culture flasks and culture plates were purchased from Corning company; doxycycline (DOX) was purchased from Shanghai Shenggong Biological Engineering Co., Ltd .; Puromycin (Puromycin ).
  • Blasticidin was purchased from Chemicon; restriction enzymes were purchased from Takara and NEB; ligase was purchased from NEB; DNA polymerase was purchased from Takara; plasmid extraction kits and gel recovery kits were purchased from OmegaBiotech; primers The synthesis was completed by Shanghai Shenggong Biological Engineering Co., Ltd .; the gene synthesis was completed by Nanjing Kingsray; the ELISA kit was purchased from Baoshi De Company.
  • cytokines mIL12, mGMCSF, mFLT3L, mIL2, mIL15, mIL21, mIL7, hIL12, hGMCSF, hFLT3L, hIL2, hIL15, hIL21 and hIL7 were purchased from Peprotech.
  • each polyline represents the area of a tumor in a mouse.
  • 5 polylines represent data of 5 mice in one experiment; scores represent mice with tumor clearance.
  • the enzyme digestion system was as follows: 6 ⁇ g of pUC57 vector plasmid with rtTA ligated, 4 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l EcoRI, add water to a total volume of 40 ⁇ l, and stand at 37 ° C for 12 hours. . Remove the EP tube, add 4.4 ⁇ l of 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. The rtTA fragment is recovered after the electrophoresis and is ready for use.
  • the digestion system is as follows: 2 ⁇ g pLentis-CMV-IRES-Bsd vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l EcoRI, add water to a total volume of 30 ⁇ l, 37 ° C Let stand for 12 hours. Remove the EP tube, add 3.3 ⁇ l of 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-CMV-IRES-Bsd vector fragment is recovered and used.
  • the system is as follows: 2 ⁇ l pLentis-CMV-IRES-Bsd, 2 ⁇ l RTTA, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water, and place at room temperature for 4 hours.
  • the ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, placed in LB medium, and cultured overnight at 37 ° C in a shaker.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the rtTA fragment was successfully identified by enzyme digestion.
  • pLentis-CMV-IRES-Bsd vector and then send the correct vector for sequencing to confirm that the first expression vector pLentis-CMV-rtTA-IRES-Bsd was successfully constructed.
  • B16 (rtTA) After digestion cultured mouse B16 melanoma cells, at 10 5 cells / well were seeded into 6-well culture volume of 1ml, 24 hours, 10 ⁇ l of the first expression vector pLentis-CMV-rtTA-IRES- Bsd virus After continuing to culture in the incubator for 24 hours, discard the supernatant and replace it with fresh medium to continue the cultivation. After the cells are full, transfer them to the culture flask and add rice blast to the concentration suitable for the cells. Blasticidin, continue to culture, replace the medium every two days, and maintain the blasticidin concentration of 8 ⁇ g / ml. After one week of screening, the surviving cells are the cells that stably express the regulatory protein. Named B16 (rtTA).
  • a primer was used to carry out a PCR reaction with the GFP gene as a template to amplify the GFP gene.
  • the PCR conditions were performed according to the instructions of PrimeStarHSDNA polymerase.
  • the digestion system is as follows: 30 ⁇ g PCR recovery product, 4 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l EcoRI, add water to the total The volume was 40 ⁇ l, and left at 37 ° C. for 12 hours. Take out the EP tube, add 4.4 ⁇ l of 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, recover the GFP gene fragment and set aside.
  • the digestion can regulate the expression vector, the system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l EcoRI, add water to a total volume of 30 ⁇ l, and stand at 37 ° C for 12 hours. Take out the EP tube, add 3.3 ⁇ l of 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the carrier fragment is recovered and used. PLentis-PTRE-MCS-PGK-PURO and GFP were ligated.
  • the ligating system was as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l GFP, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium. The plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, send the correct vector for sequencing to confirm that the second expression vector pLentis-PTRE-GFP-PGK-PURO was successfully constructed.
  • the method of preparing the virus of the GFP expression vector is the same as that of the first expression vector, and the virus of the second expression vector pLentis-PTRE-GFP-PGK-PURO is obtained.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, after 24 hours, was added 10 ⁇ l of the second regulatable expression vector pLentis-PTRE-GFP-PGK- After the PURO virus was continuously cultured in the incubator for 24 hours, the supernatant was discarded and replaced with fresh medium to continue the culture. After the cells were full, transfer it to the culture flask and add it at a final concentration of 3 ⁇ g / ml. Puromycin. After three days of continuous culture, the surviving cells are the cells that can regulate the expression of GFP. This cell is named B16 (rtTA) -GFP.
  • the B16 (rtTA) -GFP cells in the logarithmic growth phase were digested, diluted to 2 ⁇ 10 6 cells / ml with HBSS, and injected into 8- to 10-week-old C57BL / 6 female mice (purchased at 50 ⁇ l / head using a 1 ml syringe).
  • HBSS HBSS-maleic anhydride-semiconductor
  • the results showed that induced expression of GFP had no inhibitory effect on tumor growth (where each broken line represents the area of the tumor in a mouse).
  • heterodimeric proteins comprising: mIL2IL12aIL12bGMCSF, mIL12aIL2GMCSFIL12b, mIL2IL12aGMCSFIL12b, mIL12aGMCSFIL12bIL2, mIL12aIL7IL12bGMCSF, mIL12aIL15IL12bGMCSF, mIL12aIL21IL12bGMCSF, mIL12aIL2IL12bFLT3L, mIL12aIL7IL12bFLT3L, mIL12aIL15IL12bFLT3L, mIL12aIL21IL12bFLT3L, hIL12aIL2IL12bGMCSF, hIL12aIL7IL12bGMCSF, hIL12aIL15IL12bGMCSF, hIL12aIL21IL12bGMCSF, hIL12aIL2IL12bFLT3L, hIL12aIL7IL12bFLT3L, hIL
  • the digestion system is as follows: 5 ⁇ gmIL12aIL2IL12bGMCSF plasmid, 4 ⁇ l digestion buffer, 1 ⁇ l of BamHI and 1 ⁇ l of XhoI, add water to a total volume of 40 ⁇ l, and stand at 37 ° C. for 12 hours. Take out the EP tube, add 4.4 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the mIL12aIL2IL12bGMCSF gene fragment is recovered and used.
  • the amino acid sequence of the mIL12aIL2IL12bGMCSF protein heterodimer is shown in SEQ ID No. 18, and the nucleotide sequence encoding the mIL12aIL2IL12bGMCSF is shown in SEQ ID No. 38.
  • Digestion can regulate the expression vector pLentis-PTRE-MCS-PGK-PURO.
  • the digestion system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to the total volume 30 ⁇ l, and allowed to stand for 12 hours at 37 ° C. Remove the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector fragment is recovered and used.
  • the pLentis-PTRE-MCS-PGK-PURO and mIL12aIL2IL12bGMCSF were connected.
  • the connection system was as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l mIL12aIL2IL12bGMCSF, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, send the correct vector for sequencing to confirm that the second expression vector pLentis-PTRE-mIL12aIL2IL12bGMCSF-PGK-PURO was successfully constructed.
  • the method for preparing the virus of the mIL12aIL2IL12bGMCSF expression vector is the same as the method for preparing the virus of the first expression vector to obtain the virus of the second expression vector pLentis-PTRE-mIL12aIL2IL12bGMCSF-PGK-PURO.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, after 24 hours, was added 10 ⁇ l of the second regulatable expression vector pLentis-PTRE-mIL12aIL2IL12bGMCSF-PGK- After the PURO virus was continuously cultured in the incubator for 24 hours, the supernatant was discarded and replaced with fresh medium to continue the culture. After the cells were full, transfer it to the culture flask and add it at a final concentration of 3 ⁇ g / ml. Puromycin. After three days of continuous culture, the surviving cells are cells that can regulate the expression of mIL12aIL2IL12bGMCSF. This cell is named B16 (rtTA) -mIL12aIL2IL12bGMCSF.
  • B16 (rtTA) -mIL12aIL2IL12bGMCSF cells were plated into a 24-well plate at 5 ⁇ 10 4 per well. After 24 hours, 100 ng / ml doxycycline (DOX) was added to the wells, and the culture was continued for 72 hours, and the supernatant was collected.
  • DOX doxycycline
  • the mouse IL12p70 ELISA kit was used to detect the fusion protein expression in the supernatant. The operation was performed according to the kit instructions. Wells without DOX induction served as controls. As shown in Figure 3, the addition of DOX induced the expression of the mIL12aIL2IL12bGMCSF fusion protein.
  • the mIL2IL12aIL12bGMCSFF gene coding sequence was synthesized, with BamHI, BglII and XhoI, EcoRI restriction sites at both ends, and then digested with BamHI or BglII and XhoI or EcoRI. , 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, and leave to stand at 37 ° C. for 12 hours. Remove the EP tube, add 4.4 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the mIL2IL12aIL12bGMCSF gene fragment is recovered and used.
  • amino acid sequence of the heterodimer of mIL2IL12aIL12bGMCSF protein is shown in SEQ ID No. 19, and the nucleotide sequence encoding the mIL2IL12aIL12bGMCSF is SEQ ID NO. 39.
  • Digestion can regulate the expression vector pLentis-PTRE-MCS-PGK-PURO.
  • the digestion system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to the total volume 30 ⁇ l, and allowed to stand for 12 hours at 37 ° C. Remove the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector fragment is recovered and used.
  • the pLentis-PTRE-MCS-PGK-PURO and mIL2IL12aIL12bGMCSF were connected.
  • the connection system was as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l mIL2IL12aIL12bGMCSF, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, send the correct vector for sequencing to confirm that the second expression vector pLentis-PTRE-mIL2IL12aIL12bGMCSF-PGK-PURO was successfully constructed.
  • the method for preparing the virus of the mIL2IL12aIL12bGMCSF expression vector is the same as the method for preparing the first expression vector virus, and the virus of the second expression vector pLentis-PTRE-mIL2IL12aIL12bGMCSF-PGK-PURO is obtained.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, after 24 hours, the above-described second expression vector was added 10 ⁇ l pLentis-PTRE-mIL2IL12aIL12bGMCSF-PGK- PURO of Virus, continue to be cultured in the incubator for 24 hours, discard the supernatant, and replace the culture with fresh medium. After the cells are full, transfer it to the culture flask, and add puromycin at a final concentration of 3 ⁇ g / ml. After three days of continuous culture, the surviving cells are the cells that can regulate the expression of mIL2IL12aIL12bGMCSF. This cell is named B16 (rtTA) -mIL2IL12aIL12bGMCSF.
  • the mIL12aIL2GMCSFIL12b gene coding sequence was synthesized, with BamHI, BglII and XhoI and EcoRI restriction sites at each end, and then digested with BamHI or BglII and XhoI or EcoRI.
  • the digestion system was as follows: 5 ⁇ g mIL12aIL2GMCSFIL12b plasmid, 4 ⁇ l digestion buffer , 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, and leave to stand at 37 ° C. for 12 hours. Remove the EP tube, add 4.4 ⁇ l of 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After the electrophoresis, the mIL12aIL2GMCSFIL12b gene fragment is recovered and set aside.
  • the amino acid sequence of the mIL12aIL2GMCSFIL12b protein heterodimer is shown in SEQ ID NO.20, and the nucleotide sequence encoding the mIL12aIL2GMCSFIL12b is shown in SEQ ID NO.40.
  • Digestion can regulate the expression vector pLentis-PTRE-MCS-PGK-PURO.
  • the digestion system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 30 ⁇ l , Let stand at 37 ° C for 12 hours. Remove the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector fragment is recovered and used.
  • the pLentis-PTRE-MCS-PGK-PURO and mIL12aIL2GMCSFIL12b were connected.
  • the connection system was as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l mIL12aIL2GMCSFIL12b, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, send the correct vector for sequencing to confirm that the second expression vector pLentis-PTRE-mIL12aIL2GMCSFIL12b-PGK-PURO was successfully constructed.
  • the method of preparing the virus of the mIL12aIL2GMCSFIL12b expression vector is the same as the method of preparing the virus of the first expression vector, and obtaining the second regulated expression vector of the virus pLentis-PTRE-mIL12aIL2GMCSFIL12b-PGK-PURO.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, after 24 hours, was added 10 ⁇ l of the second regulatable expression vector pLentis-PTRE-mIL12aIL2GMCSFIL12b-PGK- After the PURO virus was continuously cultured in the incubator for 24 hours, the supernatant was discarded and replaced with fresh medium to continue the culture. After the cells were full, transfer it to the culture flask and add it at a final concentration of 3 ⁇ g / ml. Puromycin. After three days of continuous culture, the surviving cells are cells that can regulate the expression of mIL12aIL2GMCSFIL12b. This cell is named B16 (rtTA) -mIL12aIL2GMCSFIL12b.
  • the mIL2IL12aGMCSFIL12b gene coding sequence was synthesized, with BamHI, BglII and XhoI, EcoRI restriction sites at both ends, and then digested with BamHI or BglII and XhoI or EcoRI.
  • the restriction system was as follows: 5 ⁇ g mIL2IL12aGMCSFIL12b plasmid, 4 ⁇ l digestion buffer , 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, and leave to stand at 37 ° C. for 12 hours. Take out the EP tube, add 4.4 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the mIL2IL12aGMCSFIL12b gene fragment is recovered and used.
  • the amino acid sequence of the mIL2IL12aGMCSFIL12b protein heterodimer is shown in SEQ ID NO.21, and the nucleotide sequence encoding the mIL2IL12aGMCSFIL12b is shown in SEQ ID NO.41.
  • Digestion can regulate the expression vector pLentis-PTRE-MCS-PGK-PURO.
  • the digestion system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI, 1 ⁇ l XhoI, add water to a total volume of 30 ⁇ l , Let stand at 37 ° C for 12 hours. Remove the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector fragment is recovered and used.
  • the pLentis-PTRE-MCS-PGK-PURO and mIL2IL12aGMCSFIL12b were connected.
  • the connection system was as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l mIL2IL12aGMCSFIL12b, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, send the correct vector for sequencing to confirm that the second expression vector pLentis-PTRE-mIL2IL12aGMCSFIL12b-PGK-PURO was successfully constructed.
  • the method of preparing the virus of the mIL2IL12aGMCSFIL12b expression vector is the same as the method for preparing the first expression vector virus to obtain the virus of the second expression vector pLentis-PTRE-mIL2IL12aGMCSFIL12b-PGK-PURO.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, after 24 hours, was added 10 ⁇ l expression control said second vector pLentis-PTRE-mIL2IL12aGMCSFIL12b-PGK- PURO After incubating the virus in the incubator for 24 hours, discard the supernatant and replace it with fresh medium. After the cells are full, transfer it to the culture flask and add purine at a final concentration of 3 ⁇ g / ml. After continued culture for three days, the surviving cells are cells that can regulate the expression of mIL2IL12aGMCSFIL12b. This cell is named B16 (rtTA) -mIL2IL12aGMCSFIL12b.
  • the mIL12aGMCSFIL12bIL2 gene coding sequence was synthesized, with BamHI, BglII and XhoI, EcoRI restriction sites at both ends, and then digested with BamHI or BglII and XhoI or EcoRI. , 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, and leave to stand at 37 ° C. for 12 hours. Remove the EP tube, add 4.4 ⁇ l of 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. The mIL12aGMCSFIL12bIL2 gene fragment was recovered after electrophoresis and was set aside.
  • amino acid sequence of the heterodimer of mIL12aGMCSFIL12bIL2 protein is shown in SEQ ID NO.22, and the nucleotide sequence encoding the mIL12aGMCSFIL12bIL2 is SEQ ID NO.42.
  • Digestion can regulate the expression vector pLentis-PTRE-MCS-PGK-PURO.
  • the digestion system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 30 ⁇ l , Let stand at 37 ° C for 12 hours. Remove the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector fragment is recovered and used.
  • connection system is as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l mIL12aGMCSFIL12bIL2, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, send the correct vector for sequencing to confirm that the second expression vector pLentis-PTRE-mIL12aGMCSFIL12bIL2-PGK-PURO was successfully constructed.
  • the method of preparing the virus of the mIL12aGMCSFIL12bIL2 expression vector is the same as the method of preparing the first expression vector virus to obtain the virus of the second expression vector pLentis-PTRE-mIL12aGMCSFIL12bIL2-PGK-PURO.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, after 24 hours, was added 10 ⁇ l of the second regulatable expression vector pLentis-PTRE-mIL12aGMCSFIL12bIL2-PGK- After the PURO virus was continuously cultured in the incubator for 24 hours, the supernatant was discarded and replaced with fresh medium to continue the culture. After the cells were full, transfer it to the culture flask and add it at a final concentration of 3 ⁇ g / ml. Puromycin, after three days of continuous culture, the surviving cells are cells that can regulate the expression of mIL12aGMCSFIL12bIL2, and this cell is named B16 (rtTA) -mIL12aGMCSFIL12bIL2.
  • the hIL12aIL2IL12bGMCSF gene coding sequence was synthesized, with BamHI, BglII and XhoI, EcoRI restriction sites at both ends, and then digested with BamHI or BglII and XhoI or EcoRI.
  • the digestion system was as follows: 5 ⁇ g hIL12aIL2IL12bGMCSF plasmid, 4 ⁇ l digestion buffer , 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, and leave to stand at 37 ° C. for 12 hours. Remove the EP tube, add 4.4 ⁇ l of 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the hIL12aIL2IL12bGMCSF gene fragment is recovered and used.
  • the amino acid sequence of hIL12aIL2IL12bGMCSF protein heterodimer is shown in SEQ ID NO.30, and the nucleotide sequence encoding the hIL12aIL2IL12bGMCSF is shown in SEQ ID NO.50.
  • Digestion can regulate the expression vector pLentis-PTRE-MCS-PGK-PURO.
  • the digestion system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 30 ⁇ l , Let stand at 37 ° C for 12 hours. Remove the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector fragment is recovered and used.
  • the pLentis-PTRE-MCS-PGK-PURO and hIL12aIL2IL12bGMCSF were connected.
  • the connection system was as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l hIL12aIL2IL12bGMCSF, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, the correct vector was sent for sequencing to confirm that the second expression vector pLentis-PTRE-hIL12aIL2IL12bGMCSF-PGK-PURO was successfully constructed.
  • the method of preparing the virus of the hIL12aIL2IL12bGMCSF expression vector is the same as the method for preparing the first expression vector virus to obtain the virus of the second expression vector pLentis-PTRE-hIL12aIL2IL12bGMCSF-PGK-PURO.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, 24 hours, and the second expression vector was added 10 ⁇ l pLentis-PTRE-hIL12aIL2IL12bGMCSF-PGK- PURO of Virus, continue to be cultured in the incubator for 24 hours, discard the supernatant, and replace the culture with fresh medium. After the cells are full, transfer it to the culture flask, and add puromycin at a final concentration of 3 ⁇ g / ml. After three days of continuous culture, the surviving cells are cells that can regulate the expression of hIL12aIL2IL12bGMCSF. This cell is named B16 (rtTA) -hIL12aIL2IL12bGMCSF.
  • the B16 (rtTA) -hIL12aIL2IL12bGMCSF cells in the logarithmic growth phase were digested, diluted to 2 ⁇ 10 6 cells / ml with HBSS, and injected into the right side of 8- to 10-week-old C57BL / 6 female mice with a 1 ml syringe at 50 ⁇ l per mouse. On the back side, a total of 10 mice were fed with water containing 2g / L doxycycline after tumor growth. The tumor growth and tumor clearance rate of the mice were recorded. As shown in Figure 9, hIL12aIL2IL12bGMCSF can inhibit mouse tumors to a certain extent. Growth.
  • Example 10 Effect of inducing expression of mIL12aIL7IL12bGMCSF on tumor growth
  • the amino acid sequence of the mIL12aIL7IL12bGMCSF protein heterodimer is shown in SEQ ID NO.23, and the nucleotide sequence encoding the mIL12aIL7IL12bGMCSF is shown in SEQ ID NO.43.
  • Digestion can regulate the expression vector pLentis-PTRE-MCS-PGK-PURO.
  • the digestion system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI, 1 ⁇ l XhoI, add water to the total volume 30 ⁇ l, and allowed to stand for 12 hours at 37 ° C. Remove the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector fragment is recovered and used.
  • the pLentis-PTRE-MCS-PGK-PURO and mIL12aIL7IL12bGMCSF were connected.
  • the connection system was as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l mIL12aIL7IL12bGMCSF, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, send the correct vector for sequencing to confirm that the second expression vector pLentis-PTRE-mIL12aIL7IL12bGMCSF-PGK-PURO was successfully constructed.
  • the method of preparing the virus of the mIL12aIL7IL12bGMCSF expression vector is the same as the method for preparing the first expression vector virus to obtain the virus of the second expression vector pLentis-PTRE-mIL12aIL7IL12bGMCSF-PGK-PURO.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, after 24 hours, was added 10 ⁇ l of the second regulatable expression vector pLentis-PTRE-mIL12aIL7IL12bGMCSF-PGK- After the PURO virus was continuously cultured in the incubator for 24 hours, the supernatant was discarded and replaced with fresh medium to continue the culture. After the cells were full, transfer it to the culture flask and add it at a final concentration of 3 ⁇ g / ml. Puromycin. After three days of continued culture, the surviving cells are cells that can regulate the expression of mIL12aIL7IL12bGMCSF. This cell is named B16 (rtTA) -mIL12aIL7IL12bGMCSF.
  • the mIL12aIL15IL12bGMCSF gene coding sequence was synthesized, with BamHI, BglII and XhoI, EcoRI restriction sites at both ends, and then digested with BamHI or BglII and XhoI or EcoRI.
  • the digestion system was as follows: 5 ⁇ g mIL12aIL15IL12bGMCSF plasmid, 4 ⁇ l digestion buffer , 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, and leave to stand at 37 ° C. for 12 hours. Remove the EP tube, add 4.4 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After the electrophoresis, the mIL12aIL15IL12bGMCSF gene fragment is recovered and used.
  • the amino acid sequence of the mIL12aIL15IL12bGMCSF protein heterodimer is shown in SEQ ID NO.24, and the nucleotide sequence encoding the mIL12aIL15IL12bGMCSF is shown in SEQ ID NO.44.
  • Digestion can regulate the expression vector pLentis-PTRE-MCS-PGK-PURO.
  • the digestion system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to the total volume 30 ⁇ l, and allowed to stand for 12 hours at 37 ° C. Remove the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector fragment is recovered and used.
  • the pLentis-PTRE-MCS-PGK-PURO and mIL12aIL15IL12bGMCSF were connected.
  • the connection system was as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l mIL12aIL15IL12bGMCSF, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, send the correct vector for sequencing to confirm that the second expression vector pLentis-PTRE-mIL12aIL15IL12bGMCSF-PGK-PURO was successfully constructed.
  • the method of preparing the virus of the mIL12aIL15IL12bGMCSF expression vector is the same as the method for preparing the first expression vector virus, and the virus of the second expression vector pLentis-PTRE-mIL12aIL15IL12bGMCSF-PGK-PURO is obtained.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, after 24 hours, was added 10 ⁇ l of the second regulatable expression vector pLentis-PTRE-mIL12aIL15IL12bGMCSF-PGK- After the PURO virus was continuously cultured in the incubator for 24 hours, the supernatant was discarded and replaced with fresh medium to continue the culture. After the cells were full, transfer it to the culture flask and add it at a final concentration of 3 ⁇ g / ml. Puromycin. After three days of continuous culture, the surviving cells are cells that can regulate the expression of mIL12aIL15IL12bGMCSF. This cell is named B16 (rtTA) -mIL12aIL15IL12bGMCSF.
  • the B16 (rtTA) -mIL12aIL15IL12bGMCSF cells in the logarithmic growth phase were digested, diluted to 2 ⁇ 10 6 cells / ml with HBSS, and injected into the right of 8-10 week-old C57BL / 6 female mice using a 1 ml syringe at 50 ⁇ l / head.
  • mIL12aIL15IL12bGMCSF can induce tumor regression in some mice.
  • Example 12 Effect of induction of expression of mIL12aIL21IL12bGMCSF on tumor growth
  • the mIL12aIL21IL12bGMCSF gene coding sequence was synthesized, with BamHI, BglII and XhoI, EcoRI restriction sites at each end, and then digested with BamHI or BglII and XhoI or EcoRI.
  • the digestion system was as follows: 5 ⁇ g mIL12aIL21IL12bGMCSF plasmid, 4 ⁇ l digestion buffer , 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, and leave to stand at 37 ° C. for 12 hours. Take out the EP tube, add 4.4 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the mIL12aIL21IL12bGMCSF gene fragment is recovered and used.
  • the amino acid sequence of the mIL12aIL21IL12bGMCSF protein heterodimer is shown in SEQ ID NO.25, and the nucleotide sequence encoding the mIL12aIL21IL12bGMCSF is shown in SEQ ID NO.45.
  • Digestion can regulate the expression vector pLentis-PTRE-MCS-PGK-PURO.
  • the digestion system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI, 1 ⁇ l XhoI, add water to the total volume 30 ⁇ l, and allowed to stand for 12 hours at 37 ° C. Remove the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector fragment is recovered and used.
  • the pLentis-PTRE-MCS-PGK-PURO and mIL12aIL21IL12bGMCSF were connected.
  • the connection system was as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l mIL12aIL21IL12bGMCSF, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, send the correct vector for sequencing to confirm that the second expression vector pLentis-PTRE-mIL12aIL21IL12bGMCSF-PGK-PURO was successfully constructed.
  • the method for preparing the virus of the mIL12aIL21IL12bGMCSF expression vector is the same as the method for preparing the first expression vector virus, and the virus of the second expression vector pLentis-PTRE-mIL12aIL21IL12bGMCSF-PGK-PURO is obtained.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, after 24 hours, the above-described second expression vector was added 10 ⁇ l pLentis-PTRE-mIL12aIL21IL12bGMCSF-PGK- PURO of Virus, continue to be cultured in the incubator for 24 hours, discard the supernatant, and replace the culture with fresh medium. After the cells are full, transfer it to the culture flask, and add puromycin at a final concentration of 3 ⁇ g / ml. After three days of continuous culture, the surviving cells are the cells that can regulate the expression of mIL12aIL21IL12bGMCSF. This cell is named B16 (rtTA) -mIL12aIL21IL12bGMCSF.
  • Example 13 Effect of induction of expression of mIL12aIL2ILI2bFLT3L on tumor growth
  • the digestion system is as follows: 5 ⁇ g mIL12aIL2IL12bFLT3L plasmid, 4 ⁇ l digestion buffer , 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, and leave to stand at 37 ° C. for 12 hours.
  • the amino acid sequence of the mIL12aIL2IL12bFLT3L protein heterodimer is shown in SEQ ID NO.26, and the nucleotide sequence encoding the mIL12aIL2IL12bFLT3L is shown in SEQ ID NO.46.
  • Digestion can regulate the expression vector pLentis-PTRE-MCS-PGK-PURO.
  • the digestion system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to the total volume 30 ⁇ l, and allowed to stand for 12 hours at 37 ° C. Remove the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector fragment is recovered and used.
  • the pLentis-PTRE-MCS-PGK-PURO and mIL12aIL2IL12bFLT3L were connected.
  • the connection system was as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l mIL12aIL2IL12bFLT3L, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, send the correct vector for sequencing to confirm that the second expression vector pLentis-PTRE-mIL12aIL2IL12bFLT3L-PGK-PURO was successfully constructed.
  • the method for preparing the virus of the mIL12aIL2IL12bFLT3L expression vector is the same as the method for preparing the first expression vector virus to obtain the virus of the second expression vector pLentis-PTRE-mIL12aIL2IL12bFLT3L-PGK-PURO.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, after 24 hours, was added 10 ⁇ l of the second regulatable expression vector pLentis-PTRE-mIL12aIL2IL12bFLT3L-PGK- After the PURO virus was continuously cultured in the incubator for 24 hours, the supernatant was discarded and replaced with fresh medium to continue the culture. After the cells were full, transfer it to the culture flask and add it at a final concentration of 3 ⁇ g / ml. Puromycin. After three days of continuous culture, the surviving cells are cells that can regulate the expression of mIL12aIL2IL12bFLT3L. This cell is named B16 (rtTA) -mIL12aIL2IL12bFLT3L.
  • Example 14 Effect of induction of expression of mIL12aIL7IL12bFLT3L on tumor growth
  • amino acid sequence of mIL12aIL7IL12bFLT3L protein heterodimer is shown in SEQ ID NO.27, and the nucleotide sequence encoding the mIL12aIL7IL12bFLT3L protein is shown in SEQ ID NO.47.
  • Digestion can regulate the expression vector pLentis-PTRE-MCS-PGK-PURO.
  • the digestion system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 30 ⁇ l, Let stand at 37 ° C for 12 hours. Take out the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector fragment is recovered and used.
  • the pLentis-PTRE-MCS-PGK-PURO and mIL12aIL7IL12bFLT3L were connected.
  • the connection system was as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l mIL12aIL7IL12bFLT3L, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, send the correct vector for sequencing to confirm that the second expression vector pLentis-PTRE-mIL12aIL7IL12bFLT3L-PGK-PURO was successfully constructed.
  • the method for preparing the virus of the mIL12aIL7IL12bFLT3L expression vector is the same as the method for preparing the first expression vector virus, and the virus of the second expression vector pLentis-PTRE-mIL12aIL7IL12bFLT3L-PGK-PURO is obtained.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, after 24 hours, the above-described second expression vector was added 10 ⁇ l pLentis-PTRE-mIL12aIL7IL12bFLT3L-PGK- PURO of Virus, continue to be cultured in the incubator for 24 hours, discard the supernatant, and replace the culture with fresh medium. After the cells are full, transfer it to the culture flask, and add puromycin at a final concentration of 3 ⁇ g / ml. After three days of continuous culture, the surviving cells are cells that can regulate the expression of mIL12aIL7IL12bFLT3L, and this cell is named B16 (rtTA) -mIL12aIL7IL12bFLT3L.
  • Example 15 Effect of induction of expression of mIL12aIL15IL12bFLT3L on tumor growth
  • the mIL12aIL15IL12bFLT3L gene coding sequence was synthesized, with BamHI, BglII and XhoI, EcoRI restriction sites at both ends, and then digested with BamHI or BglII and XhoI or EcoRI.
  • the digestion system was as follows: 5 ⁇ g mIL12aIL15IL12bFLT3L plasmid, 4 ⁇ l digestion buffer , 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 40 ⁇ l, and leave to stand at 37 ° C. for 12 hours. Remove the EP tube, add 4.4 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After the electrophoresis, the mIL12aIL15IL12bFLT3L gene fragment is recovered and used.
  • amino acid sequence of mIL12aIL15IL12bFLT3L protein heterodimer is shown in SEQ ID NO.28, and the nucleotide sequence encoding the mIL12aIL15IL12bFLT3L is shown in SEQ ID NO.48.
  • Digestion can regulate the expression vector pLentis-PTRE-MCS-PGK-PURO.
  • the digestion system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI, 1 ⁇ l XhoI, add water to a total volume of 30 ⁇ l , Let stand at 37 ° C for 12 hours. Remove the EP tube, add 3.3 ⁇ l 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector fragment is recovered and used.
  • the pLentis-PTRE-MCS-PGK-PURO and mIL12aIL15IL12bFLT3L were connected.
  • the connection system was as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l mIL12aIL15IL12bFLT3L, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, send the correct vector for sequencing to confirm that the second expression vector pLentis-PTRE-mIL12aIL15IL12bFLT3L-PGK-PURO was successfully constructed.
  • the method of preparing the virus of the mIL12aIL15IL12bFLT3L expression vector is the same as the method for preparing the first expression vector virus to obtain the virus of the second expression vector pLentis-PTRE-mIL12aIL15IL12bFLT3L-PGK-PURO.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, after 24 hours, the above-described second expression vector was added 10 ⁇ l pLentis-PTRE-mIL12aIL15IL12bFLT3L-PGK- PURO of Virus, continue to be cultured in the incubator for 24 hours, discard the supernatant, and replace the culture with fresh medium. After the cells are full, transfer it to the culture flask, and add puromycin at a final concentration of 3 ⁇ g / ml. After three days of continuous culture, the surviving cells are cells that can regulate the expression of mIL12aIL15IL12bFLT3L. This cell is named B16 (rtTA) -mIL12aIL15IL12bFLT3L.
  • Example 16 Effect of induction of expression of mIL12aIL21IL12bFLT3L on tumor growth
  • amino acid sequence of mIL12aIL21IL12bFLT3L protein heterodimer is shown in SEQ ID NO.29, and the nucleotide sequence encoding the mIL12aIL21IL12bFLT3L protein is shown in SEQ ID NO.49.
  • Digestion can regulate the expression vector pLentis-PTRE-MCS-PGK-PURO.
  • the digestion system is as follows: 2 ⁇ g pLentis-PTRE-MCS-PGK-PURO plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI, 1 ⁇ l XhoI, add water to a total volume of 30 ⁇ l, Let stand at 37 ° C for 12 hours. Remove the EP tube, add 3.3 ⁇ l of 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-PTRE-MCS-PGK-PURO vector fragment is recovered and used.
  • the pLentis-PTRE-MCS-PGK-PURO and mIL12aIL21IL12bFLT3L were connected.
  • the connection system was as follows: 2 ⁇ l pLentis-PTRE-MCS-PGK-PURO, 2 ⁇ l mIL12aIL21IL12bFLT3L, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water. Leave at room temperature for 4 hours. The ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate, and cultured in a 37 ° C shaker overnight in LB medium.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. Then, send the correct vector for sequencing to confirm that the second expression vector pLentis-PTRE-mIL12aIL21IL12bFLT3L-PGK-PURO was successfully constructed.
  • the virus of the mIL12aIL21IL12bFLT3L expression vector is prepared by the same method as the first expression vector virus preparation method, and the virus of the second expression vector pLentis-PTRE-mIL12aIL21IL12bFLT3L-PGK-PURO is obtained.
  • B16 (rtTA) tumor cells at 10 5 cells / well were seeded into 6-well culture volume of 1ml, after 24 hours, was added 10 ⁇ l of the second regulatable expression vector pLentis-PTRE-mIL12aIL21IL12bFLT3L-PGK- After the PURO virus was continuously cultured in the incubator for 24 hours, the supernatant was discarded and replaced with fresh medium to continue the culture. After the cells were full, transfer it to the culture flask and add it at a final concentration of 3 ⁇ g / ml. Puromycin. After three days of continuous culture, the surviving cells are cells that can regulate the expression of mIL12aIL21IL12bFLT3L. This cell is named B16 (rtTA) -mIL12aIL21IL12bFLT3L.
  • Example 17 Construction of human hIL12aIL2IL12bGMCSF, human hIL12aIL7IL12bGMCSF, human hIL12aIL15IL12bGMCSF, human hIL12aIL21IL12bGMCSF, human hIL12aIL2IL12bFLT3L, human hIL12aIL7IL12bFLTLT3L, human hIL121212
  • Digest the vector pLentis-CMV-MCS-IRES-PURO in the EP tube the system is as follows: 2 ⁇ g pLentis-CMV-MCS-IRES-PURO vector plasmid, 3 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, add water to a total volume of 30 ⁇ l, Let stand at 37 ° C for 12 hours. Remove the EP tube, add 3.3 ⁇ l of 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the pLentis-CMV-MCS-IRES-PURO vector fragment is recovered and used.
  • human hIL12aIL2IL12bGMCSF When human hIL12aIL2IL12bGMCSF, human hIL12aIL7IL12bGMCSF, human hIL12aIL15IL12bGMCSF, human hIL12aIL21IL12IL12bGMCSF, human hIL12aIL2IL12bFLT3L, human hIL12aILBIL3IL7IL12ILFIL3L3 and human hIL12ILLIL12IL3 Add XhoI or EcoRI restriction sites.
  • the system of enzyme-digested plasmid with target gene was as follows: 5 ⁇ g plasmid, 4 ⁇ l digestion buffer, 1 ⁇ l BamHI and 1 ⁇ l XhoI, water was added to a total volume of 40 ⁇ l, and it was left at 37 ° C. for 12 hours. Take out the EP tube, add 4.4 ⁇ l of 10 ⁇ loading buffer, and perform electrophoresis on a 1% agarose gel. After electrophoresis, the fragments are recovered and used.
  • the amino acid sequence of hIL12aIL2IL12bGMCSF protein heterodimer is shown in SEQ ID NO.30, and the nucleotide sequence encoding the hIL12aIL2IL12bGMCSF is shown in SEQ ID NO.50.
  • the amino acid sequence of hIL12aIL7IL12bGMCSF protein heterodimer is shown in SEQ ID NO.31, and the nucleotide sequence encoding the hIL12aIL7IL12bGMCSF is shown in SEQ ID NO.51.
  • the amino acid sequence of hIL12aIL15IL12bGMCSF protein heterodimer is shown in SEQ ID NO.32, and the nucleotide sequence encoding the hIL12aIL15IL12bGMCSF is shown in SEQ ID NO.52.
  • the amino acid sequence of hIL12aIL21IL12bGMCSF protein heterodimer is shown in SEQ ID NO.33, and the nucleotide sequence encoding the hIL12aIL21IL12bGMCSF is shown in SEQ ID NO.53.
  • the amino acid sequence of hIL12aIL2IL12bFLT3L protein heterodimer is shown in SEQ ID NO.34, and the nucleotide sequence encoding the hIL12aIL2IL12bFLT3L is shown in SEQ ID NO.54.
  • the amino acid sequence of hIL12aIL7IL12bFLT3L protein heterodimer is shown in SEQ ID NO.35, and the nucleotide sequence encoding the hIL12aIL7IL12bFLT3L is shown in SEQ ID NO.55.
  • the amino acid sequence of hIL12aIL15IL12bFLT3L protein heterodimer is shown in SEQ ID NO.36, and the nucleotide sequence encoding the hIL12aIL15IL12bFLT3L is shown in SEQ ID NO.56.
  • the amino acid sequence of hIL12aIL21IL12bFLT3L protein heterodimer is shown in SEQ ID NO.37, and the nucleotide sequence encoding the hIL12aIL21IL12bFLT3L protein is shown in SEQ ID NO.57.
  • pLentis-CMV-MCS-IRES-PURO and human hIL12aIL2IL12bGMCSF respectively connected, people hIL12aIL7IL12bGMCSF, people hIL12aIL15IL12bGMCSF, people hIL12aIL21IL12bGMCSF, people hIL12aIL2IL12bFLT3L, people hIL12aIL7IL12bFLT3L, human hIL12aIL15IL12bFLT3L and human hIL12aIL21IL12bFLT3L, the following system: 2 ⁇ l pLentis-CMV-MCS-IRES-PURO vector Fragment, 2 ⁇ l gene fragment, 1 ⁇ l ligase buffer, 0.5 ⁇ l T4 DNA ligase and 4.5 ⁇ l water.
  • the ligation system was then subjected to competent transformation of E. coli. The next day, the colonies were picked from the transformed plate and placed in a 37 ° C shaker in LB medium for overnight culture.
  • the plasmid was extracted from the cultured bacteria using a plasmid extraction kit, and the fragment was successfully ligated into the vector by enzyme digestion. And then sequence the correct vector to confirm that the build was successful.
  • vector pLentis-CMV-hIL12aIL2IL12bGMCSF-IRES-PURO the vector pLentis-CMV-hIL12aIL7IL12bGMCSF-IRES-PURO, the vector pLentis-CMV-hIL12aIL15IL12bGMCSF-IRES-PURO, the vector pLhILs-CMV pLentis-CMV-hIL12aIL2IL12bFLT3L-IRES-PURO, the vector pLentis-CMV-hIL12aIL7IL12bFLT3L-IRES-PURO, the vector pLentis-CMV-hIL12aIL15IL12bFLT3L-IRES-PURO, and the vector pLentis-LTV21-IL12-ILV-IL12.
  • the cultured 293A cells were digested and seeded at 10 5 cells / well into a 6-well plate with a culture volume of 1 ml. After 24 hours, 10 ⁇ l of the virus expressing the above-mentioned gene of interest was added, and the culture was continued in the incubator for 24 hours. The supernatant was discarded and replaced with fresh medium to continue the culture. After the cells are full, transfer them to a culture flask, add a final concentration of 3 ⁇ g / ml puromycin, continue the culture, change the medium every two days, and maintain the concentration of puromycin.
  • the surviving Cells are cells that stably express the cytokines and are named 293A-hIL12aIL2IL12bGMCSF, 293A-hIL12aIL7IL12bGMCSF, 293A-hIL12aIL15IL12bGMCSF, 293A-hIL12aIL21ILbbLTF3L3A, 293A-hIL12IL2IL12IL12bLTLT3L3A, 293A-hIL12
  • the constructed expressing cells were plated into 24 well plates at 5 ⁇ 10 4 per well, and cultured for 96 hours. The supernatant was collected, and the expression of the fusion protein in the supernatant was detected using a human IL12p70 ELISA kit. The operation was performed according to the instructions. As shown in Figure 17, these cells were able to produce a large amount of IL12p70 and successfully constructed cells expressing the fusion protein.

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Abstract

蛋白质异二聚体及其用途，蛋白质异二聚体包含第一多肽链以及不同于第一多肽链的第二多肽链，其中第一多肽链包含IL12a以及与IL12a融合的第一因子，第二多肽链包含IL12b以及与IL12b融合的第二因子，且其中第一因子及第二因子各自独立地选自下组：IL2、GMCSF、IL7、IL15、IL21和FLT3L。蛋白质异二聚体可用于治疗肿瘤。

Description

蛋白质异二聚体及其用途 技术领域
本申请涉及肿瘤治疗领域，具体而言，本申请涉及蛋白质异二聚体及其用途。
背景技术
肿瘤是一种严重威胁人类健康的疾病，近年来，免疫治疗作为一种新疗法，在肿瘤治疗中显示出了巨大的潜力。细胞因子(Cytokine)是体内非常重要的免疫信号，细胞因子融合蛋白技术是当今肿瘤免疫疗法的另一个热点方向。该方法是基于这些细胞因子具有相同或相关的功能活性而各自作用靶点不同，利用基因工程技术将两种或多种细胞因子融合在一起。但是目前利用细胞因子融合蛋白技术进行肿瘤治疗的效果仍不尽人意，有较多需要改进之处。
发明内容
本申请提供了一种蛋白质异二聚体及其用途，所述蛋白质异二聚体至少具有抗肿瘤效果好的有益效果。
一方面，本申请提供了一种蛋白质异二聚体，其包含第一多肽链以及不同于所述第一多肽链的第二多肽链，其中所述第一多肽链包含IL12a以及与所述IL12a融合的第一因子，所述第二多肽链包含IL12b以及与所述IL12b融合的第二因子，且其中所述第一因子及所述第二因子各自独立地选自下组：IL2、GMCSF、IL7、IL15、IL21和FLT3L。
在某些实施方式中，所述第一因子与所述第二因子不同。
在某些实施方式中，所述第一因子和所述第二因子彼此不同地分别选自细胞因子组A和细胞因子组B，所述细胞因子组A选自下组：IL2、IL7、IL15和IL21，且细胞因子组B选自下组：GMCSF和FLT3L。
在某些实施方式中，在所述第一多肽链中，所述IL12a的C末端与所述第一因子的N末端直接或间接融合。在某些实施方式中，在所述第一多肽链中，所述IL12a的N末端与所述第一因子的C末端直接或间接融合。在某些实施方式中，在所述第二多肽链中，所述IL12b的C末端与所述第二因子的N末端直接或间接融合。在某些实施方式中，在所述第二多肽链中，所述IL12b的N末端与所述第二因子的C末端直接或间接融合。
在某些实施方式中，所述第一因子和所述第二因子选自下组：
1)第一因子为IL2，且第二因子为GMCSF；
2)第一因子为IL7，且第二因子为GMCSF；
3)第一因子为IL15，且第二因子为GMCSF；
4)第一因子为IL21，且第二因子为GMCSF；
5)第一因子为IL2，且第二因子为FLT3L；
6)第一因子为IL7，且第二因子为FLT3L；
7)第一因子为IL15，且第二因子为FLT3L；
8)第一因子为IL21，且第二因子为FLT3L；
9)第一因子为GMCSF，且第二因子为IL2；
10)第一因子为GMCSF，且第二因子为IL7；
11)第一因子为GMCSF，且第二因子为IL15；
12)第一因子为GMCSF，且第二因子为IL21；
13)第一因子为FLT3L，且第二因子为IL2；
14)第一因子为FLT3L，且第二因子为IL7；
15)第一因子为FLT3L，且第二因子为IL15；以及，
16)第一因子为FLT3L，且第二因子为IL21。
在某些实施方式中，所述IL12a、所述IL12b、所述第一因子和所述第二因子来源自哺乳动物。
在某些实施方式中，所述IL12a、所述IL12b、所述第一因子和所述第二因子的来源相同。
在某些实施方式中，所述第一多肽链包括如SEQ ID NO.60-65和SEQ ID NO.70-73中任一项所示的序列。
在某些实施方式中，所述第二多肽链包括如SEQ ID NO.66-69和SEQ ID NO.74-75中任一项所示的序列。
在某些实施方式中，所述蛋白质异二聚体包括如SEQ ID NO.18-37中任一项所示的序列。
另一方面，本申请提供一种或多种分离的核酸分子，其编码所述的蛋白质异二聚体。
在某些实施方式中，所述核酸分子包括如SEQ ID NO.38-57中任一项所示的序列。
另一方面，本申请提供一种表达载体，其包括编码所述的蛋白质异二聚体的核苷酸序列。
另一方面，本申请提供一种宿主细胞，所述宿主细胞包括所述的表达载体。
另一方面，本申请提供一种药物组合物，所述药物组合物包括所述的蛋白质异二聚体，所述的核酸分子、所述的载体和/或所述的宿主细胞，以及药学上可接受的载体。
另一方面，本申请提供一种制备所述的蛋白质异二聚体的方法，其包括以下的步骤：在能够表达所述的蛋白质异二聚体的情况下，培养所述的宿主细胞。
另一方面，本申请提供一种所述的蛋白质异二聚体和所述的药物组合物在制备治疗肿瘤药物中的应用。
在某些实施方式中，所述肿瘤包括黑色素瘤。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示所述肿瘤的生长情况；
图2显示所述蛋白质异二聚体的结构示意图；
图3显示所述蛋白质异二聚体的表达情况；
图4显示所述蛋白质异二聚体诱导肿瘤消退的情况；
图5显示所述蛋白质异二聚体诱导肿瘤消退的情况；
图6显示所述蛋白质异二聚体诱导肿瘤消退的情况；
图7显示所述蛋白质异二聚体诱导肿瘤消退的情况；
图8显示所述蛋白质异二聚体诱导肿瘤消退的情况；
图9显示所述蛋白质异二聚体诱导肿瘤消退的情况；
图10显示所述蛋白质异二聚体诱导肿瘤消退的情况；
图11显示所述蛋白质异二聚体诱导肿瘤消退的情况；
图12显示所述蛋白质异二聚体诱导肿瘤消退的情况；
图13显示所述蛋白质异二聚体诱导肿瘤消退的情况；
图14显示所述蛋白质异二聚体诱导肿瘤消退的情况；
图15显示所述蛋白质异二聚体诱导肿瘤消退的情况；
图16显示所述蛋白质异二聚体诱导肿瘤消退的情况；
图17显示所述蛋白质异二聚体的构建情况。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书 所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“融合蛋白”通常指由两种或更多种多肽构成的蛋白质。在所述融合蛋白中，所述多肽可以具备不同的特性，该特性可以是生物学特性，如体外或体内活性；也可以是简单的化学特性或物理特性，如与靶分子结合、催化反应等。在所述融合蛋白中，所述多肽可以直接结合，也可以通过接头(例如，肽连接子)或间隔物(spacer)而间接结合所述融合蛋白可以由融合基因表达，在融合基因中编码一种多肽的核苷酸序列可以在框内附有编码与该多肽不同的另一多肽的核苷酸序列。所述融合基因可以由重组宿主细胞表达为单一的蛋白质。
在本申请中，术语“细胞因子融合蛋白”通常是指包含两种以上细胞因子的融合蛋白。所述细胞因子融合蛋白可以通过基因重组技术获得。所述细胞因子融合蛋白可以既具有所包含的细胞因子的独特的生物学活性，或者使其中某些活性显著提高，还可以通过生物学活性的互补及协同效应发挥出较单一细胞因子简单配伍所不具备的复合生物学功能，甚至还可能会产生一些新的结构及生物学功能。在本申请中，所述细胞因子融合蛋白可以为蛋白质异二聚体。
在本申请中，术语“蛋白质异二聚体”是由两个不同的多肽链组成的二聚体。在本申请中，所述蛋白质异二聚体可以包括第一多肽链和第二多肽链，所述第一多肽链不同于所述第二多肽链，其中所述第一多肽链可包含IL12a以及与所述IL12a融合的第一因子，所述第二多肽链可包含IL12b以及与所述IL12b融合的第二因子。
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蛋白质。所述细胞因子对于细胞间相互作用、细胞的生长和分化有重要调节作用。例如，所述细胞因子可以选自下组：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。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。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，所述集落刺激因子可以为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“IL12”通常是指白细胞介素-12。在本申请中，IL12和IL-12可以互换 使用。IL12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为异二聚体，其包括p40亚基(40kd)和p35亚基(35kd)，这两个亚基可以通过二硫键相连接。例如，小鼠IL12(mIL12)中的p35亚基可包含SEQ ID NO：1所示的氨基酸序列，p40亚基可包含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SEQ ID NO：9所示的氨基酸序列，p40亚基可包含SEQ ID NO：10所示的氨基酸序列。
在本申请中，术语“IL2”通常是指白细胞介素-2。IL2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SEQ ID NO：3所示的氨基酸序列。又例如，人细胞因子IL2(hIL2)可包含SEQ ID NO：11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。例如，小鼠IL15(mIL15)可包含SEQ ID NO：4所示的氨基酸序列。又例如，人IL15(hIL15)可包含SEQ ID NO：12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SEQ ID NO：5所示的氨基酸序列。又例如，人IL7(hIL7)可包含SEQ ID NO：13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SEQ ID NO：6所示的氨基酸序列。又例如，人IL21(hIL21)可包含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。FTL3L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SEQ ID NO：7所示的氨基酸序列。又例如，人FTL3L(hFTL3L)可包含SEQ ID NO：15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。GMCSF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SEQ ID NO：8所示的氨基酸序列。又例如，人GMCSF(hGMCSF)可包含SEQ ID NO：16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，所述核酸分子可编码所述的蛋白质异二聚体，其包括SEQ ID NO.38-57中任一项所示的核苷酸序列。
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。所述载体可以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“宿主细胞”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蛋白质异二聚体的个体细胞、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蛋白质异二聚体组合即可。所述宿主细胞可以通过使用本申请所述的载体体外转染该宿主细胞而得到。在本申请中，通过使用表达载体的病毒转染B16(rtTA)肿瘤细胞或293A细胞，可以得到能够表达本申请所述的蛋白质异二聚体的宿主细胞。
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、滑石粉、海藻酸钙凝胶、壳聚糖和纳米微球等。例如，所述药学上可接受的载体还可以选自下组：pH调节剂、渗透压调节剂、增溶剂和抑菌剂。
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，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。
蛋白质异二聚体
一方面，本申请提供一种蛋白质异二聚体，其可以包含第一多肽链以及不同于所述第一多肽链的第二多肽链，其中所述第一多肽链可以包含IL12a以及与所述IL12a融合的第一因子，所述第二多肽链可以包含IL12b以及与所述IL12b融合的第二因子，且其中所述第一因子及所述第二因子可以各自独立地选自下组：IL2、GMCSF、IL7、IL15、IL21和FLT3L。
在本申请中，所述第一因子可以与所述第二因子不同。在本申请中，所述蛋白质异二聚体可以是一种细胞因子融合蛋白，其将所述细胞因子，即IL12和IL2、GMCSF、IL7、IL15、IL21和FLT3L中的两种或多种，通过基因重组技术融合在一起。所述蛋白质异二聚体可以既具有组成其因子独特的生物学活性，又可以通过生物学活性的互补及协同效应发挥出较单一细胞因子所不具备的生物学功能，甚至还可以产生一些新的结构及生物学功能。
在本申请中，所述第一因子和所述第二因子可以彼此不同地分别选自细胞因子组A和细胞因子组B，所述细胞因子组A可以选自下组：IL2、IL7、IL15和IL21，且细胞因子组B可以选自下组：GMCSF和FLT3L。
例如，第一因子可以选择细胞因子组A中的IL2，且第二因子可以选择细胞因子组B中的GMCSF；或第一因子可以选择细胞因子组A中的IL7，且第二因子可以选择细胞因子组B中的GMCSF；或第一因子可以选择细胞因子组A中的IL15，且第二因子可以选择细胞因子组B中的GMCSF；或第一因子可以选择细胞因子组A中的IL21，且第二因子可以选择细胞因子组B中的GMCSF；或第一因子可以选择细胞因子组A中的IL2，且第二因子可以选择细胞因子组B中的FLT3L；或第一因子可以选择细胞因子组A中的IL7，且第二因子可以选择细胞因子组B中的FLT3L；或第一因子可以选择细胞因子组A中的IL15，且第二因子可以选择细胞因子组B中的FLT3L；或第一因子可以选择细胞因子组A中的IL21，且第二因子可以选择细胞因子组B中的FLT3L；或第二因子可以选择细胞因子组A中的IL2，且第一因子可以选择细胞因子组B中的GMCSF；或第二因子可以选择细胞因子组A中的IL7，且第一因子可以选择细胞因子组B中的GMCSF；或第二因子可以选择细胞因子组A中的IL15，且第一因子可以选择细胞因子组B中的GMCSF；或第二因子可以选择细胞因子组A中的IL21，且第一因子可以选择细胞因子组B中的GMCSF；或第二因子可以选择细胞因子组A中的IL2，且第一因子可以选择细胞因子组B中的FLT3L；或第二因子可以选择细胞因子组A中的IL7，且第一因子可以选择细胞因子组B中的FLT3L；或第二因子可以选择细胞因子组A中的IL15， 且第一因子可以选择细胞因子组B中的FLT3L；或第二因子可以选择细胞因子组A中的IL21，且第一因子可以选择细胞因子组B中的FLT3L。
在本申请中，在所述第一多肽链中，所述IL12a的C末端可以与所述第一因子的N末端直接或间接融合。例如，在所述第一多肽链中，所述IL12a的C末端可以与IL2的N端直接或间接融合；或在所述第一多肽链中，所述IL12a的C末端可以与IL7的N端直接或间接融合；或在所述第一多肽链中，所述IL12a的C末端可以与IL15的N端直接或间接融合；或在所述第一多肽链中，所述IL12a的C末端可以与IL21的N端直接或间接融合；或在所述第一多肽链中，所述IL12a的C末端可以与GMCSF的N端直接或间接融合；或在所述第一多肽链中，所述IL12a的C末端可以与FLT3L的N端直接或间接融合。
在本申请中，在所述第一多肽链中，所述IL12a的N末端可以与所述第一因子的C末端直接或间接融合。例如，在所述第一多肽链中，所述IL12a的N末端可以与IL2的C端直接或间接融合；或在所述第一多肽链中，所述IL12a的N末端可以与IL7的C端直接或间接融合；或在所述第一多肽链中，所述IL12a的N末端可以与IL15的C端直接或间接融合；或在所述第一多肽链中，所述IL12a的N末端可以与IL21的C端直接或间接融合；或在所述第一多肽链中，所述IL12a的N末端可以与GMCSF的C端直接或间接融合；或在所述第一多肽链中，所述IL12a的N末端可以与FLT3L的C端直接或间接融合。
在本申请中，在所述第二多肽链中，所述IL12b的C末端可以与所述第二因子的N末端直接或间接融合。例如，在所述第二多肽链中，所述IL12b的C末端可以与IL2的N端直接或间接融合；或在所述第二多肽链中，所述IL12b的C末端可以与IL7的N端直接或间接融合；或在所述第二多肽链中，所述IL12b的C末端可以与IL15的N端直接或间接融合；或在所述第二多肽链中，所述IL12b的C末端可以与IL21的N端直接或间接融合；或在所述第二多肽链中，所述IL12b的C末端可以与GMCSF的N端直接或间接融合；或在所述第二多肽链中，所述IL12b的C末端可以与FLT3L的N端直接或间接融合。
在本申请中，在所述第二多肽链中，所述IL12b的N末端可以与所述第二因子的C末端直接或间接融合。例如，在所述第二多肽链中，所述IL12b的N末端可以与IL2的C端直接或间接融合；或在所述第二多肽链中，所述IL12b的N末端可以与IL7的C端直接或间接融合；或在所述第二多肽链中，所述IL12b的N末端可以与IL15的C端直接或间接融合；或在所述第二多肽链中，所述IL12b的N末端可以与IL21的C端直接或间接融合；或在所述第二多肽链中，所述IL12b的N末端可以与GMCSF的C端直接或间接融合；或在所述第二多肽链中，所述IL12b的N末端可以与FLT3L的C端直接或间接融合。
在本申请中，所述蛋白质异二聚体可以包含以上所有所述细胞因子的位置连接关系的任 意的排列组合。
在本申请中，所述第一因子和所述第二因子可以选自下组：
1)第一因子可以为IL2，且第二因子可以为GMCSF；
2)第一因子可以为IL7，且第二因子可以为GMCSF；
3)第一因子可以为IL15，且第二因子可以为GMCSF；
4)第一因子可以为IL21，且第二因子可以为GMCSF；
5)第一因子可以为IL2，且第二因子可以为FLT3L；
6)第一因子可以为IL7，且第二因子可以为FLT3L；
7)第一因子可以为IL15，且第二因子可以为FLT3L；
8)第一因子可以为IL21，且第二因子可以为FLT3L；
9)第一因子可以为GMCSF，且第二因子可以为IL2；
10)第一因子可以为GMCSF，且第二因子可以为IL7；
11)第一因子可以为GMCSF，且第二因子可以为IL15；
12)第一因子可以为GMCSF，且第二因子可以为IL21；
13)第一因子可以为FLT3L，且第二因子可以为IL2；
14)第一因子可以为FLT3L，且第二因子可以为IL7；
15)第一因子可以为FLT3L，且第二因子可以为IL15；以及，
16)第一因子可以为FLT3L，且第二因子可以为IL21。
在本申请中，所述IL12a和所述第一因子之间，和/或所述IL12b和所述第二因子之间可通过连接肽连接。例如，所述连接肽的氨基酸序列可以如SEQ ID.17所示。
在本申请中，所述IL12a和所述IL12b可以通过二硫键共价结合。在本申请中，所述第一多肽链和所述第二多肽链之间可以通过IL12a和IL12b之间的二硫键结合形成所述蛋白质异二聚体。在某些实施方式中，编码所述第一多肽链和所述第二多肽链的核苷酸序列之间可以包含编码自切割肽的核苷酸序列。例如，当包含上述核苷酸序列的表达载体转录并完成翻译后，所述自切割肽的位置会被切开，从而构成包括所述第一多肽链和所述第二多肽链且通过二硫键形成的所述蛋白质异二聚体。在某些实施方式中，构建表达载体时，所述第一多肽链和所述第二多肽链之间可以通过2A自剪切肽或柔性氨基酸接头肽连接。例如，所述2A自剪切肽的氨基酸序列可以如SEQ ID NO.58所示，编码所述2A自剪切肽的核苷酸序列可以如SEQ ID NO.59所示。又例如，所述柔性氨基酸接头肽的氨基酸序列可以如SEQ ID.17所示。
某些实施方式中，所述第一多肽链和所述第二多肽链之间也可以包含所述连接肽。
例如，在所述蛋白质异二聚体中，IL12a的C端可以和IL2的N端融合形成第一多肽链， 且IL12b的C端可以和GMCSF的N端融合形成第二多肽链，从而形成IL12a-IL2-IL12b-GMCSF蛋白质异二聚体。
例如，在所述蛋白质异二聚体中，IL2的C端可以和IL12a的N端融合形成第一多肽链，且IL12b的C端可以和GMCSF的N端融合形成第二多肽链，从而形成IL2-IL12a-IL12b-GMCSF蛋白质异二聚体。
例如，在所述蛋白质异二聚体中，IL12a的C端可以和IL2的N端融合形成第一多肽链，且GMCSF的C端可以和IL12b的N端融合形成第二多肽链，从而形成IL12a-IL2-GMCSF-IL12b蛋白质异二聚体。
例如，在所述蛋白质异二聚体中，IL2的C端可以和IL12a的N端融合形成第一多肽链，且GMCSF的C端可以和IL12b的N端融合形成第二多肽链，从而形成IL2-IL12a-GMCSF-IL12b蛋白质异二聚体。
例如，在所述蛋白质异二聚体中，IL12a的C端可以和GMCSF的N端融合形成第一多肽链，且IL12b的C端可以和IL2的N端融合形成第二多肽链，从而形成IL12a-GMCSF-IL12b-IL2蛋白质异二聚体。
例如，在所述蛋白质异二聚体中，IL12a的C端可以和IL7的N端融合形成第一多肽链，且IL12b的C端可以和GMCSF的N端融合形成第二多肽链，从而形成IL12a-IL7-IL12b-GMCSF蛋白质异二聚体。
例如，在所述蛋白质异二聚体中，IL12a的C端可以和IL15的N端融合形成第一多肽链，且IL12b的C端可以和GMCSF的N端融合形成第二多肽链，从而形成IL12a-IL15-IL12b-GMCSF蛋白质异二聚体。
例如，在所述蛋白质异二聚体中，IL12a的C端可以和IL21的N端融合形成第一多肽链，且IL12b的C端可以和GMCSF的N端融合形成第二多肽链，从而形成IL12a-IL21-IL12b-GMCSF蛋白质异二聚体。
例如，在所述蛋白质异二聚体中，IL12a的C端可以和IL2的N端融合形成第一多肽链，且IL12b的C端可以和FLT3L的N端融合形成第二多肽链，从而形成IL12a-IL2-IL12b-FLT3L蛋白质异二聚体。
例如，在所述蛋白质异二聚体中，IL12a的C端可以和IL7的N端融合形成第一多肽链，且IL12b的C端可以和FLT3L的N端融合形成第二多肽链，从而形成IL12a-IL7-IL12b-FLT3L蛋白质异二聚体。
例如，在所述蛋白质异二聚体中，IL12a的C端可以和IL15的N端融合形成第一多肽链，且IL12b的C端可以和FLT3L的N端融合形成第二多肽链，从而形成IL12a-IL15-IL12b-FLT3L 蛋白质异二聚体。
例如，在所述蛋白质异二聚体中，IL12a的C端可以和IL21的N端融合形成第一多肽链，且IL12b的C端可以和FLT3L的N端融合形成第二多肽链，从而形成IL12a-IL21-IL12b-FLT3L蛋白质异二聚体。
在本申请中，所述IL12a、所述IL12b、所述第一因子和所述第二因子可以来源自哺乳动物。例如，所述哺乳动物可以选自下组：人、小鼠、大鼠、猴、犬、猪、羊、牛和猫。在本申请中，所述哺乳动物可以选自下组：人、小鼠。例如，所述IL12a、IL12b、IL2、GMCSF、IL7、IL15、IL21和FLT3L可以选自小鼠，所述小鼠来源的IL12a、IL12b、IL2、GMCSF、IL7、IL15、IL21和FLT3L可以简写作mIL12a、mIL12b、mIL2、mGMCSF、mIL7、mIL15、mIL21和mFLT3L。例如，所述IL12a、IL12b、IL2、GMCSF、IL7、IL15、IL21和FLT3L可以选自人，所述人来源的IL12a、IL12b、IL2、GMCSF、IL7、IL15、IL21和FLT3L可以简写作hIL12a、hIL12b、hIL2、hGMCSF、hIL7、hIL15、hIL21和hFLT3L。
在本申请中，所述IL12a、所述IL12b、所述第一因子和所述第二因子的来源可以相同。
在本申请中，所述第一多肽链可以包括如SEQ ID NO.60-65和SEQ ID NO.70-73中任一项所示的序列。
在本申请中，所述第二多肽链可以包括如SEQ ID NO.66-69和SEQ ID NO.74-75中任一项所示的序列。
在本申请中，所述蛋白质异二聚体可以包括如SEQ ID NO.18-37中任一项所示的序列。
例如，在所述蛋白质异二聚体中，mIL12a的C端和mIL2的N端可以融合形成mIL12a-mIL2第一多肽链(序列如SEQ ID NO.60所示)，且mIL12b的C端和mGMCSF的N端可以融合形成mIL12b-mGMCSF第二多肽链(序列如SEQ ID NO.66所示)，可以形成mIL12a-mIL2-mIL12b-mGMCSF蛋白质异二聚体(序列如SEQ ID NO.18所示)。
例如，在所述蛋白质异二聚体中，mIL2的C端和mIL12a的N端可以融合形成mIL2-mIL12a第一多肽链(序列如SEQ ID NO.61所示)，且mIL12b的C端和mGMCSF的N端可以融合形成mIL12b-mGMCSF第二多肽链(序列如SEQ ID NO.66所示)，可以形成mIL2-mIL12a-mIL12b-mGMCSF蛋白质异二聚体(序列如SEQ ID NO.19所示)。
例如，在所述蛋白质异二聚体中，mIL12a的C端和mIL2的N端可以融合形成mIL12a-mIL2第一多肽链(序列如SEQ ID NO.60所示)，且mGMCSF的C端和mIL12b的N端可以融合形成mGMCSF-mIL12b第二多肽链(序列如SEQ ID NO.67所示)，可以形成mIL12a-mIL2-mGMCSF-mIL12b蛋白质异二聚体(序列如SEQ ID NO.20所示)。
例如，在所述蛋白质异二聚体中，mIL2的C端和mIL12a的N端可以融合形成 mIL2-mIL12a第一多肽链(序列如SEQ ID NO.61所示)，且mGMCSF的C端和mIL12b的N端可以融合形成mGMCSF-mIL12b第二多肽链(序列如SEQ ID NO.67所示)，可以形成mIL2-mIL12a-mGMCSF-mIL12b蛋白质异二聚体(序列如SEQ ID NO.21所示)。
例如，在所述蛋白质异二聚体中，mIL12a的C端和mGMCSF的N端可以融合形成mIL12a-mGMCSF第一多肽链(序列如SEQ ID NO.62所示)，且mIL12b的C端和mIL2的N端可以融合形成mIL12b-mIL2第二多肽链(序列如SEQ ID NO.68所示)，可以形成mIL12a-mGMCSF-mIL12b-mIL2蛋白质异二聚体(序列如SEQ ID NO.22所示)。
例如，在所述蛋白质异二聚体中，mIL12a的C端和mIL7的N端可以融合形成mIL12a-mIL7第一多肽链(序列如SEQ ID NO.63所示)，且mIL12b的C端和mGMCSF的N端可以融合形成mIL12b-mGMCSF第二多肽链(序列如SEQ ID NO.66所示)，可以形成mIL12a-mIL7-mIL12b-mGMCSF蛋白质异二聚体(序列如SEQ ID NO.23所示)。
例如，在所述蛋白质异二聚体中，mIL12a的C端和mIL15的N端可以融合形成mIL12a-mIL15第一多肽链(序列如SEQ ID NO.64所示)，且mIL12b的C端和mGMCSF的N端可以融合形成mIL12b-mGMCSF第二多肽链(序列如SEQ ID NO.66所示)，可以形成mIL12a-mIL15-mIL12b-mGMCSF蛋白质异二聚体(序列如SEQ ID NO.24所示)。
例如，在所述蛋白质异二聚体中，mIL12a的C端和mIL21的N端可以融合形成mIL12a-mIL21第一多肽链(序列如SEQ ID NO.65所示)，且mIL12b的C端和mGMCSF的N端可以融合形成mIL12b-mGMCSF第二多肽链(序列如SEQ ID NO.66所示)，可以形成mIL12a-mIL21-mIL12b-mGMCSF蛋白质异二聚体(序列如SEQ ID NO.25所示)。
例如，在所述蛋白质异二聚体中，mIL12a的C端和mIL2的N端可以融合形成mIL12a-mIL2第一多肽链(序列如SEQ ID NO.60所示)，且mIL12b的C端和mFLT3L的N端可以融合形成mIL12b-mFLT3L第二多肽链(序列如SEQ ID NO.69所示)，可以形成mIL12a-mIL2-mIL12b-mFLT3L蛋白质异二聚体(序列如SEQ ID NO.26所示)。
例如，在所述蛋白质异二聚体中，mIL12a的C端和mIL7的N端可以融合形成mIL12a-mIL7第一多肽链(序列如SEQ ID NO.63所示)，且mIL12b的C端和mFLT3L的N端可以融合形成mIL12b-mFLT3L第二多肽链(序列如SEQ ID NO.69所示)，可以形成mIL12a-mIL7-mIL12b-mFLT3L蛋白质异二聚体(序列如SEQ ID NO.27所示)。
例如，在所述蛋白质异二聚体中，mIL12a的C端和mIL15的N端可以融合形成mIL12a-mIL15第一多肽链(序列如SEQ ID NO.64所示)，且mIL12b的C端和mFLT3L的N端可以融合形成mIL12b-mFLT3L第二多肽链(序列如SEQ ID NO.69所示)，可以形成mIL12a-mIL15-mIL12b-mFLT3L蛋白质异二聚体(序列如SEQ ID NO.28所示)。
例如，在所述蛋白质异二聚体中，mIL12a的C端和mIL21的N端可以融合形成mIL12a-mIL21第一多肽链(序列如SEQ ID NO.65所示)，且mIL12b的C端和mFLT3L的N端可以融合形成mIL12b-mFLT3L第二多肽链(序列如SEQ ID NO.69所示)，可以形成mIL12a-mIL21-mIL12b-mFLT3L蛋白质异二聚体(序列如SEQ ID NO.29所示)。
例如，在所述蛋白质异二聚体中，hIL12a的C端和hIL2的N端可以融合形成hIL12a-hIL2第一多肽链(序列如SEQ ID NO.70所示)，且hIL12b的C端和hGMCSF的N端可以融合形成hIL12b-hGMCSF第二多肽链(序列如SEQ ID NO.74所示)，可以形成hIL12a-hIL2-hIL12b-hGMCSF蛋白质异二聚体(序列如SEQ ID NO.30所示)。
例如，在所述蛋白质异二聚体中，hIL12a的C端和hIL7的N端可以融合形成hIL12a-hIL7第一多肽链(序列如SEQ ID NO.71所示)，且hIL12b的C端和hGMCSF的N端可以融合形成hIL12b-hGMCSF第二多肽链(序列如SEQ ID NO.74所示)，可以形成hIL12a-hIL7-hIL12b-hGMCSF蛋白质异二聚体(序列如SEQ ID NO.31所示)。
例如，在所述蛋白质异二聚体中，hIL12a的C端和hIL15的N端可以融合形成hIL12a-hIL15第一多肽链(序列如SEQ ID NO.72所示)，且hIL12b的C端和hGMCSF的N端可以融合形成hIL12b-hGMCSF第二多肽链(序列如SEQ ID NO.74所示)，可以形成hIL12a-hIL15-hIL12b-hGMCSF蛋白质异二聚体(序列如SEQ ID NO.32所示)。
例如，在所述蛋白质异二聚体中，hIL12a的C端和hIL21的N端可以融合形成hIL12a-hIL21第一多肽链(序列如SEQ ID NO.73所示)，且hIL12b的C端和hGMCSF的N端可以融合形成hIL12b-hGMCSF第二多肽链(序列如SEQ ID NO.74所示)，可以形成hIL12a-hIL21-hIL12b-hGMCSF蛋白质异二聚体(序列如SEQ ID NO.33所示)。
例如，在所述蛋白质异二聚体中，hIL12a的C端和hIL2的N端可以融合形成hIL12a-hIL2第一多肽链(序列如SEQ ID NO.70所示)，且hIL12b的C端和hFLT3L的N端可以融合形成hIL12b-hFLT3L第二多肽链(序列如SEQ ID NO.75所示)，可以形成hIL12a-hIL2-hIL12b-hFLT3L蛋白质异二聚体(序列如SEQ ID NO.34所示)。
例如，在所述蛋白质异二聚体中，hIL12a的C端和hIL7的N端可以融合形成hIL12a-hIL7第一多肽链(序列如SEQ ID NO.71所示)，且hIL12b的C端和hFLT3L的N端可以融合形成hIL12b-hFLT3L第二多肽链(序列如SEQ ID NO.75所示)，可以形成hIL12a-hIL7-hIL12b-hFLT3L蛋白质异二聚体(序列如SEQ ID NO.35所示)。
例如，在所述蛋白质异二聚体中，hIL12a的C端和hIL15的N端可以融合形成hIL12a-hIL15第一多肽链(序列如SEQ ID NO.72所示)，且hIL12b的C端和hFLT3L的N端可以融合形成hIL12b-hFLT3L第二多肽链(序列如SEQ ID NO.75所示)，可以形成 hIL12a-hIL15-hIL12b-hFLT3L蛋白质异二聚体(序列如SEQ ID NO.36所示)。
例如，在所述蛋白质异二聚体中，hIL12a的C端和hIL21的N端可以融合形成hIL12a-hIL21第一多肽链(序列如SEQ ID NO.73所示)，且hIL12b的C端和hFLT3L的N端可以融合形成hIL12b-hFLT3L第二多肽链(序列如SEQ ID NO.75所示)，可以形成hIL12a-hIL21-hIL12b-hFLT3L蛋白质异二聚体(序列如SEQ ID NO.37所示)。
在本申请中，所述mIL12a-mIL2-mIL12b-mGMCSF、mIL2-mIL12a-mIL12b-mGMCSF、mIL12a-mIL2-mGMCSF-mIL12b、mIL2-mIL12a-mGMCSF-mIL12b、mIL12a-mGMCSF-mIL12b-mIL2、mIL12a-mIL7-mIL12b-mGMCSF、mIL12a-mIL15-mIL12b-mGMCSF、mIL12a-mIL21-mIL12b-mGMCSF、mIL12a-mIL2-mIL12b-mFLT3L、mIL12a-mIL7-mIL12b-mFLT3L、mIL12a-mIL15-mIL12b-mFLT3L、mIL12a-mIL21-mIL12b-mFLT3L、hIL12a-hIL2-hIL12b-hGMCSF、hIL12a-hIL7-hIL12b-hGMCSF、hIL12a-hIL15-hIL12b-hGMCSF、hIL12a-hIL21-hIL12b-hGMCSF、hIL12a-hIL2-hIL12b-hFLT3L、hIL12a-hIL7-hIL12b-hFLT3L、hIL12a-hIL15-hIL12b-hFLT3L和hIL12a-hIL21-hIL12b-hFLT3L可以分别依次简称为mIL12aIL2IL12bGMCSF、mIL2IL12aIL12bGMCSF、mIL12aIL2GMCSFIL12b、mIL2IL12aGMCSFIL12b、mIL12aGMCSFIL12bIL2、mIL12aIL7IL12bGMCSF、mIL12aIL15IL12bGMCSF、mIL12aIL21IL12bGMCSF、mIL12aIL2IL12bFLT3L、mIL12aIL7IL12bFLT3L、mIL12aIL15IL12bFLT3L、mIL12aIL21IL12bFLT3L、hIL12aIL2IL12bGMCSF、hIL12aIL7IL12bGMCSF、hIL12aIL15IL12bGMCSF、hIL12aIL21IL12bGMCSF、hIL12aIL2IL12bFLT3L、hIL12aIL7IL12bFLT3L、hIL12aIL15IL12bFLT3L和hIL12aIL21IL12bFLT3L。上述蛋白质异二聚体的结构可以如图2所示。
在本申请中，所述的蛋白质异二聚体可以用于治疗肿瘤。
另一方面，本申请提供了一种所述的蛋白质异二聚体在制备药物中的用途，所述药物可以用于治疗肿瘤。
所述肿瘤可以选自以下组：黑色素瘤、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。
核酸分子、表达载体、宿主细胞、药物组合物及制备方法
另一方面，本申请提供了一种或多种分离的核酸分子，其可以编码所述的蛋白质异二聚 体。例如，所述核酸分子可以包括如SEQ ID NO.38-57中任一项所示的序列。例如，编码所述mIL12aIL2IL12bGMCSF、mIL2IL12aIL12bGMCSF、mIL12aIL2GMCSFIL12b、mIL2IL12aGMCSFIL12b、mIL12aGMCSFIL12bIL2、mIL12aIL7IL12bGMCSF、mIL12aIL15IL12bGMCSF、mIL12aIL21IL12bGMCSF、mIL12aIL2IL12bFLT3L、mIL12aIL7IL12bFLT3L、mIL12aIL15IL12bFLT3L、mIL12aIL21IL12bFLT3L、hIL12aIL2IL12bGMCSF、hIL12aIL7IL12bGMCSF、hIL12aIL15IL12bGMCSF、hIL12aIL21IL12bGMCSF、hIL12aIL2IL12bFLT3L、hIL12aIL7IL12bFLT3L、hIL12aIL15IL12bFLT3L和hIL12aIL21IL12bFLT3L的核苷酸序列可以分别为SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、SEQ ID NO.51、SEQ ID NO.52、SEQ ID NO.53、SEQ ID NO.54、SEQ ID NO.55、SEQ ID NO.56和SEQ ID NO.57。
本申请中，所述核酸分子可以用于治疗肿瘤。
另一方面，本申请提供了一种所述核酸分子在制备药物中的用途，所述药物可以用于治疗肿瘤。
所述肿瘤可以选自以下组：黑色素瘤、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。
另一方面，本申请提供了一种表达载体，其可以包括编码所述的蛋白质异二聚体的核苷酸序列。在本申请中，可以通过分别将编码mIL12aIL2IL12bGMCSF、mIL2IL12aIL12bGMCSF、mIL12aIL2GMCSFIL12b、mIL2IL12aGMCSFIL12b、mIL12aGMCSFIL12bIL2、mIL12aIL7IL12bGMCSF、mIL12aIL15IL12bGMCSF、mIL12aIL21IL12bGMCSF、mIL12aIL2IL12bFLT3L、mIL12aIL7IL12bFLT3L、mIL12aIL15IL12bFLT3L和mIL12aIL21IL12bFLT3L的核苷酸序列插入到pLentis-PTRE-MCS-PGK-PURO核苷酸序列中，和，从而构建可调控表达载体pLentis-PTRE-mIL12aIL2IL12bGMCSF-PGK-PURO、pLentis-PTRE-mIL2IL12aIL12bGMCSF-PGK-PURO、pLentis-PTRE-mIL12aIL2GMCSFIL12b-PGK-PURO、pLentis-PTRE-mIL2IL12aGMCSFIL12b-PGK-PURO、pLentis-PTRE-mIL12aGMCSFIL12bIL2-PGK-PURO、pLentis-PTRE-mIL12aIL7IL12bGMCSF-PGK-PURO、pLentis-PTRE-mIL12aIL15IL12bGMCSF-PGK-PURO、pLentis-PTRE-mIL12aIL21IL12bGMCSF-PGK-PURO、pLentis-PTRE-mIL12aIL2IL12bFLT3L-PGK-PURO、pLentis-PTRE- mIL12aIL7IL12bFLT3L-PGK-PURO、pLentis-PTRE-mIL12aIL15IL12bFLT3L-PGK-PURO和pLentis-PTRE-mIL12aIL21IL12bFLT3L-PGK-PURO。例如，可以通过分别将hIL12aIL2IL12bGMCSF、hIL12aIL7IL12bGMCSF、hIL12aIL15IL12bGMCSF、hIL12aIL21IL12bGMCSF、hIL12aIL2IL12bFLT3L、hIL12aIL7IL12bFLT3L、hIL12aIL15IL12bFLT3L和hIL12aIL21IL12bFLT3L插入到pLentis-CMV-MCS-IRES-PURO中，从而构建表达载体pLentis-CMV-hIL12aIL2IL12bGMCSF-IRES-PURO、pLentis-CMV-hIL12aIL7IL12bGMCSF-IRES-PURO、pLentis-CMV-hIL12aIL15IL12bGMCSF-IRES-PURO、pLentis-CMV-hIL12aIL21IL12bGMCSF-IRES-PURO、pLentis-CMV-hIL12aIL2IL12bFLT3L-IRES-PURO、pLentis-CMV-hIL12aIL7IL12bFLT3L-IRES-PURO、pLentis-CMV-hIL12aIL15IL12bFLT3L-IRES-PURO和pLentis-CMV-hIL12aIL21IL12bFLT3L-IRES-PURO。
本申请中，所述表达载体可以用于治疗肿瘤。
另一方面，本申请提供了一种所述表达载体在制备药物中的用途，所述药物可以用于治疗肿瘤。所述肿瘤可以选自以下组：黑色素瘤、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。
在本申请中，构建载体和质粒的方法，例如将编码蛋白的基因插入到载体和质粒的方法或将质粒引入宿主细胞的方法，对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook，J.，Fritsch，E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。
另一方面，本申请提供了一种宿主细胞，其可以包括所述的表达载体。在本申请中，所述宿主细胞可以是使用所述表达载体转染B16(rtTA)肿瘤细胞制备而得到的。例如，可以分别使用pLentis-PTRE-mIL12aIL2IL12bGMCSF-PGK-PURO、pLentis-PTRE-mIL2IL12aIL12bGMCSF-PGK-PURO、pLentis-PTRE-mIL12aIL2GMCSFIL12b-PGK-PURO、pLentis-PTRE-mIL2IL12aGMCSFIL12b-PGK-PURO、pLentis-PTRE-mIL12aGMCSFIL12bIL2-PGK-PURO、pLentis-PTRE-mIL12aIL7IL12bGMCSF-PGK-PURO、pLentis-PTRE-mIL12aIL15IL12bGMCSF-PGK-PURO、pLentis-PTRE-mIL12aIL21IL12bGMCSF-PGK-PURO、pLentis-PTRE-mIL12aIL2IL12bFLT3L-PGK-PURO、pLentis-PTRE-mIL12aIL7IL12bFLT3L -PGK-PURO、pLentis-PTRE-mIL12aIL15IL12bFLT3L-PGK-PURO和pLentis-PTRE-mIL12aIL21IL12bFLT3L-PGK-PURO表达载体的病毒转染B16(rtTA)肿瘤细胞，从而得到B16(rtTA)-mIL12aIL2IL12bGMCSF、B16(rtTA)-mIL2IL12aIL12bGMCSF、B16(rtTA)-mIL12aIL2GMCSFIL12b、B16(rtTA)-mIL2IL12aGMCSFIL12b、B16(rtTA)-mIL12aGMCSFIL12bIL2、B16(rtTA)-mIL12aIL7IL12bGMCSF、B16(rtTA)-mIL12aIL15IL12bGMCSF、B16(rtTA)-mIL12aIL21IL12bGMCSF、B16(rtTA)-mIL12aIL2IL12bFLT3L、B16(rtTA)-mIL12aIL7IL12bFLT3L、B16(rtTA)-mIL12aIL15IL12bFLT3L和B16(rtTA)-mIL12aIL21IL12bFLT3L宿主细胞。例如，可以分别使用pLentis-CMV-hIL12aIL2IL12bGMCSF-IRES-PURO、pLentis-CMV-hIL12aIL7IL12bGMCSF-IRES-PURO、pLentis-CMV-hIL12aIL15IL12bGMCSF-IRES-PURO、pLentis-CMV-hIL12aIL21IL12bGMCSF-IRES-PURO、pLentis-CMV-hIL12aIL2IL12bFLT3L-IRES-PURO、pLentis-CMV-hIL12aIL7IL12bFLT3L-IRES-PURO和pLentis-CMV-hIL12aIL15IL12bFLT3L-IRES-PURO、pLentis-CMV-hIL12aIL21IL12bFLT3L-IRES-PURO表达载体的病毒转染293A细胞，从而得到293A-hIL12aIL2IL12bGMCSF、293A-hIL12aIL7IL12bGMCSF、293A-hIL12aIL15IL12bGMCSF、293A-hIL12aIL21IL12bGMCSF、293A-hIL12aIL2IL12bFLT3L、293A-hIL12aIL7IL12bFLT3L、293A-hIL12aIL15IL12bFLT3L和293A-hIL12aIL21IL12bFLT3L宿主细胞。
本申请中，所述宿主细胞可以用于治疗肿瘤。所述肿瘤可以选自以下组：黑色素瘤、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。
另一方面，本申请提供了一种所述宿主细胞在制备药物中的用途，所述药物可以用于治疗肿瘤。所述肿瘤可以选自以下组：黑色素瘤、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。
本申请提供了蛋白质异二聚体、核酸分子、表达载体、宿主细胞、药物组合物，其用于治疗肿瘤。
治疗肿瘤的方法，其包括以下的步骤：向有需要的受试者施用本申请所述的蛋白质异二 聚体、核酸分子、表达载体、宿主细胞和/或药物组合物。
所述肿瘤可以选自以下组：黑色素瘤、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。
另一方面，本申请提供了一种制备所述的蛋白质异二聚体的方法，其可以包括以下的步骤：在能够表达所述的蛋白质异二聚体的情况下，培养所述的宿主细胞。
药物组合物及其用途
另一方面，本申请提供了一种药物组合物，其可以包括所述的蛋白质异二聚体、所述的核酸分子、所述的载体和/或所述的宿主细胞，以及药学上可接受的载体。
所述药学上可接受的载体可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。例如，所述药学上可接受的载体可以包括赋形剂，例如，所述赋形剂可以选自下组：淀粉、糊精、蔗糖、乳糖、硬脂酸镁、硫酸钙、羧甲基素、滑石粉、海藻酸钙凝胶、壳聚糖和纳米微球等。例如，所述药学上可接受的载体还可以选自下组：pH调节剂、渗透压调节剂、增溶剂和抑菌剂。
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。
在本申请中，所述药物组合物可以用于治疗肿瘤。所述肿瘤可以选自以下组：黑色素瘤、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，可以通过促进细胞因子的表达减小肿瘤的大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。
在本申请中，所述药物组合物可以含有治疗有效量的所述的蛋白质异二聚体、所述的核酸分子、所述的载体和/或所述的宿主细胞。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。
另一方面，本申请提供一种缓解或治疗肿瘤的方法，所述方法可包括施用所述蛋白质异二聚体、所述的核酸分子、所述的表达载体、所述宿主细胞和/或所述药物组合物。
在本申请中，所述施用的方法可包括口服给药，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。
下面显示的实施例意在说明本发明的具体实施方案，并且不意在以任何方式限制本说明书或权利要求书的范围。
实施例
试剂：DMEM培养基、1640培养基、胎牛血清购自lifetechnologies公司；细胞培养瓶、培养板购自Corning公司；强力霉素(DOX)购自上海生工生物工程有限公司；嘌呤霉素(Puromycin)、Blasticidin购自Chemicon公司；限制性内切酶购自Takara和NEB公司；连接酶购自NEB公司；DNA聚合酶购自Takara公司；质粒提取试剂盒、胶回收试剂盒购自OmegaBiotech公司；引物合成由上海生工生物工程有限公司完成；基因合成由南京金斯瑞公司完成；ELISA试剂盒购于博士德公司。细胞因子mIL12、mGMCSF、mFLT3L、mIL2、mIL15、mIL21、mIL7、hIL12、hGMCSF、hFLT3L、hIL2、hIL15、hIL21和hIL7购于Peprotech公司。
实施例的部分试验结果参见图1、4-6。在图1和图4-图16中，每条折线各代表一只小鼠体内肿瘤的面积状况，例如，5条折线表示一次实验的5只小鼠的数据；分数则表示肿瘤清除的小鼠/接种肿瘤的小鼠的比例，如10/10表示肿瘤清除的小鼠/接种肿瘤的小鼠是10/10＝100％。
实施例1表达可调控蛋白的肿瘤细胞的制备
1.1构建第一表达载体pLentis-CMV-rtTA-IRES-Bsd
合成两端带有BamHI和EcoRI位点的rtTA(GenBank：ALK44378.1)的DNA序列，合成产物连接在载体pUC57上。酶切所述连接有rtTA的pUC57载体，酶切体系如下：6μg连接有rtTA的pUC57载体质粒、4μl酶切缓冲液、1μl BamHI和1μl EcoRI，加水至总体积为40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收rtTA片段，待用。
在EP管内酶切载体pLentis-CMV-IRES-Bsd，酶切体系如下：2μg pLentis-CMV-IRES-Bsd载体质粒、3μl酶切缓冲液、1μl BamHI和1μl EcoRI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-CMV-IRES-Bsd载体片段，待用。
连接pLentis-CMV-IRES-Bsd和rtTA，体系如下：2μl pLentis-CMV-IRES-Bsd、2μl rtTA、 1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水，置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中，37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定rtTA片段是否成功连入pLentis-CMV-IRES-Bsd载体中，然后将正确的载体送测序，确定第一表达载体pLentis-CMV-rtTA-IRES-Bsd构建成功。
1.2制备第一表达载体pLentis-CMV-rtTA-IRES-Bsd的病毒
1)消化培养的293FT细胞，计数后将3×10 6细胞/孔铺入10cm培养皿中，培养液体积为10ml。
2)第二天晚上，观察细胞状态，如果细胞状态合适良好，进行转染。在培养板中加入氯喹至终浓度25μM，取一只试管，加入灭菌水及以下质粒(5μg pMD2.G+15μg pSPAX2+20μg pLentis-CMV-rtTA-IRES-Bsd)，总体积为1045μl，然后加入155μl 2MCaCl 2，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500μlHBSS重悬沉淀，溶解2小时后分装成小管，-70℃保存，得到第一表达载体pLentis-CMV-rtTA-IRES-Bsd的病毒。
1.3使用第一表达载体pLentis-CMV-rtTA-IRES-Bsd的病毒转染B16肿瘤细胞
消化培养的小鼠黑色素瘤细胞B16，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl第一表达载体pLentis-CMV-rtTA-IRES-Bsd的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按适合此细胞的浓度加入杀稻瘟菌素(blasticidin)，继续培养，每两天更换一次培养基，并保持杀稻瘟菌素的浓度为8μg/ml，筛选一周后，存活的细胞即为稳定表达调控蛋白的细胞，将此细胞命名为B16(rtTA)。
实施例2诱导表达绿色荧光蛋白(GFP)对肿瘤生长的影响
2.1构建编码绿色荧光蛋白(GFP)的可调控表达载体
使用引物，以带有GFP基因为模板进行PCR反应扩增GFP基因，PCR条件按PrimeStarHSDNApolymerase说明书进行。PCR进行琼脂糖凝胶电泳后，用胶回收试剂盒回收，然后用BamHI和EcoRI进行酶切，酶切体系如下：30μg PCR回收产物、4μl酶切缓冲液、1μl  BamHI和1μl EcoRI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收GFP基因片段，待用。
酶切可调控表达载体，体系如下：2μg pLentis-PTRE-MCS-PGK-PURO质粒、3μl酶切缓冲液、1μl BamHI和1μl EcoRI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl10×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。连接pLentis-PTRE-MCS-PGK-PURO和GFP，连接体系如下：2μl pLentis-PTRE-MCS-PGK-PURO、2μl GFP、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-GFP-PGK-PURO构建成功。
2.2制备可调控表达GFP的细胞
制备GFP表达载体的病毒，方法与第一表达载体的病毒制备方法一致，得到第二表达载体pLentis-PTRE-GFP-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二可调控表达载体pLentis-PTRE-GFP-PGK-PURO的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素(puromycin)，继续培养三天后，存活的细胞即为可调控表达GFP的细胞，此细胞命名为B16(rtTA)-GFP。
2.3调控表达GFP对肿瘤生长的影响
将对数生长期的B16(rtTA)-GFP细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠(购于北京华阜康生物科技股份有限公司)的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况，如图1所示，结果显示诱导表达GFP对肿瘤生长无抑制作用(其中每条折线各代表一只小鼠体内肿瘤的面积状况)。
实施例3设计蛋白质异二聚体
设计包括如下蛋白质异二聚体：mIL2IL12aIL12bGMCSF、mIL12aIL2GMCSFIL12b、mIL2IL12aGMCSFIL12b、mIL12aGMCSFIL12bIL2、mIL12aIL7IL12bGMCSF、mIL12aIL15IL12bGMCSF、mIL12aIL21IL12bGMCSF、mIL12aIL2IL12bFLT3L、mIL12aIL7IL12bFLT3L、mIL12aIL15IL12bFLT3L、mIL12aIL21IL12bFLT3L、 hIL12aIL2IL12bGMCSF、hIL12aIL7IL12bGMCSF、hIL12aIL15IL12bGMCSF、hIL12aIL21IL12bGMCSF、hIL12aIL2IL12bFLT3L、hIL12aIL7IL12bFLT3L、hIL12aIL15IL12bFLT3L和hIL12aIL21IL12bFLT3L。上述蛋白质异二聚体的结构如图2所示。
实施例4诱导表达mIL12aIL2IL12bGMCSF对肿瘤生长的影响
4.1构建mIL12aIL2IL12bGMCSF可调控表达载体
合成mIL12aIL2IL12bGMCSF编码序列，两端分别带有BamHI、BglII和XhoI、EcoRI酶切位点，然后用BamHI或BglII和XhoI或EcoRI进行酶切，酶切体系如下：5μg mIL12aIL2IL12bGMCSF质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12aIL2IL12bGMCSF基因片段，待用。
mIL12aIL2IL12bGMCSF蛋白质异二聚体的氨基酸序列如SEQ ID NO.18所示，编码所述mIL12aIL2IL12bGMCSF的核苷酸序列如SEQ ID NO.38所示。
酶切可调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：2μg pLentis-PTRE-MCS-PGK-PURO载体质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和mIL12aIL2IL12bGMCSF，连接体系如下：2μl pLentis-PTRE-MCS-PGK-PURO、2μl mIL12aIL2IL12bGMCSF、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-mIL12aIL2IL12bGMCSF-PGK-PURO构建成功。
4.2制备可调控表达mIL12aIL2IL12bGMCSF的细胞
制备所述mIL12aIL2IL12bGMCSF表达载体的病毒，方法与第一表达载体的病毒制备方法一致，得到第二表达载体pLentis-PTRE-mIL12aIL2IL12bGMCSF-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二可调控表达载体pLentis-PTRE-mIL12aIL2IL12bGMCSF-PGK-PURO的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素，继续培养三天后，存活的细胞即为可调控表达mIL12aIL2IL12bGMCSF的细 胞，此细胞命名为B16(rtTA)-mIL12aIL2IL12bGMCSF。
4.3体外检测mIL12aIL2IL12bGMCSF的诱导表达效果
将B16(rtTA)-mIL12aIL2IL12bGMCSF细胞按5×10 4每孔铺入24孔板中，24小时后，在孔内加入100ng/ml的强力霉素(DOX)，继续培养72小时，收集上清。使用小鼠IL12p70ELISA试剂盒检测上清中的融合蛋白表达量，操作按试剂盒说明书进行。未加DOX诱导的孔作为对照。如图3所示，DOX的加入诱导了mIL12aIL2IL12bGMCSF融合蛋白的表达。
4.4诱导表达mIL12aIL2IL12bGMCSF对肿瘤生长的影响
将对数生长期的B16(rtTA)-mIL12aIL2IL12bGMCSF细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况以及肿瘤清除率，肿瘤清除小鼠数/总的小鼠数＝10/10，如图4所示，mIL12aIL2IL12bGMCSF能够在全部小鼠中诱导肿瘤消退。
实施例5诱导表达mIL2IL12aIL12bGMCSF对肿瘤生长的影响
5.1构建mIL2IL12aIL12bGMCSF可调控表达载体
合成mIL2IL12aIL12bGMCSFF基因编码序列，两端分别带有BamHI、BglII和XhoI、EcoRI酶切位点，然后用BamHI或BglII和XhoI或EcoRI进行酶切，酶切体系如下：5μg mIL2IL12aIL12bGMCSF质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL2IL12aIL12bGMCSF基因片段，待用。
mIL2IL12aIL12bGMCSF蛋白质异二聚体的氨基酸序列如SEQ ID NO.19所示，编码所述mIL2IL12aIL12bGMCSF的核苷酸序列是SEQ ID NO.39。
酶切可调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：2μg pLentis-PTRE-MCS-PGK-PURO载体质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和mIL2IL12aIL12bGMCSF，连接体系如下：2μl pLentis-PTRE-MCS-PGK-PURO、2μl mIL2IL12aIL12bGMCSF、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测 序，确定第二表达载体pLentis-PTRE-mIL2IL12aIL12bGMCSF-PGK-PURO构建成功。
5.2制备可调控表达mIL2IL12aIL12bGMCSF的细胞
制备所述mIL2IL12aIL12bGMCSF表达载体的病毒，方法与第一表达载体病毒制备方法一致，得到第二表达载体pLentis-PTRE-mIL2IL12aIL12bGMCSF-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二表达载体pLentis-PTRE-mIL2IL12aIL12bGMCSF-PGK-PURO的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素，继续培养三天后，存活的细胞即为可调控表达mIL2IL12aIL12bGMCSF的细胞，此细胞命名为B16(rtTA)-mIL2IL12aIL12bGMCSF。
5.3诱导表达mIL2IL12aIL12bGMCSF对肿瘤生长的影响
将对数生长期的B16(rtTA)-mIL2IL12aIL12bGMCSF细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况以及肿瘤清除率，肿瘤清除小鼠数/总的小鼠数＝10/10，如图5所示，mIL2IL12aIL12bGMCSF能够在全部小鼠中诱导肿瘤消退。
实施例6诱导表达mIL12aIL2GMCSFIL12b对肿瘤生长的影响
6.1构建mIL12aIL2GMCSFIL12b可调控表达载体
合成mIL12aIL2GMCSFIL12b基因编码序列，两端分别带有BamHI、BglII和XhoI、EcoRI酶切位点，然后用BamHI或BglII和XhoI或EcoRI进行酶切，酶切体系如下：5μg mIL12aIL2GMCSFIL12b质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12aIL2GMCSFIL12b基因片段，待用。
mIL12aIL2GMCSFIL12b蛋白质异二聚体的氨基酸序列如SEQ ID NO.20所示，编码所述mIL12aIL2GMCSFIL12b的核苷酸序列如SEQ ID NO.40所示。
酶切可调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：2μgpLentis-PTRE-MCS-PGK-PURO载体质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和mIL12aIL2GMCSFIL12b，连接体系如下：2μl  pLentis-PTRE-MCS-PGK-PURO、2μl mIL12aIL2GMCSFIL12b、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-mIL12aIL2GMCSFIL12b-PGK-PURO构建成功。
6.2制备可调控表达mIL12aIL2GMCSFIL12b的细胞
制备所述mIL12aIL2GMCSFIL12b表达载体的病毒，方法与第一表达载体的病毒制备方法一致，得到第二可调控表达载体pLentis-PTRE-mIL12aIL2GMCSFIL12b-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二可调控表达载体pLentis-PTRE-mIL12aIL2GMCSFIL12b-PGK-PURO的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素，继续培养三天后，存活的细胞即为可调控表达mIL12aIL2GMCSFIL12b的细胞，此细胞命名为B16(rtTA)-mIL12aIL2GMCSFIL12b。
6.3诱导表达mIL12aIL2GMCSFIL12b对肿瘤生长的影响
将对数生长期的B16(rtTA)-mIL12aIL2GMCSFIL12b细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况以及肿瘤清除率，肿瘤清除小鼠数/总的小鼠数＝10/10，如图6所示，mIL12aIL2GMCSFIL12b能够在全部小鼠中诱导肿瘤消退。
实施例7诱导表达mIL2IL12aGMCSFIL12b对肿瘤生长的影响
7.1构建mIL2IL12aGMCSFIL12b可调控表达载体
合成mIL2IL12aGMCSFIL12b基因编码序列，两端分别带有BamHI、BglII和XhoI、EcoRI酶切位点，然后用BamHI或BglII和XhoI或EcoRI进行酶切，酶切体系如下：5μg mIL2IL12aGMCSFIL12b质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL2IL12aGMCSFIL12b基因片段，待用。
mIL2IL12aGMCSFIL12b蛋白质异二聚体的氨基酸序列如SEQ ID NO.21所示，编码所述mIL2IL12aGMCSFIL12b的核苷酸序列如SEQ ID NO.41所示。
酶切可调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：2μg  pLentis-PTRE-MCS-PGK-PURO质粒、3μl酶切缓冲液、1μl BamHI、1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和mIL2IL12aGMCSFIL12b，连接体系如下：2μl pLentis-PTRE-MCS-PGK-PURO、2μl mIL2IL12aGMCSFIL12b、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-mIL2IL12aGMCSFIL12b-PGK-PURO构建成功。
7.2制备可调控表达mIL2IL12aGMCSFIL12b的细胞
制备所述mIL2IL12aGMCSFIL12b表达载体的病毒，方法与第一表达载体病毒制备方法一致，得到第二表达载体pLentis-PTRE-mIL2IL12aGMCSFIL12b-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二表达调控载体pLentis-PTRE-mIL2IL12aGMCSFIL12b-PGK-PURO的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素，继续培养三天后，存活的细胞即为可调控表达mIL2IL12aGMCSFIL12b的细胞，此细胞命名为B16(rtTA)-mIL2IL12aGMCSFIL12b。
7.3诱导表达mIL2IL12aGMCSFIL12b对肿瘤生长的影响
将对数生长期的B16(rtTA)-mIL2IL12aGMCSFIL12b细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况以及肿瘤清除率，肿瘤清除小鼠数/总的小鼠数＝10/10，如图7所示，mIL2IL12aGMCSFIL12b能够在全部小鼠中诱导肿瘤消退。
实施例8诱导表达mIL12aGMCSFIL12bIL2对肿瘤生长的影响
8.1构建mIL12aGMCSFIL12bIL2可调控表达载体
合成mIL12aGMCSFIL12bIL2基因编码序列，两端分别带有BamHI、BglII和XhoI、EcoRI酶切位点，然后用BamHI或BglII和XhoI或EcoRI进行酶切，酶切体系如下：5μg mIL12aGMCSFIL12bIL2质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电 泳后回收mIL12aGMCSFIL12bIL2基因片段，待用。
mIL12aGMCSFIL12bIL2蛋白质异二聚体的氨基酸序列如SEQ ID NO.22所示，编码所述mIL12aGMCSFIL12bIL2的核苷酸序列是SEQ ID NO.42。
酶切可调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：2μg pLentis-PTRE-MCS-PGK-PURO质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和mIL12aGMCSFIL12bIL2，连接体系如下：2μl pLentis-PTRE-MCS-PGK-PURO、2μl mIL12aGMCSFIL12bIL2、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-mIL12aGMCSFIL12bIL2-PGK-PURO构建成功。
8.2制备可调控表达mIL12aGMCSFIL12bIL2的细胞
制备所述mIL12aGMCSFIL12bIL2表达载体的病毒，方法与第一表达载体病毒制备方法一致，得到第二表达载体pLentis-PTRE-mIL12aGMCSFIL12bIL2-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二可调控表达载体pLentis-PTRE-mIL12aGMCSFIL12bIL2-PGK-PURO的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素，继续培养三天后，存活的细胞即为可调控表达mIL12aGMCSFIL12bIL2的细胞，此细胞命名为B16(rtTA)-mIL12aGMCSFIL12bIL2。
8.3诱导表达mIL12aGMCSFIL12bIL2对肿瘤生长的影响
将对数生长期的B16(rtTA)-mIL12aGMCSFIL12bIL2细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况以及肿瘤清除率，肿瘤清除小鼠数/总的小鼠数＝10/10，如图8所示，结果显示mIL12aGMCSFIL12bIL2能够在全部小鼠中诱导肿瘤消退。
实施例9诱导表达hIL12aIL2IL12bGMCSF对肿瘤生长的影响
9.1构建hIL12aIL2IL12bGMCSF可调控表达载体
合成hIL12aIL2IL12bGMCSF基因编码序列，两端分别带有BamHI、BglII和XhoI、EcoRI酶切位点，然后用BamHI或BglII和XhoI或EcoRI进行酶切，酶切体系如下：5μg hIL12aIL2IL12bGMCSF质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收hIL12aIL2IL12bGMCSF基因片段，待用。
hIL12aIL2IL12bGMCSF蛋白质异二聚体的氨基酸序列如SEQ ID NO.30所示，编码所述hIL12aIL2IL12bGMCSF的核苷酸序列如SEQ ID NO.50所示。
酶切可调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：2μg pLentis-PTRE-MCS-PGK-PURO质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和hIL12aIL2IL12bGMCSF，连接体系如下：2μl pLentis-PTRE-MCS-PGK-PURO、2μl hIL12aIL2IL12bGMCSF、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-hIL12aIL2IL12bGMCSF-PGK-PURO构建成功。
9.2制备可调控表达hIL12aIL2IL12bGMCSF的细胞
制备所述hIL12aIL2IL12bGMCSF表达载体的病毒，方法与第一表达载体病毒制备方法一致，得到第二表达载体pLentis-PTRE-hIL12aIL2IL12bGMCSF-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二表达载体pLentis-PTRE-hIL12aIL2IL12bGMCSF-PGK-PURO的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素，继续培养三天后，存活的细胞即为可调控表达hIL12aIL2IL12bGMCSF的细胞，此细胞命名为B16(rtTA)-hIL12aIL2IL12bGMCSF。
9.3诱导表达hIL12aIL2IL12bGMCSF对肿瘤生长的影响
将对数生长期的B16(rtTA)-hIL12aIL2IL12bGMCSF细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况以及肿瘤清除率，如图9所示，hIL12aIL2IL12bGMCSF能够一定程度上抑制小鼠肿瘤的生长。
实施例10诱导表达mIL12aIL7IL12bGMCSF对肿瘤生长的影响
10.1构建mIL12aIL7IL12bGMCSF可调控表达载体
合成mIL12aIL7IL12bGMCSF基因编码序列，两端分别带有BamHI、BglII和XhoI、EcoRI酶切位点，然后用BamHI或BglII和XhoI或EcoRI进行酶切，酶切体系如下：5μg mIL12aIL7IL12bGMCSF质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12aIL7IL12bGMCSF基因片段，待用。
mIL12aIL7IL12bGMCSF蛋白质异二聚体的氨基酸序列如SEQ ID NO.23所示，编码所述mIL12aIL7IL12bGMCSF的核苷酸序列如SEQ ID NO.43所示。
酶切可调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：2μg pLentis-PTRE-MCS-PGK-PURO载体质粒、3μl酶切缓冲液、1μl BamHI、1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和mIL12aIL7IL12bGMCSF，连接体系如下：2μl pLentis-PTRE-MCS-PGK-PURO、2μl mIL12aIL7IL12bGMCSF、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-mIL12aIL7IL12bGMCSF-PGK-PURO构建成功。
10.2制备可调控表达mIL12aIL7IL12bGMCSF的细胞
制备所述mIL12aIL7IL12bGMCSF表达载体的病毒，方法与第一表达载体病毒制备方法一致，得到第二表达载体pLentis-PTRE-mIL12aIL7IL12bGMCSF-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二可调控表达载体pLentis-PTRE-mIL12aIL7IL12bGMCSF-PGK-PURO的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素，继续培养三天后，存活的细胞即为可调控表达mIL12aIL7IL12bGMCSF的细胞，此细胞命名为B16(rtTA)-mIL12aIL7IL12bGMCSF。
10.3诱导表达mIL12aIL7IL12bGMCSF对肿瘤生长的影响
将对数生长期的B16(rtTA)-mIL12aIL7IL12bGMCSF细胞消化，用HBSS稀释到2×10 6 个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况以及肿瘤清除率，肿瘤清除小鼠数/总的小鼠数＝10/10，如图10所示，mIL12aIL7IL12bGMCSF能够在全部小鼠中诱导肿瘤消退。
实施例11诱导表达mIL12aIL15IL12bGMCSF对肿瘤生长的影响
11.1构建mIL12aIL15IL12bGMCSF可调控表达载体
合成mIL12aIL15IL12bGMCSF基因编码序列，两端分别带有BamHI、BglII和XhoI、EcoRI酶切位点，然后用BamHI或BglII和XhoI或EcoRI进行酶切，酶切体系如下：5μg mIL12aIL15IL12bGMCSF质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12aIL15IL12bGMCSF基因片段，待用。
mIL12aIL15IL12bGMCSF蛋白质异二聚体的氨基酸序列如SEQ ID NO.24所示，编码所述mIL12aIL15IL12bGMCSF的核苷酸序列如SEQ ID NO.44所示。
酶切可调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：2μg pLentis-PTRE-MCS-PGK-PURO载体质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和mIL12aIL15IL12bGMCSF，连接体系如下：2μl pLentis-PTRE-MCS-PGK-PURO、2μl mIL12aIL15IL12bGMCSF、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-mIL12aIL15IL12bGMCSF-PGK-PURO构建成功。
11.2制备可调控表达mIL12aIL15IL12bGMCSF的细胞
制备所述mIL12aIL15IL12bGMCSF表达载体的病毒，方法与第一表达载体病毒制备方法一致，得到第二表达载体pLentis-PTRE-mIL12aIL15IL12bGMCSF-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二可调控表达载体pLentis-PTRE-mIL12aIL15IL12bGMCSF-PGK-PURO的病毒，在培养箱内继续培养24小时后， 弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素，继续培养三天后，存活的细胞即为可调控表达mIL12aIL15IL12bGMCSF的细胞，此细胞命名为B16(rtTA)-mIL12aIL15IL12bGMCSF。
11.3诱导表达mIL12aIL15IL12bGMCSF对肿瘤生长的影响
将对数生长期的B16(rtTA)-mIL12aIL15IL12bGMCSF细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况以及肿瘤清除率，肿瘤清除小鼠数/总的小鼠数＝8/10，如图11所示，mIL12aIL15IL12bGMCSF能够在部分小鼠中诱导肿瘤消退。
实施例12诱导表达mIL12aIL21IL12bGMCSF对肿瘤生长的影响
12.1构建mIL12aIL21IL12bGMCSF可调控表达载体
合成mIL12aIL21IL12bGMCSF基因编码序列，两端分别带有BamHI、BglII和XhoI、EcoRI酶切位点，然后用BamHI或BglII和XhoI或EcoRI进行酶切，酶切体系如下：5μg mIL12aIL21IL12bGMCSF质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12aIL21IL12bGMCSF基因片段，待用。
mIL12aIL21IL12bGMCSF蛋白质异二聚体的氨基酸序列如SEQ ID NO.25所示，编码所述mIL12aIL21IL12bGMCSF的核苷酸序列如SEQ ID NO.45所示。
酶切可调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：2μg pLentis-PTRE-MCS-PGK-PURO载体质粒、3μl酶切缓冲液、1μl BamHI、1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和mIL12aIL21IL12bGMCSF，连接体系如下：2μl pLentis-PTRE-MCS-PGK-PURO、2μl mIL12aIL21IL12bGMCSF、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-mIL12aIL21IL12bGMCSF-PGK-PURO构建成功。
12.2制备可调控表达mIL12aIL21IL12bGMCSF的细胞
制备所述mIL12aIL21IL12bGMCSF表达载体的病毒，方法与第一表达载体病毒制备方法一致，得到第二表达载体pLentis-PTRE-mIL12aIL21IL12bGMCSF-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二表达载体pLentis-PTRE-mIL12aIL21IL12bGMCSF-PGK-PURO的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素，继续培养三天后，存活的细胞即为可调控表达mIL12aIL21IL12bGMCSF的细胞，此细胞命名为B16(rtTA)-mIL12aIL21IL12bGMCSF。
12.3诱导表达mIL12aIL21IL12bGMCSF对肿瘤生长的影响
将对数生长期的B16(rtTA)-mIL12aIL21IL12bGMCSF细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况以及肿瘤清除率，肿瘤清除小鼠数/总的小鼠数＝10/10，如图12所示，mIL12aIL21IL12bGMCSF能够在全部小鼠中诱导肿瘤消退。
实施例13诱导表达mIL12aIL2ILI2bFLT3L对肿瘤生长的影响
13.1构建mIL12aIL2IL12bFLT3L可调控表达载体
合成mIL12aIL2IL12bFLT3L基因编码序列，两端分别带有BamHI、BglII和XhoI、EcoRI酶切位点，然后用BamHI或BglII和XhoI或EcoRI进行酶切，酶切体系如下：5μg mIL12aIL2IL12bFLT3L质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12aIL2IL12bFLT3L基因片段，待用。
mIL12aIL2IL12bFLT3L蛋白质异二聚体的氨基酸序列如SEQ ID NO.26所示，编码所述mIL12aIL2IL12bFLT3L的核苷酸序列如SEQ ID NO.46所示。
酶切可调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：2μg pLentis-PTRE-MCS-PGK-PURO载体质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和mIL12aIL2IL12bFLT3L，连接体系如下：2μl pLentis-PTRE-MCS-PGK-PURO、2μl mIL12aIL2IL12bFLT3L、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二 天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-mIL12aIL2IL12bFLT3L-PGK-PURO构建成功。
13.2制备可调控表达mIL12aIL2IL12bFLT3L的细胞
制备所述mIL12aIL2IL12bFLT3L表达载体的病毒，方法与第一表达载体病毒制备方法一致，得到第二表达载体pLentis-PTRE-mIL12aIL2IL12bFLT3L-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二可调控表达载体pLentis-PTRE-mIL12aIL2IL12bFLT3L-PGK-PURO的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素，继续培养三天后，存活的细胞即为可调控表达mIL12aIL2IL12bFLT3L的细胞，此细胞命名为B16(rtTA)-mIL12aIL2IL12bFLT3L。
13.3诱导表达mIL12aIL2IL12bFLT3L对肿瘤生长的影响
将对数生长期的B16(rtTA)-mIL12aIL2IL12bFLT3L细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况以及肿瘤清除率，肿瘤清除小鼠数/总的小鼠数＝9/10，如图13所示，mIL12aIL2IL12bFLT3L能够在部分小鼠中诱导肿瘤消退。
实施例14诱导表达mIL12aIL7IL12bFLT3L对肿瘤生长的影响
14.1构建mIL12aIL7IL12bFLT3L可调控表达载体
合成mIL12aIL7IL12bFLT3L基因编码序列，两端分别带有BamHI、BglII和XhoI、EcoRI酶切位点，然后用BamHI或BglII和XhoI或EcoRI进行酶切，酶切体系如下：5μg mIL12aIL7IL12bFLT3L质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12aIL7IL12bFLT3L基因片段，待用。
mIL12aIL7IL12bFLT3L蛋白质异二聚体的氨基酸序列如SEQ ID NO.27所示，编码所述mIL12aIL7IL12bFLT3L的核苷酸序列如SEQ ID NO.47所示。
酶切可调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：2μgpLentis-PTRE-MCS-PGK-PURO质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进 行电泳，电泳后回收pLentis-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和mIL12aIL7IL12bFLT3L，连接体系如下：2μl pLentis-PTRE-MCS-PGK-PURO、2μl mIL12aIL7IL12bFLT3L、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-mIL12aIL7IL12bFLT3L-PGK-PURO构建成功。
14.2制备可调控表达mIL12aIL7IL12bFLT3L的细胞
制备所述mIL12aIL7IL12bFLT3L表达载体的病毒，方法与第一表达载体病毒制备方法一致，得到第二表达载体pLentis-PTRE-mIL12aIL7IL12bFLT3L-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二表达载体pLentis-PTRE-mIL12aIL7IL12bFLT3L-PGK-PURO的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素，继续培养三天后，存活的细胞即为可调控表达mIL12aIL7IL12bFLT3L的细胞，此细胞命名为B16(rtTA)-mIL12aIL7IL12bFLT3L。
14.3诱导表达mIL12aIL7IL12bFLT3L对肿瘤生长的影响
将对数生长期的B16(rtTA)-mIL12aIL7IL12bFLT3L细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况以及肿瘤清除率，肿瘤清除小鼠数/总的小鼠数＝9/10，如图14所示，mIL12aIL7IL12bFLT3L能够在部分小鼠中诱导肿瘤消退。
实施例15诱导表达mIL12aIL15IL12bFLT3L对肿瘤生长的影响
15.1构建mIL12aIL15IL12bFLT3L可调控表达载体
合成mIL12aIL15IL12bFLT3L基因编码序列，两端分别带有BamHI、BglII和XhoI、EcoRI酶切位点，然后用BamHI或BglII和XhoI或EcoRI进行酶切，酶切体系如下：5μg mIL12aIL15IL12bFLT3L质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12aIL15IL12bFLT3L基因片段，待用。
mIL12aIL15IL12bFLT3L蛋白质异二聚体的氨基酸序列如SEQ ID NO.28所示，编码所述 mIL12aIL15IL12bFLT3L的核苷酸序列如SEQ ID NO.48所示。
酶切可调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：2μgpLentis-PTRE-MCS-PGK-PURO载体质粒、3μl酶切缓冲液、1μl BamHI、1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl 10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和mIL12aIL15IL12bFLT3L，连接体系如下：2μl pLentis-PTRE-MCS-PGK-PURO、2μl mIL12aIL15IL12bFLT3L、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-mIL12aIL15IL12bFLT3L-PGK-PURO构建成功。
15.2制备可调控表达mIL12aIL15IL12bFLT3L的细胞
制备所述mIL12aIL15IL12bFLT3L表达载体的病毒，方法与第一表达载体病毒制备方法一致，得到第二表达载体pLentis-PTRE-mIL12aIL15IL12bFLT3L-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二表达载体pLentis-PTRE-mIL12aIL15IL12bFLT3L-PGK-PURO的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素，继续培养三天后，存活的细胞即为可调控表达mIL12aIL15IL12bFLT3L的细胞，此细胞命名为B16(rtTA)-mIL12aIL15IL12bFLT3L。
15.3诱导表达mIL12aIL15IL12bFLT3L对肿瘤生长的影响
将对数生长期的B16(rtTA)-mIL12aIL15IL12bFLT3L细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况以及肿瘤清除率，肿瘤清除小鼠数/总的小鼠数＝7/10，如图15所示，mIL12aIL15IL12bFLT3L能够在部分小鼠中诱导肿瘤消退。
实施例16诱导表达mIL12aIL21IL12bFLT3L对肿瘤生长的影响
16.1构建mIL12aIL21IL12bFLT3L可调控表达载体
合成mIL12aIL21IL12bFLT3L基因编码序列，两端分别带有BamHI、BglII和XhoI、EcoRI酶切位点，然后用BamHI或BglII和XhoI或EcoRI进行酶切，酶切体系如下：5μg  mIL12aIL21IL12bFLT3L质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收mIL12aIL21IL12bFLT3L基因片段，待用。
mIL12aIL21IL12bFLT3L蛋白质异二聚体的氨基酸序列如SEQ ID NO.29所示，编码所述mIL12aIL21IL12bFLT3L的核苷酸序列如SEQ ID NO.49所示。
酶切可调控表达载体pLentis-PTRE-MCS-PGK-PURO，酶切体系如下：2μgpLentis-PTRE-MCS-PGK-PURO质粒、3μl酶切缓冲液、1μl BamHI、1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-PTRE-MCS-PGK-PURO载体片段，待用。
连接pLentis-PTRE-MCS-PGK-PURO和mIL12aIL21IL12bFLT3L，连接体系如下：2μl pLentis-PTRE-MCS-PGK-PURO、2μl mIL12aIL21IL12bFLT3L、1μl连接酶缓冲液、0.5μl T4DNA连接酶和4.5μl水。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37℃摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体送测序，确定第二表达载体pLentis-PTRE-mIL12aIL21IL12bFLT3L-PGK-PURO构建成功。
16.2制备可调控表达mIL12aIL21IL12bFLT3L的细胞
制备所述mIL12aIL21IL12bFLT3L表达载体的病毒，方法与第一表达载体病毒制备方法一致，得到第二表达载体pLentis-PTRE-mIL12aIL21IL12bFLT3L-PGK-PURO的病毒。
消化培养的B16(rtTA)肿瘤细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml，24小时后，加入10μl上述第二可调控表达载体pLentis-PTRE-mIL12aIL21IL12bFLT3L-PGK-PURO的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养，待细胞长满后，将其传出到培养瓶中，按终浓度3μg/ml加入嘌呤霉素，继续培养三天后，存活的细胞即为可调控表达mIL12aIL21IL12bFLT3L的细胞，此细胞命名为B16(rtTA)-mIL12aIL21IL12bFLT3L。
16.3诱导表达mIL12aIL21IL12bFLT3L对肿瘤生长的影响
将对数生长期的B16(rtTA)-mIL12aIL21IL12bFLT3L细胞消化，用HBSS稀释到2×10 6个/ml，使用1ml注射器按50μl/只注射到8-10周龄的C57BL/6雌性小鼠的右背侧，共10只，待肿瘤长出后使用含2g/L强力霉素的水喂养，记录小鼠肿瘤生长情况以及肿瘤清除率，肿瘤清除小鼠数/总的小鼠数＝9/10，如图16所示，mIL12aIL21IL12bFLT3L能够在部分小鼠中诱导肿瘤消退。
实施例17构建表达人hIL12aIL2IL12bGMCSF、人hIL12aIL7IL12bGMCSF、人hIL12aIL15IL12bGMCSF、人hIL12aIL21IL12bGMCSF、人hIL12aIL2IL12bFLT3L、人hIL12aIL7IL12bFLT3L、人hIL12aIL15IL12bFLT3L、人hIL12aIL21IL12bFLT3L的细胞
17.1构建可调控表达目的基因的载体
在EP管内酶切载体pLentis-CMV-MCS-IRES-PURO，体系如下：2μg pLentis-CMV-MCS-IRES-PURO载体质粒、3μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积30μl，37℃静置12小时。取出EP管，加入3.3μl10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收pLentis-CMV-MCS-IRES-PURO载体片段，待用。
分别合成人hIL12aIL2IL12bGMCSF、人hIL12aIL7IL12bGMCSF、人hIL12aIL15IL12bGMCSF、人hIL12aIL21IL12bGMCSF、人hIL12aIL2IL12bFLT3L、人hIL12aIL7IL12bFLT3L、人hIL12aIL15IL12bFLT3L和人hIL12aIL21IL12bFLT3L的DNA序列，合成时在其5’端加上BamHI或BglII酶切位点，在其3’端加上XhoI或EcoRI酶切位点。酶切合成的带有目的基因的质粒，体系如下：5μg质粒、4μl酶切缓冲液、1μl BamHI和1μl XhoI，加水至总体积40μl，37℃静置12小时。取出EP管，加入4.4μl10×上样缓冲液，用1％琼脂糖凝胶进行电泳，电泳后回收片段，待用。
hIL12aIL2IL12bGMCSF蛋白质异二聚体的氨基酸序列如SEQ ID NO.30所示，编码所述hIL12aIL2IL12bGMCSF的核苷酸序列如SEQ ID NO.50所示。
hIL12aIL7IL12bGMCSF蛋白质异二聚体的氨基酸序列如SEQ ID NO.31所示，编码所述hIL12aIL7IL12bGMCSF的核苷酸序列如SEQ ID NO.51所示。
hIL12aIL15IL12bGMCSF蛋白质异二聚体的氨基酸序列如SEQ ID NO.32所示，编码所述hIL12aIL15IL12bGMCSF的核苷酸序列如SEQ ID NO.52所示。
hIL12aIL21IL12bGMCSF蛋白质异二聚体的氨基酸序列如SEQ ID NO.33所示，编码所述hIL12aIL21IL12bGMCSF的核苷酸序列如SEQ ID NO.53所示。
hIL12aIL2IL12bFLT3L蛋白质异二聚体的氨基酸序列如SEQ ID NO.34所示，编码所述hIL12aIL2IL12bFLT3L的核苷酸序列如SEQ ID NO.54所示。
hIL12aIL7IL12bFLT3L蛋白质异二聚体的氨基酸序列如SEQ ID NO.35所示，编码所述hIL12aIL7IL12bFLT3L的核苷酸序列如SEQ ID NO.55所示。
hIL12aIL15IL12bFLT3L蛋白质异二聚体的氨基酸序列如SEQ ID NO.36所示，编码所述hIL12aIL15IL12bFLT3L的核苷酸序列如SEQ ID NO.56所示。
hIL12aIL21IL12bFLT3L蛋白质异二聚体的氨基酸序列如SEQ ID NO.37所示，编码所述hIL12aIL21IL12bFLT3L的核苷酸序列如SEQ ID NO.57所示。
分别连接pLentis-CMV-MCS-IRES-PURO和人hIL12aIL2IL12bGMCSF、人hIL12aIL7IL12bGMCSF、人hIL12aIL15IL12bGMCSF、人hIL12aIL21IL12bGMCSF、人hIL12aIL2IL12bFLT3L、人hIL12aIL7IL12bFLT3L、人hIL12aIL15IL12bFLT3L和人hIL12aIL21IL12bFLT3L，体系如下：2μl pLentis-CMV-MCS-IRES-PURO载体片段、2μl基因片段、1μl连接酶缓冲液、0.5μl T4 DNA连接酶和水4.5μl。置于室温连接4小时。然后将连接体系进行大肠杆菌感受态的转化。第二天从转化的平板上挑取菌落，置于LB培养基中37度摇床内过夜培养，使用质粒提取试剂盒从培养的细菌中提取质粒，通过酶切鉴定片段是否成功连入载体中，然后将正确的载体测序，确定构建成功。获得表达目的基因的载体pLentis-CMV-hIL12aIL2IL12bGMCSF-IRES-PURO、载体pLentis-CMV-hIL12aIL7IL12bGMCSF-IRES-PURO、载体pLentis-CMV-hIL12aIL15IL12bGMCSF-IRES-PURO、载体pLentis-CMV-hIL12aIL21IL12bGMCSF-IRES-PURO、载体pLentis-CMV-hIL12aIL2IL12bFLT3L-IRES-PURO、载体pLentis-CMV-hIL12aIL7IL12bFLT3L-IRES-PURO、载体pLentis-CMV-hIL12aIL15IL12bFLT3L-IRES-PURO和载体pLentis-CMV-hIL12aIL21IL12bFLT3L-IRES-PURO。
17.2制备可调控表达目的基因的细胞
1)消化培养的293FT细胞，计数后将3×10 6个细胞/孔铺入10cm培养皿中，培养液体积为10ml，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+表达载体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。
17.3制备可调控表达目的基因的细胞
消化培养的293A细胞，按10 5个细胞/孔接种到6孔板中，培养体积为1ml。24小时后，加入10μl表达上述目的基因的病毒，在培养箱内继续培养24小时后，弃去上清，换为新鲜的培养基继续培养。待细胞长满后，将其传出到培养瓶中，加入终浓度3μg/ml嘌呤霉素， 继续培养，每两天更换一次培养基，并保持嘌呤霉素的浓度，筛选一周后，存活的细胞即为稳定表达所述细胞因子的细胞，分别命名为293A-hIL12aIL2IL12bGMCSF、293A-hIL12aIL7IL12bGMCSF、293A-hIL12aIL15IL12bGMCSF、293A-hIL12aIL21IL12bGMCSF、293A-hIL12aIL2IL12bFLT3L、293A-hIL12aIL7IL12bFLT3L、293A-hIL12aIL15IL12bFLT3L、293A-hIL12aIL21IL12bFLT3L。
将构建的表达细胞，按5×10 4个每孔铺入到24孔板中，培养96小时后，收集上清，用人IL12p70 ELISA试剂盒检测上清中融合蛋白的表达，操作按说明书进行。如图17所示，这些细胞均能够产生大量的IL12p70成功构建了表达融合蛋白的细胞。
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 (19)

  1. 蛋白质异二聚体，其包含第一多肽链以及不同于所述第一多肽链的第二多肽链，其中所述第一多肽链包含IL12a以及与所述IL12a融合的第一因子，所述第二多肽链包含IL12b以及与所述IL12b融合的第二因子，且其中所述第一因子及所述第二因子各自独立地选自下组：IL2、GMCSF、IL7、IL15、IL21和FLT3L。
  2. 根据权利要求1所述的蛋白质异二聚体，其中所述第一因子与所述第二因子不同。
  3. 根据权利要求1-2中任一项所述的蛋白质异二聚体，其中，所述第一因子和所述第二因子彼此不同地分别选自细胞因子组A和细胞因子组B，所述细胞因子组A选自下组：IL2、IL7、IL15和IL21，且细胞因子组B选自下组：GMCSF和FLT3L。
  4. 根据权利要求1-3中任一项所述的蛋白质异二聚体，其中，在所述第一多肽链中，所述IL12a的C末端与所述第一因子的N末端直接或间接融合；和/或，在所述第一多肽链中，所述IL12a的N末端与所述第一因子的C末端直接或间接融合。
  5. 根据权利要求1-4中任一项所述的蛋白质异二聚体，其中，在所述第二多肽链中，所述IL12b的C末端与所述第二因子的N末端直接或间接融合；和/或，在所述第二多肽链中，所述IL12b的N末端与所述第二因子的C末端直接或间接融合。
  6. 根据权利要求1-5中任一项所述的蛋白质异二聚体，其中，所述第一因子和所述第二因子选自下组：
    1)第一因子为IL2，且第二因子为GMCSF；
    2)第一因子为IL7，且第二因子为GMCSF；
    3)第一因子为IL15，且第二因子为GMCSF；
    4)第一因子为IL21，且第二因子为GMCSF；
    5)第一因子为IL2，且第二因子为FLT3L；
    6)第一因子为IL7，且第二因子为FLT3L；
    7)第一因子为IL15，且第二因子为FLT3L；
    8)第一因子为IL21，且第二因子为FLT3L；
    9)第一因子为GMCSF，且第二因子为IL2；
    10)第一因子为GMCSF，且第二因子为IL7；
    11)第一因子为GMCSF，且第二因子为IL15；
    12)第一因子为GMCSF，且第二因子为IL21；
    13)第一因子为FLT3L，且第二因子为IL2；
    14)第一因子为FLT3L，且第二因子为IL7；
    15)第一因子为FLT3L，且第二因子为IL15；以及，
    16)第一因子为FLT3L，且第二因子为IL21。
  7. 根据权利要求1-6中任一项所述的蛋白质异二聚体，其中所述IL12a、所述IL12b、所述第一因子和所述第二因子来源自哺乳动物。
  8. 根据权利要求7所述的蛋白质异二聚体，其中所述IL12a、所述IL12b、所述第一因子和所述第二因子的来源相同。
  9. 根据权利要求1-8中任一项所述的蛋白质异二聚体，其中所述第一多肽链包括如SEQ ID NO.60-65和SEQ ID NO.70-73中任一项所示的序列。
  10. 根据权利要求1-9中任一项所述的蛋白质异二聚体，其中所述第二多肽链包括如SEQ ID NO.66-69和SEQ ID NO.74-75中任一项所示的序列。
  11. 根据权利要求1-10中任一项所述的蛋白质异二聚体，其中所述蛋白质异二聚体包括如SEQ ID NO.18-37中任一项所示的序列。
  12. 一种或多种分离的核酸分子，其编码根据权利要求1-11中任一项所述的蛋白质异二聚体。
  13. 根据权利要求12所述的核酸分子，其包括如SEQ ID NO.38-57中任一项所示的序列。
  14. 表达载体，其包括编码根据权利要求1-11中任一项所述的蛋白质异二聚体的核苷酸序列。
  15. 宿主细胞，其包括根据权利要求14所述的表达载体。
  16. 药物组合物，其包括根据权利要求1-11中任一项所述的蛋白质异二聚体、根据权利要求11-13所述的核酸分子、根据权利要求14所述的载体和/或根据权利要求15所述的宿主细胞，以及药学上可接受的载体。
  17. 制备根据权利要求1-11中任一项所述的蛋白质异二聚体的方法，其包括以下的步骤：在能够表达根据权利要求1-11中任一项所述的蛋白质异二聚体的情况下，培养根据权利要求15所述的宿主细胞。
  18. 根据权利要求1-11中任一项所述的蛋白质异二聚体和根据权利要求16所述的药物组合物在制备治疗肿瘤药物中的应用。
  19. 根据权利要求18所述的应用，其中所述肿瘤包括黑色素瘤。
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