WO2010099637A1 - 增殖抗原特异性t细胞的方法 - Google Patents

增殖抗原特异性t细胞的方法 Download PDF

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WO2010099637A1
WO2010099637A1 PCT/CN2009/000481 CN2009000481W WO2010099637A1 WO 2010099637 A1 WO2010099637 A1 WO 2010099637A1 CN 2009000481 W CN2009000481 W CN 2009000481W WO 2010099637 A1 WO2010099637 A1 WO 2010099637A1
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antigen
cells
immunogen
specific
cell
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高斌
丁洁
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中国科学院微生物研究所
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    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A61K39/464492—Glycoprotein 100 [Gp100]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a method for amplifying proliferation of antigen-specific T cells.
  • immunotherapy As an effective treatment, immunotherapy has been widely used in recent years. Among them, cellular immunotherapy is widely used in the treatment of cancer and has a surprising effect.
  • immune cells capable of killing abnormal cells are difficult to produce in sufficient quantities.
  • amplification of immune cells is usually achieved by antibody stimulation and addition of cytokine culture.
  • amplification of T cells is usually achieved by anti-CD3 antibody plus interleukin-2.
  • this method can only non-specifically amplify immune cells, and the number of effective target cells obtained is small and cannot meet the needs.
  • Another method is to perform selective amplification by repeatedly stimulating immune cells with antigen-presenting cells (such as dendritic cells, B cells, etc.) that bind to the antigen of interest, to obtain a large number of antigen-specific immune cells, but this
  • antigen-presenting cells such as dendritic cells, B cells, etc.
  • the method is suitable for the amplification of specific immune cells by antigens with strong immunogenicity, and the weak immunogenic antigen is difficult to stimulate the immune cells to multiply and specifically proliferate, which cannot meet the therapeutic needs. Therefore, there is a need for a method for efficiently producing a large number of antigen-specific immune cells.
  • the method for proliferating antigen-specific tau cells comprises the steps of: transfecting a gene encoding an immunological recognition molecule of a polypeptide-MHC molecule complex of a target antigen B into a polypeptide-MHC molecule complex against immunogen A Specific T cells of the substance, obtain a double-antibody-specific T cell which recognizes the polypeptide-MHC molecule complex of the antigen B of interest and the polypeptide-MHC molecule complex of the immunogen A, thereby obtaining the target antigen B-specific T cell; Progenitor A stimulates proliferation to amplify the antigen B-specific T cells;
  • the immunogen A is a virus, a bacterium, a polypeptide of 7 amino acids or more and 35 amino acids or less, a chimeric protein, or an allogeneic antigen; the virus may be any one having strong immunogenicity and capable of promptly and effectively stimulating immunity.
  • the cellular immune response, the induction of immune cell proliferation, and the immune response mechanism are relatively well studied, and viruses that are not highly pathogenic to mammalian cells may specifically be influenza virus, Epstein-Barr virus, CMV virus, and the like.
  • the immunological recognition molecule recognizing the antigen B polypeptide-MHC molecular complex of the target antigen includes a T cell antigen receptor (TCR), a T cell antigen receptor (TCR)-like antibody, and a killing activated receptor that binds to the MHC molecule on the surface of the natural killer cell. (KAR) or a killer-inhibiting receptor (KIR) that binds to MHC molecules on the surface of natural killer cells.
  • Another object of the invention is to provide an antigen-specific T cell.
  • the antigen-specific tau cells provided by the present invention are prepared according to the method comprising the following steps:
  • the gene encoding the immunoreactive molecule of the polypeptide-MHC molecule complex of the antigen B of interest is transferred to a specific T cell of the polypeptide-MHC molecule complex against immunogen A, and a polypeptide-MHC molecule complex for identifying the antigen B of interest is obtained.
  • the bispecific antibody-specific T cells of the polypeptide and the MHC molecule complex of immunogen A, that is, the target antigen B-specific T cells are obtained;
  • the immunogen A is a virus, a bacterium, a polypeptide of 7 amino acids or more and 35 amino acids or less, a chimeric protein, or an allogeneic antigen; the virus may be any one having strong immunogenicity and capable of promptly and effectively stimulating immunity.
  • the immunological recognition molecules of the molecular complex include a T cell antigen receptor, a tau cell antigen receptor-like antibody, a killer-activated receptor that binds to the surface of the natural killer cell and the C-molecule, or a killer inhibitory receptor that binds to the MHC molecule on the surface of the natural killer cell. .
  • the specific T cell of the polypeptide-MHC molecule complex against immunogen A is obtained by immunizing an animal with the immunogen A.
  • the method for stimulating proliferation may be as follows 1), 2) or 3) :
  • the method for stimulating proliferation comprises the steps of: introducing the antigen B-specific T cells into an animal, immunizing with the immunogen A multiple times, and proliferating and amplifying the antigen B-specific T cells in vivo;
  • the method for stimulating proliferation comprises the steps of: co-culturing the antigen B-specific T cells with the immunogen A, and proliferating and amplifying the antigen B-specific T cells in vitro;
  • the method for stimulating proliferation comprises the steps of: co-culturing the antigen B-specific T cells with a feeder cell that binds to the immunogen A, and allowing the antigen B-specific T cells to proliferate in vitro.
  • the transfection is effected by a viral vector, liposome, cationic polymer, or electroporation; the viral vector is a retroviral vector, a lentiviral vector or an adenovirus-associated vector.
  • the method of transferring is as follows 1), 2), 3) or 4):
  • the recombinant virus comprises the immunological recognition score of the target antigen B polypeptide-MHC molecular complex The coding gene of the child.
  • the antigen B is a tumor differentiation antigen.
  • the tumor can be a melanoma.
  • the antigen B may specifically be a melanoma differentiation antigen gpl00.
  • the immunological recognition molecule which recognizes the polypeptide of the antigen B of interest - the MHC molecular complex is a receptor protein against the melanoma differentiation antigen gplOO.
  • the gene encoding the receptor protein of the anti-melanoma differentiation antigen gplOO includes fragment 1 and fragment 2.
  • the nucleotide sequence of fragment 1 is shown in sequence 1 of the sequence listing, and the nucleotide sequence of fragment 2 is as in the sequence of the sequence listing. 2 is shown.
  • the feeder cells of any of the above-described immunogen A binding are prepared according to the method comprising the steps of: co-cultivating the immunogen A with the feeder cells, and then treating the cells with mitomycin to obtain the binding immunity.
  • the original A word cell is prepared according to the method comprising the steps of: co-cultivating the immunogen A with the feeder cells, and then treating the cells with mitomycin to obtain the binding immunity.
  • the original A word cell is prepared according to the method comprising the steps of: co-cultivating the immunogen A with the feeder cells, and then treating the cells with mitomycin to obtain the binding immunity.
  • the original A word cell is prepared according to the method comprising the steps of: co-cultivating the immunogen A with the feeder cells, and then treating the cells with mitomycin to obtain the binding immunity.
  • any of the antigen-specific T cells described above in the treatment of diseases is also within the scope of protection of the present invention.
  • the treatment may specifically be an immunological input treatment.
  • the principle of the present invention is to stimulate T cells with immunogen A to obtain specific T cells against the immunogen A polypeptide-MHC (major histocompatibility complex) molecular complex; and to recognize the target antigen B polypeptide - MHC molecular complex a gene encoding an immunological recognition molecule, transferred into the immunogen A-specific T cell, and obtained a double-antibody-specific T cell that is transferred to an immunological recognition molecule that recognizes the antigen B polypeptide-MHC complex; using immunogen A Proliferation amplifies the above-mentioned double-antibody-specific T cells.
  • the method of the invention is suitable for the proliferation of specific T cells with strong immunogenic or weak immunogenic antigens, and is more suitable for the proliferation of specific T cells of weak immunogenic antigens, such as the specificity of tumors with weak immunogenicity. More specifically, the cells may be melanoma antigen-specific T cells.
  • the double-antibody-specific T cells obtained by the method of the present invention and which are recognized by the immunoreactive molecule of the target antigen B polypeptide-Gu C complex are induced by the immunogen A by using the specificity against the immunogen A, and the proliferation thereof is greatly proliferated.
  • the invention overcomes the defects that the weak immunogenic target antigen cannot induce the proliferation of the specific T cells in vitro, and the amplification effect is good, and the amplified immune cells have high killing rate to the target antigen; in addition, the proliferation method of the invention is simple in operation, low cost. Therefore, the proliferation method of the present invention is suitable for mass production of a plurality of antigen-specific immune cells of interest for use in immunological input therapy for diseases such as various tumors.
  • Figure 1 is a screening of a packaging cell line producing retroviruses.
  • Figure 2 shows the results of tetramer staining of gplOO peptide or influenza virus MP peptide of bispecific T cells.
  • Figure 3 shows the results of intracellular cytokine staining of gplOO peptide or influenza virus MP peptide of bispecific T cells.
  • Figure 4 shows the proliferation results of bispecific T cells after co-culture with different stimuli in vitro.
  • Figure 5 shows the results of proliferation of bispecific T cells in HLA-A2/Kb transgenic mice.
  • Figure 6 shows the results of in vitro killing of target cells and tumor cells that bind tumor antigens by bispecific T cells.
  • Figure 7 is a graph showing the results of bispecific T cells killing target cells that bind tumor antigens in HLA-A2/Kb transgenic mice.
  • Figure 8 shows the results of melanoma assay in bispecific T cells in nude mice.
  • APB plasmid (Morgan, RA, et al., High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens. J Immunol, 2003. 171 (6): p. 3287-95) (Institute of Microbiology, Chinese Academy of Sciences), the APB plasmid contains the gene encoding the melanoma gplOO TCR protein, including fragment 1 and fragment 2.
  • the nucleotide sequence of fragment 1 is shown in sequence 1 of the sequence listing.
  • the nucleotide sequence of fragment 2 is shown in SEQ ID NO: 2 in the sequence listing.
  • influenza virus is influenza A virus PR/8/34. (de Wit, E. , et al. , Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments. Virus Res, 2004. 103 (1-2): p. 155-61. ) ( Institute of Microbiology, Chinese Academy of Sciences).
  • HLA-A2/Kb transgenic mice Zhou, M., et al., Screening and identification of severe acute respiratory syndrome-associated corona virus-specific CTL Epitopes. J Immunol, 2006. 177 (4) : p. 2138-45. ) (Institute of Microbiology, Chinese Academy of Sciences). Interleukin-2 was purchased from Chiron BV, Amsterdam, The Netherlands.
  • Co-cultivation medium RPMI 1640 medium containing 10% (by volume) fetal bovine serum, 2 mM glutamine, 10 OOU/ml penicillin, 100 ug/ml streptomycin, 5 x 1 (T 2 mM 2-mercaptoethanol).
  • the APB plasmid was transfected into Phoenix-Eco cells, and the resulting viral supernatant was infected with PT67 packaging cells.
  • the infected monoclonal PT67 virus supernatants were infected with SupTl human lymphoma cell line, respectively, and the expression of human CD3 was detected. Any infected SupTl human lymphoma cell line was a blank control. This method is used to screen for packaging cell lines that produce the highest virus titer.
  • Fig. 1A The results are shown in Fig. 1A, which indicates that the selected PT67 packaging cell line produced the highest virus titer (i.e., maximally increased the expression of human CD3 on the surface of SupT1 cells), and can be used as a packaging cell line for production in subsequent experiments.
  • Influenza virus-immunized mice HLA-A2/Kb transgenic mice were immunized with influenza virus nasally.
  • mouse spleen lymphocytes After 2 to 4 weeks of immunization, the mouse spleen cells were taken (after each spleen was ground, 2 times through a 200 mesh filter to make a cell suspension), and red blood cells were added to the cell suspension. The lysate lysed the red blood cells, the red blood cell lysed fragments and platelets were removed by centrifugation, and the lymphocytes were collected; after washing 3 times with 1640 cell culture medium containing 10% fetal bovine serum, the lymphocytes were suspended in the co-culture medium.
  • the lymphocytes obtained in the step 2) were inoculated into the co-cultivation medium, and the retrovirus-producing virus (the TCR expressing the anti-melanoma gplOO) was added thereto.
  • PT67 packaging cells PT67 packaging cell with a ratio of the number of lymphocytes was 8 X10 4: 1X10 6) and influenza virus (influenza virus ratio of T cells to 2xl0 6 EID50: 1X10 6 th), at 37 ° C, 5% C0
  • influenza virus influenza virus ratio of T cells to 2xl0 6 EID50: 1X10 6 th
  • the culture was carried out for 5 days under the conditions of 2 , wherein after 24 to 48 hours of culture, interleukin-2 was added to the system (the concentration of interleukin-2 in the system was 50 IU/ml), and then added every other day; Specific T cells (anti-melanoma specific and anti-influenza virus specific).
  • Fig. 1C 1 indicates 0 days, and 2 indicates 2 Days, 3 means 4 days, 4 means 5 days.
  • the suspension cells are aspirated, dead cells are removed, and the influenza virus is stimulated and fed with mitomycin (80 g/ml, 37 ° C, 2 h).
  • mitomycin 80 g/ml, 37 ° C, 2 h.
  • the mouse spleen cells and 50 IU/ml of interleukin-2 were further cultured at 37 ° C, 5% C0 2 for 3-5 days to obtain more bispecific T cells.
  • HLA-A2/Kb transgenic mice were immunized with influenza virus nasally, and mouse spleen lymphocytes were placed in co-culture medium after 2 to 4 weeks, and simultaneously added thereto.
  • Influenza virus, influenza virus concentration in the culture system is 2xlO3 ⁇ 4ID50/ml, co-culture for 5-10 days at 37 ° C, 5% ⁇ 2 ; during the period of 24-48 hours, add 50 IU / ml interleukin - 2, every other day afterwards; get anti-influenza virus-specific T cells.
  • Figure 1A shows the expression of human CD3 on the cell surface after SupT1 infection by the retroviral virus produced by the packaging cell line PT67. Control indicates that there is no SupT1 infected with retrovirus, and APB indicates SupTl infected with retrovirus APB.
  • the abscissa is human CD3 (human leukocyte differentiation antigen -3), which indicates the relative fluorescence intensity value, and the ordinate is the number of cells.
  • Figure 1B shows retrovirus (APB) infection and influenza virus-stimulated mouse T cell surface human TCR
  • T cell antigen receptor The abscissa is human TCR, which represents the relative intensity of green fluorescence, and the ordinate is the number of cells.
  • FIG. 1C shows the expression of human TCR (T cell antigen receptor) on the surface of mouse T cells at different times after retroviral (APB) infection and influenza virus stimulation.
  • the abscissa is human TCR, which represents the relative intensity of green fluorescence, and the ordinate is the number of cells.
  • the bispecific T cells obtained in Example 1 were subjected to the following experiment.
  • FITC-labeled anti-mouse CD8 was purchased from BD, PharMingen, CA, USA;
  • Anti-mouse IFN- ⁇ - ⁇ was purchased from eBioscience, catalog number 12-7311-71;
  • Monacin was purchased from eBioscience, catalog number 00-4505-51;
  • the penetrating buffer was purchased from eBioscience, catalog number 00-8333.
  • Initial T cells Spleen T cells of HLA-A2 transgenic mice that were not immunized were isolated, i.e., primary T cells.
  • bispecific T cells Experiments were carried out with bispecific T cells, anti-influenza virus-specific T cells, and primary T cells as experimental cells.
  • Tetramer staining Double-specific T cells were washed twice with PBS, resuspended in 100 ⁇ l of PBS, added with MP peptide tetramer (PE) or gplOO peptide tetramer (PE), stained at 37 ° C for 20 min and then added. Anti-mouse CD8 mAb was stained for 20 min at room temperature. After washing with PBS for 2 times, it was resuspended in 500 ⁇ l of PBS and counted by an upflow cytometer.
  • PE MP peptide tetramer
  • PE gplOO peptide tetramer
  • Intracellular cytokine staining Inoculate bispecific sputum cells in co-culture medium, and add gplOO peptide to each treatment group (gplOO peptide concentration in culture system is 20 ⁇ M), influenza virus MP peptide (flu) The virus MP peptide was stimulated in the culture system at a concentration of 20 ⁇ M for 1-2 hours, and then monensin was added thereto (the concentration of monensin in the culture system was 5 ⁇ M), at 37 ° C, After 4-5 hours of treatment under 5% C0 2 conditions, paraformaldehyde (1%) was fixed, added to the penetration buffer and resuspended, stained with anti-mouse IFN- ⁇ -PE monoclonal antibody and anti-mouse CD8 monoclonal antibody. After washing with PBS for 2 times, it was resuspended in 500 ⁇ l of PBS and counted by an upflow cytometer. No peptide was used as a control.
  • the experiment was set up twice.
  • Fig. 2 (1 indicates initial sputum cells, 2 indicates specific sputum cells against influenza virus, and 3 indicates bispecific sputum cells).
  • the results showed that 10-15% of the bispecific sputum cells reacted with the MP tetramer, 15-20% reacted with the gplOO tetramer, and the control anti-influenza virus-specific T cells hardly reacted with the gplOO tetramer. , indicating that the bispecific T cells express both the TCR against the melanoma antigen gplOO and the TCR against the influenza virus antigen MP, which is bispecific.
  • the abscissa of Fig. 2A is the mouse leukocyte differentiation antigen - 8, indicating the relative intensity of green fluorescence; the ordinate is the relative intensity of red fluorescence of the MP peptide tetramer (PE).
  • the abscissa is mouse leukocyte differentiation antigen-8, indicating the relative intensity of green fluorescence; and the ordinate is the relative intensity of red fluorescence of gplOO peptide tetramer (PE).
  • Fig. 3A shows the results of intracellular cytokine staining of bispecific T cells
  • Fig. 3B shows the results of intracellular cytokine staining of anti-influenza virus-specific T cells.
  • 1 indicates an influenza virus antigen MP peptide reaction group
  • 2 indicates a reaction group with a melanoma antigen gplOO peptide
  • 3 indicates a control group without a peptide.
  • the abscissa represents the cell surface mouse leukocyte differentiation antigen-8, indicating the relative intensity of green fluorescence; the ordinate is intracellular mouse IFN- Y (PE) red fluorescence relative intensity value.
  • B16 - AAD melanoma cells (Mullins, DW, et al. Route of Immunization with Peptide - pulsed Dendritic Cells Controls the Distribution of Memory and Effector T Cells in Lymphoid Tissues and Determines the Pattern of Regional Tumor Control. J Exp Med, 2003 . 198 (7): p. 1023 - 34. ) (Institute of Microbiology, Chinese Academy of Sciences).
  • Bispecific T cells were stained with green fluorescent dye (CFSE) and different stimuli were added, and interleukin-2 (interleukin-2 in the system at a concentration of 50 IU/ml) was added (added every other day) ), cultured in a co-cultivation medium at 37 ° C, 5% CO 2 for 3 days, and then observed by a flow cytometer for cell proliferation.
  • CFSE green fluorescent dye
  • Influenza virus-feeder cells were added, and the ratio of the number of cells to bispecific T cells was 1:1; the method for obtaining influenza virus-feeder cells: The spleen T of HLA-A2/Kb transgenic mice without any immunization was taken. The cells were inoculated into the co-culture medium, and the influenza virus (influenza virus concentration in the culture system was 2 ⁇ 10 6 EID50/ml) was added thereto for 2 hours, and then mitomycin (the concentration of mitomycin in the culture system) was added. It was 80 g / ml), treated at 37 ° C for 2 h, and washed three times with the medium, and the obtained cells were influenza virus-conjugated feeder cells.
  • influenza virus influenza virus concentration in the culture system was 2 ⁇ 10 6 EID50/ml
  • mitomycin the concentration of mitomycin in the culture system
  • the ratio of the ratio of the bispecific T cells to the mitomycin-treated B16-MD melanoma cells is 0. 5: 1;
  • B16-MD melanoma cells were seeded in a co-cultivation medium, and mitomycin was added thereto (the concentration of mitomycin in the culture system was 200 g/ml), treated at 37 2 for 2 h, washed 3 times with medium to obtain mitomycin-treated B16-MD melanoma cells.
  • the experiment was set to repeat 3 times.
  • Bispecific T cells proliferated significantly after incubation with influenza virus-folending feeder cells and anti-CD3 antibodies, while B16-MD melanoma cells only weakly stimulated proliferation, indicating that tumor antigens are weaker immunogens and are not effective. It stimulates the proliferation of bispecific T cells, while the influenza virus antigen is a strong antigen, which can strongly stimulate the proliferation of bispecific tau cells.
  • A denotes bispecific T cells
  • B denotes anti-influenza virus-specific T cells.
  • 1 indicates that Influenza virus-feeder cell co-culture group
  • 2 indicates co-culture group with B16-AAD melanoma cells
  • 3 indicates that only culture medium was used for co-culture medium
  • 4 indicates co-culture group with anti-CD3 antibody.
  • Bispecific T cells (3x10 6 cells/cell) were intravenously administered to mice, and mice were immunized with influenza virus 24 hours later (intraperitoneal immunization once, 10 7 9 EID 50 ).
  • Mouse spleen cells were taken at different time points to detect gplOO tetramer and CD8 double positive cells, and the expansion of T cells stimulated by influenza virus in mice was observed. The experiment was set to repeat twice.
  • Splenocytes from HLA-A2/Kb transgenic mice were stained with CFSE (10 ⁇ ), and then melanoma antigen ⁇ 100 peptide (1 ( ⁇ 3 ⁇ 41, 37 ° ⁇ , 30 min), influenza virus antigen MP peptide (10) ⁇ ⁇ , 37°C, 30min), influenza virus (2EID 5 ./cell, 37°C, 120min) After stimulation, mixed with effector cells (bispecific T cells) at different target ratios, 37°C, After 4 h, staining was performed with PI (propidium iodide, 10 ⁇ ⁇ / ⁇ 1, Sigma-Aldrich, 81845), and the killing rate was calculated by up-flow cytometry. The experiment was repeated three times.
  • Fig. 6 The results of in vitro killing are shown in Fig. 6. A indicates that the bispecific T cells kill the target cells bound to the specific antigen in vitro, and B indicates that the bispecific T cells kill the tumor cells in vitro.
  • Fig. 6A 1 indicates that the target cell is a homologous mouse spleen cell which binds to the gplOO peptide
  • 2 indicates that the target cell is a homologous mouse spleen cell which binds to the MP peptide
  • 3 indicates that the target cell is a homologous mouse spleen cell which binds influenza virus
  • 4 indicates The target cells are homologous mouse spleen cells without any stimulation.
  • ⁇ indicates that the effector cells are bispecific T cells, and ⁇ indicates that the effector cells are homologous mouse spleen cells (control) that are not antigen-stimulated.
  • Fig. 6B shows the results of killing B16-AAD melanoma cells, ⁇ indicates the result of killing EG7 cells, and ⁇ indicates the result of killing K41 cells.
  • bispecific T cells significantly kill spleen cells of syngeneic mice that bind gplOO peptide, MP peptide or influenza virus without killing unbound mouse spleen cells (Fig. 6A). Moreover, bispecific T cells can significantly kill B16-AAD melanoma cells (Fig. 6 ⁇ ) without killing control tumor target cells (Fig. 6B ⁇ , )).
  • lxlO 7 bispecific T cells were intravenously administered into HLA-A2/Kb transgenic mice, and 2 hours later, mice were immunized with influenza virus (intraperitoneal immunization, 10 7 ' 9 EID 5() ). After 7 days, the target cells were intravenously injected, and 4 hours later, the mouse spleen cells were subjected to flow cytometry to select PKH26-positive cells to observe the killing condition.
  • the target cells were prepared as follows: The spleen cells of HLA-A2/Kb transgenic mice were stained with PKH26 (4 ⁇ ⁇ ) and CFSE (2 ⁇ ⁇ , or 0.2 ⁇ M), respectively, and then CFSE was highly stained (2 ⁇ ⁇ ).
  • the target cell population binds to gplOO or influenza virus; while the CFSE low-stained (0.2 ⁇ ) target cell population is not stimulator as a control.
  • the CFSE high-stained and low-stained target cell population was mixed at a ratio of 1:1 (2.5 x lO 6 cells/group) and the experimental mice were intravenously used as target cells. The experiment was set to repeat 3 times.
  • FIG. 7 The results of in vivo killing are shown in Figure 7.
  • 1 is the experimental control group, which indicates the results of in vivo killing experiments on mice 7 days after injection of PBS; 2 is the experimental group, indicating that mice are injected with bispecific T cells (2A, 2B), or influenza-specific T cells (2C), After 24 hours, the virus was immunized with the influenza virus, and the result of the in vivo killing experiment was performed after 7 days.
  • A, C indicates that the target cell that binds to the gplOO tumor antigen is killed, and B indicates that the target cell that binds to the influenza virus is killed.
  • the control influenza virus-specific T cells were unable to kill target cells that bind gplOO tumor antigen (Fig. 7C).
  • mice Balb/c nu/nu nude mice were purchased from the Experimental Animal Research Center of Peking University Medical School.
  • the double-antibody-specific T cells obtained by the method of the present invention and transferred to the immunological recognition molecule of the antigen B polypeptide-MHC complex of the target antigen are induced by the immunogen A by using the specificity against the immunogen A, and the cells are proliferated in a large amount.
  • the invention overcomes the defects that the weak immunogenic target antigen cannot induce the proliferation of the specific T cells in vitro, and the amplification effect is good, and the amplified immune cells have high killing rate to the target antigen; in addition, the proliferation method of the invention is simple in operation and cost. low. Therefore, the proliferation method of the present invention is suitable for mass production of a plurality of antigen-specific immune cells of interest for use in immunological input therapy for diseases such as various tumors.

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Description

增殖抗原特异性 T细胞的方法
技术领域
本发明涉及一种使抗原特异性 T细胞放大增殖的方法。
背景技术
免疫治疗作为一种有效的治疗手段， 近年来得到了广泛的应用。 其中， 细胞 免疫疗法被广泛应用于肿瘤癌症的治疗中， 并取的了令人惊喜的效果。 但是在细 胞免疫治疗实践中， 存在一个重大障碍， 即能够杀死异常细胞的免疫细胞难以足 够数量生产。 目前， 免疫细胞的扩增通常是通过抗体刺激以及加入细胞因子培养 实现的， 如 T细胞的扩增通常是用抗 CD3抗体加白细胞介素- 2来实现的。 但这种 方法只能非特异性地扩增免疫细胞， 得到的有效目的细胞数量不多， 不能满足需 要。 还有一种方法就是进行选择性扩增， 即用结合目的抗原的抗原递呈细胞 （如 树突状细胞， B细胞等）重复刺激免疫细胞，得到大量的目的抗原特异性免疫细胞， 但是这种方法比较适用于强免疫原性的抗原对特异性免疫细胞的扩增， 弱免疫原 性抗原很难刺激免疫细胞使之大量特异性增殖， 无法满足治疗上的需要。 因此， 亟待需要一种有效大量扩增制备抗原特异性免疫细胞的方法。
发明公开
本发明的一个目的是提供一种增殖抗原特异性 T细胞的方法。
本发明所提供的增殖抗原特异性 τ细胞的方法， 包括如下步骤： 将识别目的 抗原 B的多肽 -MHC分子复合物的免疫识别分子的编码基因， 转入针对免疫原 A的 多肽 -MHC分子复合物的特异性 T细胞， 得到识别目的抗原 B的多肽 -MHC分子复合 物和免疫原 A的多肽 -MHC分子复合物的双抗特异性 T细胞， 即得到目的抗原 B特 异性 T细胞； 用免疫原 A刺激增殖放大所述抗原 B特异性 T细胞；
所述免疫原 A为病毒、 细菌、 7个氨基酸以上 35个氨基酸以下的多肽、 嵌合 蛋白、 或同种异体抗原； 所述病毒可以是任何具有强免疫原性、 能迅速而有效地 激发免疫细胞免疫反应、 诱导免疫细胞增殖， 免疫反应机制研究得比较清楚， 不 对哺乳动物细胞高度致病的病毒， 具体可如流感病毒、 EB病毒、 CMV病毒等。
所述识别目的抗原 B多肽- MHC分子复合物的免疫识别分子包括 T细胞抗原受 体 (TCR)、 T细胞抗原受体 (TCR)样抗体、 自然杀伤细胞表面与 MHC分子结合的杀伤 活化受体 （KAR) 或自然杀伤细胞表面与 MHC分子结合的杀伤抑制受体 (KIR)。
本发明的另一个目的是提供一种抗原特异性 T细胞。
本发明所提供的抗原特异性 τ细胞， 是按照包括如下步骤的方法制备得到的: 将识别目的抗原 B的多肽 -MHC分子复合物的免疫识别分子的编码基因， 转入针对 免疫原 A的多肽 -MHC分子复合物的特异性 T细胞，得到识别目的抗原 B的多肽 -MHC 分子复合物和免疫原 A的多肽 -MHC分子复合物的双抗特异性 T细胞， 即得到目的 抗原 B特异性 T细胞；
所述免疫原 A为病毒、 细菌、 7个氨基酸以上 35个氨基酸以下的多肽、 嵌合 蛋白、 或同种异体抗原； 所述病毒可以是任何具有强免疫原性、 能迅速而有效地 激发免疫细胞免疫反应、 诱导免疫细胞增殖， 免疫反应机制研究得比较清楚， 不 对哺乳动物细胞高度致病的病毒， 具体可如流感病毒、 EB病毒、 CMV病毒等； 所述识别目的抗原 B的多肽 - MHC分子复合物的免疫识别分子包括 T细胞抗原 受体、 τ细胞抗原受体样抗体、 自然杀伤细胞表面与丽 c分子结合的杀伤活化受体 或自然杀伤细胞表面与 MHC分子结合的杀伤抑制受体。
上述方法和细胞中， 所述针对免疫原 A的多肽 -MHC分子复合物的特异性 T细 胞是通过用所述免疫原 A免疫动物得到的。
所述刺激增殖的方法可以为如下 1 ) 、 2 ) 或 3 ) :
1 )所述刺激增殖的方法包括如下步骤： 将所述抗原 B特异性 T细胞导入动物 体内， 用免疫原 A多次免疫， 在体内增殖放大所述抗原 B特异性 T细胞；
2 )所述刺激增殖的方法包括如下步骤： 将所述抗原 B特异性 T细胞与所述免 疫原 A共培养， 使所述抗原 B特异性 T细胞在体外培养中增殖放大；
3)所述刺激增殖的方法包括如下步骤： 将所述抗原 B特异性 T细胞与结合所 述免疫原 A的饲养细胞共培养， 使所述抗原 B特异性 T细胞在体外培养中增殖放 大。
所述转入是通过病毒载体、 脂质体、 阳离子聚合物， 或电穿孔实现的； 所述 病毒载体为逆转录病毒载体、 慢病毒载体或腺病毒相关载体。
所述转入的方法为如下 1 ) 、 2 ) 、 3 ) 或 4) ：
1 )将包装重组病毒的包装细胞、所述针对免疫原 A多肽 -MHC分子复合物的免 疫原 A特异性 T细胞与所述免疫原 A共培养；
2)将包装重组病毒的包装细胞、所述针对免疫原 A多肽- MHC分子复合物的免 疫原 A特异性 T细胞、 所述结合免疫原 A的饲养细胞共培养；
3 )将重组病毒、 所述针对免疫原 A多肽 -MHC分子复合物的免疫原 A特异性 T 细胞、 所述免疫原 A共培养；
4)将重组病毒、 所述针对免疫原 A多肽- MHC分子复合物的免疫原 A特异性 T 细胞、 所述结合免疫原 A的饲养细胞共培养；
所述重组病毒中含有所述识别目的抗原 B多肽- MHC分子复合物的免疫识别分 子的编码基因。
上述方法和细胞中， 所述抗原 B为肿瘤分化抗原。 所述肿瘤可为黑色素瘤。 所述抗原 B具体可为黑色素瘤分化抗原 gpl00。
所述识别目的抗原 B的多肽 - MHC分子复合物的免疫识别分子为抗所述黑色素 瘤分化抗原 gplOO的受体蛋白。
所述抗黑色素瘤分化抗原 gplOO的受体蛋白的编码基因包括片段 1和片段 2， 片段 1的核苷酸序列如序列表中序列 1所示， 片段 2的核苷酸序列如序列表中序 列 2所示。
上述任一所述结合免疫原 A的饲养细胞是按照包括如下步骤的方法制备的： 先将所述免疫原 A与所述饲养细胞共培养， 再用丝裂霉素处理细胞得到所述结合 免疫原 A的词养细胞。
上述任一所述抗原特异性 T细胞在疾病治疗中的应用也属于本发明的保护范 围。 其中所述治疗具体可为免疫输入治疗。
本发明的原理是用免疫原 A刺激 T细胞， 获得针对免疫原 A多肽 -MHC (主要组 织相容性复合物)分子复合物的特异性 T细胞；将能识别目的抗原 B多肽- MHC分子 复合物的免疫识别分子的编码基因， 转入所述的免疫原 A特异性 T细胞， 得到转 入识别目的抗原 B多肽 -MHC复合物的免疫识别分子的双抗特异性 T细胞； 用免疫 原 A增殖放大上述双抗特异性 T细胞。
本发明方法适合于强免疫原性或弱免疫原性抗原的特异性 T细胞的增殖， 更 适合于弱免疫原性抗原的特异性 T细胞的增殖， 如免疫原性弱的肿瘤的特异性 T 细胞， 更具体可为黑色素瘤抗原特异性 T细胞。
本发明方法得到的转入识别目的抗原 B多肽-顧 C复合物的免疫识别分子的双 抗特异性 T细胞， 利用其抗免疫原 A的特异性， 用免疫原 A进行诱导， 使其大量 增殖， 克服了以往弱免疫原性目的抗原无法体外诱导其特异性 T细胞增殖的缺陷， 扩增效果好， 扩增出的免疫细胞对目的抗原杀伤率高； 另外， 本发明的增殖方法 操作简单、 成本低。 因此， 本发明的增殖方法适用于大量制备多种目的抗原特异 性免疫细胞， 以用于多种肿瘤等疾病的免疫输入治疗。
附图说明
图 1为筛选产逆转录病毒的包装细胞系。
图 2为双特异性 T细胞的 gplOO肽或流感病毒 MP肽的四聚体染色结果。
图 3为双特异性 T细胞的 gplOO肽或流感病毒 MP肽细胞内细胞因子染色结果。 图 4为双特异性 T细胞在体外与不同刺激物共培养后的增殖结果。
图 5为双特异性 T细胞在 HLA-A2/Kb转基因小鼠体内增殖的结果。 图 6为双特异性 T细胞体外杀伤结合肿瘤抗原的靶细胞及肿瘤细胞的结果。 图 7为双特异性 T细胞在 HLA- A2/Kb转基因小鼠体内杀伤结合肿瘤抗原的靶 细胞的结果。
图 8为双特异性 T细胞在裸鼠体内的抑黑色素瘤实验结果。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明， 均为常规方法。
下述实施例中所用的材料、 试剂等， 如无特殊说明， 均可从生物公司购买。 Phoenix-Eco逆转录病毒包装细胞 (Morgan, R. A. , et al.， High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens. J Immunol, 2003. 171 (6) : p. 3287-95) (中国科学院微生物研究所） 。
PT67逆转录病毒包装细胞 （Quan， S.， et al. Regulation of human heme oxygenase in endothelial cells by using sense and ant i sense retroviral constructs. PNAS, 2001. 98 (21)： p. 12203-08) (中国科学院微生物研究所） 。
SupTl人淋巴细胞瘤细胞系（Morgan, R. A, , et al.， High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens. J Immunol, 2003. 171 (6) : p. 3287-95 ) (中国科学院微生物研究所) 。
APB质粒 (Morgan, R. A. , et al.， High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens. J Immunol, 2003. 171 (6)： p. 3287-95 ) (中国科学院微 生物研究所） ， APB质粒中含有抗黑色素瘤 gplOO TCR蛋白的编码基因， 包括片段 1和片段 2， 片段 1的核苷酸序列如序列表中序列 1所示， 片段 2的核苷酸序列如 序列表中序列 2所示。
流感病毒为甲型流感病毒 PR/8/34。 (de Wit, E. , et al. , Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments. Virus Res, 2004. 103 (1-2)： p. 155-61. ) (中国科学院微生物研 究所） 。
HLA-A2/Kb转基因小鼠（Zhou, M.， et al. , Screening and identification of severe acute respiratory syndrome-associa ted corona virus-specific CTL epitopes. J Immunol, 2006. 177 (4) : p. 2138-45. ) (中国科学院微生物研究所) 。 白细胞介素- 2购自 Chiron B. V.， Amsterdam, The Netherlands。
共培养培养基： 含 10% (体积百分含量）胎牛血清、 2mM谷氨酰胺、 lOOU/ml青 霉素、 100ug/ml 链霉素、 5xl(T2 mM 2-巯基乙醇的 RPMI1640培养基。
实施例 1、双特异性 T细胞（抗肿瘤特异性和抗流感病毒特异性 T细胞）的制 备
一、 表达重组病毒 （含有抗黑色素瘤 gplOO TCR蛋白的编码基因） 的包装细 胞系的制备
用 APB质粒转染 Phoenix- Eco细胞， 产生的病毒上清感染 PT67包装细胞， 将 感染后的各单克隆 PT67病毒上清分别感染 SupTl人淋巴细胞瘤细胞系，检测人 CD3 的表达， 以未经任何感染的 SupTl人淋巴细胞瘤细胞系为空白对照。 该法用来筛 选产生病毒滴度最高的包装细胞系。
结果如图 1A所示， 表明， 所筛选出的该 PT67包装细胞系产生病毒滴度最高 (即能最大程度提高 SupTl 细胞表面人 CD3表达） ， 可以作为后续实验中产毒的 包装细胞系。
二、 双特异性 T细胞的制备
( 1 ) 流感病毒免疫小鼠： 用流感病毒鼻腔免疫 HLA-A2/Kb转基因小鼠。
(2)小鼠脾脏淋巴细胞的获得：免疫 2- 4周后取小鼠脾细胞（每个脾研磨后， 2 次通过 200目滤网制成细胞悬液） ， 向细胞悬液中加入红细胞裂解液裂解红细胞， 离心去除红细胞裂解碎片和血小板， 收集淋巴细胞； 用含 10%胎牛血清的 1640细 胞培养基洗 3遍后， 用共培养培养基悬浮淋巴细胞。
(3 ) 双特异性 T细胞的获得： 将步骤 2 ) 获得的淋巴细胞接种于共培养培养 基中， 再向其中加入步骤一筛选得到的产逆转录病毒 （表达抗黑色素瘤 gplOO的 人 TCR) PT67包装细胞 （PT67包装细胞与淋巴细胞的数目比为 8 X104: 1X106) 和 流感病毒 （流感病毒与 T细胞的比例为 2xl06EID50: 1X106个） ， 在 37°C， 5% C02 条件下培养 5天， 其中， 培养 24- 48 小时后向体系中加入白细胞介素- 2 (白细胞 介素- 2在体系中的浓度为 50IU/ml ) ， 以后每隔一天加一次； 获得双特异性 T细 胞 （抗黑色素瘤特异性和抗流感病毒特异性） 。
在共培养的不同时间点检测 T细胞表面抗人 gplOO- TCR的表达。
实验设 3次重复，结果如图 1B和 1C所示。图 1B和 1C结果显示用 抗人 TCR ( a
/ β ) -ΡΙΤϋ (Βϋ Biosciences, PharMingen, CA, USA. Cat. 333140)检测到表面 表达人 gplOO- TCR， 表明逆转录病毒 （APB) 成功感染小鼠 T细胞， 获得双特异性 Τ细胞（抗黑色素瘤特异性和抗流感病毒特异性） 。 图 1C中 1表示 0天、 2表示 2 天、 3表示 4天、 4表示 5天。
如需进一步扩增双特异性 T 细胞， 则吸取悬浮细胞， 去除死细胞， 再加入流 感病毒剌激且用丝裂霉素 （80 g/ml， 37°C， 2h) 处理的饲养细胞 （同系小鼠的 脾细胞） 及 50IU/ml的白细胞介素 - 2在 37°C， 5% C02条件下继续培养 3- 5天， 得 到更多的双特异性 T细胞。
(4)抗流感病毒特异性 T细胞的获得： 用流感病毒鼻腔免疫 HLA-A2/Kb转基 因小鼠， 2- 4周后取小鼠脾脏淋巴细胞置于共培养培养基中， 同时向其中加入流感 病毒， 流感病毒在培养体系中的浓度为 2xlO¾ID50/ml， 在 37°C， 5%∞2条件下共 培养 5-10天； 期间， 培养 24- 48 小时后加入 50IU/ml 白细胞介素- 2， 以后每隔 一天加一次;获得抗流感病毒特异性 T细胞。
图 1A表示包装细胞系 PT67产生的逆转录病毒感染 SupTl后细胞表面人 CD3 的表达。 Control表示没有感染逆转录病毒的 SupTl， APB表示感染逆转录病毒 APB 的 SupTl。 横坐标是人 CD3 (人白细胞分化抗原 -3) ，表示相对荧光强度值， 纵坐 标是细胞数。
图 1B表示逆转录病毒 （APB) 感染且流感病毒剌激的小鼠 T细胞表面人 TCR
(T细胞抗原受体） 的表达。 横坐标是人 TCR， 表示绿色荧光相对强度值， 纵坐标 是细胞数。
图 1C表示逆转录病毒 (APB)感染且流感病毒刺激后不同时间小鼠 T细胞表面 人 TCR (T细胞抗原受体） 的表达。 横坐标是人 TCR， 表示绿色荧光相对强度值， 纵坐标是细胞数。
实施例 2、 双特异性 T细胞的增殖及功能 '
将实施例 1中得到的双特异性 T细胞进行如下实验。
黑色素瘤抗原 gplOO肽的四聚体按照文献（Estcourt, M. J.， A. J. McMichael, and T. Hanke, Altered primary CD8+ T cell response to a modified virus Ankara (MVA) -vectored vaccine in the absence of CD4+ T cell help. Eur J Immunol, 2005. 35 (12)： p. 3460-7) 中所述方法制备。
流感病毒抗原基质蛋白 MP肽的四聚体按照文献 （Estcourt, M. J. , A. J. McMichael, and T. Hanke, Altered primary CD8+ T cell response to a modified virus Ankara (MVA) -vectored vaccine in the absence of CD4+ T cell help. Eur J Immunol, 2005. 35 (12)： p. 3460-7) 中所述方法制备。
FITC标记的抗小鼠 CD8购自 BD, PharMingen, CA， USA;
抗小鼠 IFN- γ -ΡΕ购自 eBioscience, 产品目录号为 12-7311-71；
莫能霉素购自 eBioscience, 产品目录号为 00- 4505-51; 穿透缓冲液购自 eBioscience, 产品目录号为 00-8333。
初始 T细胞： 分离未进行任何免疫的 HLA-A2转基因小鼠的脾脏 T细胞， 即为 初始 T细胞。
一、 四聚体染色及细胞内细胞因子染色
分别以双特异性 T细胞、 抗流感病毒特异性 T细胞、 初始 T细胞为实验细胞 进行实验， 下面以双特异性 T细胞为例详细叙述实验方法：
四聚体染色： 将双特异性 T细胞用 PBS洗 2遍后重悬于 100 μ 1PBS， 加入 MP 肽四聚体 （PE) 或 gplOO肽四聚体 （PE) ， 37°C染色 20min后加入抗小鼠 CD8单 抗室温染色 20min。 PBS洗 2遍后重悬于 500 μ 1PBS后上流式细胞仪计数。
细胞内细胞因子染色： 将双特异性 Τ细胞接种于共培养培养基， 向不同的处 理组中分别加入 gplOO肽 (gplOO肽在培养体系中的浓度为 20 μ Μ)、 流感病毒 MP 肽 (流感病毒 MP肽在培养体系中的浓度为 20 μ Μ)刺激 1-2小时， 再向其中加入莫 能霉素（莫能霉素在培养体系中的浓度为 5 μ Μ) ， 在 37°C、 5% C02条件下处理 4-5 小时后， 多聚甲醛（1%)固定后加入穿透缓冲液清洗并重悬， 用抗小鼠 IFN- γ - PE 单抗和抗小鼠 CD8单抗染色， PBS洗 2遍后重悬于 500 μ 1PBS后上流式细胞仪计数。 以不加肽作为对照。
实验设 2次重复。
四聚体染色结果如图 2所示（1表示初始 Τ细胞， 2表示抗流感病毒的特异性 Τ细胞， 3表示双特异性 Τ细胞） 。 结果显示双特异性 Τ细胞中 10-15%能与 MP四 聚体反应， 15- 20%能与 gplOO四聚体反应， 而对照的抗流感病毒特异性 T细胞几 乎不与 gplOO四聚体反应， 表明双特异性 T细胞既表达抗黑色素瘤抗原 gplOO 的 TCR,也表达抗流感病毒抗原 MP的 TCR， 是双特异性的。
图 2A横坐标是小鼠白细胞分化抗原- 8， 表示绿色荧光相对强度值； 纵坐标为 MP肽四聚体 （PE) 的红色荧光相对强度值。
图 2B中横坐标是小鼠白细胞分化抗原 -8， 表示绿色荧光相对强度值； 纵坐标 为 gplOO肽四聚体 （PE) 的红色荧光相对强度值。
细胞内细胞因子染色结果如图 3所示，显示双特异性 T细胞中有约 10%与流感 病毒抗原 MP肽反应能分泌 γ -干扰素，约有 10%与黑色素瘤抗原 gplOO肽反应能分 泌 Y -干扰素， 从细胞因子分泌水平反映了双特异性 T细胞是双特异性的。
图 3A为双特异性 T细胞的细胞内细胞因子染色结果， 图 3B为抗流感病毒特 异性 T细胞的细胞内细胞因子染色结果。 1表示加流感病毒抗原 MP肽反应组， 2 表示与加黑色素瘤抗原 gplOO肽反应组， 3表示不加肽的对照组。横坐标代表细胞 表面小鼠白细胞分化抗原 -8，表示绿色荧光相对强度值；纵坐标为细胞内小鼠 IFN - Y (PE)的红色荧光相对强度值。
二、 双特异性 T细胞在体外和小鼠体内的增殖
B16— AAD黑色素瘤细胞 (Mullins, D. W. , et al. Route of Immunization with Peptide— pulsed Dendritic Cells Controls the Distribution of Memory and Effector T Cells in Lymphoid Tissues and Determines the Pattern of Regional Tumor Control. J Exp Med, 2003 . 198 (7)： p. 1023 - 34. ) (中国科学院微生物 研究所） 。
(一） 体外培养增殖：
将双特异性 T细胞用绿色荧光染料 （CFSE) 染色后加入不同的刺激物， 同时 加入白细胞介素- 2 (白细胞介素- 2在体系中的浓度为 50IU/ml ) (每隔一天加一 次） ， 用共培养培养基在 37°C、 5% C02条件下培养 3天， 然后上流式细胞仪观 察细胞增殖状况。
不同刺激物处理分别为：
1 ) 加入流感病毒 -饲养细胞， 其与双特异性 T细胞的数目比例为 1: 1； 流感病毒-饲养细胞的获得方法： 取未经任何免疫的 HLA-A2/Kb转基因小鼠的 脾脏 T细胞， 接种于共培养培养基， 向其中加入流感病毒 （流感病毒在培养体系 中的浓度为 2xl06EID50/ml )刺激 2h， 再加入丝裂霉素（丝裂霉素在培养体系中的 浓度为 80 g/ml ) ， 在 37°C处理 2h， 用培养基洗 3次， 得到的细胞即为流感病毒 结合的饲养细胞。
2)加入丝裂霉素处理的 B16- MD黑色素瘤细胞， 其与双特异性 T细胞的比例 为 0. 5: 1；
丝裂霉素处理的 B16-AAD黑色素瘤细胞的获得方法： 将 B16- MD黑色素瘤细 胞接种于共培养培养基， 向其中加入丝裂霉素 （丝裂霉素在培养体系中的浓度为 200 g/ml ) ， 在 37Ό处理 2h， 用培养基洗 3次， 得到丝裂霉素处理的 B16-MD 黑色素瘤细胞。
3) 只用共培养培养基培养双特异性 T细胞， 不加入任何细胞；
4) 加入抗 - CD3抗体， 抗体浓度为 200ng/ml。
实验设 3次重复。
体外培养增殖结果如图 4所示。 双特异性 T细胞与结合流感病毒的饲养细胞、 抗 CD3抗体共同孵育后都有明显增殖， 而 B16- MD黑色素瘤细胞只能微弱刺激其 增殖， 表明肿瘤抗原是较弱的免疫原， 不能有效刺激双特异性 T细胞增殖， 而流 感病毒抗原是强抗原， 能有力刺激双特异性 τ细胞增殖。
图 4中， A表示双特异性 T细胞， B表示抗流感病毒特异性 T细胞。 1表示与 流感病毒-饲养细胞共培养组， 2表示与 B16-AAD黑色素瘤细胞共培养组， 3表示 只用共培养培养基培养组， 4表示与抗- CD3抗体共培养组。
(二） 小鼠体内的增殖：
将双特异性 T细胞 （3xl06个 /只） 从静脉输入小鼠体内， 24小时后用流感病 毒免疫小鼠（腹腔免疫一次， 107 9 EID50)。在不同时间点取小鼠脾细胞检测 gplOO 四聚体和 CD8双阳性细胞，观察 T细胞被流感病毒刺激后在小鼠体内的扩增情况。 实验设 2次重复。
体内的增殖结果如图 5所示。 流感病毒免疫小鼠后， 双特异性 T细胞 （書） 在体内有明显增殖， 在免疫后第 7天抗肿瘤 TCR表达最高， 约占 CD8阳性 T细胞 的 10%， 随后由于体内免疫平衡体系的作用而下降。表明流感病毒抗原能有效刺激 双特异性 T细胞在体内增殖。 而对照的抗流感病毒特异性 T细胞 （〇） 未检测到 抗肿瘤 TCR的表达。
三、 双特异性 T细胞在体内和体外的杀伤实验
EG7细胞 (Fu, H. M. , et al. Investigation of endogenous antigen processing by delivery of an intact protein into cells. J. Immunol. Methods, 2008. 335 : p. 90-97) (中国科学院微生物研究所） 。
K41细胞 (Fu, H. M.， et al. Investigation of endogenous antigen processing by delivery of an intact protein into cells. J. Immunol. Methods, 2008. 335 : p. 90-97) (中国科学院微生物研究所） 。
(一） 体外杀伤：
将 HLA- A2/Kb转基因小鼠的脾细胞用 CFSE ( 10 μ Μ) 染色，然后分别用黑色素 瘤抗原§ 100肽（1(^ ¾1，37°〇， 30min)、流感病毒抗原 MP肽（10 μ Μ， 37°C， 30min) , 流感病毒 （2EID5。/cell, 37°C， 120min) 刺激后， 以不同的效靶比与效应细胞 （双 特异性 T细胞）混合， 37°C、4h后用 PI (碘化丙锭， 10μ§/ιη1， Sigma-Aldrich, 81845) 染色， 上流式细胞仪检测， 计算杀伤率。 实验设 3次重复。
体外杀伤结果如图 6所示， A表示双特异性 T细胞体外杀伤结合特异抗原的靶 细胞， B表示双特异性 T细胞体外杀伤肿瘤细胞。
图 6A中 1表示靶细胞为结合 gplOO肽的同系小鼠脾细胞， 2表示靶细胞为结 合 MP肽的同系小鼠脾细胞， 3表示靶细胞为结合流感病毒的同系小鼠脾细胞， 4 表示靶细胞为未经任何刺激的同系小鼠脾细胞。 眷表示效应细胞为双特异性 T细 胞， 〇表示效应细胞为未经抗原刺激的同系小鼠脾细胞 （对照） 。 图 6B中参表示杀伤 B16- AAD黑色素瘤细胞的结果， 〇表示杀伤 EG7细胞的结 果， 酾表示杀伤 K41细胞的结果。
结果表明双特异性 T细胞显著杀伤结合 gplOO肽、 MP肽或流感病毒的同系小 鼠脾细胞， 而不杀伤未结合的小鼠脾细胞 （图 6A) 。 并且， 双特异性 T细胞可以 显著杀伤 B16-AAD黑色素瘤细胞 （（图 6 Β· ) ， 而不杀伤对照肿瘤靶细胞 （（图 6B 〇、 國） 。
(二） 体内杀伤：
将 lxlO7双特异性 T细胞从静脉输入 HLA-A2/Kb转基因小鼠体内， 2 小时后用 流感病毒 (腹腔免疫一次， 107'9 EID5())免疫小鼠。 7天后从静脉输入靶细胞， 4小 时后取小鼠脾细胞上流式细胞仪选 PKH26阳性的细胞观察杀伤状况。 靶细胞制备 如下： 将 HLA-A2/Kb转基因小鼠的脾细胞分别用 PKH26 (4 μ Μ) 和 CFSE (2 μ Μ，或 0. 2 u M) 染色，然后 CFSE高染 (2 μ Μ) 的靶细胞群结合 gplOO或流感病毒； 同时 CFSE低染 （0. 2 μ Μ) 的靶细胞群不加刺激物作为对照。 将 CFSE高染和低染的靶 细胞群以 1 : 1 的比例混合后 （2. 5 xlO6个 /群） 静脉输入实验小鼠作为靶细胞。 实验设 3次重复。
体内杀伤结果如图 7所示。 1为实验对照组， 表示小鼠注射 PBS后 7d进行体 内杀伤实验的结果； 2是实验组， 表示小鼠注射双特异性 T细胞 （2A, 2B) ， 或流 感特异性 T细胞 （2C) ，24小时后用流感病毒免疫， 7d后进行体内杀伤实验的结 果。
A, C表示杀伤结合 gplOO肿瘤抗原的靶细胞， B表示杀伤结合流感病毒的靶细 胞。
结果表明， 双特异性 T细胞体内杀伤结合 gplOO肿瘤抗原的靶细胞（图 7A) , 平均杀伤率达到 65%; 杀伤结合流感病毒的靶细胞（图 B)，平均杀伤率达到 48. 5% o 而对照流感病毒特异性 T细胞不能杀伤结合 gplOO肿瘤抗原的靶细胞 （图 7C)。
四、 双特异性 T细胞在裸鼠体内的抑瘤实验
Balb/c nu/nu 裸鼠购自北京大学医学部实验动物研究中心。
用 8xl04B16- AAD鼠黑色素瘤细胞接种于 Balb/c nu/nu裸鼠皮下， 24小时后 静脉注射 lxlO6双特异性 T细胞， 同时以抗流感病毒特异性 T细胞作为对照。再 24 小时后腹腔免疫流感病毒（ 107·9 EID50,以后每 7-10天免疫一次）刺激 T细胞增殖。 观察裸鼠体内肿瘤生长状况。 肿瘤体积 (立方厘米) =肿瘤长径 (厘米 ) X [肿瘤短径 (厘米 )]2/2。
结果如图 8所示。 双特异性 T细胞（图 8疆， n=10) 能显著抑制黑色素瘤细胞 在裸鼠体内的增殖， 肿瘤体积与对照组 （未注射 T细胞暴（11=7)， 注射抗流感病毒 特异性 T细胞〇（n=7) ) 相比均有显著性差异 （p<0. 05) 。
工业应用
本发明方法得到的转入识别目的抗原 B多肽 -MHC复合物的免疫识别分子的双 抗特异性 T细胞， 利用其抗免疫原 A的特异性， 用免疫原 A进行诱导， 使其大量 增殖， 克服了以往弱免疫原性目的抗原无法体外诱导其特异性 T细胞增殖的缺陷， 扩增效果好， 扩增出的免疫细胞对目的抗原杀伤率高； 另外， 本发明的增殖方法 操作简单、 成本低。 因此， 本发明的增殖方法适用于大量制备多种目的抗原特异 性免疫细胞， 以用于多种肿瘤等疾病的免疫输入治疗。

Claims

权利要求
1、 一种增殖抗原特异性 T细胞的方法， 包括如下步骤： 将识别目的抗原 Β的 多肽- MHC分子复合物的免疫识别分子的编码基因， 转入针对免疫原 Α的多肽 -MHC 分子复合物的特异性 T细胞， 得到识别目的抗原 B的多肽 - MHC分子复合物和免疫 原 A的多肽 -MHC分子复合物的双抗特异性 T细胞， 即得到目的抗原 B特异性 T细 胞； 用免疫原 A刺激增殖放大所述抗原 B特异性 T细胞；
所述免疫原 A为病毒、 细菌、 7个氨基酸以上 35个氨基酸以下的多肽、 嵌合 蛋白、 或同种异体抗原；
所述识别目的抗原 B的多肽 MHC分子复合物的免疫识别分子包括 T细胞抗原 受体、 T细胞抗原受体样抗体、 自然杀伤细胞表面与 MHC分子结合的杀伤活化受体 或自然杀伤细胞表面与 MHC分子结合的杀伤抑制受体。
2、 根据权利要求 1所述的方法， 其特征在于： 所述针对免疫原 A的多肽 -MHC 分子复合物的特异性 T细胞是通过用所述免疫原 A免疫动物得到的。
3、根据权利要求 2所述的方法，其特征在于：所述刺激增殖的方法为如下 1 )、
2) 或 3) ：
1 )所述刺激增殖的方法包括如下步骤： 将所述抗原 B特异性 T细胞导入动物 体内， 用免疫原 A多次免疫， 在体内增殖放大所述抗原 B特异性 T细胞；
2)所述刺激增殖的方法包括如下步骤： 将所述抗原 B特异性 T细胞与所述免 疫原 A共培养， 使所述抗原 B特异性 T细胞在体外培养中增殖放大；
3)所述刺激增殖的方法包括如下步骤： 将所述抗原 B特异性 T细胞与结合所 述免疫原 A的伺养细胞共培养， 使所述抗原 B特异性 T细胞在体外培养中增殖放 大。
4、 根据权利要求 1-3中任一所述的方法， 其特征在于： 所述转入是通过病毒 载体、 脂质体、 阳离子聚合物， 或电穿孔实现的； 所述病毒载体为逆转录病毒载 体、 慢病毒载体或腺病毒相关载体。
5、根据权利要求 4所述的方法，其特征在于：所述转入的方法为如下 1 )、 2)、
3 ) 或 4) ：
1 )将包装重组病毒的包装细胞、所述针对免疫原 A多肽 -MHC分子复合物的免 疫原 A特异性 T细胞与所述免疫原 A共培养； 2)将包装重组病毒的包装细胞、所述针对免疫原 A多肽- MHC分子复合物的免 疫原 A特异性 T细胞、 所述结合免疫原 A的饲养细胞共培养；
3 )将重组病毒、 所述针对免疫原 A多肽 -MHC分子复合物的免疫原 A特异性 T 细胞、 所述免疫原 A共培养；
4)将重组病毒、 所述针对免疫原 A多肽- MHC分子复合物的免疫原 A特异性 T 细胞、 所述结合免疫原 A的饲养细胞共培养；
所述重组病毒中含有所述识别目的抗原 B多肽 - MHC分子复合物的免疫识别分 子的编码基因。
6、 根据权利要求 1-5中任一所述的方法， 其特征在于： 所述抗原 B为肿瘤分 化抗原。
7、 根据权利要求 6所述的方法， 其特征在于： 所述肿瘤为黑色素瘤。
8、 根据权利要求 7所述的方法， 其特征在于： 所述抗原 B为黑色素瘤分化抗 原 gpl00。
9、根据权利要求 8所述的方法，其特征在于：所述识别目的抗原 B的多肽 - MHC 分子复合物的免疫识别分子为抗所述黑色素瘤分化抗原 gplOO的受体蛋白。
10、根据权利要求 9所述的方法，其特征在于:所述抗黑色素瘤分化抗原 gplOO 的受体蛋白的编码基因包括片段 1和片段 2，片段 1的核苷酸序列如序列表中序列 1所示， 片段 2的核苷酸序列如序列表中序列 2所示。
11、 根据权利要求 1-10中任一所述的方法， 其特征在于： 所述病毒为流感病 毒、 EB病毒或 CMV病毒。
12、 一种抗原特异性 T细胞， 是按照包括如下步骤的方法制备得到的： 将识 别目的抗原 B的多肽 - MHC分子复合物的免疫识别分子的编码基因， 转入针对免疫 原 A的多肽 -MHC分子复合物的特异性 T细胞， 得到识别目的抗原 B的多肽 - MHC分 子复合物和免疫原 A的多肽 -MHC分子复合物的双抗特异性 T细胞， 即得到目的抗 原 B特异性 T细胞；
所述免疫原 A为病毒、 细菌、 7个氨基酸以上 35个氨基酸以下的多肽、 嵌合 蛋白、 或同种异体抗原；
所述识别目的抗原 B的多肽 -MHC分子复合物的免疫识别分子包括 T细胞抗原 受体、 T细胞抗原受体样抗体、 自然杀伤细胞表面与 MHC分子结合的杀伤活化受体 或自然杀伤细胞表面与 MHC分子结合的杀伤抑制受体。
13、根据权利要求 12所述的细胞，其特征在于：所述抗原 B为肿瘤分化抗原。
14、 根据权利要求 13所述的细胞， 其特征在于： 所述肿瘤为黑色素瘤。
15、 根据权利要求 14所述的细胞， 其特征在于： 所述抗原 B为黑色素瘤分化 抗原 gpl00。
16、 根据权利要求 15所述的细胞， 其特征在于： 所述识别目的抗原 B的多肽
-MHC分子复合物的免疫识别分子为抗所述黑色素瘤分化抗原 gplOO的受体蛋白。
17、根据权利要求 16所述的细胞，其特征在于:所述抗黑色素瘤分化抗原 gplOO 的受体蛋白的编码基因包括片段 1和片段 2，片段 1的核苷酸序列如序列表中序列 1所示， 片段 2的核苷酸序列如序列表中序列 2所示。
18、 根据权利要求 12-17中任一所述的细胞， 其特征在于： 所述病毒为流感 病毒、 EB病毒或 CMV病毒。
19、根据权利要求 12- 18中任一所述的细胞， 其特征在于： 所述针对免疫原 A 的多肽 - MHC分子复合物的特异性 T细胞是通过用所述免疫原 A免疫动物得到的。
20、 根据权利要求 12- 19中任一所述的细胞， 其特征在于： 所述转入是通过 病毒载体、 脂质体、 阳离子聚合物， 或电穿孔实现的； 所述病毒载体为逆转录病 毒载体、 慢病毒载体或腺病毒相关载体。
21、根据权利要求 20所述的细胞， 其特征在于： 所述转入的方法为如下 1 ) 、 2) 、 3 )或 4) ：
1 )将包装重组病毒的包装细胞、所述针对免疫原 A多肽 -MHC分子复合物的免 疫原 A特异性 T细胞与所述免疫原 A共培养；
2)将包装重组病毒的包装细胞、所述针对免疫原 A多肽 -MHC分子复合物的免 疫原 A特异性 T细胞、 所述结合免疫原 A的饲养细胞共培养；
3 )将重组病毒、 所述针对免疫原 A多肽 -MHC分子复合物的免疫原 A特异性 T 细胞、 所述免疫原 A共培养；
4)将重组病毒、 所述针对免疫原 A多肽 -MHC分子复合物的免疫原 A特异性 T 细胞、 所述结合免疫原 A的饲养细胞共培养；
所述重组病毒中含有所述识别目的抗原 B多肽- MHC分子复合物的免疫识别分 子的编码基因。
22、 权利要求 1-11中任一所述抗原特异性 T细胞在疾病治疗中的应用。
23、 根据权利要求 13所述的应用， 其特征在于： 所述治疗为免疫输入治疗。
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