WO2023078427A1 - 一种体外大规模扩增富含Tscm和Tcm的双阴性T细胞的方法 - Google Patents

一种体外大规模扩增富含Tscm和Tcm的双阴性T细胞的方法 Download PDF

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WO2023078427A1
WO2023078427A1 PCT/CN2022/130054 CN2022130054W WO2023078427A1 WO 2023078427 A1 WO2023078427 A1 WO 2023078427A1 CN 2022130054 W CN2022130054 W CN 2022130054W WO 2023078427 A1 WO2023078427 A1 WO 2023078427A1
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cells
dnt
recombinant human
cell
cancer
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French (fr)
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杨黎明
王丹
王留洋
李先才
童建军
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瑞创生物技术有限公司
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Definitions

  • the invention belongs to the technical field of cell therapy, and in particular relates to a method for large-scale expansion of double-negative T cells rich in Tscm and Tcm in vitro.
  • Adoptive immune cell therapy is an emerging tumor treatment method with significant curative effect, and it is a new treatment method for immune anti-cancer. It uses biotechnology and biological agents to activate and expand immune cells collected from patients or healthy donors in vitro, and then reinfuse them back into the patient's body to stimulate and enhance the patient's own immune function, so as to achieve the purpose of treating tumors.
  • Tscm stem cell-like memory T cells
  • Tcm central memory T cells
  • effector memory T cells Effector Memory T, Tem
  • Tscm cells and Tcm cells have strong stem cell-like characteristics, which enable them to maintain a certain ability of self-renewal, differentiation and long-term survival, and have a strong ability to secrete IFN- ⁇ , which can exist in the body for a long time and play a long-term anti-inflammatory role. tumor effect.
  • Double negative T (Double Negative T, DNT) cells refer to the CD3+CD4-CD8- mature T lymphocyte subsets that normally exist in peripheral blood, accounting for about 1-3% of peripheral blood mononuclear cells.
  • the surface of DNT cells expresses CD3 molecules and ⁇ - or ⁇ -T cell receptors (T Cell Receptor, TCR), but does not express CD4 and CD8 molecules, and is specific for constant natural killer T (invariant Nature Killer T, iNKT) cells ⁇ GalCer is unresponsive and thus different from conventional T cells, NK cells and NKT cells.
  • DNT cells exert cytotoxicity and antigen presentation functions through various natural mechanisms of the human body while releasing cytokines/chemokines and activating a broader immune response.
  • DNT cells have cytotoxic activity against a variety of tumors, and have good tumor killing effects in vivo and in vitro in AML, lymphoma, cervical cancer, lung cancer, liver cancer, gastric cancer and other blood or solid tumors; at the same time, DNT Cells also play important roles in immunomodulation, immunosuppression, and autoimmune diseases.
  • the killing of tumor cells by DNT cells is not restricted by the Major Histocompatibility Complex (MHC) molecules, and the allogeneic DNT cells from healthy donors expanded in vitro have no killing toxicity to normal cells after infusion into patients.
  • MHC Major Histocompatibility Complex
  • hematopoietic stem cells does not affect the further differentiation of hematopoietic stem cells, does not cause graft versus host disease (Graft versus Host Disease, GvHD)), and has no host versus graft (Host versus Graft, HvG) reaction, which makes it a clinical application with broad application prospects.
  • GvHD graft versus Host Disease
  • HvG host versus graft
  • the expansion of DNT cells was first based on the small-scale expansion method used in laboratory research.
  • the small scale of the culture and the need to add animal-derived serum limited its clinical application; the subsequently developed cell in vitro expansion system removed the animal-derived products, but still need to add autologous plasma and/or AB serum and/or human serum albumin and need to add soluble anti-human CD3 monoclonal antibody as an activator during the whole culture cycle, the latter is currently commercial GMP grade products are Rat origin (clone number: OKT3), which brings potential safety risks to the clinical application of the product.
  • the proportion of Tscm cells and Tcm cells in the DNT cells expanded in vitro in the past is also very low, which has not been mentioned in the previous literature and patents.
  • the purpose of the present invention is to provide a method for large-scale expansion of double-negative T cells rich in Tscm and Tcm cell characteristics in vitro.
  • a method for in vitro expansion of double-negative T cells enriched in Tscm cells and Tcm comprising the steps of:
  • sample II is cultivated to obtain sample III; wherein, one or more of cytokines selected from the following group is added to the culture system:
  • step (c) no anti-human CD3 antibody is added to the culture system.
  • step (c) also includes the following steps:
  • the pretreatment in step (b) includes:
  • sample II In a culture system suitable for the growth of DNT cells, the initial sample I, which was depleted of CD4+ and CD8+, was activated with an anti-human CD3 monoclonal antibody to obtain sample II.
  • the number of days for activation is 2-7 days, preferably 3-6 days.
  • the anti-human CD3 monoclonal antibody is selected from human CD3 monoclonal antibodies immobilized on media, including but not limited to those coated on culture media, magnetic beads or degradable microbeads. Human CD3 monoclonal antibody on beads.
  • step (b2) the initial sample I that removes CD4+ and CD8+ is activated using human CD3/CD28 monoclonal antibody coated on degradable microbeads, so as to obtain sample II.
  • the concentration of the anti-human CD3 monoclonal antibody is selected from 20ng/ml-20 ⁇ g/ml.
  • step (b) the number of cells in the initial sample I in which CD4+ and CD8+ are removed is N0;
  • step (c) the number of DNT cells in the sample III is N1;
  • step (d) the number of DNT cells in the sample IV is N2, wherein,
  • N1/N0 ⁇ 50 preferably ⁇ 70; more preferably ⁇ 100; more preferably ⁇ 200;
  • N2/N0 ⁇ 200 preferably ⁇ 500; more preferably ⁇ 1000; more preferably ⁇ 10000.
  • the density of DNT cells in the sample II is selected from 1 ⁇ 10 5 -1 ⁇ 10 7 cells/ml; preferably 5 ⁇ 10 5 -8 ⁇ 10 6 cells/ml; more preferably 1 ⁇ 10 6 -4 ⁇ 10 6 cells/ml.
  • the cytokine is selected from one or more of the following groups:
  • the number of days of culture is selected from 5-21 days, preferably 6-17 days, more preferably 7-14 days.
  • the culture system of the medium suitable for the growth of DNT cells does not contain anti-human CD3 antibody, such as OKT3.
  • the culture system in steps (b), (c), and (d) contains 200-1000IU/mL (preferably 300-700IU/mL, more preferably 500IU/mL) recombinant human interleukin-2.
  • the culture system of the medium suitable for the growth of DNT cells does not contain serum.
  • the culture system of the medium suitable for DNT cell growth contains a serum substitute selected from the following group: ICSR (Immune Cell Serum Replacement), KSR (KnockOut TM Serum Replacement).
  • the concentration (v/v) of ICSR is 2%-30%.
  • the medium is selected from the group consisting of AIM-V, X-VIVO-10, X-VIVO-15, Aly505, GT551 medium.
  • molecules selected from the group consisting of 1-10 ⁇ g/ml recombinant human transferrin, 1-10 ⁇ g/ml recombinant human insulin, and 10-100 ⁇ g/ml ascorbic acid are added to the medium , 1-5 ⁇ g/ml ethanolamine, 1-5 ⁇ g/ml linoleic acid, 1-5 ⁇ g/ml oleic acid.
  • step (c) the medium comprises:
  • cytokines selected from the following group:
  • a DNT cell population is provided, and the DNT cell population is prepared by the method described in the first aspect of the present invention.
  • the DNT cell population has one or more characteristics selected from the following group:
  • the ratio of Tscm cells to Tcm cells is ⁇ 45%; preferably ⁇ 50%; preferably ⁇ 70%; more preferably ⁇ 75%.
  • the DNT cell survival rate is ⁇ 70%; preferably ⁇ 80%; more preferably ⁇ 90%; more preferably ⁇ 95%.
  • the purity (%) of the DNT cells (CD3 + ) is ⁇ 80%; preferably ⁇ 90%; more preferably ⁇ 95%; more preferably ⁇ 97%.
  • the purity (%) of the DNT cells is ⁇ 85%; preferably ⁇ 90%; more preferably ⁇ 95%; more preferably ⁇ 97%.
  • DNT cells according to the second aspect of the present invention for preparing a pharmaceutical composition or preparation, and the pharmaceutical composition or preparation is used for:
  • the tumor is a tumor that is allogeneic to the DNT cells.
  • the tumor is selected from the group consisting of blood tumors, solid tumors, or a combination thereof.
  • the blood tumor is selected from the group consisting of lymphoma (Hodgkins and non-Hodgkins), acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute Lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), or a combination thereof.
  • lymphoma Hodgkins and non-Hodgkins
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute Lymphocytic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • CML chronic myelogenous leukemia
  • CMML chronic myelomonocytic leukemia
  • MDS myelodysplastic
  • the solid tumor is selected from the group consisting of gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, melanoma, bone cancer, prostate cancer, colorectal cancer, breast cancer , colorectal cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, head and neck cancer, pancreatic cancer, or combinations thereof.
  • gastric cancer peritoneal metastasis of gastric cancer
  • liver cancer leukemia, kidney tumor, lung cancer, small intestine cancer, melanoma
  • bone cancer prostate cancer
  • colorectal cancer breast cancer
  • colorectal cancer cervical cancer
  • ovarian cancer lymphoma, nasopharyngeal cancer
  • adrenal tumor bladder tumor
  • NSCLC non-small cell lung cancer
  • the autoimmune diseases include: diabetes, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, nausea anemia, hemolytic anemia, autoimmune thrombocytopenia, autoimmune liver disease, ankylosing spondylitis, myasthenia gravis, IgA nephropathy, primary renal ice syndrome, psoriasis, vitiligo.
  • a cell preparation containing the DNT cell population as described in the second aspect of the present invention there is provided a cell preparation containing the DNT cell population as described in the second aspect of the present invention.
  • the cell preparation includes the DNT cell population and a pharmaceutically acceptable carrier.
  • a medium suitable for the growth of DNT cells comprising one or more selected from the following cytokines:
  • the medium suitable for the growth of DNT cells contains a serum replacement selected from the group consisting of ICSR (Immune Cell Serum Replacement) and KSR (KnockOut TM Serum Replacement).
  • the culture medium contains one or more selected from the following group:
  • the medium is selected from the group consisting of AIM-V, X-VIVO-10, X-VIVO-15, Aly505, GT551 medium.
  • the culture medium comprises:
  • cytokines selected from the following group:
  • a method for for (a) preventing and/or treating tumors; (b) preventing and/or treating infectious diseases; (c) preventing and/or treating autoimmune diseases; (d) Prevention and/or treatment of graft-versus-host disease; and/or (e) a method of regulating immune response, administering DNT cells as described in the second aspect of the present invention to a subject in need.
  • Figure 1 shows a process flow diagram of the present invention.
  • Figure 2 shows the DNT cell proliferation curve (Figure 2A) and cell viability curve ( Figure 2B).
  • Figure 3A and Figure 3B show the change curve of DNT cell purity (CD3 + %, CD4 - CD8 - %).
  • Figure 3C shows the day 11 flow diagram of DNT cell purity.
  • FIG. 4 shows the DNT cell differentiation curve and the DNT cell differentiation flow diagram on the 11th day
  • Figure 5 shows the antitumor activity of DNT cells and MV411 at the 10th day of in vitro expansion with an effect-to-target ratio of 4:1 and co-incubated for 2 hours.
  • Figure 6 shows the real-time tumor killing curve of the 20th hour of co-incubation of DNT cells and Hela cells with an effective target ratio of 5:1 on the 10th day of in vitro expansion.
  • Figure 7 shows the DNT cell proliferation curve (Figure 7A) and cell viability curve ( Figure 7B).
  • Figure 8A and Figure 8B show the change curve of DNT cell purity
  • Figure 8C shows the flow chart of DNT cell purity on day 11
  • FIG. 9 shows the DNT cell differentiation curve and the DNT cell differentiation flow diagram on the 11th day
  • Figure 10 shows the antitumor activity of DNT cells and MV411 with an effect-to-target ratio of 4:1 on the 10th day of in vitro expansion and co-incubation for 2 hours
  • Figure 11 shows the real-time tumor killing curve at the 20th hour of co-incubation of DNT cells and Hela cells with an effect-to-target ratio of 5:1 on the 10th day of in vitro expansion.
  • Figure 12 shows DNT cell proliferation curves (Figure 12A) and cell viability curves ( Figure 12B).
  • Figure 13 shows the change curve of DNT cell purity and the flow chart of DNT cell purity on day 11 ( Figure 13C).
  • Figure 14 shows the DNT cell differentiation curve and the DNT cell differentiation flow diagram on day 11.
  • Figure 15 shows the antitumor activity of DNT cells and MV411 at the 10th day of in vitro expansion with an effect-to-target ratio of 4:1 and co-incubated for 2 hours.
  • Figure 16 shows the real-time tumor killing curve of the 20th hour of co-incubation of DNT cells and Hela cells with an effect-to-target ratio of 5:1 on the 10th day of in vitro expansion.
  • the inventors After extensive and in-depth research, and a large number of process optimization experiments, the inventors first developed a serum-free medium containing well-defined additive factors to perform large-scale expansion of double-negative T cells rich in Tscm and Tcm characteristics in vitro Methods. Specifically, it provides a kind of peripheral blood derived from healthy donors, using one or more serum-free media, adding additive factors with clear components and no xenogeneic components, and no additional soluble antibody is added during the expansion stage of the DNT culture system. Human CD3 antibody, so as to expand the double-negative T cells rich in Tscm and Tcm cell characteristics in vitro, so as to further improve the long-term anti-tumor effect of allogeneic DNT cells in clinical application. The present invention has been accomplished on this basis.
  • the term "about” can refer to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • administration and “administration” are used interchangeably and refer to the physical introduction of the product of the present invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including Intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • DNT cells were isolated and purified from peripheral blood in vitro.
  • 20ng/ml-20 ⁇ g/ml anti-human CD3 monoclonal antibody is coated on solid medium, and 200-1000IU/mL recombinant human interleukin 2 is added to the medium
  • a medium suitable for cultivating DNT cells in which:
  • One or several cytokines selected from the following group: 10-1000IU recombinant human interleukin 2, 5-50ng/ml recombinant human interleukin 7, 5-50ng/ml recombinant human interleukin 12, 5-50ng/ml recombinant human interleukin 15 , 5-50ng/ml recombinant human interleukin 21, 1-10ng/ml recombinant human interleukin 1 ⁇ ;
  • One or more additive factors selected from the group: 1-10 ⁇ g/ml recombinant human transferrin, 1-10 ⁇ g/ml recombinant human insulin, 10-100 ⁇ g/ml ascorbic acid, 1-5 ⁇ g/ml ethanolamine, 1-5 ⁇ g/ml of linoleic acid, 1-5 ⁇ g/ml of oleic acid.
  • the anti-human CD3 monoclonal antibody is coated on solid media, including but not limited to culture media and magnetic beads.
  • the anti-human CD3 monoclonal antibody can be co-coated with other antibodies (such as anti-human CD28 monoclonal antibody) on a solid medium to activate DNT cells.
  • CD3/CD28 magnetic beads are used to activate DNT cells, wherein the CD3/CD28 magnetic beads include the same kind of magnetic beads coated with anti-human CD3 monoclonal antibody and anti-human CD28 monoclonal antibody, or two kinds of magnetic beads coated with anti-human CD3 monoclonal antibody and anti-human CD28 monoclonal antibody respectively.
  • no anti-human CD3 antibody preferably no soluble anti-human CD3 monoclonal antibody, such as OKT3, is added.
  • the present invention provides a double-negative T cell (Double Negative T, DNT) rich in Tscm (Stem Cell-Like Memory T, stem cell-like memory T) and Tcm (Central Memory T, central memory T), that is, the present invention DNT cell population.
  • DNT Double Negative T
  • Tscm Ste Cell-Like Memory T, stem cell-like memory T
  • Tcm Central Memory T, central memory T
  • the DNT cell population is prepared by the method described in the first aspect of the present invention. It has one or more characteristics selected from the group consisting of:
  • the ratio of Tscm cells to Tcm cells is ⁇ 45%; preferably ⁇ 50%; preferably ⁇ 70%; more preferably ⁇ 75%.
  • the DNT cell survival rate is ⁇ 70%; preferably ⁇ 80%; more preferably ⁇ 90%; more preferably ⁇ 95%.
  • the purity (%) of the DNT cells (CD3 + ) is ⁇ 80%; preferably ⁇ 90%; more preferably ⁇ 95%; more preferably ⁇ 97%.
  • the purity (%) of the DNT cells is ⁇ 85%; preferably ⁇ 90%; more preferably ⁇ 95%; more preferably ⁇ 97%.
  • the ratio of Tscm to Tcm cells is significantly or extremely significantly better than that in the prior art, and the ratio of Teff effect killer cells is significantly or extremely significantly lower than that in the prior art. Therefore, it has stronger renewal, differentiation and survival ability.
  • compositions and methods of administration are provided.
  • the present invention also provides the use of the DNT cells of the present invention for the preparation of a pharmaceutical composition or preparation, which can be used to treat diseases selected from the group consisting of tumors, infectious diseases, autoimmune immune diseases, graft-versus-host disease.
  • the blood tumor is selected from the group consisting of lymphoma (Hodgkins and non-Hodgkins), acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute Lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), or a combination thereof.
  • lymphoma Hodgkins and non-Hodgkins
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute Lymphocytic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • CML chronic myelogenous leukemia
  • CMML chronic myelomonocytic leukemia
  • MDS myelodysplastic
  • the solid tumor is selected from the group consisting of gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, melanoma, bone cancer, prostate cancer, colorectal cancer, breast cancer , colorectal cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, head and neck cancer, pancreatic cancer, or combinations thereof.
  • gastric cancer peritoneal metastasis of gastric cancer
  • liver cancer leukemia, kidney tumor, lung cancer, small intestine cancer, melanoma
  • bone cancer prostate cancer
  • colorectal cancer breast cancer
  • colorectal cancer cervical cancer
  • ovarian cancer lymphoma, nasopharyngeal cancer
  • adrenal tumor bladder tumor
  • NSCLC non-small cell lung cancer
  • the autoimmune diseases include: diabetes, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, nausea anemia, hemolytic anemia, autoimmune thrombocytopenia, autoimmune liver disease, ankylosing Spondylitis, myasthenia gravis, IgA nephropathy, primary renal ice syndrome, psoriasis, vitiligo
  • the present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the cell preparation of the present invention, and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, powder, dimethylsulfoxide (DMSO), and combinations thereof.
  • the pharmaceutical formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the cell preparations of the invention can also be used with other therapeutic agents.
  • composition of the present invention can be administered to desired subjects (such as humans and non-human mammals) by conventional means.
  • desired administration methods include (but are not limited to): intravenous injection, arterial injection, chest cavity, abdominal pelvic cavity, subarachnoid cavity, nasal sinuses, intracranial, injection in different tissues (such as tumor tissue, inflammatory lesion tissue), etc. wait.
  • the amplification process is simple and stable, and can realize large-scale culture of DNT cells rich in Tscm and Tcm cell characteristics in vitro.
  • the purity of the final product DNT cells (CD3+CD4-CD8-) is ⁇ 85%, which meets the needs of clinical use.
  • DNT cells rich in Tscm and Tcm cell characteristics are obtained by culturing, wherein the proportion of Tscm cells and Tcm cells in the DNT cells is ⁇ 50%.
  • mouse anti-human CD3 antibody (clone number OKT3) in the amplification stage of the DNT culture system, which further reduces the residue of foreign proteins in the final product and improves product safety and quality.
  • Cells can be expanded in vitro from freshly enriched DNT cells or recovered DNT cells after cryopreservation.
  • Example 1 In vitro expansion and culture of DNT cells activated by anti-human CD3 monoclonal antibody
  • peripheral blood 20-200ml of peripheral blood was collected from healthy donors into sodium heparin tubes. use Kit (Stem Cell Technologies Inc), to remove CD4 + and CD8 + T cells, and the cells obtained in this way are CD4 + and CD8 + depleted DNT cells.
  • step 0 coat T25 culture flask with anti-human CD3 monoclonal antibody (10 ⁇ g/mL) (clone number is OKT3), and use DNT cell-specific serum-free medium (AIM-V base) for DNT cells obtained in step 1.1 medium, add 8 ⁇ g/ml recombinant human transferrin, 8 ⁇ g/ml recombinant human insulin, 30 ⁇ g/ml ascorbic acid, 1.5 ⁇ g/ml ethanolamine, 1 ⁇ g/ml linoleic acid, 1 ⁇ g/ml oleic acid) 10% (v/v) ICSR and 500 IU/mL recombinant human interleukin 2 were adjusted to a concentration of 1 ⁇ 10 6 -4 ⁇ 10 6 cells/mL, and put into the aforementioned coated T25 culture flask. Culture in a 5% CO 2 incubator at 37°C.
  • DNT cells were harvested.
  • DNT cells were harvested if necessary. Cells were collected in a 250ml conical centrifuge bottle, centrifuged at 900 ⁇ g for 10 minutes, and washed with solvent. The collected DNT cells are adjusted to a concentration of 0.1-1 ⁇ 10 8 cells/mL with solvent, and this is the finished DNT cell preparation, which can be used clinically after passing the quality inspection.
  • the tumoricidal activity of DNT cells against MV411 cells was analyzed ( FIG. 5 ).
  • Adopt the RTCA method (through a special process, the microelectronic cell sensor chip is integrated into the bottom of the cell detection plate to construct a real-time, dynamic, quantitative tracking cell impedance detection and sensing system for changes in cell morphology, proliferation and differentiation, etc.)
  • the real-time tumor killing curve of DNT cells to Hela cells was detected ( FIG. 6 ).
  • Example 2 In vitro expansion and culture of DNT cells activated by magnetic beads
  • the isolated and purified DNT cells were mixed with CD3/CD28 magnetic beads (the same magnetic beads were coated with anti-human CD3 monoclonal antibody and anti-human CD28 Monoclonal antibody, or two kinds of magnetic beads respectively coated with anti-human CD3 monoclonal antibody and anti-human CD28 monoclonal antibody) were mixed and inoculated in T25 culture flasks, and added with DNT cell special serum-free medium (GT551 basal medium) 3 ⁇ g/ml recombinant human transferrin, 3 ⁇ g/ml recombinant human insulin, 70 ⁇ g/ml ascorbic acid, 2 ⁇ g/ml ethanolamine, 0.5 ⁇ g/ml linoleic acid, 0.5 ⁇ g/ml oleic acid) added 20% ( v/v) ICSR and 1000 IU/mL recombinant human interleukin 2 were adjusted to a concentration of 1 ⁇ 10 6
  • DNT cells were harvested.
  • DNT cells were harvested if necessary. Cells were collected in a 250ml conical centrifuge bottle, centrifuged at 900 ⁇ g for 10 minutes, and washed with solvent. The collected DNT cells are adjusted to a concentration of 0.5-1 ⁇ 10 8 cells/mL with solvent, and this is the finished DNT cell preparation, which can be used clinically after passing the quality inspection.
  • RTCA method was used to detect the real-time tumor killing curve of DNT cells against Hela cells ( FIG. 11 ).
  • Example 3 In vitro expansion and culture of DNT cells activated by anti-human CD3 monoclonal antibody
  • DNT cells use the special serum-free medium for DNT cells (Aly505 basal medium to add 3 ⁇ g/ml recombinant human transferrin, 3 ⁇ g/ml recombinant human insulin, 30 ⁇ g/ml ascorbic acid, 1 ⁇ g/ml ethanolamine, 0.5 ⁇ g/ml of linoleic acid, 0.5 ⁇ g/ml of oleic acid) adding 10% ICSR and 500IU/mL of recombinant human interleukin 2, 5ng/ml of recombinant human interleukin 7, 5ng/ml of recombinant human interleukin 15, and adjusting DNT cells Adjust the concentration of 1 ⁇ 10 6 to 4 ⁇ 10 6 cells/mL according to the volume of the cells and adjust the T175 culture flask or 2L culture bag to continue the culture.
  • DNT cells Adjust the concentration of 1 ⁇ 10 6 to 4 ⁇ 10 6 cells/mL according to the volume of the cells and adjust the T175 culture fla
  • DNT cells were harvested.
  • DNT cells were harvested if necessary. Cells were collected in a 250ml conical centrifuge bottle, centrifuged at 900 ⁇ g for 10 minutes, and washed with solvent. The collected DNT cells are adjusted to a concentration of 0.5-1 ⁇ 10 8 cells/mL with solvent, and this is the finished DNT cell preparation, which can be used clinically after passing the quality inspection.
  • RTCA method was used to detect the real-time tumor killing curve of DNT cells on Hela cells ( FIG. 16 ).
  • the patent of the present invention is the large-scale expansion of double-negative T (Double Negative T, DNT) cell characteristics rich in Tscm (Stem Cell-Like Memory T, stem cell-like memory T) and Tcm (Central Memory T, central memory T) in vitro ) cell method (hereinafter referred to as the new process) is compared with our company's existing patent (CN104109653A), that is, the method for large-scale expansion of human peripheral blood DNT cells using an animal serum-free culture system (hereinafter referred to as the old process):
  • peripheral blood 20-200ml of peripheral blood was collected from 4 healthy donors into sodium heparin tubes. use Kit (Stem Cell Technologies Inc), to remove CD4 + and CD8 + T cells, and the cells obtained in this way are CD4+ and CD8+ depleted DNT cells.
  • DNT serum-free medium AIM-V basal medium plus 500 IU/mL recombinant Human interleukin 2, 2ng/ml recombinant human interleukin 4, 8ng/ml recombinant human interleukin 7, 5ng/ml recombinant
  • DNT serum-free medium (GT551 basal medium) with 500 IU/mL recombinant human interleukin 2, 2 ng/ml recombinant human interleukin 4, 8 ng/ml recombinant human interleukin 7, 5 ng/ml recombinant human interleukin 12 , 5v% autologous serum and 6v% autologous serum) additionally add 50ng/ml soluble anti-human CD3 monoclonal antibody, adjust the concentration to 1 ⁇ 10 6 ⁇ 4 ⁇ 10 6 cells/mL, adjust the T175 culture flask according to the cell volume or 2L culture bag to continue culture.
  • GT551 basal medium 500 IU/mL recombinant human interleukin 2, 2 ng/ml recombinant human interleukin 4, 8 ng/ml recombinant human interleukin 7, 5 ng/ml recombinant human interleukin 12 , 5v% autologous serum and 6v% autolog
  • DNT cells were harvested.
  • DNT cells were harvested if necessary. Cells were collected in a 250ml conical centrifuge bottle, centrifuged at 900 ⁇ g for 10 minutes, and washed with solvent. The collected DNT cells are adjusted to a concentration of 0.5-1 ⁇ 10 8 cells/mL with solvent, and this is the finished DNT cell preparation, which can be used clinically after passing the quality inspection.
  • the ratio of Tscm to Tcm cells in the DNT cells expanded by the new process is significantly or very significantly higher than that of the old process, while the ratio of Teff effect killing cells is significantly or very significantly lower than that of the old process (as shown in Figure 19, shown in Table 4).
  • the results of the tumor-killing activity of the cells on the 10th day showed that the tumor-killing activity of DNT cells expanded by the new process on MV411 cells was significantly higher than that of the old process.
  • mouse-derived soluble anti-human CD3 antibody is not used in the amplification stage, which reduces the residue of mouse-derived components and further improves product safety;

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Abstract

本发明涉及一种体外大规模扩增富含Tscm和Tcm的双阴性T细胞的方法。具体地，包括步骤：(a)提供一获自供体的外周血起始样品I；(b)对起始样品I进行预处理，从而获得样品II；(c)在含有适合DNT细胞生长的培养基的培养体系中，培养样品II，从而获得样品III。本发明的扩增工艺简单，稳定，可实现体外大规模培养富含Tscm和Tcm细胞特征的DNT细胞，终产品DNT细胞纯度(CD3+CD4-CD8-)≥85％，满足临床使用需求。

Description

一种体外大规模扩增富含Tscm和Tcm的双阴性T细胞的方法 技术领域
本发明属于细胞治疗技术领域，具体涉及一种体外大规模扩增富含Tscm和Tcm的双阴性T细胞的方法。
背景技术
过继免疫细胞治疗是一种新兴的、具有显著疗效的肿瘤治疗手段，是免疫抗癌的新型治疗方法。它是运用生物技术和生物制剂对从病人或健康捐赠者体内采集免疫细胞进行体外激活和扩增后回输到病人体内，用以激发、增强病人自身免疫功能，从而达到治疗肿瘤目的。
已有文献和临床数据报导，决定过继细胞免疫治疗持久性和抗肿瘤作用的关键因素与输入患者前的细胞记忆分化状态即Tscm细胞和Tcm(Central Memory T)细胞在细胞产品中的比例密切相关。根据最新研究的记忆T细胞分化模式，天然T细胞在抗原的刺激下首先分化成干细胞样记忆T细胞(Tscm),然后分化成中央记忆T细胞(Tcm)，并且最后分化成效应记忆T细胞(Effector Memory T，Tem)细胞。Tscm细胞和Tcm细胞具有较强的干细胞样特性，使其保持一定的自我更新、分化和长久存活能力，且有较强的IFN-γ分泌能力，可在体内长时间存在，起到长效抗肿瘤作用。
双阴性T(Double Negative T，DNT)细胞是指外周血中正常存在的CD3+CD4-CD8-成熟T淋巴细胞亚群，约占外周血单个核细胞1-3％。DNT细胞表面表达CD3分子和αβ-或γδ-T细胞受体(T Cell Receptor，TCR)，但不表达CD4和CD8分子，且对于恒定型自然杀伤T(invariant Nature Killer T，iNKT)细胞特异性αGalCer无反应，因此不同于常规的T细胞、NK细胞和NKT细胞。DNT细胞通过多种人体天然机制发挥细胞毒性、抗原呈递功能同时释放细胞因子/趋化因子，激活更广泛的免疫应答。已报导，DNT细胞对多种肿瘤具有细胞毒活性，在AML、淋巴瘤、宫颈癌、肺癌、肝癌、胃癌等多种血液或者实体肿瘤中都有较好的体内外杀瘤效果；同时，DNT细胞在免疫调节，免疫抑制以及自身免疫病方面也有重要的作用。DNT细胞杀死肿瘤细胞不受组织相容性复合物(Major Histocompatibility Complex，MHC)分子限制，经体外扩增的健康捐赠者来源的异体DNT细胞输注到患者体内后对正常细胞无杀伤毒性且不影响造血干细 胞的进一步分化，不引起移植物抗宿主病(Graft versus Host Disease,GvHD))，也没有宿主抗移植物(Host versus Graft，HvG)反应，这使其成为具备广泛应用前景的临床治疗肿瘤的候选药物。
DNT细胞的扩增最早是基于实验室科研用小规模扩增方法，其培养规模小且需要添加动物源血清，使其临床应用受到限制；随后开发的细胞体外扩增体系中虽去除了动物源产品，但仍需要添加自体血浆和/或AB血清和/或人血白蛋白且在全培养周期都需要添加可溶性的抗人CD3单克隆抗体作为激活剂，后者目前商业化GMP级产品均为鼠源(克隆号：OKT3)，给产品的临床应用带来潜在安全风险。以往体外扩增的DNT细胞中Tscm细胞和Tcm细胞的比例也很低，在既往文献以及专利中也均未有涉及。
因此，本领域需要一种体外大规模扩增富含Tscm和Tcm细胞特征的双阴性T细胞的方法。
发明内容
本发明目的在于提供一种体外大规模扩增富含Tscm和Tcm细胞特征的双阴性T细胞的方法。
在本发明的第一方面，提供了一种富含Tscm细胞和Tcm的双阴性T细胞的体外扩增方法，包括步骤：
(a)提供一获自供体的外周血起始样品I；
(b)对起始样品I进行预处理，从而获得样品II；
(c)在含有适合DNT细胞生长的培养基的培养体系中，培养样品II，从而获得样品III；其中，所述培养体系中添加选自下组细胞因子中的一种或多种：
5-50ng/ml重组人白介素21、1-10ng/ml重组人白介素1β、5-50ng/ml重组人白介素7、5-50ng/ml重组人白介素15、5-50ng/ml重组人白介素12；
其中，步骤(c)中，所述培养体系中不添加抗人CD3抗体。
在另一优选例中，步骤(c)还包括以下步骤：
(d)在含有适合DNT细胞生长的培养基的培养体系中，培养样品III，从而获得所需量的富含Tscm和Tcm的DNT细胞，为样品IV；其中，所述培养体系中添加选自下组细胞因子中的一种或多种：
5-50ng/ml重组人白介素7、5-50ng/ml重组人白介素12或5-50ng/ml重组人白介素15；和
(e)在含有适合DNT细胞保存的溶液体系中，收集样品IV。
在另一优选例中，步骤(b)中的预处理包括：
(b1)去除起始样品I中的CD4+和CD8+T细胞，从而获得去除CD4+和CD8+的起始样品I；
(b2)在含有适合DNT细胞生长的培养体系中，用抗人CD3单克隆抗体激活去除CD4+和CD8+的起始样品I，从而获得样品II。
在另一优选例中，步骤(b2)中，所述激活的天数为2-7天，较佳地3-6天。
在另一优选例中，步骤(b2)中，所述抗人CD3单克隆抗体选自固化在介质上的人CD3单克隆抗体，包括但不限于包被在培养介质、磁珠或可降解微珠上的人CD3单克隆抗体。
在另一优选例中，步骤(b2)中，使用包被于可降解微珠上的人CD3/CD28单克隆抗体激活去除CD4+和CD8+的起始样品I，从而获得样品II。
在另一优选例中，所述抗人CD3单克隆抗体的浓度选自20ng/ml-20μg/ml。
在另一优选例中，步骤(b)中，所述去除CD4+和CD8+的起始样品I中的细胞数为N0；
步骤(c)中，样品III中DNT细胞的数量为N1；
步骤(d)中，样品IV中DNT细胞的数量为N2，其中，
N1/N0≥50；较佳地≥70；更佳地≥100；更佳地≥200；
N2/N0≥200；较佳地≥500；更佳地≥1000；更佳地≥10000。
在另一优选例中，步骤(b)中，所述样品II中DNT细胞的密度选自1×10 5-1×10 7个细胞/ml；较佳地5×10 5-8×10 6个细胞/ml；更佳地1×10 6-4×10 6个细胞/ml。
在另一优选例中，步骤(c)中，所述细胞因子选自下组中的一种或多种：
10-1000IU重组人白介素2、5-50ng/ml重组人白介素7、5-50ng/ml重组人白介素12、5-50ng/ml重组人白介素15、5-50ng/ml重组人白介素21、1-10ng/ml重组人白介素1β。
在另一优选例中，步骤(c)中，所述培养天数选自5-21天，较佳地6-17天，更佳地7-14天。
在另一优选例中，步骤(b)、(c)、和(d)中，所述适合DNT细胞生长的培养基的培养体系中不含有抗人CD3抗体，如OKT3。
在另一优选例中，步骤(b)、(c)、和(d)中的培养体系中含有200-1000IU/mL(较佳地为300-700IU/mL，更佳地为500IU/mL)的重组人白介素2。
在另一优选例中，步骤(b)、(c)、和(d)中，所述适合DNT细胞生长的培养基的培养体系中不含血清。
在另一优选例中，步骤(b)、(c)、和(d)中，所述适合DNT细胞生长的培养基的培养体系中含有选自下组的血清替代物：ICSR(Immune Cell Serum Replacement)、KSR(KnockOut TMSerum Replacement)。
在另一优选例中，步骤(b)、(c)、和(d)中，所述ICSR的浓度(v/v)为2％-30％。
在另一优选例中，所述培养基选自下组：AIM-V、X-VIVO-10、X-VIVO-15、Aly505、GT551培养基。
在另一优选例中，所述培养基中还添加了选自下组的分子：1-10μg/ml的重组人转铁蛋白，1-10μg/ml重组人胰岛素，10-100μg/ml的抗坏血酸，1-5μg/ml的乙醇胺，1-5μg/ml的亚油酸，1-5μg/ml的油酸。
在另一优选例中，步骤(c)中，所述培养基中包含：
(1)200-1000IU/mL重组人白介素2；
(2)0％-20％，较佳地5％-15％(v/v)ICSR；
(3)选自下组的一种或几种细胞因子：
10-1000IU，较佳地50-700IU重组人白介素2、
1-50ng/ml，较佳地5-25ng/ml重组人白介素7、
1-50ng/ml，较佳地5-25ng/ml重组人白介素12、
1-50ng/ml，较佳地5-25ng/ml重组人白介素15、
1-50ng/ml，较佳地5-25ng/ml重组人白介素21、
1-10ng/ml，较佳地1.5-8.5ng/ml重组人白介素1β；和
(4)选自下组的一种或几种添加因子：
0.5-15μg/ml，较佳地1-10μg/ml的重组人转铁蛋白，
0.5-15μg/ml，较佳地1-10μg/m重组人胰岛素，
10-100μg/ml，较佳地15-60μg/ml的抗坏血酸，
0.5-10μg/ml，较佳地1-5μg/ml的乙醇胺，
0.5-10μg/ml，较佳地1-5μg/ml的亚油酸，
0.1-5μg/ml，较佳地1.5-2.5μg/ml的油酸。
在本发明的第二方面，提供了一种DNT细胞群，所述DNT细胞群是由本发明第一方面所述的方法制备的。
在另一优选例中，所述DNT细胞群具有选自下组的一个或多个特征：
(a1)40％-80％的细胞为Tscm细胞；
(b1)10％-40％的细胞为Tcm细胞；或
(a2)45％-75％的细胞为Tscm细胞；
(b2)5％-35％的细胞为Tcm细胞；或
(a3)30％-60％的细胞为Tscm细胞；
(b3)15％-40％的细胞为Tcm细胞；或
(a4)20％-40％的细胞为Tscm细胞；
(b4)20％-30％的细胞为Tcm细胞。
在另一优选例中，所述DNT细胞群中，Tscm细胞和Tcm细胞比例≥45％；较佳地≥50％；较佳地≥70％；更佳地≥75％。
在另一优选例中，所述DNT细胞存活率≥70％；较佳地≥80％；更佳地≥90％；更佳地≥95％。
在另一优选例中，所述DNT细胞(CD3 +)纯度(％)≥80％；较佳地≥90％；更佳地≥95％；更佳地≥97％。
在另一优选例中，所述DNT细胞(CD3 +CD4 -CD8 -)纯度(％)≥85％；较佳地≥90％；更佳地≥95％；更佳地≥97％。
在本发明的第三方面，提供了一种如本发明第二方面所述的DNT细胞的用途，用于制备一药物组合物或制剂，所述药物组合物或制剂用于：
(a)预防和/或治疗肿瘤；
(b)预防和/或治疗感染性疾病；
(c)预防和/或治疗自身免疫性疾病；
(d)预防和/或治疗移植物抗宿主疾病；和/或
(e)调节免疫应答。
在另一优选例中，所述肿瘤为与所述DNT细胞异体的肿瘤。
在另一优选例中，所述肿瘤选自下组：血液肿瘤、实体瘤、或其组合。
在另一优选例中，所述血液肿瘤选自下组：淋巴瘤(Hodgkins和非Hodgkins)、急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、慢性髓性白血病(CML)、慢性骨髓单核细胞性白血病(CMML)、骨髓增生异常综合征(MDS)，或其组合。
在另一优选例中，所述实体瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、黑素瘤、骨癌、前列腺癌、结直肠癌、乳腺癌、大 肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、头颈癌、胰腺癌，或其组合。
在另一优选例中，所述自身免疫性疾病包括：糖尿病、类风湿关节炎、多发性硬化、系统性红斑狼疮、恶心贫血、溶血性贫血、自身免疫性血小板减少、自身免疫性肝病、强直性脊柱炎、重症肌无力、Ig A肾病、原发肾冰综合征、银屑病、白癜风。
在本发明的第四方面，提供了一种细胞制剂，所述细胞制剂含有如本发明的第二方面所述的DNT细胞群。
在另一优选例中，所述的细胞制剂包括所述的DNT细胞群和药学上可接受的载体。
在本发明的第五方面，提供了一种适合DNT细胞生长的培养基，所述培养基包含选自下组细胞因子中的一种或多种：
5-50ng/ml重组人白介素21、1-10ng/ml重组人白介素1β、5-50ng/ml重组人白介素7、5-50ng/ml重组人白介素15、5-50ng/ml重组人白介素12；
其中，所述培养基中不添加抗人CD3抗体。
在另一优选例中，所述适合DNT细胞生长的培养基中含有选自下组的血清替代物：ICSR(Immune Cell Serum Replacement)、KSR(KnockOut TMSerum Replacement)。
在另一优选例中，所述培养基包含选自下组中的一种或多种：
10-1000IU重组人白介素2、5-50ng/ml重组人白介素7、5-50ng/ml重组人白介素12、5-50ng/ml重组人白介素15、5-50ng/ml重组人白介素21、1-10ng/ml重组人白介素1β。
在另一优选例中，所述培养基选自下组：AIM-V、X-VIVO-10、X-VIVO-15、Aly505、GT551培养基。
在另一优选例中，所述培养基中包含：
(1)200-1000IU/mL重组人白介素2；
(2)0％-20％，较佳地5％-15％(v/v)ICSR；
(3)选自下组的一种或几种细胞因子：
10-1000IU，较佳地50-700IU重组人白介素2、
1-50ng/ml，较佳地5-25ng/ml重组人白介素7、
1-50ng/ml，较佳地5-25ng/ml重组人白介素12、
1-50ng/ml，较佳地5-25ng/ml重组人白介素15、
1-50ng/ml，较佳地5-25ng/ml重组人白介素21、
1-10ng/ml，较佳地1.5-8.5ng/ml重组人白介素1β；和
(4)选自下组的一种或几种添加因子：
0.5-15μg/ml，较佳地1-10μg/ml的重组人转铁蛋白，
0.5-15μg/ml，较佳地1-10μg/m重组人胰岛素，
10-100μg/ml，较佳地15-60μg/ml的抗坏血酸，
0.5-10μg/ml，较佳地1-5μg/ml的乙醇胺，
0.5-10μg/ml，较佳地1-5μg/ml的亚油酸，
0.1-5μg/ml，较佳地1.5-2.5μg/ml的油酸。
在本发明的第六方面，提供了一种(a)预防和/或治疗肿瘤；(b)预防和/或治疗感染性疾病；(c)预防和/或治疗自身免疫性疾病；(d)预防和/或治疗移植物抗宿主疾病；和/或(e)调节免疫应答的方法，给有需要的受试者施用如本发明的第二方面所述的DNT细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了本发明的工艺流程图。
图2显示了DNT细胞增殖曲线(图2A)以及细胞活率曲线(图2B)。
图3A和图3B显示了DNT细胞纯度变化曲线(CD3 +％、CD4 -CD8 -％)。
图3C显示了第11天DNT细胞纯度流式图。
图4显示了DNT细胞分化曲线以及第11天DNT细胞分化流式图
图5显示了体外扩增第10天DNT细胞与MV411效靶比4：1，共孵育2小时的细胞杀瘤活性。
图6显示了体外扩增第10天DNT细胞与Hela细胞效靶比5：1共孵育第20小时 的实时杀瘤曲线图。
图7显示了DNT细胞增殖曲线(图7A)以及细胞活率曲线(图7B)。
图8A和图8B显示了DNT细胞纯度变化曲线，以及图8C显示了第11天DNT细胞纯度流式图
图9显示了DNT细胞分化曲线以及第11天DNT细胞分化流式图
图10显示了体外扩增第10天DNT细胞与MV411效靶比4：1，共孵育2小时的细胞杀瘤活性
图11显示了体外扩增第10天DNT细胞与Hela细胞效靶比5：1共孵育第20小时的实时杀瘤曲线图。
图12显示了DNT细胞增殖曲线(图12A)以及细胞存活率曲线(图12B)。
图13显示了DNT细胞纯度变化曲线以及第11天DNT细胞纯度流式图(图13C)。
图14显示了DNT细胞分化曲线以及第11天DNT细胞分化流式图。
图15显示了体外扩增第10天DNT细胞与MV411效靶比4：1，共孵育2小时的细胞杀瘤活性。
图16显示了体外扩增第10天DNT细胞与Hela细胞效靶比5：1共孵育第20小时的实时杀瘤曲线图。
图17显示了4个供者新工艺与旧工艺DNT细胞增殖(图17A)、细胞存活率变化曲线(图17B)(n＝4)。
图18显示了4个供者新工艺与旧工艺DNT细胞纯度变化曲线差异分析(n＝4)(18A-B)；以及第10天供者2的新工艺(18C)和旧工艺(18D)DNT细胞纯度流式图。
图19显示了荧光素标记的抗人CD45RA/CD62L抗体，采用流式细胞仪检测4个供者新工艺与旧工艺DNT细胞扩增第7天、第10天、第14天Tscm/Tcm/Tem/Teff细胞比例变化曲线、差异分析(n＝4)；以及第10天供者2的DNT细胞Tscm/Tcm/Tem/Teff细胞分化流式图。
图20显示了4个供者体外扩增第10天DNT细胞与MV411效靶比4：1共孵育2小时的细胞杀瘤活性(n＝4)。
具体实施方式
本发明人经过广泛而深入地研究，经过大量的工艺优化实验，首次开发了 通过含有成分明确的添加因子的无血清培养基，进行体外大规模扩增富含Tscm和Tcm特征的双阴性T细胞的方法。具体地，提供一种源自健康捐赠者外周血，用一种或多种无血清培养基，加入成分明确、无异种成分的添加因子，并且在DNT培养体系的扩增阶段不额外添加可溶性抗人CD3抗体，从而体外大规模扩增富含Tscm和Tcm细胞特征的双阴性T细胞，以进一步提高异体DNT细胞临床应用时的长效抗肿瘤作用。在此基础上完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
如本文所用，术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
如本文所用，术语“给予”、“施用”可互换使用，是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者，包括静脉内、肌内、皮下、腹膜内、脊髓或其它肠胃外给药途径，例如通过注射或输注。
本发明的体外扩增方法
本发明的工艺流程如下(图1)：
(1)从外周血中体外分离纯化DNT细胞。
(2)DNT细胞的激活
D0-2天，
20ng/ml-20μg/ml的抗人CD3单克隆抗体包被在固体介质，培养基中加入200-1000IU/mL重组人白介素2
2％-30％(v/v)ICSR；
1-10μg/ml的重组人转铁蛋白，
1-10μg/ml重组人胰岛素，
10-100μg/ml的抗坏血酸，
1-5μg/ml的乙醇胺，
1-5μg/ml的亚油酸，
0.1-5μg/ml的油酸。
(3)体外扩增富含Tscm/Tcm的DNT细胞
D3-14天，适合培养DNT细胞的培养基，其中加入：
200-1000IU/mL重组人白介素2；
0％-20％(v/v)ICSR；
选自下组的一种或几种细胞因子：10-1000IU重组人白介素2、5-50ng/ml重组人白介素7、5-50ng/ml重组人白介素12、5-50ng/ml重组人白介素15、5-50ng/ml重组人白介素21、1-10ng/ml重组人白介素1β；
选自下组的一种或几种添加因子：1-10μg/ml的重组人转铁蛋白，1-10μg/ml重组人胰岛素，10-100μg/ml的抗坏血酸，1-5μg/ml的乙醇胺，1-5μg/ml的亚油酸，1-5μg/ml的油酸。
(4)洗涤、收集细胞
D14-17天用250ml尖底离心瓶收集DNT细胞，900g 10Min离心，再以溶媒洗涤
(5)制备富含Tscm/Tcm的DNT细胞制剂
以溶媒调整为0.5～1×10 8个细胞/mL的浓度，此为DNT细胞制剂成品，质检合格后可供临床使用。
特别地，在DNT细胞的激活中，抗人CD3单克隆抗体包被在固体介质上，包括但不限于培养介质、磁珠上。较佳地，所述抗人CD3单克隆抗体可以与其他抗体(例如抗人CD28单克隆抗体)共同包被于固体介质来激活DNT细胞。在本发明的一个具体实施例中，使用CD3/CD28磁珠来激活DNT细胞，其中，所述CD3/CD28磁珠包括同一种磁珠表面包被有抗人CD3的单克隆抗体和抗人CD28的单克隆抗体，或两种磁珠分别包被抗人CD3的单克隆抗体和抗人CD28的单克隆抗体。
在体外扩增富含Tscm/Tcm的DNT细胞时，不添加抗人CD3抗体，较佳地不添加可溶性抗人CD3单克隆抗体，例如OKT3。
DNT细胞群
本发明提供了一种富含Tscm(Stem Cell-Like Memory T,干细胞样记忆T) 和Tcm(Central Memory T,中央记忆T)特征的双阴性T细胞(Double Negative T,DNT)，即本发明的DNT细胞群。其中，所述DNT细胞群是由本发明第一方面所述的方法制备的。其具有选自下组的一个或多个特征：
(a1)40％-80％的细胞为Tscm细胞；
(b1)10％-40％的细胞为Tcm细胞；或
(a2)45％-75％的细胞为Tscm细胞；
(b2)5％-35％的细胞为Tcm细胞；或
(a3)30％-60％的细胞为Tscm细胞；
(b3)15％-40％的细胞为Tcm细胞；或
(a4)20％-40％的细胞为Tscm细胞；
(b4)20％-30％的细胞为Tcm细胞。
在另一优选例中，所述DNT细胞群中，Tscm细胞和Tcm细胞比例≥45％；较佳地≥50％；较佳地≥70％；更佳地≥75％。
在另一优选例中，所述DNT细胞存活率≥70％；较佳地≥80％；更佳地≥90％；更佳地≥95％。
在另一优选例中，所述DNT细胞(CD3 +)纯度(％)≥80％；较佳地≥90％；更佳地≥95％；更佳地≥97％。
在另一优选例中，所述DNT细胞(CD3 +CD4 -CD8 -)纯度(％)≥85％；较佳地≥90％；更佳地≥95％；更佳地≥97％。
本发明的DNT细胞群中，Tscm与Tcm细胞比例显著或极显著的优于现有技术中的细胞比例，并且Teff效应杀伤细胞的比例显著或极显著低于现有技术。因此具有更强的更新、分化和存活能力。
药物组合物和施用方法
本发明还提供了含有本发明的的DNT细胞的用途，用于制备一药物组合物或制剂，所述药物组合物或制剂可以用于治疗选自下组的疾病：肿瘤、感染性疾病、自身免疫性疾病、移植物抗宿主疾病。
在另一优选例中，所述血液肿瘤选自下组：淋巴瘤(Hodgkins和非Hodgkins)、急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、 急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、慢性髓性白血病(CML)、慢性骨髓单核细胞性白血病(CMML)、骨髓增生异常综合征(MDS)，或其组合。
在另一优选例中，所述实体瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、黑素瘤、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、头颈癌、胰腺癌，或其组合。
在另一优选例中，所述自身免疫性疾病包括：糖尿病、类风湿关节炎、多发性硬化、系统性红斑狼疮、恶心贫血、溶血性贫血、自身免疫性血小板减少、自身免疫性肝病、强直性脊柱炎、重症肌无力、Ig A肾病、原发肾冰综合征、银屑病、白癜风
本发明还提供了一种药物组合物，它含有安全有效量的本发明的细胞制剂，以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、粉剂、二甲基亚砜(DMSO)及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。活性成分的给药量是治疗有效量。此外，本发明的细胞制剂还可与其他治疗剂一起使用。
对于本发明的药物组合物，可通过常规的方式施用于所需的对象(如人和非人哺乳动物)。代表性的施用方式包括(但并不限于)：静脉注射、动脉注射、胸腔、腹盆腔、蛛网膜下腔、鼻窦、颅内、不同组织(如肿瘤组织、炎症病变组织)等不同部位的注射等。
本发明的主要优点包括：
(1)扩增工艺简单，稳定，可实现体外大规模培养富含Tscm和Tcm细胞特征的DNT细胞，终产品DNT细胞纯度(CD3+CD4-CD8-)≥85％，满足临床使用需求。
(2)添加成分明确，整个培养过程使用成分明确的无血清培养基，且在培养过程中不添加任何成分的动物源或者人源等成分不明确的血清或血浆，为异体DNT细胞的临床应用安全性提供更佳保障，使其成为一款真正的质量可控、安全可靠的通用型细胞治疗产品。
(3)培养获得富含Tscm和Tcm细胞特征的DNT细胞，其中DNT细胞中Tscm细胞和Tcm细胞比例≥50％。
(4)在DNT培养体系的扩增阶段无需加入鼠源抗人CD3抗体(克隆号OKT3)，进一步降低终产品中的异种蛋白残留，提升产品安全性和质量。
(5)可从新鲜富集的DNT细胞或冻存后复苏的DNT细胞体外扩增细胞。
下面结合具体实施例，进一步陈述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1：抗人CD3单克隆抗体激活DNT细胞体外扩增培养
1.1健康捐赠者DNT细胞富集
从健康捐赠者收集20～200ml外周血至含肝素钠管中。利用
Figure PCTCN2022130054-appb-000001
试剂盒(Stem Cell Technologies Inc)，去除CD4 +、CD8 +T细胞，如此获得的细胞即为去除CD4 +与CD8 +的DNT细胞。
1.2 DNT细胞的激活
第0-2天，用抗人CD3单克隆抗体(10μg/mL)(克隆号为OKT3)包被T25培养瓶，将步骤1.1获得的DNT细胞用DNT细胞专用无血清培养基(AIM-V基础培养基，添加8μg/ml的重组人转铁蛋白，8μg/ml重组人胰岛素，30μg/ml的抗坏血酸，1.5μg/ml的乙醇胺，1μg/ml的亚油酸，1μg/ml的油酸)添加10％(v/v)ICSR和500IU/mL的重组人白介素2，调整成1×10 6～4×10 6个细胞/mL的浓度，放入前述包被好的T25培养瓶中。于37℃的5％CO 2培养箱中培养。
1.3体外扩增富含Tscm和Tcm细胞特征的DNT细胞
第3-6天，用DNT细胞专用无血清培养基(AIM-V基础培养基添加8μg/ml的重组人转铁蛋白，8μg/ml重组人胰岛素，30μg/ml的抗坏血酸，1.5μg/ml的乙醇胺，1μg/ml的亚油酸，1μg/ml的油酸)添加10％(v/v)ICSR和500IU/mL的重组人白介素2，2ng/ml重组人白介素1β，5ng/ml重组人白介素21，将DNT细胞调整成1×10 6～4×10 6个细胞/mL的浓度，据细胞体积调整为T75培养瓶或者T175培养瓶继续培养。
第7-14天，用DNT细胞专用无血清培养基(AIM-V基础培养基添加5μg/ml的重组人转铁蛋白，5μg/ml重组人胰岛素，50μg/ml的抗坏血酸，1.5μg/ml的乙醇胺，1μg/ml的亚油酸，1μg/ml的油酸)添加5％(v/v)ICSR和500IU/mL的重组人白介素2，5ng/ml重组人白介素7，5ng/ml重组人白介素12，将DNT细胞调整成1×10 6～4×10 6个细胞/mL的浓度，后续依据细胞体积调整T175培养瓶或者2L培养袋继续培养。
第13～14天视需要，收获DNT细胞。
1.4收获DNT细胞以制成DNT细胞制剂
第13～14天，视需要，收获DNT细胞。用250ml尖底离心瓶收集细胞，900×g离心10分钟，再以溶媒洗涤。将收集到的DNT细胞以溶媒调整为0.1～1×10 8个细胞/mL的浓度，此为DNT细胞制剂成品，质检合格后可供临床使用。
实验结果如图2-图6，表1所示。其中，
(1)通过AO/PI染色方法检测扩增第7、9、11和第14天DNT细胞的生长曲线(图2A)以及细胞存活率(图2B)。
结果表明，第7、9、11、14天时，细胞体外扩增倍数分别高达74倍、416.6倍、2233.8倍、8071.5倍；细胞的存活率分别为91.93％、95.43％、87.99％、87.55％。
(2)采用流式细胞仪检测DNT细胞扩增第7、9、11和第14天DNT细胞纯度变化曲线以及第11天DNT细胞CD3 +％和CD3 +CD4 -CD8 -％流式图。结果如图3所示，其中，图3A和图3B显示了DNT细胞纯度变化曲线(CD3 +％、CD3 +CD4 -CD8 -％)；图3C显示了第11天DNT细胞纯度流式图。
如下表所示，结果表明使用本发明的培养方法，体外扩增第11天获得较高的DNT细胞纯度(CD3 +％＝97.80％,CD3 +CD4 -CD8 -％＝93.70％)。
(3)使用荧光素标记的抗人CD45RA/CD62L抗体，用流式细胞仪检测DNT细胞扩增第7、9、11和第14天时Tscm/Tcm/Tem/Teff细胞的比例(图4A)以及第11天Tscm/Tcm/Tem/Teff细胞分化流式图(图4B)。
结果表明，体外扩增第11天获得的DNT细胞Tscm的比例为48.10％，Tcm的比例为29.60％。
表1.DNT细胞的扩增倍数、存活率、纯度以及DNT细胞分化比例
Figure PCTCN2022130054-appb-000002
Figure PCTCN2022130054-appb-000003
(4)PKH-26对MV411细胞株进行标记后与体外扩增第10天的DNT细胞效靶比4：1共孵育2小时，采用流式细胞术设门标记MV411细胞，分析靶细胞凋亡分析DNT细胞对MV411细胞的杀瘤活性(图5)。
结果表明，体外扩增第10天的DNT细胞与MV411效靶比4：1孵育2小时，对MV411细胞的杀瘤活性高达92.3％。
(5)采用RTCA方法(通过特殊工艺,将微电子细胞传感器芯片整合到细胞检测板的底部，用以构建实时、动态、定量跟踪细胞形态和增殖分化等改变的细胞阻抗检测传感系统。)检测DNT细胞对Hela细胞的实时杀瘤曲线(图6)。
结果表明，体外扩增第10天DNT细胞与Hela细胞效靶比5：1共孵育过夜，共孵育第20小时的细胞杀瘤活性为69.1％。
实施例2：磁珠激活DNT细胞体外扩增培养
2.1健康捐赠者DNT细胞富集
参考实施列1中的1.1部分
2.2 DNT细胞的激活
第0-2天，将分离纯化的DNT细胞(磁珠：DNT细胞＝1:1)与CD3/CD28磁珠(同一种磁珠表面包被有抗人CD3的单克隆抗体和抗人CD28的单克隆抗体，或两种磁珠分别包被抗人CD3的单克隆抗体和抗人CD28的单克隆抗体)混匀接种于T25培养瓶，用DNT细胞专用无血清培养基(GT551基础培养基添加3μg/ml的重组人转铁蛋白，3μg/ml重组人胰岛素，70μg/ml的抗坏血酸，2μg/ml的乙醇胺，0.5μg/ml的亚油酸，0.5μg/ml的油酸)添加20％(v/v)ICSR和1000IU/mL的重组人白介素2，调整成1×10 6～4×10 6个细胞/mL的浓度，放入前述包被好的T25培养瓶中。于37℃的5％CO 2培养箱中培养。
2.3体外扩增富含Tscm和Tcm细胞特征的DNT细胞
第3-6天，用DNT细胞专用无血清培养基(GT551基础培养基添加3μg/ml的重组人转铁蛋白，3μg/ml重组人胰岛素，70μg/ml的抗坏血酸，2μg/ml的乙醇胺，0.5μg/ml的亚油酸，0.5μg/ml的油酸)添加20％ICSR和1000IU/mL的重组人白介素2，5ng/ml重组人白介素1β，3ng/ml重组人白介素21，将DNT细胞调整成1×10 6～4×10 6个细胞/mL的浓度，据细胞体积调整为T75培养瓶或者T175培养瓶继续培养。
第7-14天，用DNT细胞专用无血清培养基(GT551基础培养基添加8μg/ml的重组人转铁蛋白，8μg/ml重组人胰岛素，50μg/ml的抗坏血酸，1μg/ml的乙醇胺，1μg/ml的亚油酸，1μg/ml的油酸)1000IU/mL的重组人白介素2，5ng/ml重组人白介素7，5ng/ml重组人白介素15，将DNT细胞调整成1×10 6～4×10 6个细胞/mL的浓度依据细胞体积调整T175培养瓶或者2L培养袋继续培养。
第13～14天视需要，收获DNT细胞。
2.4收获DNT细胞以制成DNT细胞制剂
第13～14天，视需要，收获DNT细胞。用250ml尖底离心瓶收集细胞，900×g离心10分钟，再以溶媒洗涤。将收集到的DNT细胞以溶媒调整为0.5～1×10 8个细胞/mL的浓度，此为DNT细胞制剂成品，质检合格后可供临床使用。
实验结果如图7-图11所示。其中，
(1)AO/PI染色方法检测扩增第7、9、11和第14天DNT细胞的生长曲线(图7A)以及细胞存活率(图7B)。
(2)CD3/CD4/CD8抗体标记，流式细胞仪检测DNT细胞扩增第7天、第9天、第11天、第14天DNT细胞纯度变化曲线以及第11天DNT细胞CD3 +％和CD3 +CD4 -CD8 -％流式图。图8A和图8B显示了DNT细胞纯度变化曲线，以及图8C显示了第11天DNT细胞纯度流式图。如下表所示，结果表明使用本发明的培养方法，体外扩增第11天获得的DNT细胞纯度为CD3 +％＝98.50％,CD3 +CD4 -CD8 -％＝89.20％。
(3)荧光素标记的抗人CD45RA/CD62L抗体，采用流式细胞仪检测DNT细胞扩增第7天、第9天、第11天、第14天Tscm/Tcm/Tem/Teff细胞比例(图9A)以及第11天Tscm/Tcm/Tem/Teff细胞分化流式图(图9B)。
实验结果如下表2所示：
表2.DNT细胞的扩增倍数、存活率、纯度以及DNT细胞分化比例
Figure PCTCN2022130054-appb-000004
Figure PCTCN2022130054-appb-000005
(4)PKH-26对MV411细胞株进行标记后与体外扩增第10天的DNT细胞效靶比4：1共孵育2小时，采用流式细胞术设门标记MV411，分析靶细胞凋亡分析DNT细胞对MV411细胞的杀瘤活性(图10)。
结果表明，体外扩增第10天DNT细胞与MV411效靶比4：1孵育，共孵育2小时时，对MV411细胞的杀瘤活性为34.80％。
(5)采用RTCA方法检测DNT细胞对Hela细胞的实时杀瘤曲线(图11)。
结果表明，体外扩增第10天DNT细胞与Hela细胞效靶比5：1共孵育过夜，共孵育第20小时的杀瘤性为65.40％。
实施例3：抗人CD3单克隆抗体激活DNT细胞体外扩增培养
3.1健康捐赠者DNT细胞富集
参考实施列1中的1.1部分
3.2 DNT细胞的激活
用抗人CD3单克隆抗体(10μg/mL)包被T25培养瓶，将步骤1.1获得的DNT细胞用DNT细胞专用无血清培养基(Aly505基础培养基添加5μg/ml的重组人转铁蛋白，5μg/ml重组人胰岛素，50μg/ml的抗坏血酸，1.5μg/ml的乙醇胺，1μg/ml的亚油酸，1μg/ml的油酸)添加10％ICSR和500IU/mL的重组人白介素2，调整成1×10 6～4×10 6个细胞/mL的浓度，放入前述包被好的T25培养瓶中。于37℃的5％CO 2培养箱中培养。
3.3体外扩增富含Tscm和Tcm细胞特征的DNT细胞
第3-6天，用DNT细胞专用无血清培养基(Aly505基础培养基添加5μg/ml的重组人转铁蛋白，5μg/ml重组人胰岛素，50μg/ml的抗坏血酸，1.5μg/ml的乙醇 胺，1μg/ml的亚油酸，1μg/ml的油酸)添加10％ICSR和500IU/mL的重组人白介素2，5ng/ml重组人白介素7，5ng/ml重组人白介素15，将DNT细胞调整成1×10 6～4×10 6个细胞/mL的浓度，据细胞体积调整为T75培养瓶或者T175培养瓶继续培养。
第7-14天，用DNT细胞专用无血清培养基(Aly505基础培养基添加3μg/ml的重组人转铁蛋白，3μg/ml重组人胰岛素，30μg/ml的抗坏血酸，1μg/ml的乙醇胺，0.5μg/ml的亚油酸，0.5μg/ml的油酸)添加10％ICSR和500IU/mL的重组人白介素2，5ng/ml重组人白介素7，5ng/ml重组人白介素15，将DNT细胞调整成1×10 6～4×10 6个细胞/mL的浓度据细胞体积调整T175培养瓶或者2L培养袋继续培养。
第13～14天视需要，收获DNT细胞。
3.4收获DNT细胞以制成DNT细胞制剂
第13～14天，视需要，收获DNT细胞。用250ml尖底离心瓶收集细胞，900×g离心10分钟，再以溶媒洗涤。将收集到的DNT细胞以溶媒调整为0.5～1×10 8个细胞/mL的浓度，此为DNT细胞制剂成品，质检合格后可供临床使用。
实验结果如图12-图16所示。其中，
(1)AO/PI染色方法检测扩增第7、9、11和第14天DNT细胞的生长曲线(图12A)以及细胞存活率(图12B)。
(2)荧光素标记的抗人CD3/CD4/CD8抗体，采用流式细胞仪检测DNT细胞扩增第7天、第9天、第11天、第14天DNT细胞纯度变化曲线以及第11天DNT细胞CD3 +％和CD3 +CD4 -CD8 -％流式图。图13A和图13B显示了DNT细胞纯度变化曲线，以及图13C显示了第11天DNT细胞纯度流式图。
(3)荧光素标记的抗人CD45RA/CD62L抗体，采用流式细胞仪检测DNT细胞扩增第7天、第9天、第11天、第14天Tscm/Tcm/Tem/Teff细胞比例(图14A)以及第11天Tscm/Tcm/Tem/Teff细胞分化流式图(图14B)。
实验结果如下表3所示：
表3.表1.DNT细胞的扩增倍数、存活率、纯度以及DNT细胞分化比例
Figure PCTCN2022130054-appb-000006
Figure PCTCN2022130054-appb-000007
(4)PKH-26对MV411细胞株进行标记后与体外扩增第10天的DNT细胞效靶比4：1共孵育2小时，采用流式细胞术设门标记MV411，分析靶细胞凋亡分析DNT细胞对MV411细胞的杀瘤活性(图15)。
结果表明，体外扩增第10天DNT细胞与MV411效靶比4：1孵育，共孵育2小时时，对MV411细胞的杀瘤活性为78.40％。
(5)采用RTCA方法检测DNT细胞对Hela细胞的实时杀瘤曲线(图16)。
结果表明，体外扩增第10天DNT细胞与Hela细胞效靶比5：1共孵育过夜，共孵育第20小时的杀瘤活性为54.30％。
4、对比例：
本发明专利为体外大规模扩增富含Tscm(Stem Cell-Like Memory T,干细胞样记忆T)和Tcm(Central Memory T,中央性记忆T)的细胞特征的双阴性T(Double Negative T,DNT)细胞的方法(下称新工艺)与我公司已有专利(CN104109653A)，即利用无动物血清培养体系大规模扩增人外周血DNT细胞的方法(下称旧工艺)进行对比：
从4名健康捐赠者收集20～200ml外周血到含肝素钠管中。利用
Figure PCTCN2022130054-appb-000008
试剂盒(Stem Cell Technologies Inc)，去除CD4 +、CD8 +T细胞，如此获得的细胞即为去除CD4+与CD8+的DNT细胞。
4.1新工艺方法激活扩增DNT细胞：采用实施例3方法生产扩增DNT细胞
4.2旧工艺方法激活扩增DNT细胞：
4.2.1体外DNT细胞激活
用抗人CD3单克隆抗体(10μg/mL)包被T25培养瓶，将去除CD4+与CD8+的DNT细胞在T25培养瓶中用DNT无血清培养基(AIM-V基础培养基添加500IU/mL的重组人白介素2，2ng/ml重组人白介素4，8ng/ml重组人白介素7，5ng/ml重组人白介素12，5v％自体血清和6v％自体血清)调整成1×10 6～4×10 6个细胞/mL的浓度，在37℃的5％CO 2培养箱中培养3天。
4.2.2体外扩增DNT细胞
第3-6天，用DNT无血清培养基(AIM-V基础培养基添加500IU/mL的重组 人白介素2，2ng/ml重组人白介素4，8ng/ml重组人白介素7，5ng/ml重组人白介素12，5v％自体血清和6v％自体血清)调整成1×10 6～4×10 6个细胞/mL的浓度，据细胞体积调整为T75培养瓶或者T175培养瓶继续培养。
第7天，用DNT无血清培养基(A IM-V基础培养基添加500IU/mL的重组人白介素2，2ng/ml重组人白介素4，8ng/ml重组人白介素7，5ng/ml重组人白介素12，5v％自体血清和6v％自体血清)额外添加50ng/ml可溶性抗人CD3单克隆抗体，调整成1×10 6～4×10 6个细胞/mL的浓度据细胞体积调整T175培养瓶继续培养。
第10-14天，用DNT无血清培养基(GT551基础培养基添加500IU/mL的重组人白介素2，2ng/ml重组人白介素4，8ng/ml重组人白介素7，5ng/ml重组人白介素12，5v％自体血清和6v％自体血清)额外添加50ng/ml可溶性抗人CD3单克隆抗体，调整成1×10 6～4×10 6个细胞/mL的浓度，据细胞体积调整T175培养瓶或者2L培养袋继续培养。
第13～14天视需要，收获DNT细胞。
4.2.3收获DNT细胞以制成DNT细胞制剂
第13～14天，视需要，收获DNT细胞。用250ml尖底离心瓶收集细胞，900×g离心10分钟，再以溶媒洗涤。将收集到的DNT细胞以溶媒调整为0.5～1×10 8个细胞/mL的浓度，此为DNT细胞制剂成品，质检合格后可供临床使用。
4.2.4新工艺和旧工艺扩增DNT细胞功能检测以及结果分析：
(1)用AO/PI染色方法检测4个供者新工艺与旧工艺扩增第7天、第10天、第14天DNT细胞的生长曲线以及细胞存活率变化曲线、差异分析(n＝4)(图17A-B)。
与旧工艺相比，采用新工艺制备产品培养第14天DNT细胞扩增倍数、细胞存活率无显著差异。
(2)用荧光素标记的抗人CD3/CD4/CD8抗体，采用流式细胞仪检测4个供者新工艺与旧工艺扩增第7天、第10天、第14天DNT细胞纯度(CD3 +CD4 -CD8 -％)(n＝4)(图18)。
结果如图18所示，其中图18A-B显示了CD3/CD4/CD8抗体标记，流式细胞仪检测4个供者新工艺与旧工艺DNT细胞扩增第7天、第10天、第14天DNT细胞纯度变化曲线、差异分析(n＝4)；以及供者2的新工艺(18C)和旧工艺(18D)扩增第10天DNT细胞纯度流式图(CD3 +％和CD3 +CD4 -CD8 -％)。
与旧工艺相比，采用新工艺制备产品培养第14天收获的DNT细胞纯度 (CD3 +CD4 -CD8 -％)无显著差异。
(3)用荧光素标记的抗人CD45RA/CD62L抗体，采用流式细胞仪检测4个供者新工艺与旧工艺扩增第7天、第10天、第14天Tscm/Tcm/Tem/Teff(Tscm＝CD45RA +/CD62L +、Tcm＝CD45RA -/CD62L +、Tem＝CD45RA -/CD62L -、Tem＝CD45RA +/CD62L -)细胞分化比例(n＝4)(图19)。
结果如图19和表4所示，其中图19中的A-C显示了CD45RA/CD62L抗体标记，采用流式细胞仪检测4个供者新工艺与旧工艺体外扩增第7天、第10天、第14天DNT细胞的Tscm/Tcm/Tem/Teff细胞比例变化、差异分析(n＝4)；图19中的D-E显示了体外扩增第10天供者2的DNT细胞Tscm/Tcm/Tem/Teff细胞分化流式图。
与旧工艺相比，采用新工艺扩增的DNT细胞中Tscm与Tcm细胞比例显著或极显著的高于旧工艺，而Teff效应杀伤细胞的比例显著或极显著低于旧工艺(如图19，表4所示)。
表4 DNT细胞中Tscm、Tcm、Tem细胞和Teff效应杀伤细胞比例
    第7天 第10天 第14天
Tscm％ 新工艺 27.27 25.95 29.98
  旧工艺 6.63 9.60 10.93
Tcm％ 新工艺 19.23 15.15 13.80
  旧工艺 5.80 7.23 3.28
Tem％ 新工艺 33.87 29.90 23.10
  旧工艺 53.10 35.65 19.53
Teff％ 新工艺 19.63 28.98 33.15
  旧工艺 31.75 47.23 65.88
(4)采用流式细胞术检测4个供者新工艺与旧工艺扩增第10天DNT细胞与MV411细胞效靶比4：1的细胞杀瘤活性(n＝4)(图20)。
图20显示了4个供者体外扩增第10天DNT细胞与MV411效靶比4：1共孵育2小时的细胞杀瘤活性、差异分析(n＝4)。
第10天细胞杀瘤活性结果显示，采用新工艺扩增获得的DNT细胞对MV411细胞的杀瘤活性显著高于旧工艺。
综上所述，新工艺优于旧工艺的的主要点在于：
1、添加的更适合DNT细胞生长的细胞因子及培养成分；
2、扩增阶段不使用鼠源可溶性抗人CD3抗体，减少鼠源成分残留，进一步提高产品安全性；
3、整个生产工艺中用商业来源成分明确的血清替代物(例如ICSR或KSR)替换健康捐赠者血浆以保证不同产品批间一致性；
4、培养获得富含Tscm和Tcm特征的DNT细胞，其具有更强的自我更新、分化和长久存活能力，可在体内长期存留，起到长效抗肿瘤作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 一种富含Tscm细胞和Tcm的双阴性T细胞的体外扩增方法，其特征在于，包括步骤：
    (a)提供一获自供体的外周血起始样品I；
    (b)对起始样品I进行预处理，从而获得样品II；
    (c)在含有适合DNT细胞生长的培养基的培养体系中，培养样品II，从而获得样品III；其中，所述培养体系中添加选自下组细胞因子中的一种或多种：
    5-50ng/ml重组人白介素21、1-10ng/ml重组人白介素1β、5-50ng/ml重组人白介素7、5-50ng/ml重组人白介素15、5-50ng/ml重组人白介素12；
    其中，步骤(c)中，所述培养体系中不添加抗人CD3抗体。
  2. 如权利要求1所述的方法，其特征在于，步骤(c)还包括以下步骤：
    (d)在含有适合DNT细胞生长的培养基的培养体系中，培养样品III，从而获得所需量的富含Tscm和Tcm的DNT细胞，为样品IV；其中，所述培养体系中添加选自下组细胞因子中的一种或多种：
    5-50ng/ml重组人白介素7、5-50ng/ml重组人白介素12或5-50ng/ml重组人白介素15；和
    (e)在含有适合DNT细胞保存的溶液体系中，收集样品IV。
  3. 如权利要求1所述的方法，其特征在于，步骤(b)中的预处理包括：
    (b1)去除起始样品I中的CD4+和CD8+T细胞，从而获得去除CD4+和CD8+的起始样品I；
    (b2)在含有适合DNT细胞生长的培养体系中，用抗人CD3单克隆抗体激活去除CD4+和CD8+的起始样品I，从而获得样品II。
  4. 如权利要求1所述的方法，其特征在于，步骤(b)中，所述去除CD4+和CD8+的起始样品I中的细胞数为N0；
    步骤(c)中，样品III中DNT细胞的数量为N1；
    步骤(d)中，样品IV中DNT细胞的数量为N2，其中，
    N1/N0≥50；较佳地≥70；更佳地≥100；更佳地≥200；
    N2/N0≥200；较佳地≥500；更佳地≥1000；更佳地≥10000。
  5. 如权利要求1所述的方法，其特征在于，步骤(c)中，所述培养天数选自5-21天，较佳地6-17天，更佳地7-14天。
  6. 如权利要求1所述的方法，其特征在于，步骤(b)、(c)、和(d)中，所述适合DNT细胞生长的培养基的培养体系中含有选自下组的血清替代物：ICSR(Immune Cell Serum Replacement)、KSR(KnockOut TMSerum Replacement)。
  7. 一种DNT细胞群，其特征在于，所述DNT细胞群是由权利要求1所述的方法制备的。
  8. 如权利要求7所述的DNT细胞群，其特征在于，所述DNT细胞群具有选自下组的一个或多个特征：
    (a1)40％-80％的细胞为Tscm细胞；
    (b1)10％-40％的细胞为Tcm细胞；或
    (a2)45％-75％的细胞为Tscm细胞；
    (b2)5％-35％的细胞为Tcm细胞；或
    (a3)30％-60％的细胞为Tscm细胞；
    (b3)15％-40％的细胞为Tcm细胞；或
    (a4)20％-40％的细胞为Tscm细胞；
    (b4)20％-30％的细胞为Tcm细胞。
  9. 如权利要求7所述的DNT细胞群，其特征在于，所述DNT细胞(CD3 +)纯度(％)≥80％；较佳地≥90％；更佳地≥95％；更佳地≥97％；和/或
    所述DNT细胞(CD3 +CD4 -CD8 -)纯度(％)≥85％；较佳地≥90％；更佳地≥95％；更佳地≥97％。
  10. 如权利要求7所述的DNT细胞在制备一药物组合物或制剂中的用途，所述药物组合物或制剂用于：
    (a)预防和/或治疗肿瘤；
    (b)预防和/或治疗感染性疾病；
    (c)预防和/或治疗自身免疫性疾病；
    (d)预防和/或治疗移植物抗宿主疾病；和/或
    (e)调节免疫应答。
  11. 如权利要求10所述的用途，其特征在于，所述肿瘤选自下组：血液肿瘤、实体瘤、或其组合。
  12. 如权利要求10所述的用途，其特征在于，所述血液肿瘤选自下组：淋巴瘤(Hodgkins和非Hodgkins)、急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、慢性髓性白血病(CML)、慢性骨髓单核细胞性白血病(CMML)、骨髓增生异常综合征(MDS)，或其组合。
  13. 如权利要求10所述的用途，其特征在于，所述实体瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、黑素瘤、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、头颈癌、胰腺癌，或其组合。
  14. 如权利要求11所述的用途，其特征在于，所述自身免疫性疾病包括：糖尿病、类风湿关节炎、多发性硬化、系统性红斑狼疮、恶心贫血、溶血性贫血、自身免疫性血小板减少、自身免疫性肝病、强直性脊柱炎、重症肌无力、Ig A肾病、原发肾冰综合征、银屑病、白癜风。
  15. 一种细胞制剂，其特征在于，所述细胞制剂含有如权利要求7所述的DNT细胞群。
  16. 一种适合DNT细胞生长的培养基，其特征在于，所述培养基包含选自下组细胞因子中的一种或多种：
    5-50ng/ml重组人白介素21、1-10ng/ml重组人白介素1β、5-50ng/ml重组人白介素7、5-50ng/ml重组人白介素15、5-50ng/ml重组人白介素12；
    其中，所述培养基中不添加抗人CD3抗体。
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